Yaakov N., Cohen R., Ravi Y., Mani K. A., Harush I., Dadosh T., Brontvein O., Zelinger E. & Mechrez G. (2025) Colloids and Surfaces A: Physicochemical and Engineering Aspects. 705, 135684
We have developed a new and facile approach for synthesis, labeling, and characterizing amphiphilic nanoscale Janus particles. The aqueous dispersion of the Aerodisp W1226 fumed silica particles were embedded into polycarbonate (P.C.) microspheres surfaces via inverse solvent displacement method and further primary functionalization by an amine group. Subsequently, the polymer was dissolved, and a second modification was preferred to introduce a thiol group on the revealed embedded side of the particles, forming silica-based Janus/amphiphilic particles. We first characterized the Janus particles by laser scanning confocal microscopy and then with direct stochastic optical reconstruction microscopy (dSTORM) to detect the labeling distribution of the different functional groups. Super-resolution imaging results confirmed that the fabricated particles are Janus particles with different chemical properties on each hemisphere. The particles were also characterized by scanning and transmission electron microscopy for size and shape. The results showed aspherical particles with two hemispheres, confirming successful fabrication and chemical modification. Also, this work successfully demonstrated the synthesis, characterization and analysis of nanoscale Janus/amphiphilic particles nanoscale Janus particle.
Sigal N., Lichtenstein-Wolfheim R., Schlussel S., Azulay G., Borovok I., Holdengraber V., Elad N., Wolf S. G., Zalk R., Zarivach R., Frank G. A. & Herskovits A. A. (2024) Nature Microbiology. 9, 10, p. 2727-2737
Tailocins are phage tail-like bacteriocins produced by various bacterial species to kill kin competitors. Given that tailocin release is dependent upon cell lysis, regulation of tailocin production at the single-cell and population level remains unclear. Here we used flow cytometry, competition assays and structural characterization of tailocin production in a human bacterial pathogen, Listeria monocytogenes. We revealed that a specialized subpopulation, constituting less than 1% of the total bacterial population, differentiates to produce, assemble and store thousands of tailocin particles. Tailocins are packed in a highly ordered manner, clustered in a liquid crystalline phase that occupies a substantial volume of the cell. Tailocin production confers a competitive growth advantage for the rest of the population. This study provides molecular insights into tailocin production as a form of altruism, showing how cell specialization within bacterial populations can confer competitive advantages at the population level.
Aram L., de Haan D., Varsano N., Gilchrist J. B., Heintze C., Rotkopf R., Rechav K., Elad N., Kröger N. & Gal A. (2024) Nature Communications. 15, 7888
Silica cell-wall formation in diatoms is a showcase for the ability of organisms to control inorganic mineralization. The process of silicification by these unicellular algae is tightly regulated within a membrane-bound organelle, the silica deposition vesicle (SDV). Two opposing scenarios were proposed to explain the tight regulation of this intracellular process: a template-mediated process that relies on preformed scaffolds, or a template-independent self-assembly process. The present work points to a third scenario, where the SDV membrane is a dynamic mold that shapes the forming silica. We use in-cell cryo-electron tomography to visualize the silicification process in situ, in its native-state, and with a nanometer-scale resolution. This reveals that the plasma membrane interacts with the SDV membrane via physical tethering at membrane contact sites, where the curvature of the tethered side of the SDV membrane mirrors the intricate silica topography. We propose that silica growth and morphogenesis result from the biophysical properties of the SDV and plasma membranes.
Orekhov N., Bukhtiiarova N., Brushevich Z. A., Muravev A. A., Nadav E., Tsarfati Y., Kossoy A., Feldman I., Zelenina A., Rubekina A. A., Semenov S. N. & Skorb E. V. (2024) Chemical Communications. 60, 77, p. 10680-10683
Herein, we obtained two supramolecular assemblies with layered structures from melamine, N-methylmelamine, and hexynyl-cyanuric acid in water. By combination of X-ray diffraction, electron microscopy, and molecular dynamics studies, we found that introducing one methyl group in melamine alters the arrangement of the layers in these structures.
Eyal Z., Gorelick-Ashkenazi A., Deis R., Barzilay Y., Broder Y., Kellum A. P., Varsano N., Hartstein M., Sorrentino A., Kaplan-Ashiri I., Rechav K., Metzler R., Houben L., Kronik L., Rez P. & Gur D. (2024) BioRxiv. 2024.07.20
Many animals exhibit remarkable colors produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These systems are utilized for various purposes, including vision, camouflage, communication, and thermal regulation. Each guanine crystal forms within a membrane-bound organelle called an iridosome, where precise control over crystal formation occurs. While the presence of guanine crystals in iridosomes is well-documented, the mechanisms facilitating the accumulation of water-insoluble guanine and driving its crystallization remain unclear. Here, we employ advanced imaging and spectroscopy techniques to characterize the maturation of iridosomes in zebrafish iridophores during development. Using cryo-electron microscopy, we found that amorphous guanine accumulates in early-stage iridosomes. Synchrotron-based soft X-ray microscopy studies revealed that, unlike mature crystals, the accumulated guanine is initially in its protonated state. Live imaging with a pH sensor demonstrated that early-stage iridosomes are acidic and that their pH gradually approaches neutrality during maturation. Additionally, the application of a V-ATPase inhibitor reduced the acidity of iridosomes and significantly decreased crystal formation, suggesting the involvement of V-ATPase in regulating the organelle pH. Our findings reveal new insights into the molecular mechanisms facilitating guanine accumulation and crystallization within iridosomes, emphasizing the pivotal role of pH alternations in the precise formation of biogenic crystals.Competing Interest StatementThe authors have declared no competing interest.
Gallina P., Kvapil M., Konečná A., Horák M., Bitton O., Houben L., Křápek V., Haran G., Idrobo J. C. & Šikola T. (2024) Microscopy and Microanalysis. 30, Supplement_1, ozae044.73
In recent years, strong coupling between different types of surface excitations, for instance, surface plasmon polaritons with excitons or phonon polaritons, has become a subject of growing interest. It has been caused by both fundamental and application research reasons covering new quantum nano-optics effects, electromagnetically induced transparency (EMT), chemical dynamics and reactivity, and other novel issues. Such an interesting physical phenomenon associated with the Rabi splitting and creation of new hybrid modes has been mostly studied by conventional reflection/transmission optical spectroscopic methods. However, using these techniques not all modes can be generated and observed. Therefore, there are attempts to study these coupling effects by application of electron beams capable of generating sub-radiant dark modes and being suitable for their detection by electron energy loss spectroscopy (EELS). To do it both at sufficient spatial (< 10 nm) and energy (≤ 100 eV) resolutions, STEM-EELS systems with monochromatized probe electron beams have to be used.
Song M. S., Houben L., Zhao Y., Bae H., Rothem N., Gupta A., Yan B., Kalisky B., Zaluska-Kotur M., Kacman P., Shtrikman H. & Beidenkopf H. (2024) Nature Nanotechnology.
Due to quasi-one-dimensional confinement, nanowires possess unique electronic properties, which can promote specific device architectures. However, nanowire growth presents paramount challenges, limiting the accessible crystal structures and elemental compositions. Here we demonstrate solid-state topotactic exchange that converts wurtzite InAs nanowires into Zintl Eu<sub>3</sub>In<sub>2</sub>As<sub>4</sub>. Molecular-beam-epitaxy-based in situ evaporation of Eu and As onto InAs nanowires results in the mutual exchange of Eu from the shell and In from the core. Therefore, a single-phase Eu<sub>3</sub>In<sub>2</sub>As<sub>4</sub> shell grows, which gradually consumes the InAs core. The mutual exchange is supported by the substructure of the As matrix, which is similar across the wurtzite InAs and Zintl Eu<sub>3</sub>In<sub>2</sub>As<sub>4</sub> and therefore is topotactic. The Eu<sub>3</sub>In<sub>2</sub>As<sub>4</sub> nanowires undergo an antiferromagnetic transition at a Néel temperature of ~6.5 K. Ab initio calculations confirm the antiferromagnetic ground state and classify Eu<sub>3</sub>In<sub>2</sub>As<sub>4</sub> as a C<sub>2</sub>T axion insulator, hosting both chiral hinge modes and unpinned Dirac surface states. The topotactic mutual-exchange nanowire growth will, thus, enable the exploration of intricate magneto-topological states in Eu<sub>3</sub>In<sub>2</sub>As<sub>4</sub> and potentially in other exotic compounds.
Klar P. B., Waterman D. G., Gruene T., Mullick D., Song Y., Gilchrist J. B., Owen C. D., Wen W., Biran I., Houben L., Regev-Rudzki N., Dzikowski R., Marom N., Palatinus L., Zhang P., Leiserowitz L. & Elbaum M. (2024) ACS Central Science. 10, 8, p. 1504-1514
Detoxification of heme in Plasmodium depends on its crystallization into hemozoin. This pathway is a major target of antimalarial drugs. The crystalline structure of hemozoin was established by X-ray powder diffraction using a synthetic analog, β-hematin. Here, we apply emerging methods of in situ cryo-electron tomography and 3D electron diffraction to obtain a definitive structure of hemozoin directly from ruptured parasite cells. Biogenic hemozoin crystals take a striking polar morphology. Like β-hematin, the unit cell contains a heme dimer, which may form four distinct stereoisomers: two centrosymmetric and two chiral enantiomers. Diffraction analysis, supported by density functional theory analysis, reveals a selective mixture in the hemozoin lattice of one centrosymmetric and one chiral dimer. Absolute configuration has been determined by morphological analysis and confirmed by a novel method of exit-wave reconstruction from a focal series. Atomic disorder appears on specific facets asymmetrically, and the polar morphology can be understood in light of water binding. Structural modeling of the heme detoxification protein suggests a function as a chiral agent to bias the dimer formation in favor of rapid growth of a single crystalline phase. The refined structure of hemozoin should serve as a guide to new drug development.
Kadam S. R., Krishnappa M., Ghosh S., Sreedhara M. B., Neyman A., Upcher A., Nativ Roth E., Houben L., Zak A., Enyashin A. N., Bar-Ziv R. & Bar-Sadan M. (2024) Applied Materials Today. 39, 102288
Vanadium sulfide (VS<sub>2</sub>) is a layered transition metal dichalcogenide (TMD), comparable in crystal structure to the well-known MoS<sub>2</sub> and WS<sub>2</sub>. Theoretical predictions attribute much potential to VS<sub>2</sub>, since it is metallic-like and has an active basal plane, essential for catalytic performance. However, it is much less studied than other members of the TMD family due to the difficulties in synthesizing specific structures with controlled properties. Here we present unique structures of VS<sub>2</sub> nanotubes and conduct a comparative study with other well-known inorganic nanotubes and nanostructures of MoS<sub>2</sub> and WS<sub>2</sub>. We evaluate the effect of the curvature and strain, the abundance of surface defects, and the availability of surface sites in various structures by electrochemical methods. We show that MoS<sub>2</sub> has the best intrinsic activity, which is enhanced by an extensive electrochemical surface area. The woven-like structure of the MoS<sub>2</sub> nanotube walls provides a combined effect of strain, crystallinity, and defects. For WS<sub>2</sub> structures, the strained surface of the nanotubes results in sites with higher intrinsic activity than the edge sites, but structures such as the nano-triangles, which provide a higher number of edge sites, exhibit competing activity. As for the VS<sub>2</sub> structures, although theoretical calculations predict optimal active sites for the hydrogen evolution reaction (HER), they are extremely sensitive to stoichiometry variations that hamper their catalytic activity. Our findings contribute insights to the improvement and design of VS<sub>2</sub>based nanocatalysts for the HER and shed light on the general factors that govern the activity in the unique TMD nanotubes family.
Palakurthy S., Houben L., Elbaum M. & Elbaum R. (2024) Biomacromolecules. 25, 6, p. 3409-3419
Plants undergo substantial biomineralization of silicon, which is deposited primarily in cell walls as amorphous silica. The mineral formation could be moderated by the structure and chemistry of lignin, a polyphenol polymer that is a major constituent of the secondary cell wall. However, the reactions between lignin and silica have not yet been well elucidated. Here, we investigate silica deposition onto a lignin model compound. Polyphenyl propanoid was synthesized from coniferyl alcohol by oxidative coupling with peroxidase in the presence of acidic tetramethyl orthosilicate, a silicic acid precursor. Raman, Fourier transform infrared, and X-ray photoelectron spectroscopies detected changes in lignin formation in the presence of silicic acid. Bonds between the Si-O/Si-OH residues and phenoxyl radicals and lignin functional groups formed during the first 3 h of the reaction, while silica continued to form over 3 days. Thermal gravimetric analysis indicated that lignin yields increased in the presence of silicic acid, possibly via the stabilization of phenolic radicals. This, in turn, resulted in shorter stretches of the lignin polymer. Silica deposition initiated within a lignin matrix via the formation of covalent Si-O-C bonds. The silica nucleants grew into 2-5 nm particles, as observed via scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy. Additional silica precipitated into an extended gel. Collectively, our results demonstrate a reciprocal relation by which lignin polymerization catalyzes the formation of silica, and at the same time silicic acid enhances lignin polymerization and yield.
Alus L., Houben L., Shaked N., Niazov-Elkan A., Pinkas I., Oron D. & Addadi L. (2024) Advanced Materials. 36, 28, 2308832
Spherical particles with diameters within the wavelength of visible light, known as spherulites, manipulate light uniquely due to their spatial organization and their structural birefringence. Most of the known crystalline spherulites are branched, and composed of metals, alloys, and semi-crystalline polymers. Recently, a different spherulite architecture is discovered in the vision systems of decapod crustaceans - core-shell spherulites composed of highly birefringent ((Formula presented.)) organic single-crystal platelets, with exceptional optical properties. These metastructures, which efficiently scatter light even in dense aqueous environments, have no synthetic equivalence and serve as a natural proof-of-concept as well as synthetic inspiration for thin scattering media. Here, the synthesis of core-shell spherulites composed of guanine crystal platelets (((Formula presented.)) is presented in a two-step emulsification process in which a water/oil/water emulsion and induced pH changes are used to promote interfacial crystallization. Carboxylic acids neutralize the dissolved guanine salts to form spherulites composed of single, radially stacked, β-guanine platelets, which are oriented tangentially to the spherulite surface. Using Mie theory calculations and forward scattering measurements from single spherulites, it is found that due to the single-crystal properties and orientation, the synthetic spherulites possess a high tangential refractive index, similarly to biogenic particles.
Freidzon D., Wachtel E., Cohen H., Houben L., Kossoy A., Brontvein O., Varenik M., Frenkel A. I., Ehre D. & Lubomirsky I. (2024) Solid State Ionics. 411, 116572
Coupling between an electrochemical reaction and a functional material property has been termed electro-chemo-X, or EC-X, where X can refer to mechanical, optical, magnetic or thermal properties. Recently, our group has demonstrated a two-terminal electro-chemo-mechanical (ECM) membrane actuator operating under ambient conditions and containing a Ce<sub>0.8</sub>Gd<sub>0.2</sub>O<sub>1.9</sub> solid electrolyte layer sandwiched between two Gd-doped ceria/TiO<sub>x</sub> nanocomposite thin films. Reducing one nanocomposite film while oxidizing the other was observed to produce reversible volume change thereby driving membrane actuator operation. Here, we use the same electrolyte and nanocomposite layer pair (the upper one as the ion reservoir and the lower, as the active layer) to further explore the EC-X effect. We demonstrate the suitability of the nanocomposite for a three-terminal, thin film-based resistivity switch. We find that application of ±6 V ( Ce<sup>+4</sup> is similarly effective in leading to increased nanocomposite conductivity, while reduction produces the opposite effect. With the expectation that the response time can be significantly shortened, the proposed resistivity switch may be suitable for future applications such as sensors, neuromorphic computing or spintronics.
Biran I., Houben L., Kossoy A. & Rybtchinski B. (2024) Journal of Physical Chemistry C. 128, 14, p. 5988-5995
The crystallinity of polymeric materials defines their properties, in particular, the mechanical ones. High-resolution transmission electron microscopy (TEM) imaging of polymers would be critical to address intricate polymer crystallinity, yet it is challenging due to polymer sensitivity to the electron beam. We performed high-resolution TEM imaging of polycaprolactone (PCL) thin films employing low-dose focal series reconstruction (LDFSR). LDFSR enabled submolecular resolution imaging of polymer crystals. The direct imaging study was augmented by scanning nanobeam electron diffraction (NBED) using the 4D STEM technique to map micro- and nanoscale crystalline domains. Employing LDFSR combined with 4D STEM, we directly observed interacting polymer chains in the crystal lattice, elucidating the crystal structure with a high degree of precision including lattice deformations. We also imaged PCL lamella using conventional TEM. Our methodology enables long-sought insights into the polymer structure, introducing a new tool for high resolution studies of polymer crystallinity that fills a critical gap in the structural science of polymer materials.
Hegner F. S., Cohen A., Rudel S. S., Kronawitter S. M., Grumet M., Zhu X., Korobko R., Houben L., Jiang C. M., Schnick W., Kieslich G., Yaffe O., Sharp I. D. & Egger D. A. (2024) Advanced Energy Materials. 14, 19, 2303059
Ternary nitride semiconductors are rapidly emerging as a promising class of materials for energy conversion applications, offering an appealing combination of strong light absorption in the visible range, desirable charge transport characteristics, and good chemical stability. In this work, it is shown that finite-temperature lattice dynamics in CuTaN<sub>2</sub> a prototypical ternary nitride displaying particularly strong visible light absorption exhibit a pronounced anharmonic character that plays an essential role in defining its macroscopic optoelectronic and thermal properties. Low-frequency vibrational modes that are Raman-inactive from symmetry considerations of the average crystal structure and unstable in harmonic phonon calculations are found to appear as intensive Raman features near room temperature. The atomic contributions to the anharmonic vibrations are characterized by combining Raman measurements with molecular dynamics and density functional theory calculations. This analysis reveals that anharmonic lattice dynamics have large ramifications on the fundamental properties of this compound, resulting in uniaxial negative thermal expansion and the opening of its bandgap to a near-optimal value for solar energy harvesting. The atomic-level understanding of anharmonic lattice dynamics, as well as the finding that they strongly influence key properties of this semiconductor at room temperature, have important implications for design of new functional materials, especially within the emerging class of ternary nitride semiconductors.
Seifer S., Houben L. & Elbaum M. (2024) Ultramicroscopy. 259, 113936
We demonstrate the use of a 4-dimensional scanning transmission electron microscope (4D-STEM) to extract atomic cross section information in amorphous materials. We measure the scattering amplitudes of 200 keV electrons in several representative specimens: amorphous carbon, silica, amorphous ice of pure water, and vitrified phosphate buffer solution. Diffraction patterns are recorded by 4D-STEM with or without energy filter at the zero-loss peak. In addition, Electron Energy Loss Spectroscopy (EELS) data are acquired at several thicknesses and energies. Mixed elastic and inelastic contributions for thick samples can be decoupled based on a convolution model. Measured differential cross sections between 1 and 3 mrad are due primarily to plasmon excitations and follow precisely a 1/θ<sup>2</sup> angular distribution. The measured intensities match Inokuti's calculations of total dipole matrix elements for discrete dipole transitions alone, i.e., transitions to bound states of the atom and not to continuum states. We describe the fundamental mechanism of plasmon excitation in insulators as a two-step interaction process with a fast electron. First, a target electron in the specimen is excited, the probability for which follows from the availability of atomic transitions, with a strong dependence on the column of the periodic table. Second, the dielectric response of the material determines the energy loss. The energy of the loss peak depends primarily on the valence electrons. Elastic scattering is dominant at higher angles, and can be fitted conveniently to 1/θ<sup>3.7</sup> with a linear dependence on atomic number for light atoms. In order to facilitate the interpretation of 4D STEM measurements in terms of material composition, we introduce two key parameters. Zeta is an analytical equivalent of classical STEM Z-contrast, determined by the ratio of elastic to inelastic scattering coefficients, while eta is the elastic coefficient divided by thickness. The two parameters may serve for identification of basic classes of materials in biological and other amorphous organic specimens.
Shalom B. O., Andrés M. A., Head A. R., Epstein B. Z., Brontvein O., Pérez-Dieste V., Villar-Garcia I. J., Walton A. S., Polus K., Weatherup R. S. & Eren B. (2024) Cell Reports Physical Science. 102165
The chemical state of nickel anodes during the oxygen evolution reaction can impact their electrocatalytic performance. Here, X-ray photoelectron and absorption spectroscopies reveal the chemical state of nickel nanoparticles under oxygen evolution reaction conditions in a mildly alkaline carbonate-bicarbonate buffer solution. Ni<sup>2+</sup> and Ni<sup>3+</sup> species are observed at the reaction onset potential with a 7:4 ratio, with no remaining metallic nickel. These species include NiO, which increasingly converts to other Ni<sup>2+</sup> and Ni<sup>3+</sup> species once the potential is increased above the onset potential. Conversely, when a 20-nm-thick nickel film is used instead of nickel nanoparticles, a significant amount of metallic nickel remains in the inner layers. Nickel nanoparticles also undergo significant morphological and structural changes during the reaction, as evidenced by ex situ transmission electron microscopy. Amorphization of the nanoparticles is attributed to significant H<sub>2</sub>O incorporation, with the oxygen intensity increasing both in operando and ex situ measurements.
Zhu Q., Cohen S. R., Brontvein O., Fransson J. & Naaman R. (2024) Small. 2406631
Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted wide attention due to their promising applications in biomedicine, chemical catalysis, and magnetic memory devices. In this work, the force is measured between a single SPION coated with chiral molecules and a ferromagnetic substrate by atomic force microscopy (AFM), with the substrate magnetized either toward or away from the approaching AFM tip. The force between the coated SPION and the magnetic substrate depends on the handedness of the molecules adsorbed on the SPION and on the direction of the magnetization of the substrate. By inserting nm-scale spacing layers between the coated SPION and the magnetic substrate it is shown that the SPION has a short-range magnetic monopole-like magnetic field. A theoretical framework for the nature of this field is provided.
Israel L., Furer V., Levin-Zaidman S., Dezorella N., Brontvein O., Ablin J. N. & Gross A. (2024) Clinical and Experimental Rheumatology. 42, 6, p. 1215-1223
Objectives: The pathogenesis of fibromyalgia (FM), characterised by chronic widespread pain and fatigue, remains notoriously elusive, hampering attempts to develop disease modifying treatments. Mitochondria are the headquarters of cellular energy metabolism, and their malfunction has been proposed to contribute to both FM and chronic fatigue. Thus, the aim of the current pilot study, was to detect structural changes in mitochondria of peripheral blood mononuclear cells (PBMCs) of FM patients, using transmission electron microscopy (TEM).Methods: To detect structural mitochondrial alterations in FM, we analysed PBMCs from seven patients and seven healthy controls, using TEM. Patients were recruited from a specialised Fibromyalgia Clinic at a tertiary medical centre. After providing informed consent, participants completed questionnaires including the widespread pain index (WPI), symptoms severity score (SSS), fibromyalgia impact questionnaire (FIQ), beck depression inventory (BDI), and visual analogue scale (VAS), to verify a diagnosis of FM according to ACR criteria. Subsequently, blood samples were drawn and PBMCs were collected for EM analysis.Results: TEM analysis of PBMCs showed several distinct mitochondrial cristae patterns, including total loss of cristae in FM patients. The number of mitochondria with intact cristae morphology was reduced in FM patients and the percentage of mitochondria that completely lacked cristae was increased. These results correlated with the WPI severity. Moreover, in the FM patient samples we observed a high percentage of cells containing electron dense aggregates, which are possibly ribosome aggregates. Cristae loss and possible ribosome aggregation were intercorrelated, and thus may represent reactions to a shared cellular stress condition. The changes in mitochondrial morphology suggest that mitochondrial dysfunction, resulting in inefficient oxidative phosphorylation and ATP production, metabolic and redox disorders, and increased reactive oxygen species (ROS) levels, may play a pathogenetic role in FM.Conclusions: We describe novel morphological changes in mitochondria of FM patients, including loss of mitochondrial cristae. While these observations cannot determine whether the changes are pathogenetic or represent an epiphenomenon, they highlight the possibility that mitochondrial malfunction may play a causative role in the cascade of events leading to chronic pain and fatigue in FM. Moreover, the results offer the possibility of utilising changes in mitochondrial morphology as an objective biomarker in FM. Further understanding the connection between FM and dysfunction of mitochondria physiology, may assist in developing both novel diagnostic tools as well as specific treatments for FM, such as approaches to improve/strengthen mitochondria function.
Zhao Z., Pinkas I., Zhang C., Xiao Y., Sui X., Brontvein O. & Li H. (2024) ACS Applied Nano Materials. 7, 8, p. 9609-9615
The sixth-generation wireless communication (6G) extends the electromagnetic pollution up to the terahertz band. The two-dimensional titanium carbide of intercalated structure Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> MXene attracts much attention because it exhibits high terahertz electromagnetic interference (EMI) shielding effectiveness (SE) due to the large intrinsic electrical conductivity as well as few-atom monosheet thickness. Scientists worldwide are making continuous efforts to optimize the MXene structures for EMI shielding application. Innovatively, we demonstrate a chemical method to bend the nanoflake by grafting two types of alkane, octane (C<sub>8</sub>H<sub>18</sub>) and dodecane (C<sub>12</sub>H<sub>26</sub>), onto the surface terminals. The chain length of alkane exceeds the bond length of surface functionalities T<sub>x</sub> (=O, −OH, −F) to introduce intrananoflake and internanoflake strains into Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> MXene. The nanoflakes deformation leads to Raman peak redshift and broader line width. The element distribution shows that the alkane increases the oxygen-fluorine ratio of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> MXene from 3:2 to 3:1 (Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>-C<sub>8</sub>H<sub>18</sub>) and even up to 4:1 (Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>-C<sub>12</sub>H<sub>26</sub>). Electronic microscopy (SEM/TEM) shows obvious edge-fold and tensile/compressive deformation of the nanoflake. The EMI SE of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>-C<sub>12</sub>H<sub>26</sub> achieves 35 dB, which is higher than those of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>-C<sub>8</sub>H<sub>18</sub> (26 dB) and Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> MXene (22 dB). The alkane grafting increases the absorption coefficient of the MXene thin film by more than 50% but has negligible contribution to the refractive index. Meanwhile, the conductivity of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>-C<sub>8</sub>H<sub>18</sub> MXene is over twice higher than that of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> MXene, whereas the conductivity of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub>-C<sub>12</sub>H<sub>26</sub> is three times higher than that of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> MXene. The nanoflake curvature of alkane grafted Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> MXene enlarges the specific surface area and causes topological defects, which increase the absorption as well as the conductivity so that the terahertz EMI SE is enhanced correspondingly. The realization of Ti<sub>3</sub>C<sub>2</sub>T<sub>x</sub> MXene of curved nanoflake for the enhancement of terahertz EMI SE is valuable for 6G electromagnetic protection.
Cohen D., Brontvein O. & Bar-Shir A. (2024) ACS Applied Nano Materials. 7, 7, p. 6791-6796
MgF<sub>2</sub>-based formulations have unique physicochemical properties, making them attractive for diverse applications. Here, we show the ability to control the morphology of Sm<sup>3+</sup>-doped, paramagnetic MgF<sub>2</sub> nanocrystals (Sm:MgF<sub>2</sub> NCs) for use as nanosized imaging agents for <sup>19</sup>F-magnetic resonance imaging (<sup>19</sup>F-MRI). By reducing the temperature of the reaction mixture from 160 to 110 °C and shortening the reaction time from 16 to 3 h, the morphology of the fabricated oleate-coated Sm:MgF<sub>2</sub> NCs was transitioned from rod-shaped to spherical-shaped nanofluorides, both with a characteristic high-resolution liquid-state <sup>19</sup>F-NMR resonance at −198 ppm. Further coating the surface of the spheric Sm:MgF<sub>2</sub> NCs with a phospholipid layer resulted in a water-soluble formulation that was used to show its detectability with <sup>19</sup>F-MRI.
Danieli Y., Sanders E., Brontvein O. & Joselevich E. (2024) ACS Applied Materials and Interfaces. 16, 2, p. 2637-2648
Infrared photodetectors are essential devices for telecommunication and night vision technologies. Two frequently used materials groups for this technology are III-V and II-VI semiconductors, notably, mercury-cadmium-telluride alloys (MCT). However, growing them usually requires expensive substrates that can only be provided on small scales, and their large-scale production as crystalline nanostructures is challenging. In this paper, we present a two-stage process for creating aligned MCT nanowires (NWs). First, we report the growth of planar CdTe nanowires with controlled orientations on flat and faceted sapphire substrates via the vapor-liquid-solid (VLS) mechanism. We utilize this guided growth approach to parallelly integrate the NWs into fast near-infrared photodetectors with characteristic rise and fall times of ∼100 μs at room temperature. An epitaxial effect of the planar growth and the unique structure of the NWs, including size and composition, are suggested to explain the high performance of the devices. In the second stage, we show that cation exchange with mercury can be applied, resulting in a band gap narrowing of up to 55 meV, corresponding to an exchange of 2% Cd with Hg. This work opens new opportunities for creating small, fast, and sensitive infrared detectors with an engineered band gap operating at room temperature.
Shatz O., Fraiberg M., Isola D., Das S., Gogoi O., Polyansky A., Shimoni E., Dadosh T., Dezorella N., Wolf S. G. & Elazar Z. (2024) Developmental Cell. 59, 7, p. 911-923.e4
Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle expansion. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy.
Ben-Hur S., Sernik S., Afar S., Kolpakova A., Politi Y., Gal L., Florentin A., Golani O., Sivan E., Dezorella N., Morgenstern D., Pietrokovski S., Schejter E., Yacobi-Sharon K. & Arama E. (2024) Nature Communications. 15, 5715
Mitochondria are maternally inherited, but the mechanisms underlying paternal mitochondrial elimination after fertilization are far less clear. Using Drosophila, we show that special egg-derived multivesicular body vesicles promote paternal mitochondrial elimination by activating an LC3-associated phagocytosis-like pathway, a cellular defense pathway commonly employed against invading microbes. Upon fertilization, these egg-derived vesicles form extended vesicular sheaths around the sperm flagellum, promoting degradation of the sperm mitochondrial derivative and plasma membrane. LC3-associated phagocytosis cascade of events, including recruitment of a Rubicon-based class III PI(3)K complex to the flagellum vesicular sheaths, its activation, and consequent recruitment of Atg8/LC3, are all required for paternal mitochondrial elimination. Finally, lysosomes fuse with strings of large vesicles derived from the flagellum vesicular sheaths and contain degrading fragments of the paternal mitochondrial derivative. Given reports showing that in some mammals, the paternal mitochondria are also decorated with Atg8/LC3 and surrounded by multivesicular bodies upon fertilization, our findings suggest that a similar pathway also mediates paternal mitochondrial elimination in other flagellated sperm-producing organisms.
Su M., Fleischer T., Grosheva I., Horev M. B., Olszewska M., Mattioli C. C., Barr H., Plotnikov A., Carvalho S., Moskovich Y., Minden M. D., Chapal-Ilani N., Wainstein A., Papapetrou E. P., Dezorella N., Cheng T., Kaushansky N., Geiger B. & Shlush L. I. (2024) iScience. 27, 4, 109443
Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.
Deis R., Lerer-Goldshtein T., Baiko O., Eyal Z., Brenman-Begin D., Goldsmith M., Kaufmann S., Heinig U., Dong Y., Lushchekina S., Varsano N., Olender T., Kupervaser M., Porat Z., Levin-Zaidman S., Pinkas I., Mateus R. & Gur D. (2024) Nature Chemical Biology.
Organisms evolve mechanisms that regulate the properties of biogenic crystals to support a wide range of functions, from vision and camouflage to communication and thermal regulation. Yet, the mechanism underlying the formation of diverse intracellular crystals remains enigmatic. Here we unravel the biochemical control over crystal morphogenesis in zebrafish iridophores. We show that the chemical composition of the crystals determines their shape, particularly through the ratio between the nucleobases guanine and hypoxanthine. We reveal that these variations in composition are genetically controlled through tissue-specific expression of specialized paralogs, which exhibit remarkable substrate selectivity. This orchestrated combination grants the organism with the capacity to generate a broad spectrum of crystal morphologies. Overall, our findings suggest a mechanism for the morphological and functional diversity of biogenic crystals and may, thus, inspire the development of genetically designed biomaterials and medical therapeutics. (Figure presented.).
Garty Y., Bussi Y., Levin-Zaidman S., Shimoni E., Kirchhoff H., Charuvi D., Nevo R. & Reich Z. (2024) Nature Plants. 10, 5, p. 830
Correction to: Nature Plantshttps://doi.org/10.1038/s41477-024-01628-9, published online 23 February 2024 In the version of the article initially published, there was an error in the protein orientations in Fig. 6, where the bulk orientation of the PSI did not face the stroma. This has now been amended in the HTML and PDF versions of the article.
Garty Y., Bussi Y., Levin-Zaidman S., Shimoni E., Kirchhoff H., Charuvi D., Nevo R. & Reich Z. (2024) Nature Plants. 10, 3, p. 512-524
The balance between linear electron transport (LET) and cyclic electron transport (CET) plays an essential role in plant adaptation and protection against photo-induced damage. This balance is largely maintained by phosphorylation-driven alterations in the PSIILHCII assembly and thylakoid membrane stacking. During the dark-to-light transition, plants shift this balance from CET, which prevails to prevent overreduction of the electron transport chain and consequent photo-induced damage, towards LET, which enables efficient CO2 assimilation and biomass production. Using freeze-fracture cryo-scanning electron microscopy and transmission electron microscopy of Arabidopsis leaves, we reveal unique membrane regions possessing characteristics of both stacked and unstacked regions of the thylakoid network that form during this transition. A notable consequence of the morphological attributes of these regions, which we refer to as stacked thylakoid doublets, is an overall increase in the proximity and connectivity of the two photosystems (PSI and PSII) that drive LET. This, in turn, reduces diffusion distances and barriers for the mobile carriers that transfer electrons between the two PSs, thereby maximizing LET and optimizing the plants ability to utilize light energy. The mechanics described here for the shift between CET and LET during the dark-to-light transition are probably also used during chromatic adaptation mediated by state transitions.
Goldman A., Mullokandov M., Zaltsman Y., Regev L., Levin-Zaidman S. & Gross A. (2024) EMBO Reports. 25, 1, p. 45-67
Fusion of the outer mitochondrial membrane (OMM) is regulated by mitofusin 1 (MFN1) and 2 (MFN2), yet the differential contribution of each of these proteins is less understood. Mitochondrial carrier homolog 2 (MTCH2) also plays a role in mitochondrial fusion, but its exact function remains unresolved. MTCH2 overexpression enforces MFN2-independent mitochondrial fusion, proposedly by modulating the phospholipid lysophosphatidic acid (LPA), which is synthesized by glycerol-phosphate acyl transferases (GPATs) in the endoplasmic reticulum (ER) and the OMM. Here we report that MTCH2 requires MFN1 to enforce mitochondrial fusion and that fragmentation caused by loss of MTCH2 can be specifically counterbalanced by overexpression of MFN2 but not MFN1, partially independent of its GTPase activity and mitochondrial localization. Pharmacological inhibition of GPATs (GPATi) or silencing ER-resident GPATs suppresses MFN2's ability to compensate for the loss of MTCH2. Loss of either MTCH2, MFN2, or GPATi does not impair stress-induced mitochondrial fusion, whereas the combined loss of MTCH2 and GPATi or the combined loss of MTCH2 and MFN2 does. Taken together, we unmask two cooperative mechanisms that sustain mitochondrial fusion.
Lansky Z., de Haan D., Piven Y., Rechav K. & Gal A. (2024) Advanced Science. 11, 41, 2402492
Organisms are able to control material patterning down to the nanometer scale. This is exemplified by the intricate geometrical patterns of the silica cell wall of diatoms, a group of unicellular algae. Theoretical and modeling studies propose putative physical and chemical mechanisms to explain morphogenesis of diatom silica. Nevertheless, direct investigations of the underlying formation process are challenging because this process occurs within the confines of the living cell. Here, a method is developed for in situ 3D visualization of silica development in the diatom Stephanopyxis turris, using electron microscopy slice-and-view techniques. The formation of an isotropic hexagonal pattern made of nanoscale pores is documented. Surprisingly, these data reveal a directional process that starts with elongation of silica rods along one of the three equivalent orientations of the hexagonal lattice. Only as a secondary step, these rods are connected by crisscrossing bridges that give rise to the complete hexagonal pattern. These in situ observations combine two known properties of diatom silica, close packing of pores and branching of rods, to a unified process that yields isotropic patterns from an anisotropic background. Future research into diatom morphogenesis should focus on rod elongation and branching as the key for pattern formation.
Zelinger E., Brumfeld V., Rechav K., Waiger D., Kossovsky T. & Heifetz Y. (2024) Communications Biology. 7, 155
In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. We address this using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, we were able to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. Our findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues.
Yonai E., Weiner S., Shimoni E. & Blonder R. (2024) International Journal of Science Education.
Authentic science learning has a significant potential for contributing to students self-efficacy (SE) and science career aspirations (SCA) by introducing contemporary science. However, the design of authentic learning remains challenging due to the elusive and subjective nature of experiencing authenticity and the interdisciplinarity and complexity of contemporary science. This study aimed to transform these challenges into opportunities by integrating theoretical frameworks of authentic design with a unique setting, including: (a) authentic pedagogy highlighting social experiences, (b) authentic scientific equipment including hands-on operation of a scanning electron microscope (SEM) situated in a research institute, (c) contemporary science theme (biomineralization). This integrated design was investigated by collecting data from 70 secondary science students on their perceived-authenticity, SE, and SCA using prepost Likert-type questionnaires. After discovering that authenticity was intensely perceived, we used inferential statistics to examine the relationships between different authentic aspects related to both hard and soft science skills and SE and SCA. We identified a significant connection between students experience of acquiring authentic knowledge (i.e. hard skills) and their SCA, as well as between the social experience (i.e. soft skills) and students SE. This study contributes to understanding the design and impact of authentic science activities in research institutes.
Brookstein O., Shimoni E., Eliaz D., Kaplan-Ashiri I., Carmel I. & Shimanovich U. (2024) Nature Communications. 15, 6671
Silk fibers unique mechanical properties have made them desirable materials, yet their formation mechanism remains poorly understood. While ions are known to support silk fiber production, their exact role has thus far eluded discovery. Here, we use cryo-electron microscopy coupled with elemental analysis to elucidate the changes in the composition and spatial localization of metal ions during silk evolution inside the silk gland. During the initial protein secretion and storage stages, ions are homogeneously dispersed in the silk gland. Once the fibers are spun, the ions delocalize from the fibroin core to the sericin-coating layer, a process accompanied by protein chain alignment and increased feedstock viscosity. This change makes the protein more shear-sensitive and initiates the liquid-to-solid transition. Selective metal ion doping modifies silk fibers mechanical performance. These findings enhance our understanding of the silk fiber formation mechanism, laying the foundations for developing new concepts in biomaterial design.
Kaslasi H., Rakita Y., Kaplan-Ashiri I., Hodes G. & Bendikov T. (2024) Crystal Growth and Design. 24, 15, p. 6421-6430
Interest in halide perovskites (HaPs) is motivated by the combination of superior optoelectronic properties, ease of synthesis, and a surprisingly low density of electrically active defects. HaPs possess high chemical sensitivity, especially those having an organic cation at their A position (AMX<sub>3</sub>). X-ray photoelectron spectroscopy (XPS) is a surface technique with sensitivity that goes down to a single atomic layer and provides unique information that relates the elemental composition with the chemical and electronic states of the elements in the material. Our study focuses on XPS imaging in combination with selected small-area spectra and uses aged (3 years old) solution-grown single crystals of mixed A-cation Cs<sub>x</sub>MA<sub>1-x</sub>PbBr<sub>3</sub> (MA = CH<sub>3</sub>NH<sub>3</sub><sup>+</sup>) HaPs as a candidate for investigating intracrystal heterogeneity. With XPS, we followed the variations in chemical composition by measuring crystals at different regions down to 50 μm diameter of the samples. By comparing the surface of the crystals with their cross-section, we found significant changes in the Cs<sup>+</sup> and Br<sup>-</sup> concentrations, which increase toward the interior of the crystal. Contrarily, concentrations of carbon and nitrogen predominate on the top surface and especially at crystal edges, which form a partial covering of the crystals beyond the visible crystal boundaries, something that is not seen by electron microscopy analysis and shows the advantage of the XPS for measurements of light elements. Besides demonstrating the utility of the XPS technique, this compositional heterogeneity within the Cs<sub>x</sub>MA<sub>1-x</sub>PbBr<sub>3</sub> crystals reveals novel insights into the complex chemical nature of what may be seen as uniform single crystals and brings crucial information for their understanding.
Kuperman O. A., de Andrade P., Sui X. M., Maria R., Kaplan-Ashiri I., Jiang Q., Terlier T., Kirkensgaard J. J. K., Field R. A. & Natalio F. (2024) Cell Reports Physical Science. 5, 5, 101963
Cotton ovule in vitro cultures are a promising platform for exploring biofabrication of fibers with tailored properties. When the ovules' growth medium is supplemented with chemically synthesized cellulose precursors, it results in their integration into the developing fibers, thereby tailoring their end properties. Here, we report the feeding of synthetic glucosyl phosphate derivative, 6-deoxy-6-fluoro-glucose-1-phosphate (6F-Glc-1P) to cotton ovules growing in vitro, demonstrating the metabolic incorporation of 6F-Glc into the fibers with enhanced mechanical properties and moisture-retention capacity while emphasizing the role of molecular hierarchical architecture in defining functional characteristics and mechanical properties. This incorporation strategy bypasses the early steps of conventional metabolic pathways while broadening the range of functionalities that can be employed to customize fiber end properties. Our approach combines materials science, chemistry, and plant sciences to illustrate the innovation required to find alternative solutions for sustainable production of functional cotton fibers with enhanced and emergent properties.
Cohen A., Li J., Cohen H., Kaplan-Ashiri I., Khodorov S., Wachtel E. J., Lubomirsky I., Frenkel A. I. & Ehre D. (2024) ACS Applied Electronic Materials. 6, 2, p. 853-861
The local environments of Sc and Y in predominantly ⟨002⟩ textured, Al<sub>1-x</sub>Do<sub>x</sub>N (Do = Sc, x = 0.25, 0.30 or Y, x = 0.25) sputtered thin films with wurtzite symmetry were investigated using X-ray absorption (XAS) and photoelectron (XPS) spectroscopies. We present evidence from the X-ray absorption fine structure (XAFS) spectra that, when x = 0.25, both Sc<sup>3+</sup> and Y<sup>3+</sup> ions are able to substitute for Al<sup>3+</sup>, thereby acquiring four tetrahedrally coordinated nitrogen ligands, i.e., coordination number (CN) of 4. On this basis, the crystal radius of the dopant species in the wurtzite lattice, not available heretofore, could be calculated. By modeling the scandium local environment, extended XAFS (EXAFS) analysis suggests that when x increases from 0.25 to 0.30, CN for a fraction of the Sc ions increases from 4 to 6, signaling octahedral coordination. This change occurs at a dopant concentration significantly lower than the reported maximum concentration of Sc (42 mol % Sc) in wurtzite (Al, Sc)N. XPS spectra provide support for our observation that the local environment of Sc in (Al, Sc)N may include more than one type of coordination.
Snarski L., Biran I., Bendikov T., Pinkas I., Iron M. A., Kaplan-Ashiri I., Weissman H. & Rybtchinski B. (2024) Advanced Functional Materials. 34, 7, 2309742
While individual single-wall carbon nanotubes (SWCNTs) have remarkable strength and electrical conductivity, SWCNT networks fabricated from dispersions have inferior properties due to nanotube bundling, limiting the potential applications of SWCNT materials. Herein, a common dye molecule (purpurin) is used to exfoliate SWCNTs via noncovalent functionalization and to fabricate SWCNT materials by a simple solution-based process. The advantageous noncovalent interactions result in efficient exfoliation and metallic SWCNT enrichment, affording SWCNT materials with high mechanical robustness and electrical conductivity. This method is used to prepare mechanically robust SWCNT films and flexible transparent conductive electrodes.Purpurin, a common dye molecule, is utilized to efficiently exfoliate single-wall carbon nanotubes (SWCNTs) in an aqueous solution. The advantageous noncovalent interactions result in metallic SWCNT enrichment, affording the fabrication of flexible, robust, and highly electrical conductive SWCNT networks. Transparent conductive electrodes based on those networks show excellent optoelectronic performance suitable for application in touch screens and LEDs.image
Hör J., Wolf S. G. & Sorek R. (2024) Nature. 631, 8022, p. 850-856
Several immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defence<sup>15</sup>. Here we studied an antiphage defence system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fibre, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defence system release a mixture of partially assembled, tailless phage particles and fully assembled phages in which the central tail fibre is obstructed by the covalently attached ubiquitin-like protein. These phages show severely impaired infectivity, explaining how the defence system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.
de Haan D., Aram L., Peled-Zehavi H., Addadi Y., Ben-Joseph O., Rotkopf R., Elad N., Rechav K. & Gal A. (2023) Nature Communications. 14, 1, 480
Diatoms are unicellular algae characterized by silica cell walls. These silica elements are known to be formed intracellularly in membrane-bound silica deposition vesicles and exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements remains unknown. Here we study the membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during its formation, the mineral phase is in tight association with the silica deposition vesicle membranes, which form a precise mold of the delicate geometrical patterns. We find that during exocytosis, the distal silica deposition vesicle membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. We demonstrate that within the cell, the proximal silica deposition vesicle membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.
Kundrat V., Cohen H., Kossoy A., Bonani W., Houben L., Zalesak J., Wu B., Sofer Z., Popa K. & Tenne R. (2023) Small. 20, 14, 2307684
Uranium is a high-value energy element, yet also poses an appreciable environmental burden. The demand for a straightforward, low energy, and environmentally friendly method for encapsulating uranium species can be beneficial for long-term storage of spent uranium fuel and a host of other applications. Leveraging on the low melting point (60 °C) of uranyl nitrate hexahydrate and nanocapillary effect, a uranium compound is entrapped in the hollow core of WS<sub>2</sub> nanotubes. Followingly, the product is reduced at elevated temperatures in a hydrogen atmosphere. Nanocrystalline UO<sub>2</sub> nanoparticles anchor within the WS<sub>2</sub> nanotube lumen are obtained through this procedure. Such methodology can find utilization in the processing of spent nuclear fuel or other highly active radionuclides as well as a fuel for deep space missions. Moreover, the low melting temperatures of different heavy metal-nitrate hydrates, pave the way for their encapsulation within the hollow core of the WS<sub>2</sub> nanotubes, as demonstrated herein.
Kundrat V., Bukvisova K., Novak L., Prucha L., Houben L., Zalesak J., Vukusic A., Holec D., Tenne R. & Pinkas J. (2023) Crystal Growth and Design. 24, 1, p. 378-390
Tungsten suboxide W<sub>18</sub>O<sub>49</sub> nanowhiskers are a material of great interest due to their potential high-end applications in electronics, near-infrared light shielding, catalysis, and gas sensing. The present study introduces three main approaches for the fundamental understanding of W<sub>18</sub>O<sub>49</sub> nanowhisker growth and structure. First, W<sub>18</sub>O<sub>49</sub> nanowhiskers were grown from γ-WO<sub>3</sub>/a-SiO<sub>2</sub> nanofibers in situ in a scanning electron microscope (SEM) utilizing a specially designed microreactor (μReactor). It was found that irradiation by the electron beam slows the growth kinetics of the W<sub>18</sub>O<sub>49</sub> nanowhisker, markedly. Following this, an in situ TEM study led to some new fundamental understanding of the growth mode of the crystal shear planes in the W<sub>18</sub>O<sub>49</sub> nanowhisker and the formation of a domain (bundle) structure. High-resolution scanning transmission electron microscopy analysis of a cross-sectioned W<sub>18</sub>O<sub>49</sub> nanowhisker revealed the well-documented pentagonal Magnéli columns and hexagonal channel characteristics for this phase. Furthermore, a highly crystalline and oriented domain structure and previously unreported mixed structural arrangement of tungsten oxide polyhedrons were analyzed. The tungsten oxide phases found in the cross section of the W<sub>18</sub>O<sub>49</sub> nanowhisker were analyzed by nanodiffraction and electron energy loss spectroscopy (EELS), which were discussed and compared in light of theoretical calculations based on the density functional theory method. Finally, the knowledge gained from the in situ SEM and TEM experiments was valorized in developing a multigram synthesis of W<sub>18</sub>O<sub>49</sub>/a-SiO<sub>2</sub> urchin-like nanofibers in a flow reactor.
Niazov-Elkan A., Shepelenko M., Alus L., Kazes M., Houben L., Rechav K., Leitus G., Kossoy A., Feldman Y., Kronik L., Vekilov P. G. & Oron D. (2023) Advanced Materials. 36, 8, 2306996
Numerous bio-organisms employ template-assisted crystallization of molecular solids to yield crystal morphologies with unique optical properties that are difficult to reproduce synthetically. Here, a facile procedure is presented to deposit bio-inspired birefringent crystals of xanthine derivatives on a template of single-crystal quartz. Crystalline sheets that are several millimeters in length, several hundred micrometers in width, and 300600 nm thick, are obtained. The crystal sheets are characterized with a well-defined orientation both in and out of the substrate plane, giving rise to high optical anisotropy in the plane parallel to the quartz surface, with a refractive index difference Δn ≈ 0.25 and a refractive index along the slow axis of n ≈ 1.7. It is further shown that patterning of the crystalline stripes with a tailored periodic grating leads to a thin organic polarization-dependent diffractive meta-surface, opening the door to the fabrication of various optical devices from a platform of small-molecule based organic dielectric crystals.
Bandyopadhyay D., Ghosh S., Houben L., Bar-Ziv R. & Bar-Sadan M. (2023) ACS Applied Energy Materials. 6, 21, p. 10987-10995
Forming complex structures of functional materials in a controlled and reproducible fashion is a well-known challenge. Specifically, bimetallic phosphides are of interest as electrocatalysts for energy-related applications; however, a satisfactory structure-function relationship has not yet been fully deciphered yet. Here, we show that a colloidal chemistry approach produces bimetallic phosphides of Co and Cu, where segregation and phase transformation induce significant changes in morphology compared with solid solutions. Their complexity permits tuning of the catalytic sites to the hydrogen and oxygen evolution reactions (HER and OER), allowing the bimetallic phosphides to catalyze the full water-splitting reaction. The experimental results show that water cleavage, H-OH + e<sup>-</sup> → H* + OH (Volmer), is particularly favorable on Cu<sub>x</sub>Co<sub>y</sub>P catalysts (and especially when x = 50%), enhancing their HER performance. As for the OER enhancement, the results show that the bimetallic phosphides undergo a surface transformation during the OER, whereby (oxy)hydroxides form at anodic potentials in alkaline solutions and serve as the actual electrocatalysts. Thus, the use of Cu<sub>x</sub>Co<sub>y</sub> phosphides as starting materials can lead to optimized oxide growth. In summary, the higher catalytic activity of the bimetallic phosphides is attributed to their altered morphology, surface area, adsorption sites, and valence state, which also make them efficient bifunctional electrocatalysts for the overall water-splitting reaction.
Cohen D., Houben L., Galisova A., Feldman Y., Fox S., Biton I. E. & Bar-Shir A. (2023) ACS Applied Nano Materials. 6, 14, p. 13107-13115
Nanostructured metal fluorides (nanofluorides) are an emerging type of inorganic nanocrystals (NCs) with unique physiochemical properties for advanced applications. One recent demonstration used water-dispersed ultrasmall CaF2 nanofluorides as imaging agents that combined the advantages of inorganic NCs with the benefit of background-free 19F-magnetic resonance imaging (19F-MRI). Nevertheless, obtaining small nanofluorides with a face-centered cubic crystal structure, where all fluorides are magnetically equivalent to result in a single 19F NMR signal, is challenging for other types of nanofluorides, preventing their use in 19F-MRI. Here, we show the development of ultrasmall, water-dispersed, barium fluoride (BaF2) NCs for bioimaging applications. By doping BaF2 with two types of lanthanides, diamagnetic-La3+ and paramagnetic-Sm3+, we were able to control the morphology and 19F-MR properties of the final La,Sm:BaF2 (termed LaSamBa) formulation. The fine-tuning of the La3+ content enabled us to obtain monodispersed 4.5 nm NCs, and control over the Sm3+ content provided LaSamBa with very short T1 relaxation properties (ca. 100 ms) needed for enhanced 19F-MRI sensitivity. This type of nanofluorides was examined in two different imaging modalities (i.e., 19F-MRI and CT), benefiting from their single 19F-NMR signal and the high atomic number of barium atoms, respectively. As their 19F chemical shift significantly differs from that of other nanofluorides (e.g., CaF2 and SrF2), LaSamBa expanded the nanofluoride library for future multitarget 19F-MRI studies.
Reuven B., Movsesyan A., Santiago E. Y., Houben L., Wang Z., Govorov A. O. & Markovich G. (2023) Advanced Optical Materials.
The anisotropic chiroptical activity of chiral tellurium nanorods (Te NRs) is studied experimentally via circular dichroism (CD) measurements of macroscopically aligned samples. Te NRs with chiral, twisted prismatic shapes, and aspect ratio of ~4 are synthesized using the chiral ligand D-penicillamine and the surfactant sodium dodecyl sulfate. The anisotropy of their optical activity is studied at two different configurations with the long axis of the NRs oriented parallel and perpendicular to the CD probe beam. The parallel alignment is achieved in aqueous suspension using an alternating electric field that acts on their polarizability anisotropy, estimated to be 3.8 × 10<sup>−30</sup> F m<sup>2</sup>. The perpendicular orientation is measured in a film of the NRs, prepared at a liquid waterorganic interface such that the NRs are deposited on a substrate with their long-axis in-plane. Finite-element electromagnetic simulations reproduce well the experimental CD spectra, demonstrating that the optical activity is dominated by the chiral shape-related Mie-type resonances. Both the experimental results and simulations show that the CD spectrum is practically inverted between the two light incidence orientations, a phenomenon that appears general to twisted anisotropic nano-objects.
Basha A., Levi G., Houben L., Amrani T., Goldfarb I. & Kohn A. (2023) Ultramicroscopy. 249, 113737
The introduction of direct electron detectors (DEDs) to transmission electron microscopy has set off the resolution revolution, especially for cryoTEM low-dose imaging of soft matter. In comparison to traditional indirect electron detectors such as Charged-Coupled Devices (CCD), DEDs show an improved modulation transfer function (MTF) and detective quantum efficiency (DQE) across all spatial frequencies, as well as faster frame rates which enable single electron counting. The benefits of such characteristics for imaging, spectroscopy and electron holography have been demonstrated previously. However, studies are lacking on the application of DEDs for localized characterization of short- to medium- range-order (SRO, MRO) in amorphous materials using electron scattering. Therefore, we evaluate the performance of a Monolithic Active Pixel Sensor DED for the characterization of SRO and MRO in nanoscale volumes of amorphous materials, using SiO<sub>2</sub> and Ta<sub>2</sub>O<sub>5</sub> thin films as test cases. The performance of the detector is compared systematically to electron scattering measurements recorded on an indirect detector (CCD) using 200 keV electrons and electron doses starting at approximately 500[Formula presented]. In addition, the effects of sample cooling and energy-filtering on the measured SRO of the oxides were investigated. We demonstrate that the performance of the DED resulted in improved SRO characterization in comparison to that obtained from the CCD measurements. The DED enabled to achieve a larger measured maximal scattering vector, ∼16.5[Formula presented] compared to ∼15[Formula presented], for the CCD. Furthermore, an improved signal-to-noise ratio of approximately two-fold was observed across all spatial frequencies for both 200 keV and 80 keV electrons. These improvements are shown to result from the superior DQE of the DED. Consequently, the DED measurements enabled to determine the coordination numbers of atomic bonds more accurately. We expect that further benefits of the DED for S/MRO characterization will be highlighted for ultra- sensitive materials that cannot withstand electron doses above several [Formula presented].
Urban K. W., Barthel J., Houben L., Jia C. L., Jin L., Lentzen M., Mi S. B., Thust A. & Tillmann K. (2023) Progress in Materials Science. 133, 101037
Transmission electron microscopy is an indispensable tool in modern materials science. It enables the structure of materials to be studied with high spatial resolution, and thus makes a decisive contribution to the fact that it is now possible to understand the microstructure-related physical and chemical characteristics and to correlate these with the macroscopic materials properties. It was tantamount to a paradigm shift when electron microscopy reached atomic resolution in the late 1990s due to the invention of aberration-corrected electron optics. It is now generally accepted practice to perform picometer-scale measurements and chemical analyses with reference to single atomic units. This review has three objectives. Microscopy in atomic dimensions is applied quantum physics. The consequences of this for practical work and for the understanding and application of the results shall be worked out. Typical applications in materials science will be used to show what can be done with this kind of microscopy and where its limitations lie. In the absence of relevant monographs, the aim is to provide an introduction to this new type of electron microscopy and to enable the reader to access the literature in which special issues are addressed. The paper begins with a brief presentation of the principles of optical aberration correction. It then discusses the fundamentals of atomic imaging and covers typical examples of practical applications to problems in modern materials science. It is emphasized that in atomic-resolution electron microscopy the quantitative interpretation of the images must always be based on the solution of the quantum physical and optical problem on a computer.
Liao J., Wang X., Danieli Y., Houben L., Rechav K., Song J., Song J., Zhao Z., Zhang L., Zhou G., Joselevich E. & Xu J. (2023) Advanced Materials Technologies. 8, 14, 2202179
Large-scale on-chip integration of organic nanowire-based devices requires the deterministic assembly of organic small molecules into highly-aligned nanowires. In this work, phthalocyanine molecules are self-assembled into horizontally-aligned nanowires after generating parallel hydrophobic nanogrooves on a sapphire surface. In contrast to previous self-oriented inorganic nanowires, these molecular nanowires are separated from their supporting sapphire by an ultrathin amorphous layer, indicating a complete elimination of lattice matching between nanowires and substrates. Therefore, small molecules beyond phthalocyanines hold promise to form aligned nanowires using this graphoepitaxial self-assembly strategy. The excellent alignment and high crystallinity of these nanowires enable the desired in-situ integration of nanowire-based devices without additional postgrowth processing steps. As a proof of concept, self-oriented CuPc nanowires are integrated into photodetector arrays directly on their growth substrate after electrode arrays are transferred onto the nanowires. Compared to previous CuPc photodetectors constructed using other approaches, these detectors exhibit a faster response to the spectrum in the 488-780 nm range (rise and fall times are 0.05-0.43 s and 0.38-2.34 s, respectively) while offering comparable detectivities (2.49 x 10(10) Jones on average). This graphoepitaxial self-assembly offers new opportunities for the aligned growth of organic crystalline nanowires and their large-scale in-situ integration into functional devices.
Balakrishnan S. K., Parambil P. C., Houben L., Asher M., Yaffe O. & Edri E. (2023) Cell Reports Physical Science. 4, 3, 101298
The prevalence of self-healing semiconductors is restricted to a few select cases, limiting understanding of the self-healing phenomenon. Herein, we report self-healing after photo-induced damage in antimony trichalcogenides and chalcoiodides, which are quasi-one-dimensional semiconductors with prospective applications in photovoltaics and electronic devices. We relate the self-healing to a photo-induced phase transition and the damaged state to a hidden phase from which the damaged semiconductors recover (self-heal). By using vibrational spectroscopy, we elucidate the intermediate species in the reaction. We suggest that the occurrence of bonding states at the bottom of the conduction band is central to promoting the hidden phase. Comparing four different materials with a similar crystal structure enables us to uncover the common structural feature that leads to the self-healing in these materials. Expanding the type and number of self-healing semiconductors, and uncovering the underlying solid-state reactions, will facilitate the development of self-healing electronic building blocks and devices.
Kumar S., Damle V. H., Bendikov T., Itzhak A., Elbaum M., Rechav K., Houben L., Tischler Y. & Cahen D. (2023) ACS applied materials & interfaces. 15, 19, p. 23908-23921
Two-dimensional (2D) halide perovskites, HaPs, can provide chemical stability to three-dimensional (3D) HaP surfaces, protecting them from exposure to ambient species and from reacting with contacting layers. Both actions occur with 2D HaPs, with the general stoichiometry R2PbI4 (R: long or bulky organic amine) covering the 3D ones. Adding such covering films can also boost power conversion efficiencies of photovoltaic cells by passivating surface/interface trap states. For maximum benefit, we need conformal ultrathin and phase-pure (n = 1) 2D layers to enable efficient tunneling of photogenerated charge carriers through the 2D film barrier. Conformal coverage of ultrathin (
Rosentsveig R., Sreedhara M. B., Sinha S. S., Kaplan-Ashiri I., Brontvein O., Feldman Y., Pinkas I., Zheng K., Castelli I. E. & Tenne R. (2023) Inorganic Chemistry. 62, 44, p. 18267-18279
The synthesis of complex new nanostructures is challenging but also bears the potential for observing new physiochemical properties and offers unique applications in the long run. High-temperature synthesis of ternary WSe2xS2(1-x) (denoted as WSSe) nanotubes in a pure phase and in substantial quantities is particularly challenging, requiring a unique reactor design and control over several parameters, simultaneously. Here, the growth of WSSe nanotubes with the composition 0 ≤ x < 1 from W18O49 nanowhiskers in an atmospheric chemical vapor deposition (CVD) flow reactor is investigated. The oxide precursor powder is found to be heavily agglomerated, with long nanowhiskers decorating the outer surface of the agglomerates and their core being enriched with oxide microcrystallites. The reaction kinetics with respect to the chalcogen vapors varies substantially between the two kinds of oxide morphologies. Insights into the chemical reactivity and diffusion kinetics of S and Se within W18O49 nanowhishkers and the micro-oxide crystallites were gained through detailed microscopic, spectroscopic analysis of the reaction products and also through density functional theory (DFT) calculations. For safety reasons, the reaction duration was limited to half an hour each. Under these circumstances, the reaction was completed for some 50% of the nanotubes and the other half remained with thick oxide core producing new WOx@WSSe core-shell nanotubes. Furthermore, the selenium reacted rather slowly with the WOx nanowhiskers, whereas the more ionic and smaller sulfur atoms were shown to diffuse and react faster. The yield of the combined hollow and core-shell nanotubes on the periphery of the agglomerated oxide was very high, approaching 100% in parts of the reactor boat. The nanotubes were found to be very thin (∼80% with a diameter
Yosef Tal N., Dodiuk H., Amir E., Brontvein O., Tenne R. & Kenig S. (2023) Nano-Structures and Nano-Objects. 36, 101034
Surface modification of tungsten disulfide (WS<sub>2</sub>) nanoparticles (NPs) by a photo-initiator (PI) for radical curing of acrylate nano-structured systems has been studied with the purpose of acquiring covalent bonding of the acrylate matrix to the NPs surface. This surface functionalization was expected to photosensitize both the PI and the NPs thereby enhance the photocuring of the acrylate as well as improving its mechanical properties. The studied PI was the commonly used Bis(acyl) phosphane oxides (BAPOs). For surface modification of the WS<sub>2</sub> NPs, the BAPO was functionalized first with Tri(methoxy) silane (TMESI) to yield TMESI-BAPO moieties. Subsequently, the functionalized BAPO was chemisorbed to the WS<sub>2</sub> NPs resulting in WS<sub>2</sub> NPs surface modified by TMESI-BAPO pendant moieties. The functionalization and surface modification were confirmed using spectroscopic techniques as well as electron microscopy, and thermal measurements. Thermal gravimetric analysis (TGA) displayed 40 wt% weight loss for TMESI-BAPO modified WS<sub>2</sub> upon heating to 400 °C. The modified WS<sub>2</sub> NPs were incorporated into acrylate resin to study the curing kinetics and resulting mechanical properties compared to neat WS<sub>2</sub>. Nanostructured acrylate with TEMSI-BAPO modified WS<sub>2</sub> NPs exhibited an increase of 83% in storage modulus and toughness compared to neat acrylate.
Erez-Cohen O., Brontvein O. & Bar-Joseph I. (2023) Nano Letters. 23, 6, p. 2233-2238
We investigate electrically driven plasmon (EDP) emission in metal-insulator-semiconductor tunnel junctions. We find that amorphization of the silicon crystal at a narrow region near the junction due to the applied voltage plays a critical role in determining the nature of the emission. Furthermore, we suggest that the change in the properties of the insulating layer above a threshold voltage determines the EDP spatial properties, from being spatially uniform when the device is subjected to low voltages, to a spotty pattern peaking at high voltages. We emphasize the role of the high-energy emission as an unambiguous tool for distinguishing between EDP and radiative recombination of electrons and holes in the semiconductor.
Garg S., Taragin S., Saha A., Brontvein O., Leung K., Noked M. & Rosy (2023) Journal of Power Sources. 569, 233017
Despite having the ability to deliver 650 W h kg<sup>−1</sup> in addition to the impressive rate capability, superior thermal stability, and facilitated electronic and ionic lithium conduction, LiNi<sub>0.5</sub>Mn<sub>1.5</sub>O<sub>4</sub> (LNMO) is far from commercial applications. LNMO suffers from irreversible electrolytic degradation on its surface under high voltage operations leading to capacity fading and poor battery life. Therefore, this work aims to improve the stability and electrochemical behavior of LNMO by creating a Zn-enriched cathode layer interface via eccentric and facile diethyl zinc-assisted atomic surface reduction (Zn-ASR). In-depth surface characterization tools and computational calculations demonstrates a conformal 7-8 nm thin Zn-O and C-O enriched layer encapsulating the cathode particles resulting from Zn-ASR. The intensive comparative electrochemical and spectroscopic analysis, indicates superior electrochemical performance of the surface-reduced LNMO w.r.t rate capability (14% higher at 4C), cycling stability, and capacity retention (87% retention). A decrease in gaseous evolution on the surface-treated sample arising from the electrolyte degradation further explains the improvement in the stability and electrochemical behavior of Zn-ASR LNMO. This work proves that electrode material can be substantially improved and incentivized by the chemo-mechanical benefits of rationally designed surface layers.
Haber S., Solomatin N., Shapira A., Bendikov T., Brontvein O., Ein-Eli Y. & Leskes M. (2023) Journal of Power Sources. 560, 232693
Surface degradation is a major limitation in the utilization of high energy cathodes. Cathode coatings provide a promising route for achieving interfacial stability. In this case, there is interest in developing coatings which will provide efficient ion transport across the cathode-electrolyte-interface (CEI). Aluminum based coatings, particularly metal fluorides, have shown great promise for stabilizing the high voltage cathode, LiNi0.5Mn1.5O4. Despite the clear practical advantages of coatings, the chemical basis for their improved performance remains ambiguous. Here we present an in-depth investigation of a nominal AlF3 coating on LiNi0.5Mn1.5O4 based on solid state NMR spectroscopy to determine the composition and function of the Al/F coating layer. NMR results, supported by X-ray photoelectron spectroscopy and electron microscopy, reveal that the actual composition of the deposited layer is an amorphous AlOF phase. Isotope exchange is used to follow the spontaneous exchange of lithium ions across the CEI, revealing improved transport in coated cathodes. Finally, NMR experiments provide evidence for insertion of lithium ions in the Al/F coating following electrochemical cycling. The results suggest that lithium insertion into a nominally lithium-less coating plays a role in its improved performance, highlighting lithium content as an important factor in designing beneficial coatings for higher energy cathodes.
Barbe T., Rosentsveig R., Brontvein O., Sreedhara M., Zheng K., Bataille F., Vossier A., Flamant G., Castelli I., Gordon J. & Tenne R. (2023) Advanced Materials Interfaces. 10, 3, 2201930
The synthesis of fundamentally small MoS<sub>2</sub> nanotubes and nanocones(horns) that have proven elusive in prior studies has been achieved via ablation of a precursor mixture of crystallites of MoS<sub>2</sub> + MoO<sub>3</sub> by highly concentrated solar radiation. The special far-from-equilibrium conditions achieved in the solar furnace prove conducive to the generation of these singular nanostructures. Extensive electron microscopy and characterization results (transmission electron microscopy (TEM), electron diffraction (ED), X-ray diffraction (XRD), scanning TEM (STEM), and high angle annular dark field (HAADF)) reveal a range of nanoparticle shapes and sizes based on which reaction mechanisms are proposed. Molecular dynamics simulations indicate that the sizable thermal fluctuations intrinsically produced in the high-temperature solar reactor soften the MoS<sub>2</sub> nanostructures, yielding corrugated layers that favor nanostructures with only a few layers, in agreement with the experimental observations.
Hanopolskyi A. I., Mikhnevich T. A., Paikar A., Nutkovich B., Pinkas I., Dadosh T., Smith B. S., Orekhov N., Skorb E. V. & Semenov S. N. (2023) Chem. 9, 12, p. 3666-3684
Our ability to synthesize life-inspired systems and materials is intimately connected to our understanding of the interplay between reactions, diffusion, phase separation, and self-assembly. The interaction between non-linear reactions (autocatalysis) and liquid-liquid phase separation is particularly interesting because of the emergence of functional properties and structures through instabilities, which are hard to predict theoretically without the help of experimental model systems. In this work, we studied systems where chemical autocatalysis is coupled to complex coacervation and the formation of oil-in-water droplets. The autocatalysis is driven by a nucleophilic chain reaction and is coupled to complex coacervation through the formation of tri- and tetracationic species. Interestingly, we observed the formation of hierarchical colloids when we used reactants that can form oil droplets in addition to coacervate droplets. This work illustrates a mechanism for the formation of complex, hierarchical microstructures by kinetically controlled self-assembly regulated by non-linear chemical reaction networks.
Szoke T., Goldberger O., Albocher-Kedem N., Barsheshet M., Dezorella N., Nussbaum-Shochat A., Wiener R., Schuldiner M. & Amster-Choder O. (2023) Cell Reports. 42, 11, 113393
TmaR, the only known pole-localizer protein in Escherichia coli, was shown to cluster at the cell poles and control localization and activity of the major sugar regulator in a tyrosine phosphorylation-dependent manner. Here, we show that TmaR assembles by phase separation (PS) via heterotypic interactions with RNA in vivo and in vitro. An unbiased automated mutant screen combined with directed mutagenesis and genetic manipulations uncovered the importance of a predicted nucleic-acid-binding domain, a disordered region, and charged patches, one containing the phosphorylated tyrosine, for TmaR condensation. We demonstrate that, by protecting flagella-related transcripts, TmaR controls flagella production and, thus, cell motility and biofilm formation. These results connect PS in bacteria to survival and provide an explanation for the linkage between metabolism and motility. Intriguingly, a point mutation or increase in its cellular concentration induces irreversible liquid-to-solid transition of TmaR, similar to human disease-causing proteins, which affects cell morphology and division.
Berman P., de Haro L. A., Jozwiak A., Panda S., Pinkas Z., Dong Y., Cveticanin J., Barbole R., Livne R., Scherf T., Shimoni E., Levin-Zaidman S., Dezorella N., Petrovich-Kopitman E., Meir S., Rogachev I., Sonawane P. D. & Aharoni A. (2023) Nature Plants. 9, 5, p. 817-831
Modulation of the endocannabinoid system is projected to have therapeutic potential in almost all human diseases. Accordingly, the high demand for novel cannabinoids stimulates the discovery of untapped sources and efficient manufacturing technologies. Here we explored Helichrysum umbraculigerum, an Asteraceae species unrelated to Cannabis sativa that produces Cannabis-type cannabinoids (for example, 4.3% cannabigerolic acid). In contrast to Cannabis, cannabinoids in H. umbraculigerum accumulate in leaves' glandular trichomes rather than in flowers. The integration of de novo whole-genome sequencing data with unambiguous chemical structure annotation, enzymatic assays and pathway reconstitution in Nicotiana benthamiana and in Saccharomyces cerevisiae has uncovered the molecular and chemical features of this plant. Apart from core biosynthetic enzymes, we reveal tailoring ones producing previously unknown cannabinoid metabolites. Orthology analyses demonstrate that cannabinoid synthesis evolved in parallel in H. umbraculigerum and Cannabis. Our discovery provides a currently unexploited source of cannabinoids and tools for engineering in heterologous hosts.
Rein A., Geron I., Kugler E., Fishman H., Gottlieb E., Abramovich I., Giladi A., Amit I., Mulet-Lazaro R., Delwel R., Gröschel S., Levin-Zaidman S., Dezorella N., Holdengreber V., Rao T. N., Yacobovich J., Steinberg-Shemer O., Huang Q., Tan Y., Chen S., Izraeli S. & Birger Y. (2023) Haematologica. 108, 9, p. 2316-2330
Mono-allelic germline disruptions of the transcription factor GATA2 result in a propensity for developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), affecting more than 85% of carriers. How a partial loss of GATA2 functionality enables leukemic transformation years later is unclear. This question has remained unsolved mainly due to the lack of informative models, as Gata2 heterozygote mice do not develop hematologic malignancies. Here we show that two different germline Gata2 mutations (TgErg/Gata2
<sup>het</sup> and TgErg/Gata2
<sup>L359V</sup>) accelerate AML in mice expressing the human hematopoietic stem cell regulator ERG. Analysis of Erg/Gata2
<sup>het</sup> fetal liver and bone marrow-derived hematopoietic cells revealed a distinct pre-leukemic phenotype. This was characterized by enhanced transition from stem to progenitor state, increased proliferation, and a striking mitochondrial phenotype, consisting of highly expressed oxidative-phosphorylation-related gene sets, elevated oxygen consumption rates, and notably, markedly distorted mitochondrial morphology. Importantly, the same mitochondrial gene-expression signature was observed in human AML harboring GATA2 aberrations. Similar to the observations in mice, non-leukemic bone marrows from children with germline GATA2 mutation demonstrated marked mitochondrial abnormalities. Thus, we observed the tumor suppressive effects of GATA2 in two germline Gata2 genetic mouse models. As oncogenic mutations often accumulate with age, GATA2 deficiency-mediated priming of hematopoietic cells for oncogenic transformation may explain the earlier occurrence of MDS/AML in patients with GATA2 germline mutation. The mitochondrial phenotype is a potential therapeutic opportunity for the prevention of leukemic transformation in these patients.
Ben-Joseph O., de Haan D., Rechav K., Shimoni E., Levin-Zaidman S., Langer G., Probert I., Wheeler G. L. & Gal A. (2023) Small structures. 4, 7, 2200353
Coccolithophores are a group of unicellular marine algae that shape global geochemical cycles via the production of calcium carbonate crystals. Interestingly, different life-cycle phases of the same coccolithophore species produce very different calcitic scales, called coccoliths. In the widely studied diploid phase, the crystals have anisotropic and complex morphologies, while haploid cells produce coccoliths consisting solely of calcite crystals with simple rhombohedral morphology. Understanding how these two life-cycle phases control crystallization is a highly sought-after goal, yet, haploid phase crystallization has rarely been studied, and the process by which they form is unknown. Herein, advanced electron microscopy is employed to elucidate the cellular architecture of the calcification process in haploid cells. The results show that in contrast to diploid-phase calcification, the coccolith-forming vesicle of haploid-phase cells is voluminous. In this solution-like environment, the crystals nucleate and grow asynchronously in a process that resembles calcite growth in bulk solution, leading to the simple morphologies of the crystals. The two distinct mineralization regimes of coccolithophore life-cycle phases suggest that cellular architecture, and specifically confinement of the crystallization process, is a pivotal determinant of biomineral morphology and assembly.
Capua-Shenkar J., Kaestner A., Rechav K., Brumfeld V., Kaplan-Ashiri I., Avinoam O., Speter C., Halak M., Kruth H. & Addadi L. (2023) BioRxiv.
Atherosclerosis is a pathology affecting the arteries, characterized by the buildup of plaques in the blood vessel walls. Atherosclerosis is the main cause of cardiovascular diseases, which constitute the leading cause of death in the world. Cholesterol crystals are the main components of the plaques, which actively participate in plaque growth and rupture and do not dissolve in aqueous environments. Employing novel cryo-scanning electron microscopy techniques, we examined human atherosclerotic plaques at high resolution, in 3D, and in close to native conditions. We show that cholesterol crystal clearance occurs in advanced human plaques through the activity of cells. We suggest that this occurs by enzymatic esterification of cholesterol to cholesteryl ester, which aggregates into intra- and extra-cellular pools. This discovery provides further understanding of the disease process in atherosclerosis, and may inspire new therapeutic approaches.
Buss D. J., Rechav K., Reznikov N. & McKee M. D. (2023) Bone. 174, 116818
The hallmark of enthesis architecture is the 3D compositional and structural gradient encompassing four tissue zones - tendon/ligament, uncalcified fibrocartilage, calcified fibrocartilage and bone. This functional gradient accommodates the large stiffness differential between calcified bone and uncalcified tendon/ligament. Here we analyze in 3D the organization of the mouse Achilles enthesis and mineralizing Achilles tendon in comparison to lamellar bone. We use correlative, multiscale high-resolution volume imaging methods including & mu;CT with submicrometer resolution and FIB-SEM tomography (both with deep learning-based image segmentation), and TEM and SEM imaging, to describe ultrastructural features of physiologic, age-related and aberrant mineral patterning. We applied these approaches to murine wildtype (WT) Achilles enthesis tissues to describe in normal calcifying fibrocartilage a crossfibrillar mineral tessellation pattern similar to that observed in lamellar bone, but with greater variance in mineral tesselle morphology and size. We also examined Achilles enthesis structure in Hyp mice, a murine model for the inherited osteomalacic disease X-linked hypophosphatemia (XLH) with calcifying enthesopathy. In Achilles enthesis fibrocartilage of Hyp mice, we show defective crossfibrillar mineral tessellation similar to that which occurs in Hyp lamellar bone. At the cellular level in fibrocartilage, unlike in bone where enlarged osteocyte mineral lacunae are found as peri-osteocytic lesions, mineral lacunar volumes for fibrochondrocytes did not differ between WT and Hyp mice. While both WT and Hyp aged mice demonstrate Achilles tendon midsubstance ectopic mineralization, a consistently defective mineralization pattern was observed in Hyp mice. Strong immunostaining for osteopontin was observed at all mineralization sites examined in both WT and Hyp mice. Taken together, this new 3D ultrastructural information describes details of common mineralization trajectories for enthesis, tendon and bone, which in Hyp/XLH are defective.
Ibrahim J., Rechav K., Boaretto E. & Weiner S. (2023) Journal of Structural Biology. 215, 3, 107998
We report on the 3D ultrastructure of the mineralized petrous bone of mature pig using focused ion beam scanning electron microscopy (FIB-SEM). We divide the petrous bone into two zones based on the degree of mineralization; one zone close to the otic chamber has higher mineral density than the second zone further away from the otic chamber. The hypermineralization of the petrous bone results in the collagen D-banding being poorly revealed in the lower mineral density zone (LMD), and absent in the high mineral density zone (HMD). We therefore could not use D-banding to decipher the 3D structure of the collagen assembly. Instead we exploited the anisotropy option in the Dragonfly image processing software to visualize the less mineralized collagen fibrils and/or nanopores that surround the more mineralized zones known as tesselles. This approach therefore indirectly tracks the orientations of the collagen fibrils in the matrix itself. We show that the HMD bone has a structure similar to that of woven bone, and the LMD is composed of lamellar bone with a plywood-like structural motif. This agrees with the fact that the bone close to the otic chamber is fetal bone and is not remodeled. The lamellar structure of the bone further away from the otic chamber is consistent with modeling/remodeling. The absence of the less mineralized collagen fibrils and nanopores resulting from the confluence of the mineral tesselles may contribute to shielding DNA during diagenesis. We show that anisotropy evaluation of the less mineralized collagen fibrils could be a useful tool to analyze bone ultrastructures and in particular the directionality of collagen fibril bundles that make up the bone matrix.
Kahil K., Kaplan-Ashiri I., Wolf S. G., Rechav K., Weiner S. & Addadi L. (2023) Acta Biomaterialia. 155, p. 482-490
During spicule formation in sea urchin larvae, calcium ions translocate within the primary mesenchymal cells (PMCs) from endocytosed seawater vacuoles to various organelles and vesicles where they accumulate, and subsequently precipitate. During this process, calcium ions are concentrated by more than three orders of magnitude, while other abundant ions (Na, Mg) must be removed. To obtain information about the overall ion composition in the vesicles, we used quantitative cryo-SEM-EDS and cryo-STEM-EDS analyzes. For cryo-STEM-EDS, thin (500 nm) frozen hydrated lamellae of PMCs were fabricated using cryo-focused ion beam-SEM. The lamellae were then loaded into a cryo-TEM, imaged and the ion composition of electron dense bodies was measured. Analyzes performed on 18 Ca-rich particles/particle clusters from 6 cells contained Ca, Na, Mg, S and P in different ratios. Surprisingly, all the Ca-rich particles contained P in amounts up to almost 1:1 of Ca. These cryo-STEM-EDS results were qualitatively confirmed by cryo-SEM-EDS analyzes of 310 vesicles, performed on high pressure frozen and cryo-planed samples. We discuss the advantages and limitations of the two techniques, and their potential applicability, especially to study ion transport pathways and ion trafficking in cells involved in mineralization.
Karger S., Miali M. E., Solomonov A., Eliaz D., Varsano N. & Shimanovich U. (2023) Small. 20, 22, 2308069
A notable feature of complex cellular environments is protein-rich compartments that are formed via liquidliquid phase separation. Recent studies have shown that these biomolecular condensates can play both promoting and inhibitory roles in fibrillar protein self-assembly, a process that is linked to Alzheimer's, Parkinson's, Huntington's, and various prion diseases. Yet, the exact regulatory role of these condensates in protein aggregation remains unknown. By employing microfluidics to create artificial protein compartments, the self-assembly behavior of the fibrillar protein lysozyme within them can be characterized. It is observed that the volumetric parameters of protein-rich compartments can change the kinetics of protein self-assembly. Depending on the change in compartment parameters, the lysozyme fibrillation process either accelerated or decelerated. Furthermore, the results confirm that the volumetric parameters govern not only the nucleation and growth phases of the fibrillar aggregates but also affect the crosstalk between the protein-rich and protein-poor phases. The appearance of phase-separated compartments in the vicinity of natively folded protein complexes triggers their abrupt percolation into the compartments' core and further accelerates protein aggregation. Overall, the results of the study shed more light on the complex behavior and functions of protein-rich phases and, importantly, on their interaction with the surrounding environment.
Avrahami E. M., Eyal Z., Varsano N., Zagoriy I., Mahamid J. & Gal A. (2023) Advanced Materials. 36, 11, 2309547
Biogenic crystals present a variety of complex morphologies that form with exquisite fidelity. In the case of the intricate morphologies of coccoliths, calcite crystals produced by marine algae, only a single set of crystallographic facets is utilized. It is unclear which growth process can merge this simple crystallographic habit with the species-specific architectures. Here, a suite of state-of-the-art electron microscopies is used to follow both the growth trajectories of the crystals ex situ, and the cellular environment in situ, in the species Emiliania huxleyi. It is shown that crystal growth alternates between a space filling and a skeletonized growth mode, where the crystals elongate via their stable crystallographic facets, but the final morphology is a manifestation of growth arrest. This process is reminiscent of the balance between reaction-limited and transport-limited growth regimes underlying snowflake formation. It is suggested that localized ion transport regulates the kinetic instabilities that are required for transport-limited growth, leading to reproducible morphologies.
Addadi L., Varsano N. & Moshe A. B. (2023) Helvetica Chimica Acta. 106, 3, e202200173
We review in this short perspective the history of cholesterol crystals and crystal structures. We address in particular the helical crystals that form in vitro and in pathology from environments rich in bile acids or from phospholipid membranes. We review the known mechanisms leading to crystals with chiral morphology, from screw-dislocation mediated growth to mechanisms involving asymmetric mechanical strain. We propose a mechanism for cholesterol helical crystal development based on the monoclinic cholesterol monohydrate crystal structure. We suggest that curvature arises in few layers thick crystals due to the tension induced between the hydrophobic layer and the ice-like H-bonded lattice of the water molecules with the cholesterol hydroxy groups. Helicity would ensue through a combination of the curvature and the fast growth of a thin ribbon in one crystal direction.
Zhai H., Fan Y., Zhang W., Varsano N. & Gal A. (2023) ACS biomaterials science & engineering. 9, 2, p. 601-607
Multistep mineralization processes are pivotal in the fabrication of functional materials and are often characterized by far from equilibrium conditions and high supersaturation. Interestingly, such 'nonclassical' mineralization pathways are widespread in biological systems, even though concentrating molecules well beyond their saturation level is incompatible with cellular homeostasis. Here, we show how polymer phase separation can facilitate bioinspired silica formation by passively concentrating the inorganic building blocks within the polymer dense phase. The high affinity of the dense phase to mobile silica precursors generates a diffusive flux against the concentration gradient, similar to dynamic equilibrium, and the resulting high supersaturation leads to precipitation of insoluble silica. Manipulating the chemistry of the dense phase allows to control the delicate interplay between polymer chemistry and silica precipitation. These results connect two phase transition phenomena, mineralization and coacervation, and offer a framework to achieve better control of mineral formation.
Ilani T., Reznik N., Yeshaya N., Feldman T., Vilela P., Lansky Z., Javitt G., Shemesh M., Brenner O., Elkis Y., Varsano N., Jaramillo A. M., Evans C. M. & Fass D. (2023) The EMBO Journal. 42, 2, e111869
Mucus is made of enormous mucin glycoproteins that polymerize by disulfide crosslinking in the Golgi apparatus. QSOX1 is a catalyst of disulfide bond formation localized to the Golgi. Both QSOX1 and mucins are highly expressed in goblet cells of mucosal tissues, leading to the hypothesis that QSOX1 catalyzes disulfide-mediated mucin polymerization. We found that knockout mice lacking QSOX1 had impaired mucus barrier function due to production of defective mucus. However, an investigation on the molecular level revealed normal disulfide-mediated polymerization of mucins and related glycoproteins. Instead, we detected a drastic decrease in sialic acid in the gut mucus glycome of the QSOX1 knockout mice, leading to the discovery that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases. Sialylation defects in the colon are known to cause colitis in humans. Here we show that QSOX1 redox control of sialylation is essential for maintaining mucosal function.
Niazov-Elkan A., Weissman H., Shimoni E., Sui X. M., Feldman Y., Wagner H. D. & Rybtchinski B. (2023) ACS Nano. 17, 21, p. 20962-20967
Development of biodegradable plastic materials is of primary importance in view of acute environmental and health problems associated with the accumulation of plastic waste. We fabricated a biodegradable composite material based on hydroxyethyl cellulose polymer and tyrosine nanocrystals, which demonstrates enhanced strength and ductility (typically mutually excluding properties), superior to most biodegradable plastics. This emergent behavior results from an assembly pattern that leads to a uniform nanoscale morphology and strong interactions between the components. Water-resistant biodegradable composites encapsulated with hydrophobic polycaprolactone as a protection layer were also fabricated. Self-assembly of robust sustainable plastics with emergent properties by using readily available building blocks provides a valuable toolbox for creating sustainable materials.
Cohen H., Wani N. A., Ben Hur D., Migliolo L., Cardoso M. H., Porat Z., Shimoni E., Franco O. L. & Shai Y. (2023) ACS Omega. 8, 20, p. 17856-17868
Antibiotic-resistant bacterial infections have increased the prevalence of sepsis and septic shock mortality worldwide and have become a global concern. Antimicrobial peptides (AMPs) show remarkable properties for developing new antimicrobial agents and host response modulatory therapies. A new series of AMPs derived from pexiganan (MSI-78) were synthesized. The positively charged amino acids were segregated at their N- and C-termini, and the rest of the amino acids created a hydrophobic core surrounded by positive charges and were modified to simulate the lipopolysaccharide (LPS). The peptides were investigated for their antimicrobial activity and LPS-induced cytokine release inhibition profile. Various biochemical and biophysical methods were used, including attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, microscale thermophoresis (MST), and electron microscopy. Two new AMPs, MSI-Seg-F2F and MSI-N7K, preserved their neutralizing endotoxin activity while reducing toxicity and hemolytic activity. Combining all of these properties makes the designed peptides potential candidates to eradicate bacterial infection and detoxify LPS, which might be useful for sepsis treatment.
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NEW ESR DATES FROM LOVEDALE, FREE STATE, SOUTH AFRICA: IMPLICATIONS FOR THE STUDY OF TOOTH DIAGENESIS
Richard M., Kaplan-Ashiri I., Alonso M. J., Pons-Branchu E., Dapoigny A., Rossouw L. & Toffolo M. B. (2023) South African Archaeological Bulletin. 78, 219, p. 95-103
Lovedale is the only open-air Middle Stone Age site in the Free State dated to early Marine Isotope Stage 4. Framing the chronology of this context, especially by taking into account diagenetic processes that may affect age results, is thus fundamental to understand modern human dynamics in the interior of South Africa. In a recent study, we investigated the effect of diagenesis on teeth samples collected for combined electron spin resonance and uranium-series dating at the site. By combining different characterisation methods, it was shown that the uranium (U) content of enamel varied in the specimens, and that it was positively correlated with the degree of crystallinity of carbonate hydroxyapatite, whereby larger amounts of U are associated with highly crystalline enamel. The large variability in U content was in contrast with the fact that teeth were found in the same depositional context. High levels of U in some of the samples limit the accuracy of age determinations, since several uncertainties remain regarding U uptake and leaching, which both affect dose rate modelling. In such complex cases, calculating minimum ages is the most cautious option. New samples were collected at the site during the excavation campaign in 2021. Enamel was analysed using Fourier transform infrared spectroscopy and scanning electron microscopy coupled with cathodoluminescence in order to determine its degree of atomic order and the presence of foreign ions (especially U), and the correlation between the two. We discuss here the contribution of U-uptake modelling on the age calculation, and present new ESR ages calculated assuming an early uptake of U, ranging from 84 ± 9 ka to 56 ± 5 ka. Together with previous ages obtained on the gravel layer, a weighted mean age of 64 ka can be used as a minimum age estimate for the base of the sequence.
Galili N., Kaplan-Ashiri I. & Halevy I. (2023) American Mineralogist. 108, 8, p. 1436-1448
Iron oxides and oxyhydroxides show promise as superconductor materials and as repositories of paleo-environmental information. However, there are no microscale non-destructive analytical techniques to characterize their combined mineralogy, chemical composition, and crystal properties. We address this by developing cathodoluminescence mounted on a scanning electron microscope (SEM-CL) as an in situ, non-destructive method for the crystallographic and petrographic study of iron oxides and oxyhydroxides. We show that goethite, hematite, and magnetite display different SEM-CL spectra, which may be used for mineral identification. We further show that different formation pH, manganese substitution for iron in goethite and hematite, and titanium substitution for iron in magnetite cause shifts in the SEM-CL spectra of these minerals. These spectral shifts are not always detectable as a change in the emission color but are easily discernable by quantitative analysis of the spectra. Together with subtle but observable variations in the SEM-CL spectra of natural goethite and hematite, we suggest that these dependences of the SEM-CL spectra on pH and chemical composition may be used as a means of identifying multiple episodes of mineralization and recrystallization. We apply the newly developed SEM-CL methods to two polished sections of natural samples and show that quantitative analysis of the spectra obtained allows the identification of differences between varieties of the same mineral that are not observable by other means. Like the application of SEM-CL to geologic samples in this study, we suggest that this approach may be used to explore the in situ chemistry and crystallinity of various natural and manufactured iron oxides and oxyhydroxides.
Gouveia A. G., Salgueiro B. A., Ranmar D. O., Antunes W. D., Kirchweger P., Golani O., Wolf S. G., Elbaum M., Matias P. M. & Romão C. V. (2023) Frontiers in Microbiology. 14, 1240798
Arsenic (As) is a toxic heavy metal widely found in the environment that severely undermines the integrity of water resources. Bioremediation of toxic compounds is an appellative sustainable technology with a balanced cost-effective setup. To pave the way for the potential use of Deinococcus indicus, an arsenic resistant bacterium, as a platform for arsenic bioremediation, an extensive characterization of its resistance to cellular insults is paramount. A comparative analysis of D. indicus cells grown in two rich nutrient media conditions (M53 and TGY) revealed distinct resistance patterns when cells are subjected to stress via UV-C and methyl viologen (MV). Cells grown in M53 demonstrated higher resistance to both UV-C and MV. Moreover, cells grow to higher density upon exposure to 25 mM As(V) in M53 in comparison with TGY. This analysis is pivotal for the culture of microbial species in batch culture bioreactors for bioremediation purposes. We also demonstrate for the first time the presence of polyphosphate granules in D. indicus which are also found in a few Deinococcus species. To extend our analysis, we also characterized DiArsC2 (arsenate reductase) involved in arsenic detoxification and structurally determined different states, revealing the structural evidence for a catalytic cysteine triple redox system. These results contribute for our understanding into the D. indicus resistance mechanism against stress conditions.
Kirchweger P., Mullick D., Swain P. P., Wolf S. G. & Elbaum M. (2023) Journal of Structural Biology. 215, 3, 107982
Visualization of organelles and their interactions with other features in the native cell remains a challenge in modern biology. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access 3D volumes on the scale of 1 micron with a resolution of nanometers, making it ideal for this task. Here we introduce two relevant advances: (a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and (b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy. Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to-isotropic resolution in the reconstruction without averaging. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.
Kirchweger P., Mullick D., Wolf S. G. & Elbaum M. (2023) Journal of Visualized Experiments. 2023, 196, e65052
Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two-and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.
Katz M., Weinstein J., Eilon-Ashkenazy M., Gehring K., Cohen-Dvashi H., Elad N., Fleishman S. J. & Diskin R. (2022) Nature. 603, 7899, p. 174-179
Lassa virus (LASV) is a human pathogen, causing substantial morbidity and mortality1,2. Similar to other Arenaviridae, it presents a class-I spike complex on its surface that facilitates cell entry. The viruss cellular receptor is matriglycan, a linear carbohydrate that is present on α-dystroglycan3,4, but the molecular mechanism that LASV uses to recognize this glycan is unknown. In addition, LASV and other arenaviruses have a unique signal peptide that forms an integral and functionally important part of the mature spike5,6,7,8; yet the structure, function and topology of the signal peptide in the membrane remain uncertain9,10,11. Here we solve the structure of a complete native LASV spike complex, finding that the signal peptide crosses the membrane once and that its amino terminus is located in the extracellular region. Together with a double-sided domain-switching mechanism, the signal peptide helps to stabilize the spike complex in its native conformation. This structure reveals that the LASV spike complex is preloaded with matriglycan, suggesting the mechanism of binding and rationalizing receptor recognition by α-dystroglycan-tropic arenaviruses. This discovery further informs us about the mechanism of viral egress and may facilitate the rational design of novel therapeutics that exploit this binding site.
Jayaraman V., Lee D. J., Elad N., Vimer S., Sharon M., Fraser J. S. & Tawfik D. S. (2022) Nature Chemical Biology. 18, 2, p. 161-170
Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite (counter-enzymes) rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations. [Figure not available: see fulltext.]
Shtrikman H., Song M. S., Załuska-Kotur M. A., Buczko R., Wang X., Kalisky B., Kacman P., Houben L. & Beidenkopf H. (2022) Nano Letters. 22, 22, p. 8925-8931
In the pursuit of magneto-electronic systems nonstoichiometric magnetic elements commonly introduce disorder and enhance magnetic scattering. We demonstrate the growth of (EuIn)As shells, with a unique crystal structure comprised of a dense net of Eu inversion planes, over InAs and InAs<sub>1-x</sub>Sb<sub>x</sub>core nanowires. This is imaged with atomic and elemental resolution which reveal a prismatic configuration of the Eu planes. The results are supported by molecular dynamics simulations. Local magnetic and susceptibility mappings show magnetic response in all nanowires, while a subset bearing a DC signal points to ferromagnetic order. These provide a mechanism for enhancing Zeeman responses, operational at zero applied magnetic field. Such properties suggest that the obtained structures can serve as a preferred platform for time-reversal symmetry broken one-dimensional states including intrinsic topological superconductivity.
Eyal Z., Deis R., Varsano N., Dezorella N., Rechav K., Houben L. & Gur D. (2022) Journal of the American Chemical Society. 144, 49, p. 22440-22445
Controlling the morphology of crystalline materials is challenging, as crystals have a strong tendency toward thermodynamically stable structures. Yet, organisms form crystals with distinct morphologies, such as the plate-like guanine crystals produced by many terrestrial and aquatic species for light manipulation. Regulation of crystal morphogenesis was hypothesized to entail physical growth restriction by the surrounding membrane, combined with fine-tuned interactions between organic molecules and the growing crystal. Using cryo-electron tomography of developing zebrafish larvae, we found that guanine crystals form via templated nucleation of thin leaflets on preassembled scaffolds made of 20-nm-thick amyloid fibers. These leaflets then merge and coalesce into a single plate-like crystal. Our findings shed light on the biological regulation of crystal morphogenesis, which determines their optical properties.
Biran I., Rosenne S., Weissman H., Tsarfati Y., Houben L. & Rybtchinski B. (2022) Crystal Growth & Design. 22, 11, p. 6647-6655
Organic crystal nucleation and growth are complex processes that often do not fit into the framework of the existing crystallization theories. We investigated a crystal growth mechanism of an organic dye, perylene diimide, using high -resolution cryogenic transmission electron microscopy and optical spectroscopy. The elucidated mechanism involves classical (monomer attachments) and nonclassical pathways, exhibiting a self-assembly sequence where all steps are interconnected. It starts from the assembly of molecular pi-stacks that are initially disordered. They gradually optimize their structure, rigidify, and interact to form crystalline domains. The latter further evolve via the addition of individual molecules, and crystal fusion (via oriented attachment). All the observed supramolecular trans-formations are connected and follow a clear hierarchy starting from the molecular-scale interactions. The elucidation of the complex pathway of organic crystallization as a series of coordinated supramolecular transformations at multiple scales conceptually advances the understanding of order evolution in organic matter.
Sreedhara M. B., Miroshnikov Y., Zheng K., Houben L., Hettler S., Arenal R., Pinkas I., Sinha S. S., Castelli I. E. & Tenne R. (2022) Journal of the American Chemical Society. 144, 23, p. 10530-10542
Nanotubes of transition metal dichalcogenides such as WS2 and MoS2 offer unique quasi-1D properties and numerous potential applications. Replacing sulfur by selenium would yield ternary WS2(1x)Se2x (0 ≤ x ≤ 1; WSSe) nanotubes, which are expected to reveal strong modulation in their absorption edge as a function of selenium content, x Se. Solid WO2.72 oxide nanowhiskers were employed as a sacrificial template to gain a high yield of the nanotubes with a rather uniform size distribution. Though sulfur and selenium belong to the same period, their chemical reactivity with oxide nanowhiskers differed appreciably. Here, the closed ampoule technique was utilized to achieve the completion of the solidvapor reaction in short time scales instead of the conventional flow reactor method. The structure and chemical composition of the nanotubes were analyzed in detail. X-ray and electron diffractions indicated a systematic modulation of the WSSe lattice upon increasing the selenium content. Detailed chemical mapping showed that the sulfur and selenium atoms are distributed in random positions on the anion lattice site of the nanotubes. The optical excitonic features and absorption edges of the WSSe nanotubes do not vary linearly with the composition x Se, which was further confirmed by density functional theory calculations. The WSSe nanotubes were shown to exhibit strong lightmatter interactions forming excitonpolariton quasiparticles, which was corroborated by finite-difference time-domain simulations. Transient absorption analysis permitted following the excited state dynamics and elucidating the mechanism of the strong coupling. Thus, nanotubes of the ternary WSSe alloys offer strong band gap tunability, which would be useful for multispectral vision devices and other optoelectronic applications.
Biran I., Houben L., Weismann H., Hildebrand M., Kronik L. & Rybtchinski B. (2022) Advanced materials (Weinheim). 34, 26, 2202088
Structural analysis of beam-sensitive materials by transmission electron microscopy (TEM) represents a significant challenge, as high resolution TEM (HRTEM) requires high electron doses that limits its applicability to stable inorganic materials. Beam sensitive materials, such as organic crystals (of key importance in pharmaceuticals, organic electronics, and biology) must be imaged under low dose conditions, leading to problematic contrast interpretation and the loss of fine structural details. Here, we describe HRTEM imaging of organic crystalline materials with near-atomic resolution of up to 1.6 Å that enabled the real-space study of crystal structures, as well as observation of co-existing polymorphs, crystal defects, and atoms. This is made possible by a low-dose focal series reconstruction (LD-FSR) methodology developed by us, which provides HRTEM images where contrast reflects true object structure and can be performed on contemporary cryo-EM instruments available to many research institutions. We imaged copper phthalocyanine (CuPc), perchlorinated analogue of CuPc, and indigo crystalline films. In the case of indigo crystals, we were able to observe co-existing polymorphs and individual atoms (carbonyl oxygen). In the case of CuPc, we observed several polymorphs, including a new one, for which we elucidated the crystal structure based on direct in-focus imaging, accomplishing real-space crystal structure elucidation. Direct structural analysis of beam sensitive materials with high resolution that enables the real-space study of crystals can be transformative for structural science of organic materials.
Avrahami E. M., Houben L., Aram L. & Gal A. (2022) Science. 376, 6590, p. 312-316
Directing crystal growth into complex morphologies is challenging, as crystals tend to adopt thermodynamically stable morphologies. However, many organisms form crystals with intricate morphologies, as exemplified by coccoliths, microscopic calcite crystal arrays produced by unicellular algae. The complex morphologies of the coccolith crystals were hypothesized to materialize from numerous crystallographic facets, stabilized by fine-tuned interactions between organic molecules and the growing crystals. Using electron tomography, we examined multiple stages of coccolith development in three dimensions. We found that the crystals express only one set of symmetry-related crystallographic facets, which grow differentially to yield highly anisotropic shapes. Morphological chirality arises from positioning the crystals along specific edges of these same facets. Our findings suggest that growth rate manipulations are sufficient to yield complex crystalline morphologies.
Kumar S., Houben L., Rechav K. & Cahen D. (2022) Proceedings of the National Academy of Sciences - PNAS. 119, 10, e211474011
Ultra-thin hydrophobic capping layers of two-dimensional (2D) onto three-dimensional (3D) metal halide perovskites (HaPs) are an attractive strategy for preventing ambient-induced degradation and minimizing interfacial non-idealities of 3D HaPs. However, it is not obvious in how far the unusual 3D HaP lattice dynamics affect 2D-on-3D HaP composites' stability, especially at their interface, an issue important for devices made with such composites. Using low electron-fluence, four-dimensional scanning transmission electron microscopy and nanobeam electron diffraction, we show formation of the 2D (n = 1) phase on top of 3D perovskite, using focused-ion beam-prepared cross-sections, under conditions that minimize radiation damage. The 2D-on-3D HaP composites were prepared by controlled gas-phase surface cation exchange of 3D MAPbI3 films to form A2PbI4, where A = (fluoro- )phenyl-ethyl-ammonium, (F)PEA. We provide direct evidence for 2D phase formation also inside the 3D matrix, likely via A cation grain boundary diffusion, and, over time, of quasi-2D phases near the surface. These results show that the 2D/3D heterointerface is dynamic; they imply that not only small, but also large A cations, (F)PEA+, migrate. Structural rearrangements, leading to quasi-2D phase formation can be followed with the electron beam, which provides hitherto unknown atomistic insights into such interfaces, needed to assess their (in)stability. Apart from directly illustrating effects of HaP lattice dynamics, our results help understanding extensive (semi)empirical data on engineering 2D-on-3D composites and provide guidance for enhancing stability of such systems. Critically, our direct observation of electron beam-induced loss of long-range periodicity defines conditions for damage-free atomicresolution studies of HaP samples, also in device-relevant configurations.
Alam M. H., Chowdhury S., Roy A., Wu X., Ge R., Rodder M. A., Chen J., Lu Y., Stern C., Houben L., Chrostowski R., Burlison S. R., Yang S. J., Serna M. I., Dodabalapur A., Mangolini F., Naveh D., Lee J. C., Banerjee S. K., Warner J. H. & Akinwande D. (2022) ACS Nano. 16, 3, p. 3756-3767
Molybdenum trioxide (MoO<sub>3</sub>), an important transition metal oxide (TMO), has been extensively investigated over the past few decades due to its potential in existing and emerging technologies, including catalysis, energy and data storage, electrochromic devices, and sensors. Recently, the growing interest in two-dimensional (2D) materials, often rich in interesting properties and functionalities compared to their bulk counterparts, has led to the investigation of 2D MoO<sub>3</sub>. However, the realization of large-area true 2D (single to few atom layers thick) MoO<sub>3</sub>is yet to be achieved. Here, we demonstrate a facile route to obtain wafer-scale monolayer amorphous MoO<sub>3</sub>using 2D MoS<sub>2</sub>as a starting material, followed by UV-ozone oxidation at a substrate temperature as low as 120 °C. This simple yet effective process yields smooth, continuous, uniform, and stable monolayer oxide with wafer-scale homogeneity, as confirmed by several characterization techniques, including atomic force microscopy, numerous spectroscopy methods, and scanning transmission electron microscopy. Furthermore, using the subnanometer MoO<sub>3</sub>as the active layer sandwiched between two metal electrodes, we demonstrate the thinnest oxide-based nonvolatile resistive switching memory with a low voltage operation and a high ON/OFF ratio. These results (potentially extendable to other TMOs) will enable further exploration of subnanometer stoichiometric MoO<sub>3</sub>, extending the frontiers of ultrathin flexible oxide materials and devices.
Sreedhara M. B., Bukvišová K., Khadiev A., Citterberg D., Cohen H., Balema V., K. Pathak A., Novikov D., Leitus G., Kaplan-Ashiri I., Kolíbal M., Enyashin A. N., Houben L. & Tenne R. (2022) Chemistry of Materials. 34, 4, p. 1838-1853
Misfit layered compounds (MLCs) MX-TX2, where M, T = metal atoms and X = S, Se, or Te, and their nanotubes are of significant interest due to their rich chemistry and unique quasi-1D structure. In particular, LnX-TX2 (Ln = rare-earth atom) constitute a relatively large family of MLCs, from which nanotubes have been synthesized. The properties of MLCs can be tuned by the chemical and structural interplay between LnX and TX2 sublayers and alloying of each of the Ln, T, and X elements. In order to engineer them to gain desirable performance, a detailed understanding of their complex structure is indispensable. MLC nanotubes are a relative newcomer and offer new opportunities. In particular, like WS2 nanotubes before, the confinement of the free carriers in these quasi-1D nanostructures and their chiral nature offer intriguing physical behavior. High-resolution transmission electron microscopy in conjunction with a focused ion beam are engaged to study SmS-TaS2 nanotubes and their cross-sections at the atomic scale. The atomic resolution images distinctly reveal that Ta is in trigonal prismatic coordination with S atoms in a hexagonal structure. Furthermore, the position of the sulfur atoms in both the SmS and the TaS2 sublattices is revealed. X-ray photoelectron spectroscopy, electron energy loss spectroscopy, and X-ray absorption spectroscopy are carried out. These analyses conclude that charge transfer from the Sm to the Ta atoms leads to filling of the Ta 5d z 2 level, which is confirmed by density functional theory (DFT) calculations. Transport measurements show that the nanotubes are semimetallic with resistivities in the range of 104 Ω·cm at room temperature, and magnetic susceptibility measurements show a superconducting transition at 4 K.
Khatun S., Andrés M. A., Cohen S. R., Kaplan-Ashiri I., Brontvein O., Rosenhek-Goldian I., Weatherup R. S. & Eren B. (2022) Electrochimica Acta. 431, 141145
A micro-electrochemical cell is sealed with a polymer-free single-layer graphene (SLG) membrane to monitor the stability of Cu nanoparticles (NPs) attached to SLG, as well as the interfacial electronic interactions between Cu NPs and SLG both in air and in a mildly alkaline aqueous solution under electrochemical control. A combination of techniques, including in-situ Kelvin probe force microscopy (KPFM) and ex-situ electron microscopy, are applied. When Cu NPs are metallic at cathodic potentials, there is a relatively bias-independent offset in the SLG work function due to charge transfer at the Cu-SLG contact. When Cu NPs are oxidized at anodic potentials, on the other hand, the work function of SLG also depends on the applied bias in a quasi-linear fashion due to electrochemical gating, in addition to charge transfer at the CuO<sub>x</sub>-SLG contact. Furthermore, Cu NPs were found to oxidize and detach from SLG when kept under anodic potentials for a few hours, whereas they remain adhered to SLG at cathodic potentials. This is attributed to water intercalation at the CuO-SLG interface associated with the enhanced hydrophilicity of positively polarized graphene, as supported by the absence of Cu detachment following oxidation by galvanic corrosion in air.
Polyansky A., Shatz O., Fraiberg M., Shimoni E., Dadosh T., Mari M., Reggiori F. M., Qin C., Han X. & Elazar Z. (2022) EMBO Journal. 41, 23, e110771
Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.
Kluzek M., Oppenheimer-Shaanan Y., Dadosh T., Morandi M. I., Avinoam O., Raanan C., Goldsmith M., Goldberg R. & Klein J. (2022) ACS Nano. 16, 10, p. 15792-15804
Drug delivery via nanovehicles is successfully employed in several clinical settings, yet bacterial infections, forming microbial communities in the form of biofilms, present a strong challenge to therapeutic treatment due to resistance to conventional antimicrobial therapies. Liposomes can provide a versatile drug-vector strategy for biofilm treatment, but are limited by the need to balance colloidal stability with biofilm penetration. We have discovered a liposomic functionalization strategy, using membrane-embedded moieties of poly[2-(methacryloyloxy)ethyl phosphorylcholine], pMPC, that overcomes this limitation. Such pMPCylation results in liposomic stability equivalent to current functionalization strategies (mostly PEGylation, the present gold-standard), but with strikingly improved cellular uptake and cargo conveyance. Fluorimetry, cryo-electron, and fluorescence microscopies reveal a far-enhanced antibiotic delivery to model Pseudomonas aeruginosa biofilms by pMPC-liposomes, followed by faster cytosolic cargo release, resulting in significantly greater biofilm eradication than either PEGylation or free drug. Moreover, this combination of techniques uncovers the molecular mechanism underlying the enhanced interaction with bacteria, indicating it arises from bridging by divalent ions of the zwitterionic groups on the pMPC moieties to the negatively charged lipopolysaccharide chains emanating from the bacterial membranes. Our results point to pMPCylation as a transformative strategy for liposomal functionalization, leading to next-generation delivery systems for biofilm treatment.
Arbel Y. S., Bronstein Y., Dadosh T., Kamdjou T., Tsuriel S., Shapiro M., Katz B. & Herishanu Y. (2022) Frontiers in Immunology. 13, 953660
Most chronic lymphocytic leukemia (CLL) clones express B-cell receptors (BcR) of both IgM/IgD isotypes; however, 5%10% of CLL cases express isotype-switched immunoglobulin G (IgG). The early signaling and spatial patterning of the various BcRs at steady state and after activation are still fully unresolved. Herein, we show higher expression of the BcR signalosome elements and a more robust constitutive cell-intrinsic proximal BcR signaling in CLL with unmutated IGHV expressing IgM isotype (IgM U-CLL), compared with IGHV-mutated CLL (M-CLL) expressing either IgM or IgG isotypes. IgM in U-CLL is frequently located in the membrane plane in polarized patches, occasionally in caps, and sometimes inside the cells. Among M-CLL, IgM is scattered laterally in the membrane plane in a similar pattern as seen in normal B cells, whereas IgG is dispersed around the cell membrane in smaller clusters than in IgM U-CLL. Upon BcR engagement, both IgG and IgM expressing M-CLL showed attenuated signaling and only slight spatial reorganization dynamics of BcR microclusters and internalization, compared with the extensive reorganization and internalization of the BcR in IgM expressing U-CLL. The global gene signature of IgG M-CLL was closely related to that of IgM M-CLL rather than IgM U-CLL. Overall, we report fundamental differences in the basal composition, biochemical status, and spatial organization of the BcR in the three examined immunogenetic CLL subtypes that correlate with their clinical behavior. On the basis of our findings, IgG class-switched M-CLL likely represents the same disease as IgM M-CLL rather than a different biological and/or clinical entity.
Song D., Alber S., Doron-Mandel E., Schmid V., Albus C. A., Leitner O., Hamawi H., Oses-Prieto J. A., Dezorella N., Burlingame A. L., Fainzilber M. & Rishal I. (2022) Molecular & Cellular Proteomics. 21, 11, 100418
Importin β1 (KPNB1) is a nucleocytoplasmic transport factor with critical roles in both cytoplasmic and nucleocytoplasmic transport, hence there is keen interest in the characterization of its subcellular interactomes. We found limited efficiency of BioID in detection of importin complex cargos, and therefore generated a highly specific and sensitive anti-KPNB1 monoclonal antibody to enable Biotinylation by Antibody Recognition (BAR) analysis of importin β1 interactomes. The monoclonal antibody recognizes an epitope comprising residues 301-320 of human KPBN1, and strikingly is highly specific for cytoplasmic KPNB1 in diverse applications, with little reaction with KPNB1 in the nucleus. BAR with this novel antibody revealed numerous new interactors of importin β1, expanding the KPNB1 interactome to cytoplasmic and signaling complexes that highlight potential new functions for the importins complex beyond nucleocytoplasmic transport. Data are available via ProteomeXchange with identifier PXD032728.
Vinestock R. C., Felsenthal N., Assaraf E., Katz E., Rubin S., Heinemann-Yerushalmi L., Krief S., Dezorella N., Levin-Zaidman S., Tsoory M., Thomopoulos S. & Zelzer E. (2022) American Journal of Pathology. 192, 8, p. 1122-1135
Wound healing typically recruits the immune and vascular systems to restore tissue structure and function. However, injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Herein, this study established an injury model to the Achilles entheses of neonatal mice to study the effectiveness of early-age enthesis healing. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including endoplasmic reticulum stress, autophagy, and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. These findings reveal a novel healing mechanism in neonatal entheses, whereby local extracellular matrix secretion by resident cells forms an acellular extracellular matrix deposit without inflammation, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.
Biram A., Liu J., Hezroni-Bravyi H., Davidzohn N., Schmiedel D., Khatib-Massalha E., Haddad M., Grenov C. A., Lebon S., Salame T. M., Dezorella N., Hoffman D., Abou Karam P., Biton M., Lapidot T., Bemark M., Avraham R., Jung S. & Shulman Z. (2022) Immunity. 55, 3, p. 442-458.e8
Consecutive exposures to different pathogens are highly prevalent and often alter the host immune response. However, it remains unknown how a secondary bacterial infection affects an ongoing adaptive immune response elicited against primary invading pathogens. We demonstrated that recruitment of Sca-1+ monocytes into lymphoid organs during Salmonella Typhimurium (STm) infection disrupted pre-existing germinal center (GC) reactions. GC responses induced by influenza, plasmodium, or commensals deteriorated following STm infection. GC disruption was independent of the direct bacterial interactions with B cells and instead was induced through recruitment of CCR2-dependent Sca-1+ monocytes into the lymphoid organs. GC collapse was associated with impaired cellular respiration and was dependent on TNFα and IFNγ, the latter of which was essential for Sca-1+ monocyte differentiation. Monocyte recruitment and GC disruption also occurred during LPS-supplemented vaccination and Listeria monocytogenes infection. Thus, systemic activation of the innate immune response upon severe bacterial infection is induced at the expense of antibody-mediated immunity.
Halimi R., Levin-Zaidman S., Levin-Salomon V., Bialik S. & Kimchi A. (2022) Cell Death and Differentiation. 29, 6, p. 1255-1266
The role of programmed cell death during embryonic development has been described previously, but its specific contribution to peri- and post-implantation stages is still debatable. Here, we used transmission electron microscopy and immunostaining of E5.5-7.5 mouse embryos to investigate death processes during these stages of development. We report that in addition to canonical apoptosis observed in E5.5-E7.5 embryos, a novel type of cell elimination occurs in E7.5 embryos among the epiblasts at the apical side, in which cells shed membrane-enclosed fragments of cytosol and organelles into the lumen, leaving behind small, enucleated cell remnants at the apical surface. This process is caspase-independent as it occurred in Apaf1 knockout embryos. We suggest that this novel mechanism controls epiblast cell numbers. Altogether, this work documents the activation of two distinct programs driving irreversible terminal states of epiblast cells in the post-implantation mouse embryo.
Capua-Shenkar J., Varsano N., Itzhak N., Kaplan-Ashiri I., Rechav K., Jin X., Niimi M., Fan J., Kruth H. & Addadi L. (2022) Proceedings of the National Academy of Sciences of the United States of America. 119, 34
We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryoscanning electron microscopy (SEM), and cryofocused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the medialesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The medialesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density. In the three regions, thin crystals appear to be associated with intracellular or extracellular lipid droplets and multilamellar bodies. Large crystals are independently positioned in the tissue, not associated with specific cellular components. This extensive evidence strongly supports the idea that the lipid droplet surfaces and the outer membranes of multilamellar bodies play a role in cholesterol crystal nucleation and growth and that crystal formation occurs, in part, inside cells. The correlative combination of methods that allowed the direct examination of cholesterol crystals and lipid deposits in the atherosclerotic lesions may be similarly used for high-resolution examination of other tissues containing pathological or physiological cholesterol deposits.
Mullick D., Rechav K., Leiserowitz L., Regev-Rudzki N., Dzikowski R. & Elbaum M. (2022) Faraday Discussions. 240, p. 127-141
Malaria is a potentially fatal infectious disease caused by the obligate intracellular parasite Plasmodium falciparum. The parasite infects human red blood cells (RBC) and derives nutrition by catabolism of hemoglobin. As amino acids are assimilated from the protein component, the toxic heme is released. Molecular heme is detoxified by rapid sequestration to physiologically insoluble hemozoin crystals within the parasite's digestive vacuole (DV). Common antimalarial drugs interfere with this crystallization process, leaving the parasites vulnerable to the by-product of their own metabolism. A fundamental debate with important implications on drug mechanism regards the chemical environment of crystallization in situ, whether aqueous or lipid. This issue had been addressed previously by cryogenic soft X-ray tomography. We employ cryo-scanning transmission electron tomography (CSTET) to probe parasite cells throughout the life cycle in a fully hydrated, vitrified state at higher resolution. During the acquisition of CSTET data, Bragg diffraction from the hemozoin provides a uniquely clear view of the crystal boundary at nanometer resolution. No intermediate medium, such as a lipid coating or shroud, could be detected surrounding the crystals. The present study describes a unique application of CSTET in the study of malaria. The findings can be extended to evaluate new drug candidates affecting hemozoin crystal growth.
Sibony-Nevo O., Rechav K., Farstey V., Shimoni E., Varsano N., Addadi L. & Weiner S. (2022) MRS Bulletin. 47, 1, p. 18-28
Abstract: Snails of the superfamily Cavolinioidea, known as pteropods, are very abundant in the surface waters of all the oceans. Their transparent and lightweight shells are composed of densely packed, well-aligned, continuously crystalline curved aragonite fibers. Previous studies of the shell microstructure using mainly scanning electron microscopy, transmission electron microscopy, and x-ray diffraction suggested that the aragonite fibers adopt a helical motif. We mainly used focused ion beam-scanning electron microscopy to obtain three-dimensional information on the shell structure of Creseis acicula. We show that the basic structural motif in the central part of the shell (teleoconch) comprises aragonite fibers that are not helical, but are organized in nested S-shaped arcs arranged in planar arrays. This plane is oblique to the outer shell surface by approximately 20°. The planes stack in the third dimension with local displacements, to form a unique biological material. Impact statement: Of the seven basic materials used by mollusks to build their shells, the structure of one of these materials remains enigmatic, even though the snails that form this structure are by far the most abundant mollusks on earth. These so-called pteropods live in the surface waters of all the oceans and produce a significant amount of all the calcium carbonate formed in the open oceans. Since the first study of the pteropod shell structure in 1972, the basic structural motif of the arrays of highly elongated aragonite crystal fibers was inferred to be helical, although no one actually documented an entire helix. Here we resolved the 3D structure of the shell of one pteropod species using an instrument (FIB-SEM) that produces a high resolution 3D structure. We show that the basic repeating unit is a planar layer of nested S-shaped aragonite crystal fibers. Furthermore this planar layer is oblique to the shell outer surface. Besides resolving a fundamental basic question concerning mollusk shell structures, this unique organization of crystals raises fascinating questions about the mechanical properties of this most unusual curved space filling structure that will hopefully inspire materials scientists to produce superior synthetic materials.
Scher N., Rechav K., Paul-Gilloteaux P. & Avinoam O. (2022) STAR Protocols. 3, 1, 101142
We recently demonstrated how lipid droplets can serve as in situ fiducials for correlating cryo-fluorescence microscopy (cryo-FM) and cryo-focused ion beam scanning electron microscopy (cryo-FIB-SEM) datasets of mammalian cells grown on grids. Here we describe a step-by-step protocol for correlative cryo-FM and cryo-FIB-SEM, starting from sample preparation of C2C12 cell line, followed by imaging with cryo-FM and cryo-FIB-SEM. Finally, we detail how to perform the 3D-correlation with sub-micron accuracy. For complete details on the use and execution of this profile, please refer to Scher et al. (2021).
Friedman O., Böhm A., Rechav K., Pinkas I., Brumfeld V., Pass G., Weiner S. & Addadi L. (2022) Journal of Structural Biology. 214, 1, p. 107834-107834, 107834
Biogenic purine crystals function in vision as mirrors, multilayer reflectors and light scatterers. We investigated a light sensory organ in a primarily wingless insect, the jumping bristletail Lepismachilis rozsypali (Archaeognatha), an ancestral group. The visual system of this animal comprises two compound eyes, two lateral ocelli, and a median ocellus, which is located on the front of the head, pointing downwards to the ground surface. We determined that the median ocellus contains crystals of xanthine, and we obtained insights into their function. To date, xanthine biocrystals have only been found in the Archaeognatha. We performed a structural analysis, using reflection light microscopy, cryo-FIB-SEM, microCT and cryo-SEM. The xanthine crystals cover the bottom of a bowl-shaped volume in the median ocellus, in analogy to a tapetum, and reflect photons to light-sensitive receptors that are spread in the volume without apparent order or preferential orientation. We infer that the median ocellus operates as an irregular multifocal reflector, which is not capable of forming images. A possible function of this organ is to improve photon capture, and by so doing assess distances from the ground surface when jumping by determining changes in the intensity and contrast of the incident light.
Varsano N. & Wolf S. G. (2022) Current Opinion in Structural Biology. 76, 102444
Electron microscopy in three dimensions (3D) of cells and tissues can be essential for understanding the ultrastructural aspects of biological processes. The quest for 3D information reveals challenges at many stages of the workflow, from sample preparation, to imaging, data analysis and segmentation. Here, we outline several available methods, including volume SEM imaging, cryo-TEM and cryo-STEM tomography, each one occupying a different domain in the basic tradeoff between field-of-view and resolution. We discuss the considerations for choosing a suitable method depending on research needs and highlight recent developments that are essential for making 3D volume imaging of cells and tissues a standard tool for cellular and structural biologists.
Varsano N., Capua-Shenkar J., Leiserowitz L. & Addadi L. (2022) Annual Review of Materials Research. 52, p. 57-78
Cholesterol is an essential component of animal cell membranes because it influences and controls cell membrane fluidity. Cholesterol is also responsible for the most frequent lethal pathologies in developed countries because of its intimate association with atherosclerotic plaques, the rupture of which may cause heart attacks or strokes. The question is under which conditions cholesterol activity manifests itself, whether in physiology or in pathology. The answer is complex, and there is probably not one certain answer. This review article has its foundations in abundant published knowledge and evidence, but it cannot possibly be comprehensive, because the extent of cholesterol's involvement in chemistry, biology, biophysics, and medicine is so vast that we cannot embrace it all. We review cholesterol as a molecule and in its various crystalline polymorphs. We then examine cholesterol assembly pathways and, finally, cholesterol in biology and in pathology. We propose that cholesterol activity depends on its assembly states in cholesterol crystals or with other lipids in the form of more-or-less organized crystalline domains. In other words, we analyze cholesterol material properties because the assembly state of the cholesterol molecules profoundly affects the properties of the environment in which they reside.
Shepelenko M., Hirsch A., Varsano N., Beghi F., Addadi L., Kronik L. & Leiserowitz L. (2022) Journal of the American Chemical Society. 144, 12, p. 5304-5314
We revisit the important issues of polymorphism, structure, and nucleation of cholesterol·H<sub>2</sub>O using first-principles calculations based on dispersion-augmented density functional theory. For the lesser known monoclinic polymorph, we obtain a fully extended H-bonded network in a structure akin to that of hexagonal ice. We show that the energy of the monoclinic and triclinic polymorphs is similar, strongly suggesting that kinetic and environmental effects play a significant role in determining polymorph nucleation. Furthermore, we find evidence in support of various O-H···O bonding motifs in both polymorphs that may result in hydroxyl disorder. We have been able to explain, via computation, why a single cholesterol bilayer in hydrated membranes always crystallizes in the monoclinic polymorph. We rationalize what we believe is a single-crystal to single-crystal transformation of the monoclinic form on increased interlayer growth beyond that of a single cholesterol bilayer, interleaved by a water bilayer. We show that the ice-like structure is also relevant to the related cholestanol·2H<sub>2</sub>O and stigmasterol·H<sub>2</sub>O crystals. The structure of stigmasterol hydrate both as a trilayer film at the air-water interface and as a macroscopic crystal further assists us in understanding the polymorphic and thermal behavior of cholesterol·H<sub>2</sub>O. Finally, we posit a possible role for one of the sterol esters in the crystallization of cholesterol·H<sub>2</sub>O in pathological environments, based on a composite of a crystalline bilayer of cholesteryl palmitate bound epitaxially as a nucleating agent to the monoclinic cholesterol·H<sub>2</sub>O form.
Yonai E., Shimoni E., Kahil Guterman K. & Blonder R. (2022) The biophysicist.. 3, 1
Before March 2020, with the outbreak of the COVID-19 pandemic, remote instruction of science was only moderately developed compared with more traditional approaches for learning science. Since the outbreak, however, all formal education systems have been carried out in remote mode, and outreach activities that take place in a research or academic setting have usually been canceled, or there has been a search for innovative approaches to shift to digital space. Therefore, the development of learning and teaching strategies has currently focused on remote activities. In this study, a design-based approach was applied to transform an existing authentic science activity using a scanning electron microscope (SEM) from face-to-face to remote learning mode. The remote mode activity included the remote operation of the SEM by the participants. The goal was to formulate a general approach to transform authentic outreach activities from face-to-face to remote operation. To evaluate the design, we compared learners' perceived authenticity in the 2 modes and reflected on the process. Data were collected with a Likert-type questionnaire regarding participants' perceived authenticity. The results suggest that items of authenticity related to the experience of learning content have a positive potential for use in remote mode. The learners' experience of connecting with the scientists is an apparent disadvantage in remote mode. However, changes in communication technology or in the pedagogy of remote teaching is a promising direction for improving social experience.
Rothman A., Bukvišová K., Itzhak N. R., Kaplan-Ashiri I., Kossoy A. E., Sui X., Novák L., Šikola T., Kolíbal M. & Joselevich E. (2022) ACS Nano. 16, 11, p. 18757-18766
Surface-guided growth has proven to be an efficient approach for the production of nanowire arrays with controlled orientations and their large-scale integration into electronic and optoelectronic devices. Much has been learned about the different mechanisms of guided nanowire growth by epitaxy, graphoepitaxy, and artificial epitaxy. A model describing the kinetics of surface-guided nanowire growth has been recently reported. Yet, many aspects of the surface-guided growth process remain unclear due to a lack of its observation in real time. Here we observe how surface-guided nanowires grow in real time by in situ scanning electron microscopy (SEM). Movies of ZnSe surface-guided nanowires growing on periodically faceted substrates of annealed M-plane sapphire clearly show how the nanowires elongate along the substrate nanogrooves while pushing the catalytic Au nanodroplet forward at the tip of the nanowire. The movies reveal the timing between competing processes, such as planar vs nonplanar growth, catalyst-selective vapor-liquid-solid elongation vs nonselective vapor-solid thickening, and the effect of topographic discontinuities of the substrate on the growth direction, leading to the formation of kinks and loops. Contrary to some observations for nonplanar nanowire growth, planar nanowires are shown to elongate at a constant rate and not by jumps. A decrease in precursor concentration as it is consumed after long reaction time causes the nanowires to shrink back instead of growing, thus indicating that the process is reversible and takes place near equilibrium. This real-time study of surface-guided growth, enabled by in situ SEM, enables a better understanding of the formation of nanostructures on surfaces.
Cohen A., Cohen H., Cohen S., Khodorov S., Feldman Y., Kossoy A., Kaplan-Ashiri I., Frenkel A., Wachtel E., Lubomirsky I. & Ehre D. (2022) Sensors (Basel, Switzerland). 22, 18, p. 1-16, 7041
A protocol for successfully depositing [001] textured, 23 µm thick films of Al0.75Sc0.25N, is proposed. The procedure relies on the fact that sputtered Ti is [001]-textured α-phase (hcp). Diffusion of nitrogen ions into the α-Ti film during reactive sputtering of Al0.75,Sc0.25N likely forms a [111]-oriented TiN intermediate layer. The lattice mismatch of this very thin film with Al0.75Sc0.25N is ~3.7%, providing excellent conditions for epitaxial growth. In contrast to earlier reports, the Al0.75Sc0.25N films prepared in the current study are Al-terminated. Low growth stress (
Nasi H., Chiara di Gregorio M., Wen Q., Shimon L. J. W., Kaplan-Ashiri I., Bendikov T., Leitus G., Kazes M., Oron D., Lahav M. & van der Boom M. E. (2022) Angewandte Chemie (International ed.). 61, 34, e202205238
We show that metal-organic frameworks, based on tetrahedral pyridyl ligands, can be used as a morphological and structural template to form a series of isostructural crystals having different metal ions and properties. An iterative crystal-to-crystal conversion has been demonstrated by consecutive cation exchanges. The primary manganese-based crystals are characterized by an uncommon space group ( P622 ). The packing includes chiral channels that can mediate the cation exchange, as indicated by energy-dispersive X-ray spectroscopy on microtome-sectioned crystals. The observed cation exchange is in excellent agreement with the Irving-Williams series (Mn < Fe < Co < Ni < Cu > Zn) associated with the relative stability of the resulting coordination nodes. Furthermore, we demonstrate how the metal cation controls the optical and magnetic properties. The crystals maintain their morphology, allowing a quantitative comparison of their properties at both the ensemble and single-crystal level.
Richard M., Pons-Branchu E., Carmieli R., Kaplan-Ashiri I., Alvaro Gallo A., Ricci G., Caneve L., Wroth K., Dapoigny A., Tribolo C., Boaretto E. & Toffolo M. (2022) Quaternary Geochronology. 69, 101269
Teeth are usually targeted for dating archaeological sites because they are less prone to dissolution, in com-parison with bones. However, despite this apparent resistance, teeth do undergo diagenesis, which needs to beaccounted for in order to obtain accurate ages. In particular, the uptake of trace elements such as uranium indental tissues needs to be considered for dose rate determination when dated using electron spin resonance(ESR). Characterising the mineralogy and structural integrity of samples prior to dating may thus provideimportant information related to their state of preservation, especially in the case of teeth whose U content cansignificantly affect the dose rate.In this study, we dated five teeth of small-sized bovids using combined ESR/U-series dating. They werecollected at the Middle Stone Age site of Lovedale, located in the central interior of South Africa. Micromor-phology provided sedimentary context to the samples, which were recovered from a layer of gravel rich in faunalremains. Using cathodoluminescence, laser-induced fluorescence, Fourier transform infrared spectroscopy andRaman micro-spectroscopy we assessed the degree of preservation of the enamel. Results reveal that carbonatehydroxyapatite underwent post-depositional alteration, based on its molecular structure and elemental compo-sition. Although the teeth all originate from the same layer and were sampled in the same 1-m square and at asimilar elevation, U-content in the enamel differs highly from one tooth to the other, with values ranging from1.7 to 29.6 ppm. These values are correlated with equivalent doses (De) from 228 to 923 Gy and are consistentwith variations in crystallinity determined with vibrational spectroscopy. We also investigated the possiblesaturation of the ESR signal, by repeating measurements with microwave power values from 1 to 20 mW.Despite such diversity in U-content, the ages calculated assuming an early uptake of U all fall within the samerange, from 63 ± 8 ka to 68 ± 15 ka and may only represent a minimum estimate.
Paikar A., Novichkov A. I., Hanopolskyi A. I., Smaliak V. A., Sui X., Kampf N., Skorb E. V. & Semenov S. N. (2022) Advanced materials (Weinheim). 34, 13, 2106816
Regulating hydrogel actuators with chemical reaction networks is instrumental for constructing life-inspired smart materials. Herein, hydrogel actuators are engineered that are regulated by the autocatalytic front of thiols. The actuators consist of two layers. The first layer, which is regular polyacrylamide hydrogel, is in a strained conformation. The second layer, which is polyacrylamide hydrogel with disulfide crosslinks, maintains strain in the first layer. When thiols released by the autocatalytic front reduce disulfide crosslinks, the hydrogel actuates by releasing the mechanical strain in the first layer. The autocatalytic front is sustained by the reaction network, which uses thiouronium salts, disulfides of beta-aminothiols, and maleimide as starting components. The gradual actuation by the autocatalytic front enables movements such as gradual unrolling, screwing, and sequential closing of "fingers." This actuation also allows the transmission of chemical signals in a relay fashion and the conversion of a chemical signal to an electrical signal. Locations and times of spontaneous initiation of autocatalytic fronts can be preprogrammed in the spatial distribution of the reactants in the hydrogel. To approach the functionality of living matter, the actuators triggered by an autocatalytic front can be integrated into smart materials regulated by chemical circuits.
Plavec Z., Domanska A., Liu X., Laine P., Paulin L., Varjosalo M., Auvinen P., Wolf S. G., Anastasina M. & Butcher S. J. (2022) Viruses. 14, 9, 1989
Severe acute respiratory syndrome coronavirus-2 is the causative agent of COVID-19. During the pandemic of 2019-2022, at least 500 million have been infected and over 6.3 million people have died from COVID-19. The virus is pleomorphic, and due to its pathogenicity is often handled in very restrictive biosafety containments laboratories. We developed two effective and rapid purification methods followed by UV inactivation that allow easy downstream handling of the virus. We monitored the purification through titering, sequencing, mass spectrometry and electron cryogenic microscopy. Although pelleting through a sucrose cushion, followed by gentle resuspension overnight gave the best particle recovery, infectivity decreased, and the purity was significantly worse than if using the size exclusion resin Capto Core. Capto Core can be used in batch mode, and was seven times faster than the pelleting method, obviating the need for ultracentrifugation in the containment laboratory, but resulting in a dilute virus. UV inactivation was readily optimized to allow handling of the inactivated samples under standard operating conditions. When containment laboratory space is limited, we recommend the use of Capto Core for purification and UV for inactivation as a simple, rapid workflow prior, for instance, to electron cryogenic microscopy or cell activation experiments.
Keren-Paz A., Maan H., Karunker I., Olender T., Kapishnikov S., Dersch S., Kartvelishvily E., Wolf S. G., Gal A., Graumann P. L. & Kolodkin-Gal I. (2022) iScience. 25, 6, 104308
In nature, bacteria reside in biofilms multicellular differentiated communities held together by an extracellular matrix. This work identified a novel subpopulationmineral-forming cellsthat is essential for biofilm formation in Bacillus subtilis biofilms. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. Consistently, biofilm development is prevented by the inhibition of calcium uptake. Our results provide a clear demonstration of the orchestrated production of calcite exoskeleton, critical to morphogenesis in simple prokaryotes.
Takebayashi S., Iron M. A., Feller M., Rivada-Wheelaghan O., Leitus G., Diskin-Posner Y., Shimon L. J. W., Avram L., Carmieli R., Wolf S. G., Cohen-Ofri I., Sanguramath R. A., Shenhar R., Eisen M. & Milstein D. (2022) Nature Catalysis. 5, 6, p. 494-502
The olefin metathesis reaction is among the most widely applicable catalytic reactions for carboncarbon double bond formation. Currently, Mo and Rucarbene catalysts are the most common choices for this reaction. It has been suggested that an iron-based catalyst would be a desirable economical and biocompatible substitute of the Ru catalysts; however, practical solutions in this regard are still lacking. Here, we report the discovery and mechanistic studies of three-coordinate iron(II) catalysts for ring-opening metathesis polymerization of olefins. Remarkably, their reactivity enabled the formation of polynorbornene with stereoregularity and high molecular weight (>107gmol1). The polymerization in the presence of styrene revealed cross metathesis reactivity with iron catalysts. Mechanistic studies suggest the possible role of metalligand cooperation in formation of the productive catalyst. This work opens the door to the development of iron complexes that can be economical and biocompatible catalysts for olefin metathesis reactions.
Bian T., Gardin A., Gemen J., Houben L., Perego C., Lee B., Elad N., Chu Z., Pavan G. M. & Klajn R. (2021) Nature Chemistry. 13, p. 940-949
Coulombic interactions can be used to assemble charged nanoparticles into higher-order structures, but the process requires oppositely charged partners that are similarly sized. The ability to mediate the assembly of such charged nanoparticles using structurally simple small molecules would greatly facilitate the fabrication of nanostructured materials and harnessing their applications in catalysis, sensing and photonics. Here we show that small molecules with as few as three electric charges can effectively induce attractive interactions between oppositely charged nanoparticles in water. These interactions can guide the assembly of charged nanoparticles into colloidal crystals of a quality previously only thought to result from their co-crystallization with oppositely charged nanoparticles of a similar size. Transient nanoparticle assemblies can be generated using positively charged nanoparticles and multiply charged anions that are enzymatically hydrolysed into mono- and/or dianions. Our findings demonstrate an approach for the facile fabrication, manipulation and further investigation of static and dynamic nanostructured materials in aqueous environments.
Zahradník J., Marciano S., Shemesh M., Zoler E., Harari D., Chiaravalli J., Meyer B., Rudich Y., Li C., Marton I., Dym O., Elad N., Lewis M., Andersen H., Gagne M., Seder R., Douek D. & Schreiber G. (2021) Nature Microbiology. 6, 9, p. 1188-1198
SARS-CoV-2 variants of interest and concern will continue to emerge for the duration of the COVID-19 pandemic. To map mutations in the receptor-binding domain (RBD) of the spike protein that affect binding to angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, we applied in vitro evolution to affinity-mature the RBD. Multiple rounds of random mutagenic libraries of the RBD were sorted against decreasing concentrations of ACE2, resulting in the selection of higher affinity RBD binders. We found that mutations present in more transmissible viruses (S477N, E484K and N501Y) were preferentially selected in our high-throughput screen. Evolved RBD mutants include prominently the amino acid substitutions found in the RBDs of B.1.620, B.1.1.7 (Alpha), B1.351 (Beta) and P.1 (Gamma) variants. Moreover, the incidence of RBD mutations in the population as presented in the GISAID database (April 2021) is positively correlated with increased binding affinity to ACE2. Further in vitro evolution increased binding by 1,000-fold and identified mutations that may be more infectious if they evolve in the circulating viral population, for example, Q498R is epistatic to N501Y. We show that our high-affinity variant RBD-62 can be used as a drug to inhibit infection with SARS-CoV-2 and variants Alpha, Beta and Gamma in vitro. In a model of SARS-CoV-2 challenge in hamster, RBD-62 significantly reduced clinical disease when administered before or after infection. A 2.9 Å cryo-electron microscopy structure of the high-affinity complex of RBD-62 and ACE2, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.
Rosenblum G., Elad N., Rozenberg H., Wiggers F., Jungwirth J. & Hofmann H. (2021) Nature Communications. 12, 2967
Allostery is a pervasive principle to regulate protein function. Growing evidence suggests that also DNA is capable of transmitting allosteric signals. Yet, whether and how DNA-mediated allostery plays a regulatory role in gene expression remained unclear. Here, we show that DNA indeed transmits allosteric signals over long distances to boost the binding cooperativity of transcription factors. Phenotype switching in Bacillus subtilis requires an all-or-none promoter binding of multiple ComK proteins. We use single-molecule FRET to demonstrate that ComK-binding at one promoter site increases affinity at a distant site. Cryo-EM structures of the complex between ComK and its promoter demonstrate that this coupling is due to mechanical forces that alter DNA curvature. Modifications of the spacer between sites tune cooperativity and show how to control allostery, which allows a fine-tuning of the dynamic properties of genetic circuits.
Mashiach R., Weissman H., Avram-Biton L., Houben L., Diskin Posner Y., Arunachalam V., Leskes M., Rybtchinski B. & Bar-Shir A. (2021) Nano Letters. 21, 23, p. 9916-9921
Colloidal inorganic nanofluorides have aroused great interest for various applications with their development greatly accelerated thanks to advanced synthetic approaches. Nevertheless, understanding their colloidal evolution and the factors that affect their dispersion could improve the ability to rationally design them. Here, using a multimodal in situ approach that combines DLS, NMR, and cryogenic-TEM, we elucidate the formation dynamics of nanofluorides in water through a transient aggregative phase. Specifically, we demonstrate that ligand-cation interactions mediate a transient aggregation of as-formed CaF2 nanocrystals (NCs) which governs the kinetics of the colloids' evolution. These observations shed light on key stages through which CaF2 NCs are dispersed in water, highlighting fundamental aspects of nanofluorides formation mechanisms. Our findings emphasize the roles of ligands in NCs' synthesis beyond their function as surfactants, including their ability to mediate colloidal evolution by complexing cationic precursors, and should be considered in the design of other types of NCs.
Seifer S., Houben L. & Elbaum M. (2021) Microscopy and Microanalysis. 27, 6, p. 1-12
Recent advances in scanning transmission electron microscopy (STEM) have rekindled interest in multi-channel detectors and prompted the exploration of unconventional scan patterns. These emerging needs are not yet addressed by standard commercial hardware. The system described here incorporates a flexible scan generator that enables exploration of low-acceleration scan patterns, while data are recorded by a scalable eight-channel array of nonmultiplexed analog-to-digital converters. System integration with SerialEM provides a flexible route for automated acquisition protocols including tomography. Using a solid-state quadrant detector with additional annular rings, we explore the generation and detection of various STEM contrast modes. Through-focus bright-field scans relate to phase contrast, similarly to wide-field TEM. More strikingly, comparing images acquired from different off-axis detector elements reveals lateral shifts dependent on defocus. Compensation of this parallax effect leads to decomposition of integrated differential phase contrast (iDPC) to separable contributions relating to projected electric potential and to defocus. Thus, a single scan provides both a computationally refocused phase contrast image and a second image in which the signed intensity, bright or dark, represents the degree of defocus.
Malik N., Singh V., Shimon L. J. W., Houben L., Lahav M. & van der Boom M. E. (2021) Journal of the American Chemical Society. 143, 41, p. 16913-16918
We demonstrate the formation of both metallo-organic crystals and nanoscale films that have entirely different compositions and structures despite using the same set of starting materials. This difference is the result of an unexpected cation exchange process. The reaction of an iron polypyridyl complex with a copper salt by diffusion of one solution into another resulted in iron-to-copper exchange, concurrent ligand rearrangement, and the formation of metal-organic frameworks (MOFs). This observation shows that polypyridyl complexes can be used as expendable precursors for the growth of MOFs. In contrast, alternative depositions of the iron polypyridyl complex with a copper salt by automated spin coating on conductive metal oxides resulted in the formation of electrochromic coatings, and the structure and redox properties of the iron complex were retained. The possibility to form such different networks from the same set of molecular building blocks by "in solution"versus "on surface"coordination chemistry broadens the synthetic space to design functional materials.
Sreedhara M., Hettler S., Kaplan-Ashiri I., Rechav K., Feldman Y., Enyashin A., Houben L., Arenal R. & Tenne R. (2021) Proceedings of the National Academy of Sciences of the United States of America. 118, 35, e210994511
Asymmetric two-dimensional (2D) structures (often named Janus), like SeMoS and their nanotubes, have tremendous scope in material chemistry, nanophotonics, and nanoelectronics due to a lack of inversion symmetry and time-reversal symmetry. The synthesis of these structures is fundamentally difficult owing to the entropy-driven randomized distribution of chalcogens. Indeed, no Janus nanotubes were experimentally prepared, so far. Serendipitously, a family of asymmetric misfit layer superstructures (tubes and flakes), including LaX-TaX2 (where X = S/Se), were synthesized by high-temperature chemical vapor transport reaction in which the Se binds exclusively to the Ta atoms and La binds to S atoms rather than the anticipated random distribution.With increasing Se concentration, the LaS-TaX2 misfit structure gradually transformed into a new LaS-TaSe2-TaSe2 superstructure. No misfit structures were found for xSe = 1. These counterintuitive results shed light on the chemical selectivity and stability of misfit compounds and 2D alloys, in general. The lack of inversion symmetry in these asymmetric compounds induces very large local electrical dipoles. The loss of inversion and time-reversal symmetries in the chiral nanotubes offers intriguing physical observations and applications.
Maiti P. S., Ghosh S., Leitus G., Houben L. & Bar Sadan M. (2021) Chemistry of Materials. 33, 18, p. 7558-7565
Developing growth schemes for functional two-dimensional (2D) nanomaterials is of much interest, especially because synthetic conditions are often correlated with physical properties such as optical absorbance. Bi<sub>2</sub>Se<sub>3</sub>is an interesting material because of its transition from a narrow gap semiconductor, for the thinnest structures, to a topological insulator, when the structures are at least six quintuple molecular layers (QLs), and its appealing photothermal, thermoelectric, and optical properties. Here, we developed a controlled colloidal synthesis based on oriented attachment to produce Bi<sub>2</sub>Se<sub>3</sub>nanoplatelets with varying thicknesses. The synthesis scheme also enables the facile doping of the structures without noticeable intercalation. The variation in optical properties confirmed the higher concentration of charge carriers at the edge of the structures, which is due in this system to Se deficiency and low crystallinity. This research opens a way to tune the composition and thickness of nanostructured 2D Bi<sub>2</sub>Se<sub>3</sub>, an important functional material.
Elbaum M., Seifer S., Houben L., Wolf S. G. & Rez P. (2021) Accounts of Chemical Research. 54, 19, p. 3621-3631
Electron microscopy (EM) is the most versatile tool for the study of matter at scales ranging from subatomic to visible. The high vacuum environment and the charged irradiation require careful stabilization of many specimens of interest. Biological samples are particularly sensitive due to their composition of light elements suspended in an aqueous medium. Early investigators developed techniques of embedding and staining with heavy metal salts for contrast enhancement. Indeed, the Nobel Prize in 1974 recognized Claude, de Duve, and Palade for establishment of the field of cell biology, largely due to their developments in separation and preservation of cellular components for electron microscopy. A decade later, cryogenic fixation was introduced. Vitrification of the water avoids the need for dehydration and provides an ideal matrix in which the organic macromolecules are suspended; the specimen represents a native state, suddenly frozen in time at temperatures below −150 °C. The low temperature maintains a low vapor pressure for the electron microscope, and the amorphous nature of the medium avoids diffraction contrast from crystalline ice. Such samples are extremely delicate, however, and cryo-EM imaging is a race for information in the face of ongoing damage by electron irradiation. Through this journey, cryo-EM enhanced the resolution scale from membranes to molecules and most recently to atoms. Cryo-EM pioneers, Dubochet, Frank, and Henderson, were awarded the Nobel Prize in 2017 for high resolution structure determination of biological macromolecules.
Chovnik O., Cohen S. R., Pinkas I., Houben L., Gorelik T. E., Feldman Y., Shimon L. J. W., Iron M. A., Lahav M. & van der Boom M. E. (2021) ACS Nano. 15, 9, p. 14643-14652
We demonstrate the solvent-free amorphous-to-cocrystalline transformations of nanoscale molecular films. Exposing amorphous films to vapors of a haloarene results in the formation of a cocrystalline coating. This transformation proceeds by gradual strengthening of halogen-bonding interactions as a result of the crystallization process. The gassolid diffusion mechanism involves formation of an amorphous metastable phase prior to crystallization of the films. In situ optical microscopy shows mass transport during this process, which is confirmed by cross-section analysis of the final structures using focused ion beam milling combined with scanning electron microscopy. Nanomechanical measurements show that the rigidity of the amorphous films influences the crystallization process. This surface transformation results in molecular arrangements that are not readily obtained through other means. Cocrystals grown in solution crystallize in a monoclinic centrosymmetric space group, whereas the on-surface halogen-bonded assembly crystallizes into a noncentrosymmetric material with a bulk second-order nonlinear optical response.
Singh V., Houben L., Shimon L. J. W., Cohen S. R., Golani O., Feldman Y., Lahav M. & van der Boom M. E. (2021) Angewandte Chemie (International ed.). 60, 33, p. 18256-18264
We demonstrate here a unique metalloorganic material where the appearance and the internal crystal structure are in contradiction. The eggshaped (ovoid) crystals have a brainlike texture. Although these microsized crystals are monodispersed; like fingerprints their grainy surfaces are never exactly alike. Remarkably, our Xray and electron diffraction studies unexpectedly revealed that these structures are singlecrystals comprising a continuous coordination network of two differently shaped homochiral channels. By using the same building blocks under different reaction conditions, a rare series of crystals have been obtained that are uniquely rounded in their shape. In stark contrast to the brainlike crystals, these isostructural and monodispersed crystals have a comparatively smooth appearance. The sizes of these crystals vary by several orders of magnitude.The eggshaped homochiral crystals: A brainlike texture combined with single crystallinity. A series of isomorphous crystals found in a rare space group have been formed with varied morphologies. These crystals contain chiral channels and are made from achiral components.
Tsarfati Y., Biran I., Wiedenbeck E., Houben L., Colfen H. & Rybtchinski B. (2021) ACS Central Science. 7, 5, p. 900-908
The crystallization mechanisms of organic molecules in solution are not well-understood. The mechanistic scenarios where crystalline order evolves directly from the molecularly dissolved state ("classical") and from initially formed amorphous intermediates ("nonclassical") are suggested and debated. Here, we studied crystallization mechanisms of two widely used analgesics, ibuprofen (IbuH) and etoricoxib (ETO), using direct cryogenic transmission electron microscopy (cryo-TEM) imaging. In the IbuH case, parallel crystallization pathways involved diverse phases of high and low density, in which the instantaneous formation of final crystalline order was observed. ETO crystallization started from well-defined round-shaped amorphous intermediates that gradually evolved into crystals. This mechanistic diversity is rationalized by introducing a continuum crystallization paradigm: order evolution depends on ordering in the initially formed intermediates and efficiency of molecular rearrangements within them, and there is a continuum of states related to the initial order and rearrangement rates. This model provides a unified view of crystallization mechanisms, encompassing classical and nonclassical pictures.
Cohen D., Mashiach R., Houben L., Galisova A., Addadi Y., Kain D., Lubart A., Blinder P., Allouche-Arnon H. & Bar-Shir A. (2021) ACS Nano. 15, 4, p. 7563-7574
Nature-inspired nanosized formulations based on an imageable, small-sized inorganic core scaffold, on which biomolecules are assembled to form nanobiomimetics, hold great promise for both early diagnostics and developed therapeutics. Nevertheless, the fabrication of nanobiomimetics that allow noninvasive background-free mapping of pathological events with improved sensitivity, enhanced specificity, and multiplexed capabilities remains a major challenge. Here, we introduce paramagnetic glyconanofluorides as small-sized (
Jardón-Álvarez D., Kahn N., Houben L. & Leskes M. (2021) Journal of Physical Chemistry Letters. 12, 11, p. 2964-2969
Comprehending the oxygen vacancy distribution in oxide ion conductors requires structural insights over various length scales: from the local coordination preferences to the possible formation of agglomerates comprising a large number of vacancies. In Y-doped ceria, 89Y NMR enables differentiation of yttrium sites by quantification of the oxygen vacancies in their first coordination sphere. Because of the extremely low sensitivity of 89Y, longer-range information was so far not available from NMR. Herein, we utilize metal ion-based dynamic nuclear polarization, where polarization from Gd(III) dopants provides large sensitivity enhancements homogeneously throughout the bulk of the sample. This enables following 89Y89Y homonuclear dipolar correlations and probing the local distribution of yttrium sites, which show no evidence of the formation of oxygen vacancy rich regions. The presented approach can provide valuable structural insights for designing oxide ion conductors.
di Gregorio M. C., Elsousou M., Wen Q., Shimon L. J. W., Brumfeld V., Houben L., Lahav M. & van der Boom M. E. (2021) Nature Communications. 12, 1, 957
The coexistence of single-crystallinity with a multidomain morphology is a paradoxical phenomenon occurring in biomineralization. Translating such feature to synthetic materials is a highly challenging process in crystal engineering. We demonstrate the formation of metallo-organic single-crystals with a unique appearance: six-connected half-rods forming a hexagonal-like tube. These uniform objects are formed from unstable, monodomain crystals. The monodomain crystals dissolve from the inner regions, while material is anisotropically added to their shell, resulting in hollow, single-crystals. Regardless of the different morphologies and growth mechanism, the crystallographic structures of the mono- and multidomain crystals are nearly identical. The chiral crystals are formed from achiral components, and belong to a rare space group (P622). Sonication of the solvents generating radical species is essential for forming the multidomain single-crystals. This process reduces the concentration of the active metal salt. Our approach offers opportunities to generate a new class of crystals.
Bitton O., Gupta S. N., Cao Y., Vaskevich A., Houben L., Yelin T. & Haran G. (2021) Journal of Chemical Physics. 154, 1, 014703
Plasmonic cavities (PCs) made of metallic nanostructures can concentrate electromagnetic radiation into an ultrasmall volume, where it might strongly interact with quantum emitters. In recent years, there has been much interest in studying such a strong coupling in the limit of single emitters. However, the lossy nature of PCs, reflected in their broad spectra, limits their quality factors and hence their performance as cavities. Here, we study the effect of the adhesion layer used in the fabrication of metal nanostructures on the spectral linewidths of bowtie-structured PCs. Using dark-field microspectroscopy, as well as electron energy loss spectroscopy, it is found that a reduction in the thickness of the chromium adhesion layer we use from 3 nm to 0.1 nm decreases the linewidths of both bright and dark plasmonic modes. We further show that it is possible to fabricate bowtie PCs without any adhesion layer, in which case the linewidth may be narrowed by as much as a factor of 2. Linewidth reduction increases the quality factor of these PCs accordingly, and it is shown to facilitate reaching the strong-coupling regime with semiconductor quantum dots.
Mashiach R., Weissman H., Avram L., Houben L., Brontvein O., Lavie A., Arunachalam V., Leskes M., Rybtchinski B. & Bar-Shir A. (2021) Nature Communications. 12, 1, 229
Understanding inorganic nanocrystal (NC) growth dynamic pathways under their native fabrication environment remains a central goal of science, as it is crucial for rationalizing novel nanoformulations with desired architectures and functionalities. We here present an in-situ method for quantifying, in real time, NCs size evolution at sub-nm resolution, their concentration, and reactants consumption rate for studying NC growth mechanisms. Analyzing sequential high-resolution liquid-state <sup>19</sup>F-NMR spectra obtained in-situ and validating by ex-situ cryoTEM, we explore the growth evolution of fluoride-based NCs (CaF<sub>2</sub> and SrF<sub>2</sub>) in water, without disturbing the synthesis conditions. We find that the same nanomaterial (CaF<sub>2</sub>) can grow by either a particle-coalescence or classical-growth mechanism, as regulated by the capping ligand, resulting in different crystallographic properties and functional features of the fabricated NC. The ability to reveal, in real time, mechanistic pathways at which NCs grow open unique opportunities for tunning the properties of functional materials.
Cohen A., Patsha A., Mohapatra P. K., Kazes M., Ranganathan K., Houben L., Oron D. & Ismach A. (2021) ACS Nano. 15, 1, p. 526-538
Metalorganic chemical vapor deposition (MOCVD) is one of the main methodologies used for thin-film fabrication in the semiconductor industry today and is considered one of the most promising routes to achieve large-scale and high-quality 2D transition metal dichalcogenides (TMDCs). However, if special measures are not taken, MOCVD suffers from some serious drawbacks, such as small domain size and carbon contamination, resulting in poor optical and crystal quality, which may inhibit its implementation for the large-scale fabrication of atomic-thin semiconductors. Here we present a growth-etch MOCVD (GE-MOCVD) methodology, in which a small amount of water vapor is introduced during the growth, while the precursors are delivered in pulses. The evolution of the growth as a function of the amount of water vapor, the number and type of cycles, and the gas composition is described. We show a significant domain size increase is achieved relative to our conventional process. The improved crystal quality of WS2 (and WSe2) domains wasis demonstrated by means of Raman spectroscopy, photoluminescence (PL) spectroscopy, and HRTEM studies. Moreover, time-resolved PL studies show very long exciton lifetimes, comparable to those observed in mechanically exfoliated flakes. Thus, the GE-MOCVD approach presented here may facilitate their integration into a wide range of applications.
Makagon E., Wachtel E., Houben L., Cohen S. R., Li Y., Li J., Frenkel A. I. & Lubomirsky I. (2021) Advanced Functional Materials. 31, 3, 2006712
Dimensional change in a solid due to electrochemically driven compositional change is termed electro-chemo-mechanical (ECM) coupling. This effect causes mechanical instability in Li-ion batteries and solid oxide fuel cells. Nevertheless, it can generate considerable force and deformation, making it attractive for mechanical actuation. Here a Si-compatible ECM actuator in the form of a 2 mm diameter membrane is demonstrated. Actuation results from oxygen ion transfer between two 0.1 µm thick Ti oxide\Ce0.8Gd0.2O1.9 nanocomposite layers separated by a 1.5 µm thick Ce0.8Gd0.2O1.9 solid electrolyte. The chemical reaction responsible for stress generation is electrochemical oxidation/reduction in the composites. Under ambient conditions, application of 5 V DC produces actuator response within seconds, generating vertical displacement of several µm with calculated stress ≈3.5 MPa. The membrane actuator preserves its final mechanical state for more than 1 h following voltage removal. These characteristics uniquely suit ECM actuators for room temperature applications in Si-integrated microelectromechanical systems.
Haber S., Rosy, Saha A., Brontvein O., Carmieli R., Zohar A., Noked M. & Leskes M. (2021) Journal of the American Chemical Society. 143, 12, p. 4694-4704
Degradation processes at the cathodeelectrolyte interface are a major limitation in the development of high-energy lithium-ion rechargeable batteries. Deposition of protective thin coating layers on the surface of high-energy cathodes is a promising approach to control interfacial reactions. However, rational design of effective protection layers is limited by the scarcity of analytical tools that can probe thin, disordered, and heterogeneous phases. Here we propose a new structural approach based on solid-state nuclear magnetic resonance spectroscopy coupled with dynamic nuclear polarization (DNP) for characterizing thin coating layers. We demonstrate the approach on an efficient alkylated LixSiyOz coating layer. By utilizing different sources for DNP, exogenous from nitroxide biradicals and endogenous from paramagnetic metal ion dopants, we reveal the outer and inner surface layers of the deposited artificial interphase and construct a structural model for the coating. In addition, lithium isotope exchange experiments provide direct evidence for the function of the surface layer, shedding light on its role in the enhanced rate performance of coated cathodes. The presented methodology and results advance us in identifying the key properties of effective coatings and may enable rational design of protective and ion-conducting surface layers.
Rossman U., Dadosh T., Eldar Y. & Oron D. (2021) Optics Express.
Image scanning microscopy (ISM), an upgraded successor of the ubiquitous confocal microscope, facilitates up to two-fold improvement in lateral resolution, and has become an indispensable element in the toolbox of the bio-imaging community. Recently, super-resolution optical fluctuation image scanning microscopy (SOFISM) integrated the analysis of intensity-fluctuations information into the basic ISM architecture, to enhance its resolving power. Both of these techniques typically rely on pixel-reassignment as a fundamental processing step, in which the parallax of different detector elements to the sample is compensated by laterally shifting the point spread function (PSF). Here, we propose an alternative analysis approach, based on the recent high-performing sparsity-based super-resolution correlation microscopy (SPARCOM) method. Through measurements of DNA origami nano-rulers and fixed cells labeled with organic dye, we experimentally show that confocal SPARCOM (cSPARCOM), which circumvents pixel-reassignment altogether, provides enhanced resolution compared to pixel-reassigned based analysis. Thus, cSPARCOM further promotes the effectiveness of ISM, and particularly that of correlation based ISM implementations such as SOFISM, where the PSF deviates significantly from spatial invariance.
Kalaora S., Nagler A., Nejman D., Alon M., Barbolin C., Barnea E., Ketelaars S. L. C., Cheng K., Vervier K., Shental N., Bussi Y., Rotkopf R., Levy R., Benedek G., Trabish S., Dadosh T., Levin-Zaidman S., Geller L. T., Wang K., Greenberg P., Yagel G., Peri A., Fuks G., Bhardwaj N., Reuben A., Hermida L., Johnson S. B., Galloway-Peña J. R., Shropshire W. C., Bernatchez C., Haymaker C., Arora R., Roitman L., Eilam R., Weinberger A., Lotan-Pompan M., Lotem M., Levin Y., Lawley T. D., Adams D. J., Levesque M. P., Besser M. J., Schachter J., Golani O., Segal E., Ruppin E., Kvistborg P., Peterson S. N., Wargo J. A., Straussman R. & Samuels Y. (2021) Nature (London). 592, 7852, p. 138-143
A variety of species of bacteria are known to colonize human tumours<sup>111</sup>, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment<sup>1214</sup>. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours. Our analysis of 17 melanoma metastases (derived from 9 patients) revealed 248 and 35 unique HLA-I and HLA-II peptides, respectively, that were derived from 41 species of bacteria. We identified recurrent bacterial peptides in tumours from different patients, as well as in different tumours from the same patient. Our study reveals that peptides derived from intracellular bacteria can be presented by tumour cells and elicit immune reactivity, and thus provides insight into a mechanism by which bacteria influence activation of the immune system and responses to therapy.
Hatai J., Prasad P. K., Lahav-Mankovski N., Oppenheimer-Low N., Unger T., Sirkis Y. F., Dadosh T., Motiei L. & Margulies D. (2021) Chemical Communications. 57, 15, p. 1875-1878
Tri-nitrilotriacetic acid (NTA)-based fluorescent probes were developed and used to image His-tagged-labelled outer membrane protein C (His-OmpC) in liveEscherichia coli. One of these probes was designed to light up upon binding, which provided the means to assess changes in the His-OmpC expression levels by taking a simple fluorescence spectrum.
Eisenberg-Bord M., Zung N., Collado J., Drwesh L., Fenech E. J., Fadel A., Dezorella N., Bykov Y. S., Rapaport D., Fernandez-Busnadiego R. & Schuldiner M. (2021) The Journal of Cell Biology. 220, 11, e202104100
Mitochondrial functions are tightly regulated by nuclear activity, requiring extensive communication between these organelles. One way by which organelles can communicate is through contact sites, areas of close apposition held together by tethering molecules. While many contacts have been characterized in yeast, the contact between the nucleus and mitochondria was not previously identified. Using fluorescence and electron microscopy in S. cerevisiae, we demonstrate specific areas of contact between the two organelles. Using a high-throughput screen, we uncover a role for the uncharacterized protein Ybr063c, which we have named Cnm1 (contact nucleus mitochondria 1), as a molecular tether on the nuclear membrane. We show that Cnm1 mediates contact by interacting with Tom70 on mitochondria. Moreover, Cnm1 abundance is regulated by phosphatidylcholine, enabling the coupling of phospholipid homeostasis with contact extent. The discovery of a molecular mechanism that allows mitochondrial crosstalk with the nucleus sets the ground for better understanding of mitochondrial functions in health and disease.
Pardo M., Li C., Fang Z., Levin-Zaidman S., Dezorella N., Czech H., Martens P., Käfer U., Gröger T., Rüger C. P., Friederici L., Zimmermann R. & Rudich Y. (2021) Chemical Research in Toxicology. 34, 6, p. 1588-1603
Widespread smoke from wildfires and biomass burning contributes to air pollution and the deterioration of air quality and human health. A common and major emission of biomass burning, often found in collected smoke particles, is spherical wood tar particles, also known as \u201ctar balls\u201d. However, the toxicity of wood tar particles and the mechanisms that govern their health impacts and the impact of their complicated chemical matrix are not fully elucidated. To address these questions, we generated wood tar material from wood pyrolysis and isolated two main subfractions: water-soluble and organic-soluble fractions. The chemical characteristics as well as the cytotoxicity, oxidative damage, and DNA damage mechanisms were investigated after exposure of A549 and BEAS-2B lung epithelial cells to wood tar. Our results suggest that both wood tar subfractions reduce cell viability in exposed lung cells; however, these fractions have different modes of action that are related to their physicochemical properties. Exposure to the water-soluble wood tar fraction increased total reactive oxygen species production in the cells, decreased mitochondrial membrane potential (MMP), and induced oxidative damage and cell death, probably through apoptosis. Exposure to the organic-soluble fraction increased superoxide anion production, with a sharp decrease in MMP. DNA damage is a significant process that may explain the course of toxicity of the organic-soluble fraction. For both subfractions, exposure caused cell cycle alterations in the G2/M phase that were induced by upregulation of p21 and p16. Collectively, both subfractions of wood tar are toxic. The water-soluble fraction contains chemicals (such as phenolic compounds) that induce a strong oxidative stress response and penetrate living cells more easily. The organic-soluble fraction contained more polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs and induced genotoxic processes, such as DNA damage.
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Methodological focus D: FIB-SEM dual-beam microscopy for three-dimensional ultrastructural imaging of skeletal tissues
Varsano N., Kahil K., Haimov H., Rechav K., Addadi L. & Weiner S. (2021) Journal of Structural Biology. 213, 4, 107781
The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.
Scher N., Rechav K., Paul-Gilloteaux P. & Avinoam O. (2021) iScience. 24, 7, 102714
Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. Correlative light and electron microscopy workflows combining fluorescence microscopy (FM) with FIB-SEM imaging exist, whereas workflows combining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in situ fiducial markers for correlating cryo-FM and FIB-SEM datasets and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization, and mineral deposits in cells.
3D CLEM of cryo-FM and FIB-SEM datasets using fluorescently labeled lipid droplets
Cryo-FIB-SEM imaging of organelle-organelle interactions and nuclear organization
Raguin E., Rechav K., Shahar R. & Weiner S. (2021) Acta Biomaterialia. 121, p. 497-513
The mineralized collagen fibril is the basic building block of bone, and hence is the key to understanding bone structure and function. Here we report imaging of mineralized pig bone samples in 3D using the focused ion beam-scanning electron microscope (FIB-SEM) under conditions that reveal the 67 nm D-banding of mineralized collagen fibrils. We show that in adult pig osteons, the lamellar bone comprises alternating layers with either collagen fibrils predominantly aligned in one direction, and layers in which fibrils are predominantly aligned in two directions. The cement sheath contains thin layers of both these motifs, but its dominant structural component comprises a very complex layer of fibrils predominantly aligned in three or more directions. The degree of mineralization of the cement sheath is comparable to that of the osteon interior. The extent of alignment (dispersion) of the collagen fibrils in the osteonal lamellar bone is significantly higher than in the cement sheath. Canaliculi within the cement sheath are mainly aligned parallel to the cement sheath boundary, whereas in the lamellar bone they are mainly aligned perpendicular to the lamellar boundaries. This study further characterizes the presence of two types of collagen fibril arrangements previously identified in demineralized lamellar bone from other species. The simple sample preparation procedure for mineralized bone and the lower risk of introducing artifacts opens the possibility of using FIB-SEM to study more samples, to obtain automatic quantitative information on collagen fibril organization and to evaluate the degrees of mineralization all in relatively large volumes of bone.
Drake J. L., Varsano N. & Mass T. (2021) Journal of Structural Biology. 213, 4, 107782
Despite their simple body plan, stony corals (order Scleractinia, phylum Cnidaria) can produce massive and complex exoskeletal structures in shallow, tropical and subtropical regions of Earth's oceans. The species-specific macromorphologies of their aragonite skeletons suggest a highly coordinated biomineralization process that is rooted in their genomes, and which has persisted across major climatic shifts over the past 400 + million years. The mechanisms by which stony corals produce their skeletons has been the subject of interest for at least the last 160 years, and the pace of understanding the process has increased dramatically in the past decade since the sequencing of the first coral genome in 2011. In this review, we detail what is known to date about the genetic basis of the stony coral biomineralization process, with a focus on advances in the last several years as well as ways that physical and chemical tools can be combined with genetics, and then propose next steps forward for the coming decade.
Kadan Y., Tollervey F., Varsano N., Mahamid J. & Gal A. (2021) Proceedings of the National Academy of Sciences. 118, 46, e202567011
Unicellular marine microalgae are responsible for one of the largest carbon sinks on Earth. This is in part due to intracellular formation of calcium carbonate scales termed coccoliths. Traditionally, the influence of changing environmental conditions on this process has been estimated using poorly constrained analogies to crystallization mechanisms in bulk solution, yielding ambiguous predictions. Here, we elucidated the intracellular nanoscale environment of coccolith formation in the model species Pleurochrysis carterae using cryoelectron tomography. By visualizing cells at various stages of the crystallization process, we reconstructed a timeline of coccolith development. The three-dimensional data portray the native-state structural details of coccolith formation, uncovering the crystallization mechanism, and how it is spatially and temporally controlled. Most strikingly, the developing crystals are only tens of nanometers away from delimiting membranes, resulting in a highly confined volume for crystal growth. We calculate that the number of soluble ions that can be found in such a minute volume at any given time point is less than the number needed to allow the growth of a single atomic layer of the crystal and that the uptake of single protons can markedly affect nominal pH values. In such extreme confinement, the crystallization process is expected to depend primarily on the regulation of ion fluxes by the living cell, and nominal ion concentrations, such as pH, become the result, rather than a driver, of the crystallization process. These findings call for a new perspective on coccolith formation that does not rely exclusively on solution chemistry.
Mayzel B., Aram L., Varsano N., Wolf S. G. & Gal A. (2021) Nature Communications. 12, 1, 4639
Silica formation in diatoms is of interest for a range of different subjects from biomimetics to oceanography. Here the authors study the formation of silicified extensions in diatoms and find that unlike cell wall elements, that form in the cytoplasm, the extensions have a different formation mechanism outside the cytoplasm.
Grunberg N., Pevsner-Fischer M., Goshen-Lago T., Diment J., Stein Y., Lavon H., Mayer S., Levi-Galibov O., Friedman G., Ofir-Birin Y., Syu L., Migliore C., Shimoni E., Stemmer S. M., Brenner B., Dlugosz A. A., Lyden D., Regev-Rudzki N., Ben-Aharon I. & Scherz-Shouval R. (2021) Cancer Research. 81, 7, p. 1639-1653
Gastric cancer is the third most lethal cancer worldwide, and evaluation of the genomic status of gastric cancer cells has not translated into effective prognostic or therapeutic strategies. We therefore hypothesize that outcomes may depend on the tumor microenvironment (TME), in particular, cancerassociated fibroblasts (CAF). However, very little is known about the role of CAFs in gastric cancer. To address this, we mapped the transcriptional landscape of human gastric cancer stroma by microdissection and RNA sequencing of CAFs from patients with gastric cancer. A stromal gene signature was associated with poor disease outcome, and the transcription factor heat shock factor 1 (HSF1) regulated the signature. HSF1 upregulated inhibin subunit beta A and thrombospondin 2, which were secreted in CAF-derived extracellular vesicles to the TME to promote cancer. Together, our work provides the first transcriptional map of human gastric cancer stroma and highlights HSF1 and its transcriptional targets as potential diagnostic and therapeutic targets in the genomically stable tumor microenvironment.
Ghosh S., Feigelson S. W., Montresor A., Shimoni E., Roncato F., Legler D. F., Laudanna C., Haran G. & Alon R. (2021) Biophysical Journal. 120, 18, p. 4002-4012
Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.
Shprung T., Wani N., Wilmes M., Mangoni M. L., Bitler A., Shimoni E., Sahl H. & Shai Y. (2021) Biochemistry. 60, 39, p. 2943-2955
The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the two-component systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (Moskowitz, S. M.;et al. Antimicrob. Agents Chemother. 2012, 56, 1019-1030; Cheng, H. Y.;et al. J. Biomed. Sci. 2010, 17, 60). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica. The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs.
Krounbi L., Hedderick K., Eyal Z., Aram L., Shimoni E., Estroff L. A. & Gal A. (2021) Chemistry of Materials. 33, 10, p. 3534-3542
Many organisms orchestrate the controlled precipitation of minerals. This physiological process takes place at ambient conditions, using soluble ions as building blocks. A widespread strategy for such crystallization processes is using a multistep route, where the initial phase is metastable and gradually transforms into the mature mineral phase. Even though the maturation of these intermediate phases has been intensively studied, it remains unclear how the initial, far from equilibrium phase can form within the cellular context. A model system for controlled biomineralization is the production of coccoliths by marine microalgae. Coccoliths are calcium carbonate crystalline arrays that form within the intracellular environment, at very low calcium concentrations. Here, we used coccolith-derived and synthetic polymers to study, in vitro, the chemical interactions between calcium ions and organic macromolecules that precede coccolith formation. We used in situ analyses, including state-of-the-art cryo-electron tomography and liquid-cell atomic force microscopy, to study the interactions in bulk solution and on organic surfaces simultaneously. The results unveil a chemical process in which a functional surface induces the precipitation of a polymerCa dense phase, or a coacervate, at chemical conditions where precipitation in solution is kinetically inhibited. This strategy demonstrates how organisms can form dense Ca-rich phases from the submillimolar concentration of calcium within organelles. This Ca-rich phase can then transform into a mineral precursor in a subsequent step, without posing challenges to cellular homeostasis.
Yegorov Y., Sendersky E., Zilberman S., Nagar E., Ben-Asher H. W., Shimoni E., Simkovsky R., Golden S. S., Liwang A. & Schwarz R. (2021) mBio. 12, 2, 03674-20
Protein secretion as well as the assembly of bacterial motility appendages are central processes that substantially contribute to fitness and survival. This study highlights distinctive features of the mechanism that serves these functions in cyanobacteria, which are globally prevalent photosynthetic prokaryotes that significantly contribute to primary production. Our studies of biofilm development in the cyanobacterium Synechococcus elongatus uncovered a novel component required for the biofilm self-suppression mechanism that operates in this organism. This protein, which is annotated as \u201chypothetical,\u201d is denoted EbsA (essential for biofilm self-suppression A) here. EbsA homologs are highly conserved and widespread in diverse cyanobacteria but are not found outside this clade. We revealed a tripartite complex of EbsA, Hfq, and the ATPase homolog PilB (formerly called T2SE) and demonstrated that each of these components is required for the assembly of the hairlike type IV pili (T4P) appendages, for DNA competence, and affects the exoproteome in addition to its role in biofilm self-suppression. These data are consistent with bioinformatics analyses that reveal only a single set of genes in S. elongatus to serve pilus assembly or protein secretion; we suggest that a single complex is involved in both processes. A phenotype resulting from the impairment of the EbsA homolog in the cyanobacterium Synechocystis sp. strain PCC 6803 implies that this feature is a general cyanobacterial trait. Moreover, comparative exoproteome analyses of wild-type and mutant strains of S. elongatus suggest that EbsA and Hfq affect the exoproteome via a process that is independent of PilB, in addition to their involvement in a T4P/secretion machinery.
Golan O., Shalom H., Kaplan-Ashiri I., Cohen S. R., Feldman Y., Pinkas I., Ofek Almog R., Zak A. & Tenne R. (2021) Polymers. 13, 21, 3851
Poly(L-lactic acid) (PLLA) is a biocompatible, biodegradable, and semi-crystalline polymer with numerous applications including food packaging, medical implants, stents, tissue engineering scaffolds, etc. Hydroxyapatite (HA) is the major component of natural bone. Conceptually, combining PLLA and HA could produce a bioceramic suitable for implants and bone repair. However, this nanocomposite suffers from poor mechanical behavior under tensile strain. In this study, films of PLLA and HA were prepared with small amounts of nontoxic WS<sub>2</sub> nanotubes (INT-WS<sub>2</sub> ). The structural aspects of the films were investigated via electron microscopy, X-ray diffraction, Raman microscopy, and infrared absorption spectroscopy. The mechanical properties were evaluated via tensile measurements, micro-hardness tests, and nanoindentation. The thermal properties were investigated via differential scanning calorimetry. The composite films exhibited improved mechanical and thermal properties compared to the films prepared from the PLLA and HA alone, which is advantageous for medical applications.
Ceratti D. R., Cohen A. V., Tenne R., Rakita Y., Snarski L., Jasti N. P., Cremonesi L., Cohen R., Weitman M., Rosenhek-Goldian I., Kaplan-Ashiri I., Bendikov T., Kalchenko V., Elbaum M., Potenza M. A. C., Kronik L., Hodes G. & Cahen D. (2021) Materials Horizons. 8, 5, p. 1570-1586
We find significant differences between degradation and healing at the surface or in the bulk for each of the different APbBr3 single crystals (A = CH3NH3+, methylammonium (MA); HC(NH2)2+, formamidinium (FA); and cesium, Cs+). Using 1- and 2-photon microscopy and photobleaching we conclude that kinetics dominate the surface and thermodynamics the bulk stability. Fluorescence-lifetime imaging microscopy, as well as results from several other methods, relate the (damaged) state of the halide perovskite (HaP) after photobleaching to its modified optical and electronic properties. The A cation type strongly influences both the kinetics and the thermodynamics of recovery and degradation: FA heals best the bulk material with faster self-healing; Cs+ protects the surface best, being the least volatile of the A cations and possibly through O-passivation; MA passivates defects via methylamine from photo-dissociation, which binds to Pb2+. DFT simulations provide insight into the passivating role of MA, and also indicate the importance of the Br3- defect as well as predicts its stability. The occurrence and rate of self-healing are suggested to explain the low effective defect density in the HaPs and through this, their excellent performance. These results rationalize the use of mixed A-cation materials for optimizing both solar cell stability and overall performance of HaP-based devices, and provide a basis for designing new HaP variants.
Irwin R., Faust O., Petrovic I., Wolf S. G., Hofmann H. & Rosenzweig R. (2021) eLife. 10, e69601
The microtubule-associated protein, tau, is the major subunit of neurofibrillary tangles associated with neurodegenerative conditions, such as Alzheimer's disease. In the cell, however, tau aggregation can be prevented by a class of proteins known as molecular chaperones. While numerous chaperones are known to interact with tau, though, little is known regarding the mechanisms by which these prevent tau aggregation. Here, we describe the effects of ATP-independent Hsp40 chaperones, DNAJA2 and DNAJB1, on tau amyloid-fiber formation, and compare these to the small heat-shock protein HSPB1. We find that the chaperones play complementary roles, with each preventing tau aggregation differently and interacting with distinct sets of tau species. Whereas HSPB1 only binds tau monomers, DNAJB1 and DNAJA2 recognize aggregation-prone conformers and even mature fibers. In addition, we find that both Hsp40s bind tau seeds and fibers via their C-terminal domain II (CTDII), with DNAJA2 being further capable of recognizing tau monomers by a second, distinct site in CTDI. These results lay out the mechanisms by which the diverse members of the Hsp40 family counteract the formation and propagation of toxic tau aggregates, and highlight the fact that chaperones from different families/classes play distinct, yet complementary roles in preventing pathological protein aggregation.
Elad N. & Wolf S. G. (2020) Structure. 28, 11, p. 1179-1181
In this issue of Structure, breakthroughs in cryo-EM/ET research are presented. Klebl et al. (2020) demonstrate how speed in sample vitrification impacts the quality of macromolecular particles in resultant cryo-EM grids. Wu et al. (2020) combine fluorescence, ion beam milling, and tomography to unravel unique features in vitrified yeast cells.
Javitt G., Khmelnitsky L., Albert L., Bigman L. S., Elad N., Morgenstern D., Ilani T., Levy Y., Diskin R. & Fass D. (2020) Cell. 183, 3, p. 717-729.e16
The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.
Cohen-Dvashi H., Zehner M., Ehrhardt S., Katz M., Elad N., Klein F. & Diskin R. (2020) Cell Host & Microbe. 27, 3, p. 418-427.e4
Ebola virus disease is a severe health problem in Africa. Vaccines that display the Zaire ebolavirus glycoprotein spike complex are a prime component for the effort to combat it. The V(H)3-15/V(lambda)1-40-based class of antibodies was recently discovered to be a common response in individuals who received the Ebola virus vaccines. These antibodies display attractive properties, and thus likely contribute to the efficacy of the vaccines. Here, we use cryo-EM to elucidate how three V(H)3-15/V(lambda)1-40 antibodies from different individuals target the virus and found a convergent mechanism against a partially conserved site on the spike complex. Our study rationalizes the selection of the V(H)3-15/V(lambda)1-40 germline genes for specifically targeting this site and highlights Ebolavirus species-specific sequence divergences that may restrict breadth of V(H)3-15/V(lambda)1-40-based humoral response. The results from this study could help develop improved immunization schemes and further enable the design of immunogens that would be efficacious against a broader set of Ebolavirus species.
Vimer S., Ben-Nissan G., Morgenstern D., Kumar-Deshmukh F., Polkinghorn C., Quintyn R. S., Vasil'ev Y. V., Beckman J. S., Elad N., Wysocki V. H. & Sharon M. (2020) ACS Central Science. 6, 4, p. 573-588
Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (Saccharomyces cerevisiae) and mammals (rat - Rattus norvegicus, rabbit - Oryctolagus cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies.
Palmer B. A., Yallapragada V. J., Schiffmann N., Wormser E. M., Elad N., Aflalo E. D., Sagi A., Weiner S., Addadi L. & Oron D. (2020) Nature Nanotechnology. 15, 2, p. 138-144
The birefringence of isoxanthopterin crystalline spherulites enhances the reflectivity of a biological photonic crystal.Spectacular natural optical phenomena are produced by highly reflective assemblies of organic crystals. Here we show how the tapetum reflector in a shrimp eye is constructed from arrays of spherical isoxanthopterin nanoparticles and relate the particle properties to their optical function. The nanoparticles are composed of single-crystal isoxanthopterin nanoplates arranged in concentric lamellae around a hollow core. The spherulitic birefringence of the nanoparticles, which originates from the radial alignment of the plates, results in a significant enhancement of the back-scattering. This enables the organism to maximize the reflectivity of the ultrathin tapetum, which functions to increase the eye's sensitivity and preserve visual acuity. The particle size, core/shell ratio and packing are also controlled to optimize the intensity and spectral properties of the tapetum back-scattering. This system offers inspiration for the design of photonic crystals constructed from spherically symmetric birefringent particles for use in ultrathin reflectors and as non-iridescent pigments.
Mohapatra P. K., Ranganathan K., Dezanashvili L., Houben L. & Ismach A. (2020) Applied Materials Today. 20, 100734
One of the most intriguing properties of layered materials is their ability to form inherently ultra-thin atomically sharp vertical interfaces and hybrid layered compounds, in moderate environment conditions, which are ideal platforms for both, scientific research and many applications. Here, we present the selective van der Waals epitaxial formation of β-In<sub>2</sub>Se<sub>3</sub> on transition metal dichalcogenides (TMDCs). This is achieved in a two-step chemical vapor deposition (CVD) process, in which first, the monolayer TMDC, is synthesized and then the β-In<sub>2</sub>Se<sub>3</sub> is grown on top of it. The thickness of the second phase can be controlled by the growth conditions, while the crystal size is dictated by the MoS<sub>2</sub> single-crystal domain size. High-resolution transmission electron microscope (HRTEM) studies reveal a clean and sharp interface, while selected area diffraction (SAED) demonstrates a clear registry between both phases. The hybrid layered-compound exhibit better electrical transport than the intrinsic indium-selenide layer. The high crystallinity of In<sub>2</sub>Se<sub>3</sub> grown on MoS<sub>2</sub> yield fast response among the CVD-derived In<sub>2</sub>Se<sub>3</sub>-based photodetectors with rise/fall times of 4/7 ms, photoresponsivity of up to ~23 A/W and specific detectivity of ~ 5 × 10<sup>11</sup>. Our methodology allows high quality thin In<sub>2</sub>Se<sub>3</sub> layers to be formed via van der Waals epitaxy on TMDCs, forming complex vertical heterostructures for optoelectronic applications.
Ghosh S., Kadam S. R., Houben L., Bar-Ziv R. & Bar-Sadan M. (2020) Applied Materials Today. 20, 100693
Using hydrogen as fuel requires efficient production and retrieval from energy carriers. Here, we describe the synthesis of various pure phases of nickel phosphide and describe the growth mechanisms. A comparative study illustrates the phases catalytic activity towards hydrogen production through electrochemical water reduction as well as hydrogen retrieval by hydrolysis of hydrogen storage materials (ammonia-borane and NaBH<sub>4</sub>). Charge separation between the Ni<sup>δ+</sup> and P<sup>δ−</sup> sites in the various NiP phases plays a key role in achieving the desired efficacy of the catalytic reaction. Ni<sub>2</sub>P exhibited a significant enhancement towards the hydrogen evolution reaction, with an overpotential of 126 mV at J= 10 mA cm<sup>−2</sup> in acid and 180 mV in alkaline. Ni<sub>12</sub>P<sub>5</sub> was the most efficient catalyst, with a turnover frequency (TOF) = 23.0 min<sup>−1</sup> for hydrogen evolution from ammonia-borane, and TOF= 17.3 min<sup>−1</sup> from NaBH<sub>4</sub>, which is in accordance with noble metal nanoparticles.
Mashiach R., Cohen D., Avram L., Harris T., Pinkas I., Houben L., Allouche-Arnon H. & Bar-Shir A. (2020) Nano Letters. 20, 10, p. 7207-7212
Paramagnetic relaxation enhancement (PRE) is the current strategy of choice for enhancing magnetic resonance imaging (MRI) contrast and for accelerating MRI acquisition schemes. Yet, debates regarding lanthanides biocompatibility and PRE-effect on MRI signal quantification have raised the need for alternative strategies for relaxation enhancement. Herein, we show an approach for shortening the spin-lattice relaxation time (T1) of fluoride-based nanocrystals (NCs) that are used for in-vivo 19F-MRI, by inducing crystal defects in their solid-crystal core. By utilizing a phosphate-based rather than a carboxylate-based capping ligand for the synthesis of CaF2 NCs, we were able to induce grain boundary defects in the NC lattice. The obtained defects led to a ten-fold shorter T1 of the NCs fluorides. Such paramagnetic-free relaxation enhancement of CaF2 NCs, gained without affecting neither their size nor their colloidal characteristics, improved 4-fold the obtained 19F-MRI signal-to-noise ratio, allowing their use, in-vivo, with enhanced hot-spot MRI sensitivity.
Haviv E., Chen B., Carmielli R., Houben L., Cohen H., Leitus G., Avram L. & Neumann R. (2020) Journal of the American Chemical Society. 142, 34, p. 14504-14512
Host-guest solution chemistry with a wide range of organic hosts is an important and established research area, while the use of inorganic hosts is a more nascent area of research. In the recent past in a few cases, Keplerate type molybdenum oxide based porous, spherical clus-ters, shorthand notation {Mo132}, have been used as hosts for organic guests. Here we demonstrate the synthetically controlled encapsula-tion of first row transition metals (M = Mn, Fe, and Co) within a Keplerate cluster that was lined on the inner core with phosphate ani-ons, {Mo132PO4}. The resulting M2+x{Mo132PO4} host-guest complexes were characterized by 31P NMR and ENDOR spectroscopy that substantiated the encapsulation of the first-row transition metal guest. Magnetic susceptibility measurements showed that the encap-sulation of up to 10 equivalents showed little magnetic interaction between the encapsulated metals, indicating that each guest atom occupied a single site. Visualization of the capsules and differentiation of the Mo atoms of the capsule framework and the encapsulated transition metal was possible using spherical and chromatic double aberration-corrected electron microscopy combined with energy-filtered TEM (EFTEM) elemental maps. In addition, use of visible light induced XPS for chemically resolved electrical measurements (CREM) confirmed the successful encapsulation of M within {Mo132PO4} and furthermore showed photoinduced electron transfer from M to Mo. In the future such targeted electron transfer between host {Mo132} and a transition metal guest could be used as photo-initiated switches using inorganic compounds and for single site photocatalytic reactions in confined space.
Wen Q., Tenenholtz S., Shimon L. J. W., Bar-Elli O., Beck L., Houben L., Cohen S. R., Feldman Y., Oron D., Lahav M. & van der Boom M. E. (2020) Journal of the American Chemical Society. 142, 33, p. 14210-14221
We demonstrate the formation of uniform and oriented metal-organic frameworks using a combination of anion-effects and surface-chemistry. Subtle but significant morphological changes result from the nature of the coordinative counter-anion of the following metal salts: NiX2 with (X = Br-, Cl-, NO3-, and OAc-). Crystals could be obtained in solution or by template surface growth. The latter resulting in truncated crystals that resemble a half-structure of the solution-grown ones. The oriented surface-bound metal-organic frameworks (sMOFs) are obtained via a one-step solvothermal approach, rather than in a layer-by-layer approach. The MOFs are grown on Si/SiOx substrates modified with an organic monolayer or on glass substrates covered with a transparent conductive oxide (TCO). Regardless of the different morphologies, the crystallographic packing is nearly identical and is not affected by the type of anion, nor by solution versus the surface chemistry. A propeller-type arrangement of the non-chiral ligands around the metal center affords a chiral structure with two geometrically different helical channels in a 2:1 ratio with the same handedness. To demonstrate the accessibility and porosity of the macroscopically-oriented channels, a chromophore (resorufin sodium salt) was successfully embedded into the channels of the crystals by diffusion from solution, resulting in fluorescent crystals. These "colored" crystals displayed polarized emission (red) with a high polarization ratio because of the alignment of these dyes imposed by the crystallographic structure. A second-harmonic generation (SHG) study revealed Kleinman-symmetry forbidden non-linear optical properties. These surface-bound and oriented SHG-active MOFs have the potential for use as single non-linear optical (NLO) devices.
Houben L., Weissman H., Wolf S. G. & Rybtchinski B. (2020) Nature. 579, 7800, p. 540-543
Protein crystallization is important in structural biology, disease research and pharmaceuticals. It has recently been recognized that nonclassical crystallizationinvolving initial formation of an amorphous precursor phaseoccurs often in protein, organic and inorganic crystallization processes<sup>15</sup>. A two-step nucleation theory has thus been proposed, in which initial low-density, solvated amorphous aggregates subsequently densify, leading to nucleation<sup>4,6,7</sup>. This view differs from classical nucleation theory, which implies that crystalline nuclei forming in solution have the same density and structure as does the final crystalline state<sup>1</sup>. A protein crystallization mechanism involving this classical pathway has recently been observed directly<sup>8</sup>. However, a molecular mechanism of nonclassical protein crystallization<sup>915</sup> has not been established<sup>9,11,14</sup>. To determine the nature of the amorphous precursors and whether crystallization takes place within them (and if so, how order develops at the molecular level), three-dimensional (3D) molecular-level imaging of a crystallization process is required. Here we report cryogenic scanning transmission microscopy tomography of ferritin aggregates at various stages of crystallization, followed by 3D reconstruction using simultaneous iterative reconstruction techniques to provide a 3D picture of crystallization with molecular resolution. As crystalline order gradually increased in the studied aggregates, they exhibited an increase in both order and density from their surface towards their interior. We observed no highly ordered small structures typical of a classical nucleation process, and occasionally we observed several ordered domains emerging within one amorphous aggregate, a phenomenon not predicted by either classical or two-step nucleation theories. Our molecular-level analysis hints at desolvation as the driver of the continuous order-evolution mechanism, a view that goes beyond current nucleation models, yet is consistent with a broad spectrum of protein crystallization mechanisms.
Kadam S. R., Enyashin A. N., Houben L., Bar-Ziv R. & Bar-Sadan M. (2020) Journal of Materials Chemistry A. 8, 3, p. 1403-1416
Layered transition metal dichalcogenides (TMDCs) are an emerging family of catalysts. Included in this group is tungsten diselenide (WSe2), which has attracted recent attention for electro-optical applications. Tungsten is opportune because it is the heaviest transition metal in the TMDC family and is widely abundant on Earth. Moreover, WS2 and WSe2 are more benign in nature than their molybdenum counterparts. Despite this, WSe2 has been relatively unexplored as a catalyst. We report the synthesis of WSe2 doped with various transition metals (Fe, Co, Nb, Ni and Zr). Among the doped catalysts, Ni-WSe2 has been found to be the most promising electrocatalyst for the hydrogen evolution reaction (HER) as it possesses the smallest charge transfer resistance and thus facilitates a faster catalytic reaction. Upon doping with Ni, catalytic enhancement results from improved hydrogen adsorption (H-ads). Beyond this threshold of Ni loading, the improved activity in alkaline medium is due to the optimized interaction of the OH/surface active sites. Using density functional theory calculations, we identified that the catalytic sites are Se atoms either bound to a substitutional Ni dopant or constituting a small patch of NiSe grafted on the WSe2 surface.
di Gregorio M. C., Shimon L. J., Brumfeld V., Houben L., Lahav M. & van der Boom M. E. (2020) Nature Communications. 11, 1, 380
Naturally occurring single crystals having a multidomain morphology are a counterintuitive phenonomon: the macroscopic appearance is expected to follow the symmetry of the unit cell. Growing such crystals in the lab is a great challenge, especially from organic molecules. We achieve here uniform metallo-organic crystals that exhibit single crystallinity with apparently distinct domains and chirality. The chirality is present at both the molecular and macroscopic levels, although only achiral elements are used. \u201cYo-yo\u201d-like structures having opposite helical handedness evolve from initially formed seemingly achiral cylinders. This non-polyhedral morphology coexists with a continuous coordination network forming homochiral channels. This work sheds light on the enigmatic aspects of fascinating crystallization processes occurring in biological mineralization. Our findings open up opportunities to generate new porous and hierarchical chiral materials.
Oksenberg E., Merdasa A., Houben L., Kaplan-Ashiri I., Rothman A., Scheblykin I. G., Unger E. L. & Joselevich E. (2020) Nature Communications. 11, 489
Metal-halide perovskites have been shown to be remarkable and promising optoelectronic materials. However, despite ongoing research from multiple perspectives, some fundamental questions regarding their optoelectronic properties remain controversial. One reason is the high-variance of data collected from, often unstable, polycrystalline thin films. Here we use ordered arrays of stable, single-crystal cesium lead bromide (CsPbBr<sub>3</sub>) nanowires grown by surface-guided chemical vapor deposition to study fundamental properties of these semiconductors in a one-dimensional model system. Specifically, we uncover the origin of an unusually large size-dependent luminescence emission spectral blue-shift. Using multiple spatially resolved spectroscopy techniques, we establish that bandgap modulation causes the emission shift, and by correlation with state-of-the-art electron microscopy methods, we reveal its origin in substantial and uniform lattice rotations due to heteroepitaxial strain and lattice relaxation. Understanding strain and its effect on the optoelectronic properties of these dynamic materials, from the atomic scale up, is essential to evaluate their performance limits and fundamentals of charge carrier dynamics.
Bitton O., Gupta S. N., Houben L., Kvapil M., Krapek V., Sikola T. & Haran G. (2020) Nature Communications. 11, 487
Recent years have seen a growing interest in strong coupling between plasmons and excitons, as a way to generate new quantum optical testbeds and influence chemical dynamics and reactivity. Strong coupling to bright plasmonic modes has been achieved even with single quantum emitters. Dark plasmonic modes fare better in some applications due to longer lifetimes, but are difficult to probe as they are subradiant. Here, we apply electron energy loss (EEL) spectroscopy to demonstrate that a dark mode of an individual plasmonic bowtie can interact with a small number of quantum emitters, as evidenced by Rabi-split spectra. Coupling strengths of up to 85meV place the bowtie-emitter devices at the onset of the strong coupling regime. Remarkably, the coupling occurs at the periphery of the bowtie gaps, even while the electron beam probes their center. Our findings pave the way for using EEL spectroscopy to study exciton-plasmon interactions involving non-emissive photonic modes. Dark plasmonic modes fare better in some applications due to longer lifetimes but, being subradiant, are difficult to probe. The authors apply electron energy loss spectroscopy to demonstrate that a dark mode of a plasmonic cavity can couple with a few quantum emitters to exhibit vacuum Rabi splitting.
Rosy, Haber S., Evenstein E., Saha A., Brontvein O., Kratish Y., BravoZhivotovskii D., Apeloig Y., Leskes M. & Noked M. (2020) Energy Storage Materials. 33, p. 268-275
The commercialization of the high energy, lithium, and manganese-rich NCM (LMR-NCM) is impeded by its complex interfacial electrochemical processes, oxygen release, and surface degradation. Here, we introduced t-butyl-dimethylsilyllithium as a single-source precursor for depositing Li<sub>x</sub>Si<sub>y</sub>O<sub>z</sub> with an integrated network of siloxane moieties as an artificial cathode/electrolyte interphase (ACEI) which stabilizes LMR-NCM by mitigating oxygen release, electrolyte degradation and preventing fractures. Using solid-state NMR coupled with dynamic nuclear polarization, detailed molecular-level characterization of the ACEI is presented. The proposed CEI enabled improved energy-density at high rates (644 Wh.kg<sup>-1</sup>, compared to uncoated material with 457 Wh.kg<sup>-1</sup> at 4C) with suppressed parasitic reactions and O<sub>2</sub> evolution. The efficacy of the CEI is demonstrated in full graphite/LMR-NCM pouch cells with ~ 35% enhanced capacity and >80% capacity retention over 200 cycles. Altogether, these results present the importance of careful selection and design of surface chemistry for stabilizing the electrode/electrolyte interphase in challenging battery chemistries.
Marmor-Kollet H., Siany A., Kedersha N., Knafo N., Rivkin N., Danino Y. M., Moens T. G., Olender T., Sheban D., Cohen N., Dadosh T., Addadi Y., Ravid R., Eitan C., Toth Cohen B., Hofmann S., Riggs C. L., Advani V. M., Higginbottom A., Cooper-Knock J., Hanna J. H., Merbl Y., Van Den Bosch L., Anderson P., Ivanov P., Geiger T. & Hornstein E. (2020) Molecular Cell. 80, 5, p. 876-891
Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.
Golan K., Singh A. K., Kollet O., Bertagna M., Althoff M. J., Khatib-Massalha E., Petrovich-Kopitman E., Wellendorf A. M., Massalha H., Levin-Zaidman S., Dadosh T., Bohan B., V Gawali M., Dasgupta B., Lapidot T. & Cancelas J. A. (2020) Blood. 136, 23, p. 2607-2619
The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5'-monophosphate-activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.
Ulisse V., Dey S., Rothbard D. E., Zeevi E., Gokhman I., Dadosh T., Minis A. & Yaron A. (2020) Life Science Alliance. 3, 7, e202000753
During development, neurons adjust their energy balance to meet the high demands of robust axonal growth and branching. The mechanisms that regulate this tuning are largely unknown. Here, we show that sensory neurons lacking liver kinase B1 (Lkb1), a master regulator of energy homeostasis, exhibit impaired axonal growth and branching. Biochemical analysis of these neurons revealed reduction in axonal ATP levels, whereas transcriptome analysis uncovered down-regulation of Efhdl (EF-hand domain family member D1), a mitochondrial Ca2+-binding protein. Genetic ablation of Efhd1 in mice resulted in reduced axonal morphogenesis as well as enhanced neuronal death. Strikingly, this ablation causes mitochondrial dysfunction and a decrease in axonal ATP levels. Moreover, Efhd1 KO sensory neurons display shortened mitochondria at the axonal growth cones, activation of the AMP-activated protein kinase (AMPK)-Ulk (Unc-51-like autophagy-activating kinase 1) pathway and an increase in autophagic flux. Overall, this work uncovers a new mitochondrial regulator that is required for axonal morphogenesis.
Nejman D., Livyatan I., Fuks G., Gavert N., Zwang Y., Geller L. T., Rotter-Maskowitz A., Weiser R., Mallel G., Gigi E., Meltser A., Douglas G. M., Kamer I., Gopalakrishnan V., Dadosh T., Levin-Zaidman S., Avnet S., Atlan T., Cooper Z. A., Arora R., Cogdill A. P., Khan M. A. W., Ologun G., Bussi Y., Weinberger A., Lotan-Pompan M., Golani O., Perry G., Rokah M., Bahar-Shany K., Rozeman E. A., Blank C. U., Ronai A., Shaoul R., Amit A., Dorfman T., Kremer R., Cohen Z. R., Harnof S., Siegal T., Yehuda-Shnaidman E., Gal-Yam E. N., Shapira H., Baldini N., Langille M. G. I., Ben-Nun A., Kaufman B., Nissan A., Golan T., Dadiani M., Levanon K., Bar J., Yust-Katz S., Barshack I., Peeper D. S., Raz D. J., Segal E., Wargo J. A., Sandbank J., Shental N. & Straussman R. (2020) Science. 368, 6494, p. 973-980
Bacteria were first detected in human tumors more than 100 years ago, but the characterization of the tumor microbiome has remained challenging because of its low biomass. We undertook a comprehensive analysis of the tumor microbiome, studying 1526 tumors and their adjacent normal tissues across seven cancer types, including breast, lung, ovary, pancreas, melanoma, bone, and brain tumors. We found that each tumor type has a distinct microbiome composition and that breast cancer has a particularly rich and diverse microbiome. The intratumor bacteria are mostly intracellular and are present in both cancer and immune cells. We also noted correlations between intratumor bacteria or their predicted functions with tumor types and subtypes, patients' smoking status, and the response to immunotherapy.
Lahav-Mankovski N., Prasad P. K., Oppenheimer-Low N., Raviv G., Dadosh T., Unger T., Salame T. M., Motiei L. & Margulies D. (2020) Nature Communications. 11, 1, 1299
The responses of cells to their surroundings are mediated by the binding of cell surface proteins (CSPs) to extracellular signals. Such processes are regulated via dynamic changes in the structure, composition, and expression levels of CSPs. In this study, we demonstrate the possibility of decorating bacteria with artificial, self-assembled receptors that imitate the dynamic features of CSPs. We show that the local concentration of these receptors on the bacterial membrane and their structure can be reversibly controlled using suitable chemical signals, in a way that resembles changes that occur with CSP expression levels or posttranslational modifications (PTMs), respectively. We also show that these modifications can endow the bacteria with programmable properties, akin to the way CSP responses can induce cellular functions. By programming the bacteria to glow, adhere to surfaces, or interact with proteins or mammalian cells, we demonstrate the potential to tailor such biomimetic systems for specific applications.
Ghosh S., Di Bartolo V., Tubul L., Shimoni E., Kartvelishvily E., Dadosh T., Feigelson S. W., Alon R., Alcover A. & Haran G. (2020) Cell Reports. 30, 10, p. 3434-3447, 766196
T cell surfaces are covered with microvilli, actin-rich and flexible protrusions. We use super-resolution microscopy to show that ≥90% of T cell receptor (TCR) complex molecules TCRαβ and TCRζ, as well as the co-receptor CD4 (cluster of differentiation 4) and the co-stimulatory molecule CD2, reside on microvilli of resting human T cells. Furthermore, TCR proximal signaling molecules involved in the initial stages of the immune response, including the protein tyrosine kinase Lck (lymphocyte-specific protein tyrosine kinase) and the key adaptor LAT (linker for activation of T cells), are also enriched on microvilli. Notably, phosphorylated proteins of the ERM (ezrin, radixin, and moesin) family colocalize with TCRαβ as well as with actin filaments, implying a role for one or more ERMs in linking the TCR complex to the actin cytoskeleton within microvilli. Our results establish microvilli as key signaling hubs, in which the TCR complex and its proximal signaling molecules and adaptors are preassembled prior to activation in an ERM-dependent manner, facilitating initial antigen sensing.
Tamim-Yecheskel B., Fraiberg M., Kokabi K., Freud S., Shatz O., Marvaldi L., Subic N., Brenner O., Tsoory M., Eilam-Altstadter R., Biton I., Savidor A., Dezorella N., Heimer G., Behrends C., Ben-Zeev B. & Elazar Z. (2020) Autophagy. 17, 10, p. 3082-3095
Mutations in the coding sequence of human TECPR2 were recently linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder involving intellectual disability, autonomic-sensory neuropathy, chronic respiratory disease and decreased pain sensitivity. Here, we report the generation of a novel CRISPR-Cas9 tecpr2 knockout (tecpr2-/-) mouse that exhibits behavioral pathologies observed in SPG49 patients. tecpr2-/- mice develop neurodegenerative patterns in an age-dependent manner, manifested predominantly as neuroaxonal dystrophy in the gracile (GrN) and cuneate nuclei (CuN) of the medulla oblongata in the brainstem and dorsal white matter column of the spinal cord. Age-dependent correlation with accumulation of autophagosomes suggests compromised targeting to lysosome. Taken together, our findings establish the tecpr2 knockout mouse as a potential model for SPG49 and ascribe a new role to TECPR2 in macroautophagy/autophagy-related neurodegenerative disorders.
Eisenberg-Lerner A., Benyair R., Hizkiahou N., Nudel N., Maor R., Kramer M. P., Shmueli M. D., Zigdon I., Lev M. C., Ulman A., Sagiv J. Y., Dayan M., Dassa B., Rosenwald M., Shachar I., Li J., Wang Y., Dezorella N., Khan S., Shimoni E., Porat Z., Avinoam O. & Merbl Y. (2020) Nature Communications. 11, 409
The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.
Martinez S., Kolodny Y., Shemesh E., Scucchia F., Nevo R., Levin-Zaidman S., Paltiel Y., Keren N., Tchernov D. & Mass T. (2020) Frontiers in Marine Science. 7, 566663
Energy sources of corals, ultimately sunlight and plankton availability, change dramatically from shallow to mesophotic (30150 m) reefs. Depth-generalist corals, those that occupy both of these two distinct ecosystems, are adapted to cope with such extremely diverse conditions. In this study, we investigated the trophic strategy of the depth-generalist hermatypic coral Stylophora pistillata and the ability of mesophotic colonies to adapt to shallow reefs. We compared symbiont genera composition, photosynthetic traits and the holobiont trophic position and carbon sources, calculated from amino acids compound-specific stable isotope analysis (AA-CSIA), of shallow, mesophotic and translocated corals. This species harbors different Symbiodiniaceae genera at the two depths: Cladocopium goreaui (dominant in mesophotic colonies) and Symbiodinium microadriaticum (dominant in shallow colonies) with a limited change after transplantation. This allowed us to determine which traits stem from hosting different symbiont species compositions across the depth gradient. Calculation of holobiont trophic position based on amino acid δ<sup>15</sup>N revealed that heterotrophy represents the same portion of the total energy budget in both depths, in contrast to the dogma that predation is higher in corals growing in low light conditions. Photosynthesis is the major carbon source to corals growing at both depths, but the photosynthetic rate is higher in the shallow reef corals, implicating both higher energy consumption and higher predation rate in the shallow habitat. In the corals transplanted from deep to shallow reef, we observed extensive photo-acclimation by the Symbiodiniaceae cells, including substantial cellular morphological modifications, increased cellular chlorophyll a, lower antennae to photosystems ratios and carbon signature similar to the local shallow colonies. In contrast, non-photochemical quenching remains low and does not increase to cope with the high light regime of the shallow reef. Furthermore, host acclimation is much slower in these deep-to-shallow transplanted corals as evident from the lower trophic position and tissue density compared to the shallow-water corals, even after long-term transplantation (18 months). Our results suggest that while mesophotic reefs could serve as a potential refuge for shallow corals, the transition is complex, as even after a year and a half the acclimation is only partial.
Rabinovich S., Silberman A., Adler L., Agron S., Levin-Zaidman S., Bahat A., Porat Z., Ben-Zeev E., Geva I., Itkin M., Malitsky S., Buchaklian A., Helbling D., Dimmock D. & Erez A. (2020) Oncogene. 39, 1, p. 164-175
Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate-aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.
Kadan Y., Aram L., Shimoni E., Levin-Zaidman S., Rosenwasser S. & Gal A. (2020) Journal of Structural Biology. 210, 1, 107465
The formation of coccoliths, intricate calcium carbonate scales that cover the cells of unicellular marine microalgae, is a highly regulated biological process. For decades, scientists have tried to elucidate the cellular, chemical, and structural mechanisms that control the precise mineralogy and shape of the inorganic crystals. Transmission electron microscopy was pivotal in characterizing some of the organelles that orchestrate this process. However, due to the difficulties in preserving soluble inorganic phases during sample preparation, only recently, new intracellular ion-pools were detected using state-of-the-art cryo X-ray and electron microscopy techniques. Here, we combine a completely non-aqueous sample preparation procedure and room temperature electron microscopy, to investigate the presence, cellular location, and composition, of mineral phases inside mineral forming microalga species. This methodology, which fully preserves the forming coccoliths and the recently identified Ca-P-rich bodies, allowed us to identify a new class of ion-rich compartments that have complex internal structure. In addition, we show that when carefully choosing heavy metal stains, elemental analysis of the mineral phases can give accurate chemical signatures of the inorganic phases. Applying this approach to mineral forming microalgae will bridge the gap between the low-preservation power for inorganic phases of conventional chemical-fixation based electron microscopy, and the low-yield of advanced cryo techniques.
Pardo M., Li C., He Q., Levin-Zaidman S., Tsoory M., Yu Q., Wang X. & Rudich Y. (2020) Particle and Fibre Toxicology. 17, 1, 4
Background: Carbonaceous aerosols emitted from indoor and outdoor biomass burning are major risk factors contributing to the global burden of disease. Wood tar aerosols, namely, tar ball particles, compose a substantial fraction of carbonaceous emissions, especially from biomass smoldering. However, their health-related impacts and toxicity are still not well known. This study investigated the toxicity of the water-soluble fraction of pyrolyzed wood tar aerosols in exposed mice and lung epithelial cells. Results: Mice exposed to water-soluble wood tar aerosols showed increased inflammatory and oxidative stress responses. Bronchial epithelial cells exposed to the same water-soluble wood tar aerosols showed increased cell death with apoptotic characteristics. Alterations in oxidative status, including changes in reactive oxygen species (ROS) levels and reductions in the expression of antioxidant genes related to the transcription factor Nrf2, were observed and were confirmed by increased levels of MDA, a lipid peroxidation adduct. Damage to mitochondria was observed as an early event responsible for the aforementioned changes. Conclusions: The toxicity and health effect-related mechanisms of water-soluble wood tar were investigated for the first time in the context of biomass burning. Wood tar particles may account for major responses such as cell death, oxidative stress, supression of protection mechnaisms and mitochondrial damaged cause by expsoure to biomass burning aerosols.
Kumar S., Rechav K., Kaplan-Ashiri I. & Gal A. (2020) Science advances. 6, 42, p. 7554-7570, 7554
Diatoms are an abundant group of microalgae, known for their ability to form an intricate cell wall made of silica. Silicon levels in seawater are in the micromolar range, making it a challenge for diatoms to supply the rapid intracellular silicification process with the needed flux of soluble silicon. Here, we use three-dimensional cryoelectron microscopy and spectroscopy to quantitatively analyze, at submicrometer spatial resolution and sensitivity in the millimolar range, intracellular silicon in diatom cells. Our results show that the internal silicon concentration inside the cell is ~150 mM in average, three orders of magnitude higher than the external environment. The cellular silicon content is not compartmentalized, but rather unevenly distributed throughout the cell. Unexpectedly, under silicon starvation, the internal silicon pool is not depleted, reminiscent of a constitutive metabolite. Our spatially resolved approach to analyze intracellular silicon opens avenues to investigate this homeostatic trait of diatoms.
Raguin E., Rechav K., Brumfeld V., Shahar R. & Weiner S. (2020) Journal of Structural Biology. 211, 2, 107530
We examine the structure of the bone of the pharyngeal jaws of a large fish, the black drum (Pogonias cromis), that uses its tooth-jaw complex to crush hard-shelled bivalve mollusks. During mastication huge compressive forces are concentrated in a tiny zone at the tooth-bone interface. We report on the structure of this bone, with emphasis on its contact with the teeth, at different hierarchical levels and in 3D. Micro-CT shows that the molariform teeth do not have roots and are supported by a circular narrow bony rim that surrounds the periphery of the tooth base. The lower pharyngeal jaw is highly porous, as seen by reflected light microscopy and secondary electron microscopy (SE-SEM). Porosity decreases close to the bone-tooth interface and back-scattered electron (BSE-SEM) microscopy shows a slight elevation in mineral density. Focused ion beam - scanning electron microscopy (FIB-SEM) in the serial surface view (SSV) mode reveals a most surprising organization at the nanoscale level: parallel arrays of mineralized collagen fibrils surrounding channels of ~100 nm diameter, both with their long axes oriented along the load direction. The channels are filled with organic matter. These fibril-channel arrays are surrounded by a highly disordered mineralized material. This unusual structure clearly functions efficiently under compression, but the precise way by which this unique arrangement achieves this function is unknown.
Morag A., Maman N., Froumin N., Ezersky V., Rechav K. & Jelinek R. (2020) Advanced Electronic Materials. 6, 1, 1900844
The lower performance of pseudocapacitive supercapacitors in high-frequency applications such as alternating current (AC) line filtering has been ascribed to presumed slow kinetics of redox processes compared to ion diffusion in electric double layer capacitors. A nickel-deposited ruthenium/ruthenium-oxide symmetric supercapacitor exhibiting remarkable electrochemical properties, particularly very high frequency response (>1 kHz) is developed. The electrodes are prepared via a simple process consisting of electrochemical reduction of ruthenium chloride on commercially available nickel foil as the current collector. A symmetric supercapacitor comprising nickel/ruthenium/ruthenium-oxide electrodes and a polystyrene-based thin spacer exhibits particularly fast scan rates, high power density of 1500 mW cm(-2) (88 kW cm(-3)) with a maximum energy density of 0.58 mu Wh cm(-2) (34 mWh cm(-3)), and excellent capacitance retention. Notably, supercapacitors prepared by the same synthetic method albeit using conventional gold substrate instead of nickel exhibit significantly lower frequency response. The exceptional electrochemical properties of the nickel/ruthenium/ruthenium-oxide supercapacitor and simple electrode synthesis point to promising applicability in AC line filtering and power conditioning. In a broader context, this work demonstrates that, contrary to the widely held presumption, the kinetics of redox reactions at the active layers of pseudocapacitors may not be the primary barriers to high-frequency applications.
Lin W., Kluzek M., Iuster N., Shimoni E., Kampf N., Goldberg R. & Klein J. (2020) Science. 370, 6514, p. 335-338
The lubrication of hydrogels arises from fluid or solvated surface phases. By contrast, the lubricity of articular cartilage, a complex biohydrogel, has been at least partially attributed to nonfluid, lipid-exposing boundary layers. We emulated this behavior in synthetic hydrogels by incorporating trace lipid concentrations to create a molecularly thin, lipid-based boundary layer that renews continuously. We observed a 80% to 99.3% reduction in friction and wear relative to the lipid-free gel, over a wide range of conditions. This effect persists when the gels are dried and then rehydrated. Our approach may provide a method for sustained, extreme lubrication of hydrogels in applications from tissue engineering to clinical diagnostics.
Heidenreich M., Georgeson J. M., Locatelli E., Rovigatti L., Nandi S. K., Steinberg A., Nadav Y., Shimoni E., Safran S. A., Doye J. P. K. & Levy E. D. (2020) Nature Chemical Biology. 16, 9, p. 939-945
Protein self-organization is a hallmark of biological systems. Although the physicochemical principles governing protein-protein interactions have long been known, the principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remain challenging to characterize. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies. We devise a new strategy to map high-resolution phase diagrams in living cells, which provide self-assembly signatures of this system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how interaction affinity impacts the phase diagram and material state of the assemblies in vivo. The phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Finally, we find that cotranslational protein binding suffices to recruit a messenger RNA to the designed micron-scale structures.
Shtukenberg A. G., Drori R., Sturm E. V., Vidavsky N., Haddad A., Zheng J., Estroff L. A., Weissman H., Wolf S. G., Shimoni E., Li C., Fellah N., Efrati E. & Kahr B. (2020) Angewandte Chemie - International Edition. 59, 34, p. 14593-14601
The growth of spontaneously twisted crystals is a common but poorly understood phenomenon. An analysis of the formation of twisted crystals of a metastable benzamide polymorph (form II) crystallizing from highly supersaturated aqueous and ethanol solutions is given here. Benzamide, the first polymorphic molecular crystal reported (1832), would have been the first helicoidal crystal observed had the original authors undertaken an analysis by light microscopy. Polymorphism and twisting frequently concur as they are both associated with high thermodynamic driving forces for crystallization. Optical and electron microscopies as well as electron and powder X-ray diffraction reveal a complex lamellar structure of benzamide form II needle-like crystals. The internal stress produced by the overgrowth of lamellae is shown to be able to create a twist moment that is responsible for the observed non-classical morphologies.
Haimov H., Shimoni E., Brumfeld V., Shemesh M., Varsano N., Addadi L. & Weiner S. (2020) Bone. 130, 115086
Endochondral ossification in the growth plate of long bones involves cartilage mineralization, bone formation and the budding vasculature. Many of these processes take place in a complex and dynamic zone, the provisional ossification zone, of the growth plate. Here we investigate aspects of mineralization in 2D and 3D in the provisional ossification zone at different length scales using samples preserved under cryogenic or fully hydrated conditions. We use confocal light microscopy, cryo-SEM and micro-CT in the phase contrast mode. We show in 9 week old BALB/c mice the presence of vesicles containing mineral particles in the blood serum, as well as mineral particles without membranes integrated with the blood vessel walls. We also observe labeled mineral particles within cells associated with bone formation, but not in the hypertrophic cartilage cells that are involved with cartilage mineralization. High resolution micro-CT images of fresh hydrated tibiae, show that there are open continuous pathways between the blood vessel extremities and the hypertrophic chondrocyte zone. As the blood vessel extremities, the mineralizing cartilage and the forming bone are all closely associated within this narrow zone, we raise the possibility that in addition to ion transport, mineral necessary for both cartilage and bone formation is also transported through the vasculature.
Bajorowicz B., Mikolajczyk A., Pinto H. P., Miodyńska M., Lisowski W., Klimczuk T., Kaplan-Ashiri I., Kazes M., Oron D. & Zaleska-Medynska A. (2020) Journal of Physical Chemistry C. 124, 49, p. 26769-26779
Applied cutting-edge electronic structure and phonon simulations provide a reliable knowledge about the stability of perovskite structures and their electronic properties, which are crucial for design of effective nanomaterials. Gold is one of the exceptional elements, which can exist both as a monovalent and a trivalent ion in the B site of a double perovskite such as A2BIBIIIX6. However, until now, electronic properties of Cs2AuIAuIIIX6 have not been sufficiently explored and this material was never synthesized using Au1+ and Au3+ precursors in the preparation route. Here, computational simulations combined with an experimental study provide new insight into the properties and synthesis route of Cs2AuIAuIIIX6 (X = Cl, Br, and I) perovskites. First-principles calculations reveal that tetragonal Cs2AuIAuIIIX6 (X = I, Br, Cl) molecules present a band gap of 1.10, 1.15, and 1.40 eV, respectively. Application of novel approaches in the simulations of the VB-XPS for Cs2AuIAuIIICl6 allows replication of the observed spectrum and provides strong evidence of the reliability of the obtained results for the other perovskites Cs2AuIAuIIIX6, X = Br, I. Following theoretical findings, a one-step preparation route of the Cs2AuIAuIIICl6 is developed using a combination of monovalent and trivalent gold precursors at a relatively low temperature. It should be emphasized that this is the first synthesis of this material at low temperatures, allowing for obtaining highly crystalline Cs2Au2Cl6 particles with controlled morphology and without gold impurities. The band gap of synthesized Cs2AuIAuIIICl6 is extended into the NIR spectral range, where most other double perovskites are limited to higher energies, limiting their usage in single junction solar cells or in photocatalysis. The as-synthesized Cs2AuIAuIIICl6 exhibits high efficiency in a photocatalytic toluene degradation reaction under visible light irradiation. The developed approach provides information necessary for structure manipulation at the early stage of its synthesis and offers a new and useful guidance for design of novel improved lead-free inorganic halide perovskite with interesting optical and photocatalytic properties.
Ghosh S., Brüser V., Kaplan-Ashiri I., Popovitz-Biro R., Peglow S., Martínez J. I., Alonso J. A. & Zak A. (2020) Applied Physics Reviews. 7, 4, 041401
For nanoparticles with sub-10 nm diameter, the electronic bandgap becomes size dependent due to quantum confinement; this, in turn, affects their electro-optical properties. Thereby, MoS2 and WS2 monolayers acquire luminescent capability, due to the confinement-induced indirect-to-direct bandgap transition. Rolling up of individual layers results in single wall inorganic nanotubes (SWINTs). Up to the present study, their luminescence properties were expected to be auspicious but were limited to theoretical investigations only, due to the scarcity of SWINTs and the difficulties in handling them. By optimizing the conditions in the plasma reactor, relatively high yields of WS2 SWINTs 3-7 nm in diameter were obtained in this work, compared to previous reports. A correlative approach, transmission electron microscopy coupled with a scanning electron microscope, was adapted to overcome handling obstacles and for testing individual nanotubes by low-temperature cathodoluminescence. Clear cathodoluminescence spectra were obtained from WS2-SWINTs and compared with those of WS2 multiwall nanotubes and the corresponding bulk material. Uniquely, the optical properties of INTs acquired from cathodoluminescence were governed by the opposite impact from quantum size effect and strain in the bent triple S-W-S layers. The experimental findings were confirmed by the Density Functional and Time-Dependent Density Functional theoretical modeling of monolayer and bilayer nanotubes of different chiralities and diameters. This study provides experimental evidence of the quantum confinement effect in WS2 SWINTs akin to WS2 monolayer. The ability to tune the electronic structure with morphology or number of layers may be exploited toward photoelectrochemical water splitting with WS2 catalysts, devising field effect transistors, photodetectors, and so on.
Guendelman G., Lovsky Y., Yacoby E., Mor O. E., Kaplan-Ashiri I., Goldbart O. & Dayan B. (2020) Optics Express. 28, 21, p. 31297-31315
Whispering-gallery-mode (WGM) microresonators are a promising platform for highly sensitive, label-free detection and probing of individual nano-objects. Our work expands these capabilities by providing the analysis tools required for three-dimensional (3D) characterization of arbitrarily shaped nanoparticles. Specifically, we introduce a theoretical model that describes interactions between nanoparticles and WGM resonators, taking into account effects that were often not considered, such as the elliptical polarization of the transverse-magnetic (TM) mode, the possible non-spherical shape of the nanoparticle, its finite size, and the open-system nature of the modes. We also introduce a self referencing measurement method that allows the extraction of information from measurements done at arbitrary positions of the nanoparticles within the WGM. We verify our model by experimentally probing a single Tungsten-disulfide (WS2) nanotube with a silica microtoroid resonator inside a scanning electron-microscope (SEM) and perform 3D characterization of the nanotube.
Toffolo M. B., Ricci G., Chapoulie R., Caneve L. & Kaplan-Ashiri I. (2020) Radiocarbon. 62, 3, p. 545-564
Accurate radiocarbon (C-14) dating of lime mortars requires a thorough mineralogical characterization of binders in order to verify the presence of carbon-bearing contaminants. In the last 20 years, cathodoluminescence (CL) has been widely used for the identification of geologic calcium carbonate (CaCO3) aggregates and unreacted lime lumps within the particle size fraction selected for carbon recovery. These components are major sources of older and younger carbon, respectively, and should be removed to obtain accurate age determinations. More recently, laser-induced fluorescence (LIF) has provided another means of investigating the preservation state and composition of CaCO3 binders. Considered the growing interest of the mortar dating community in the latest advancements of these analytical methods, here we review the principles of CL and LIF of CaCO3, their instrument setup, and their application to the characterization of ancient lime mortars used for C-14 dating. In addition, we provide examples of SEM-CL and LIF analyses using high-resolution instrumentation, we discuss current issues and propose future lines of research.
Toffolo M. B., Regev L., Mintz E., Kaplan-Ashiri I., Berna F., Dubernet S., Yan X., Regev J. & Boaretto E. (2020) Radiocarbon. 62, 3, p. 633-655
Radiocarbon (C-14) dating of anthropogenic carbonates (CaCO3) such as ash, lime plaster and lime mortar, has proven a difficult task due to the occurrence of a number of contaminants embedded within the CaCO3 pyrogenic binder. These include C-14-free geologic components and/or secondary phases bearing an unknown amount of C-14, and thus the alteration of the original pyrogenic isotopic signature of the material results in major age offsets when carbon recovery is performed through acid hydrolysis. Here we present a characterization/quantification approach to anthropogenic carbonates that includes Fourier transform infrared spectroscopy (FTIR), X-ray diffraction, thin section petrography, thermogravimetric analysis and scanning electron microscopy coupled with high-resolution cathodoluminescence, with which we identified the pyrogenic CaCO3 fraction in an aerial lime plaster and two hydraulic mortars. The preserved pyrogenic component was then isolated by density separation and its purity checked again using FTIR. Carbon was recovered through thermal decomposition in vacuum. The resulting C-14 age matches the expected age of the lime plaster, whereas hydraulic mortars are slightly offset due to the carbonation of calcium hydroxide lumps. This approach highlights the importance of a dedicated characterization strategy prior to dating and may be applied to aerial lime plasters to obtain accurate ages.
Osherov A., Feldman Y., Kaplan-Ashiri I., Cahen D. & Hodes G. (2020) Chemistry of Materials. 32, 10, p. 4223-4231
We study halide exchange in the prototypical halide perovskite, methylammonium lead trihalide, MAPbX(3) (X = halide), to test and possibly experimentally use halide diffusion in these materials. We use macroscopic single crystals to study the fundamental exchange process(es) so as to minimize possible grain boundary and surface diffusion effects. Initially, halide exchange creates normal concentration gradients of the outgoing and incoming halides, on a scale of a few microns to a few hundred microns. The depth (from the surface) of the substituted volume depends on the halide pair and on which one is exchanged and which is exchanging. The concentration gradient of the incoming halides decreases from the crystal surface toward its inner core and vice versa for the out-going halides; the profiles roughly fit diffusion in a semi-infinite specimen. This concentration gradient changes slowly with time, with the crystal becoming more homogeneous with storage time. Using the Boltzmann-Matano method and diffusion profiles from electron-dispersive spectroscopy, we evaluate the halide diffusion coefficients; these are not constant and depend on the halide couple. Although these gradients cause a lattice parameter change and may cause a symmetry change, X-ray diffraction shows that if the exchanging halides are of similar size (e.g., Br- and Cl-, Br- and I-, but not Cl- and I-), the resulting material remains single crystalline, prima facie evidence for bulk halide diffusion. These findings are valid, irrespective of which is the exchanged halide. These results suggest that for the similar-sized halide pairs the solid-state chemical exchange is topotactic such that the resulting crystal orientation is determined by that of the initial crystal. I-Cl exchange leads to loss of single crystallinity, suggesting lack of miscibility, a finding that might bear on the difficulty in finding Cl in MAPbI(3) samples grown from Cl-containing solutions.
Waugh B., Wolf S. G., Fass D., Branlund E., Kam Z., Sedat J. W. & Elbaum M. (2020) Proceedings of the National Academy of Sciences of the United States of America. 117, 44, p. 27374-27380
The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.
Carter S. D., Hampton C. M., Langlois R., Melero R., Farino Z. J., Calderon M. J., Li W., Wallace C. T., Tran N. H., Grassucci R. A., Siegmund S. E., Pemberton J., Morgenstern T. J., Eisenman L., Aguilar J. I., Greenberg N. L., Levy E. S., Yi E., Mitchell W. G., Rice W. J., Wigge C., Pilli J., George E. W., Aslanoglou D., Courel M., Freyberg R. J., Javitch J. A., Wills Z. P., Area-gomez E., Shiva S., Bartolini F., Volchuk A., Murray S. A., Aridor M., Fish K. N., Walter P., Balla T., Fass D., Wolf S. G., Watkins S. C., Carazo J. M., Jensen G. J., Frank J. & Freyberg Z. (2020) Science Advances. 6, 14, eaay9572
The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic beta-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.
Zhang G., Hirsch A., Shmul G., Avram L., Elad N., Brumfeld V., Pinkas I., Feldman Y., Ben Asher R., Palmer B. A., Kronik L., Leiserowitz L., Weiner S. & Addadi L. (2019) Journal of the American Chemical Society. 141, 50, p. 19736-19745
The eyes of many fish contain a reflecting layer of organic crystals partially surrounding the photoreceptors of the retina, which are commonly believed to be composed of guanine. Here we study an unusual fish eye from Stizostedion lucioperca that contains two layers of organic crystals. The crystals in the outer layer are thin plates, whereas the crystals in the inner tapetum layer are block-shaped. We show that the outer layer indeed contains guanine crystals. Analyses of solutions of crystals from the inner layer indicated that the block-shaped crystals are composed of xanthopterin. A model of the structure of the block-shaped crystals was produced using symmetry arguments based on electron diffraction data followed by dispersion-augmented DFT calculations. The resulting crystal structure of xanthopterin included, however, a problematic repulsive interaction between C=O and N of two adjacent molecules. Knowing that dissolved 7,8-dihydroxanthopterin can oxidize to xanthopterin, we replaced xanthopterin with 7,8-dihydroxanthopterin in the model. An excellent fit was obtained with the powder X-ray diffraction pattern of the biogenic crystals. We then analyzed the biogenic block-shaped crystals in their solid state, using MALDI-TOF and Raman spectroscopy. All three methods unequivocally prove that the block-shaped crystals in the eye of S. lucioperca are crystals of 7,8-dihydroxanthopterin. On the basis of the eye anatomy, we deduce that the guanine crystals form a reflective layer producing the silvery color present on part of the eye surface, whereas the block-shaped crystals backscatter light into the retina in order to increase the light sensitivity of the eye.
Ehrhardt S. A., Zehner M., Kraehling V., Cohen-Dvashi H., Kreer C., Elad N., Gruell H., Ercanoglu M. S., Schommers P., Gieselmann L., Eggeling R., Dahlke C., Wolf T., Pfeifer N., Addo M. M., Diskin R., Becker S. & Klein F. (2019) Nature Medicine. 25, 10, p. 1589-1600
Recombinant vesicular stomatitis virus-Zaire Ebola virus (rVSV-ZEBOV) is the most advanced Ebola virus vaccine candidate and is currently being used to combat the outbreak of Ebola virus disease (EVD) in the Democratic Republic of the Congo (DRC). Here we examine the humoral immune response in a subset of human volunteers enrolled in a phase 1 rVSV-ZEBOV vaccination trial by performing comprehensive single B cell and electron microscopy structure analyses. Four studied vaccinees show polyclonal, yet reproducible and convergent B cell responses with shared sequence characteristics. EBOV-targeting antibodies cross-react with other Ebolavirus species, and detailed epitope mapping revealed overlapping target epitopes with antibodies isolated from EVD survivors. Moreover, in all vaccinees, we detected highly potent EBOV-neutralizing antibodies with activities comparable or superior to the monoclonal antibodies currently used in clinical trials. These include antibodies combining the IGHV3-15/IGLV1-40 immunoglobulin gene segments that were identified in all investigated individuals. Our findings will help to evaluate and direct current and future vaccination strategies and offer opportunities for novel EVD therapies.
Jantschke A., Pinkas I., Hirsch A., Elad N., Schertel A., Addadi L. & Weiner S. (2019) Journal of Structural Biology. 207, 1, p. 12-20
Guanine crystals are used by certain animals, including vertebrates, to produce structural colors or to enhance vision, because of their distinctive reflective properties. Here we use cryo-SEM, cryo-FIB SEM and Raman spectroscopic imaging to characterize crystalline inclusions in a single celled photosynthesizing marine dinoflagellate species. We demonstrate spectroscopically that these inclusions are blocky crystals of anhydrous guanine in the beta-polymorph. Two-dimensional cryo-SEM and three-dimensional cryo-FIB-SEM serial block face imaging show that the deposits of anhydrous guanine crystals are closely associated with the chloroplasts. We suggest that the crystalline deposits scatter light either to enhance light exploitation by the chloroplasts, or possibly for protection from UV radiation. This is consistent with the crystal locations within the cell, their shapes and their sizes. As the dinoflagellates are extremely abundant in the oceans and are a major group of photosynthesizing marine organisms, the presence of guanine crystals in this marine organism may have broad significance.
Chen F., Wu B., Elad N., Gal A., Liu Y., Ma Y. & Qi L. (2019) CrystEngComm. 21, 23, p. 3586-3591
As important optical devices, including diffuse scatterers, broadband reflectors, and image-forming mirrors with superior properties, biological guanine crystals have been thoroughly investigated in recent years. However, controlling the polymorph and morphology of synthetic guanine crystals is still highly challenging. Herein, a pure phase of the anhydrous guanine (AG) ss form was obtained via transformation of a hydrated amorphous guanine phase (HAmG) in solvents such as formamide, DMSO and DMF. The AG ss crystals can be stable for up to three months in the above solvents. The AG ss nano-platelets obtained in DMSO exposed the (100) plane when polyvinylpyrrolidone (PVP) was applied as an additive. The AG ss nano-platelets were about 100 nm in length, 40 nm in width, and 10-20 nm in thickness. Solid-state NMR (ssNMR) characterization indicated that the HAmG precursor had a similar short-range order as AG ss, which might be the reason for the formation of AG ss instead of the thermodynamically more stable AG a. The delicate control of the polymorph and morphology of the guanine crystals via an amorphous phase strategy may inspire the formation of highly ordered hierarchical structures of guanine crystals with unique optical properties.
Empereur-Mot C., Garcia-Seisdedos H., Elad N., Dey S. & Levy E. D. (2019) Scientific data. 6, 64
Proteins can self-associate with copies of themselves to form symmetric complexes called homomers. Homomers are widespread in all kingdoms of life and allow for unique geometric and functional properties, as reflected in viral capsids or allostery. Once a protein forms a homomer, however, its internal symmetry can compound the effect of point mutations and trigger uncontrolled self-assembly into high-order structures. We identified mutation hot spots for supramolecular assembly, which are predictable by geometry. Here, we present a dataset of descriptors that characterize these hot spot positions both geometrically and chemically, as well as computer scripts allowing the calculation and visualization of these properties for homomers of choice. Since the biological relevance of homomers is not readily available from their X-ray crystallographic structure, we also provide reliability estimates obtained by methods we recently developed. These data have implications in the study of disease-causing mutations, protein evolution and can be exploited in the design of biomaterials.
Varsano N., Beghi F., Dadosh T., Elad N., Pereiro E., Haran G., Leiserowitz L. & Addadi L. (2019) ChemPlusChem. 84, 4, p. 317-317
Invited for this month's cover are the group of Prof. Lia Addadi at the Weizmann Institute of Science, Israel and collaborators at the Università Degli Studi di Milano, Italy, and the ALBA Synchrotron Light Source, Spain. The front cover shows how cholesterol crystals form in macrophage cells and in lipid bilayers of different compositions. Cholesterol monohydrate stable triclinic crystals form in vitro as rhomb-shaped plates, whereas the monoclinic crystals fold into tubular or helical shapes. Read the full text of the article at 10.1002/cplu.201800632.
Mazal H., Iljina M., Barak Y., Elad N., Rosenzweig R., Goloubinoff P., Riven I. & Haran G. (2019) Nature Communications. 10, 1, 1438
Large protein machines are tightly regulated through allosteric communication channels. Here we demonstrate the involvement of ultrafast conformational dynamics in allosteric regulation of CIpB, a hexameric AAA+ machine that rescues aggregated proteins. Each subunit of CIpB contains a unique coiled-coil structure, the middle domain (M domain), proposed as a control element that binds the co-chaperone DnaK. Using single-molecule FRET spectroscopy, we probe the M domain during the chaperone cycle and find it to jump on the microsecond time scale between two states, whose structures are determined. The M-domain jumps are much faster than the overall activity of CIpB, making it an effectively continuous, tunable switch. Indeed, a series of allosteric interactions are found to modulate the dynamics, including binding of nucleotides, DnaK and protein substrates. This mode of dynamic control enables fast cellular adaptation and may be a general mechanism for the regulation of cellular machineries.
Varsano N., Beghi F., Dadosh T., Elad N., Pereiro E., Haran G., Leiserowitz L. & Addadi L. (2019) ChemPlusChem. 84, 4, p. 338-344
Cholesterol crystallization from mixtures of unesterified cholesterol with phospholipids and cholesterol esters is believed to be a key event in atherosclerosis progression. Not much is understood, however, about the influence of the lipid environment on cholesterol crystallization. Here we study cholesterol monohydrate crystal formation from mixed bilayers with palmitoyl-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin. We show that disordered phospholipids and sphingomyelin stabilize the formation of crystal plates of the triclinic cholesterol monohydrate polymorph, whereas saturated glycerolipids stabilize helical and tubular crystals of the metastable monoclinic polymorph. We followed the subsequent transformation of these helical crystals into the stable triclinic plates. Discovering the relations between membrane lipid composition and cholesterol crystal polymorphism may provide important clues to the understanding of cholesterol crystal formation in atherosclerosis.
Evenstein E., Rosy, Haber S., Sclar H., Houben L., Leung K., Leskes M. & Noked M. (2019) Energy Storage Materials. 19, p. 261-269
In the present work, a simple and agile methodology for atomic surface reduction of interfaces is introduced. Using a surface directed vapor phase reaction, at relatively low temperature, we show that a highly reactive and volatile molecule can be used to selectively reduce the interface, without changing the bulk of the treated material, and without the need of alternating sequence of multiple precursors, normally involved in ALD. The model system we use to demonstrate the efficacy, and potential of our approach is trimethyl aluminum, and high energy Li and Mn rich cathode (HE-NCM) as the functional material of interest. We demonstrate that with the proposed method, the particles of HE-NMC were conformally coated with similar to 3 nm amorphous layer of the reduced surface in less than 1 h (including the cooling time), as witnessed using HR-TEM. XPS and solid-state NMR, further confirmed that surface treatment was successfully achieved using the proposed method and is well explained by DFT calculations. Utilizing online electrochemical mass spectrometry (OEMS), we show in-operando that this amorphous layer helps to suppress parasitic reactions under extreme electrochemical conditions as indicated by the significant reduction in oxygen and CO2 evolution. The surface treatment further resulted in enhancement in specific capacity during the first cycle. This methodology provides a non-conventional path to achieve thin layer surface modification under facile conditions, and opens a new way to meet the requirements of surface modification strategies for improving the performance of electrode materials without utilizing expensive instrumentation and high temperature processes.
Aronovitch E., Houben L. & Bar-Sadan M. (2019) Chemistry of Materials. 31, 18, p. 7231-7237
Deposition of metal cocatalysts is a common practice to improve the activity of photocatalysts. The use of nanoalloyed nanoparticles allows the formation of diverse nanostructures, tailored for a specific application. Nevertheless, too often the spontaneous atomic scale phenomena interfere with the initial design to produce a modified structure with undesirable properties. Here, we demonstrate such a process for Pd, Ag, or their combination as metal tips mounted on seeded rods of a CdSe dot in a CdS rod (CdSe@CdS) that serve as hydrogen evolution photocatalysts. Spontaneous radial reconstruction at the metal tip brings both Pd and Ag atoms outward even when a two-stage preparation process is applied to specifically produce a core-shell structure. The diffusion of Pd outward enables hydrogen evolution even when the initial Pd tip is covered by a Ag shell, and in the opposite case, a Pd shell shows reduced activity compared with Pd-only tips, due to the surfacing of Ag atoms. In addition, we show that the tip reconstruction occurs already during synthesis; aberration-corrected high-resolution electron microscopy also reveals other processes, such as cation exchange and small clustering around the seeded rods, all quite invisible using regular TEM techniques. In addition, we studied the size effect of Pd-tipped seeded rods and showed that the %QE of seeded rods with 2.2 Pd tips is as high as 91%. These results are significant in the understanding of the structure-function relationship, as it highlights one possible hidden reconstruction pathway of nanoalloys.
Svirinovsky-Arbeli A., Rosenberg D., Krotkov D., Damari R., Kundu K., Feintuch A., Houben L., Fleischer S. & Leskes M. (2019) Solid State Nuclear Magnetic Resonance. 99, p. 7-14
In recent years dynamic nuclear polarization (DNP) has greatly expanded the range of materials systems that can be studied by solid state NMR spectroscopy. To date, the majority of systems studied by DNP were insulating materials including organic and inorganic solids. However, many technologically-relevant materials used in energy conversion and storage systems are electrically conductive to some extent or are employed as composites containing conductive additives. Such materials introduce challenges in their study by DNP-NMR which include microwave absorption and sample heating that were not thoroughly investigated so far.Here we examine several commercial carbon allotropes, commonly employed as electrodes or conductive additives, and consider their effect on the extent of solvent polarization achieved in DNP from nitroxide biradicals. We then address the effect of sample conductivity systematically by studying a series of carbons with increasing electrical conductivity prepared via glucose carbonization. THz spectroscopy measurements are used to determine the extent of vw absorption. Our results show that while the DNP performance significantly drops in samples containing the highly conductive carbons, sufficient signal enhancement can still be achieved with some compromise on conductivity. Furthermore, we show that the deleterious effect of conductive additives on DNP enhancements can be partially overcome through pulse-DNP experiments.
de Jonge N., Houben L., Dunin-Borkowski R. E. & Ross F. M. (2019) Nature Reviews Materials. 4, 1, p. 61-78
Liquid cell electron microscopy possesses a combination of spatial and temporal resolution that provides a unique view of static structures and dynamic processes in liquids. Optimizing the resolution in liquids requires consideration of both the microscope performance and the properties of the sample. In this Review, we survey the competing factors that determine spatial and temporal resolution for transmission electron microscopy and scanning transmission electron microscopy of liquids. We discuss the effects of sample thickness, stability and dose sensitivity on spatial and temporal resolution. We show that for some liquid samples, spatial resolution can be improved by spherical and chromatic aberration correction. However, other benefits offered by aberration correction may be even more useful for liquid samples. We consider the greater image interpretability offered by spherical aberration correction and the improved dose efficiency for thicker samples offered by chromatic aberration correction. Finally, we discuss the importance of detector and sample parameters for higher resolution in future experiments.
Lansky Z., Mutsafi Y., Houben L., Ilani T., Armony G., Wolf S. G. & Fass D. (2019) Journal of Structural Biology: X. 1, 100002
Cells and extracellular matrix (ECM) are mutually interdependent: cells guide self-assembly of ECM precursors, and the resulting ECM architecture supports and instructs cells. Though bidirectional signaling between ECM and cells is fundamental to cell biology, it is challenging to gain high-resolution structural information on cellular responses to the matrix microenvironment. Here we used cryo-scanning transmission electron tomography (CSTET) to reveal the nanometer- to micron-scale organization of major fibroblast ECM components in a native-like context, while simultaneously visualizing internal cell ultrastructure including organelles and cytoskeleton. In addition to extending current models for collagen VI fibril organization, three-dimensional views of thick cell regions and surrounding matrix showed how ECM networks impact the structures and dynamics of intracellular organelles and how cells remodel ECM. Collagen VI and fibronectin were seen to distribute in fundamentally different ways in the cell microenvironment and perform distinct roles in supporting and interacting with cells. This work demonstrates that CSTET provides a new perspective for the study of ECM in cell biology, highlighting labeled extracellular elements against a backdrop of unlabeled but morphologically identifiable cellular features with nanometer resolution detail.
Ben-Zvi R., Burrows H., Schvartzman M., Bitton O., Pinkas I., Kaplan-Ashiri I., Brontvein O. & Joselevich E. (2019) ACS Nano. 13, 5, p. 5572-5582
The challenge of nanowire assembly is still one of the major obstacles toward their efficient integration into functional systems. One strategy to overcome this obstacle is the guided growth approach, in which the growth of in-plane nanowires is guided by epitaxial and graphoepitaxial relations with the substrate to yield dense arrays of aligned nanowires. This method relies on crystalline substrates which are generally expensive and incompatible with silicon-based technologies. In this work, we expand the guided growth approach into noncrystalline substrates and demonstrate the guided growth of horizontal nanowires along straight and arbitrarily shaped amorphous nanolithographic open guides on silicon wafers. Nanoimprint lithography is used as a high-throughput method for the fabrication of the high-resolution guiding features. We first grow five different semiconductor materials (GaN, ZnSe, CdS, ZnTe, and ZnO) along straight ridges and trenches, demonstrating the generality of this method. Through crystallographic analysis we find that despite the absence of any epitaxial relations with the substrate, the nanowires grow as single crystals in preferred crystallographic orientations. To further expand the guided growth approach beyond straight nanowires, GaN and ZnSe were grown also along curved and kinked configurations to form different shapes, including sinusoidal and zigzag-shaped nanowires. Photoluminescence and cathodoluminescence were used as noninvasive tools to characterize the sine wave-shaped nanowires. We discuss the similarities and differences between in-plane nanowires grown by epitaxy/graphoepitaxy and artificial epitaxy in terms of generality, morphology, crystallinity, and optical properties.
Rossman U., Tenne R., Solomon O., Kaplan-Ashiri I., Dadosh T., Eldar Y. C. & Oron D. (2019) Optica. 6, 10, p. 1290-1296
The evolution of experimental superresolution microscopy has been accompanied by the development of advanced computational imaging capabilities. Recently introduced, quantum image scanning microscopy (Q-ISM) has successfully harnessed quantum correlations of light to establish an improved viable imaging modality that builds upon the preceding image scanning microscopy (ISM) superresolution method. While offering improved resolution, at present the inherently weak signal demands exhaustively long acquisition periods. Here we exploit the fact that the correlation measurement in Q-ISM is complementary to the standard ISM data, acquired simultaneously, and demonstrate joint sparse recovery from Q-ISM and ISM images. Reconstructions from images of fluorescent quantum dots are validated through correlative electron microscope measurements, and exhibit superior resolution enhancement as compared to Q-ISM images. In addition, the algorithmic fusion facilitates a drastic reduction in the requisite measurement duration, since low signal-to-noise-ratio Q-ISM measurements suffice for augmenting ISM images. Finally, we obtain enhanced superresolved reconstructions from short scans of a biological sample labeled with quantum dots, demonstrating the potential of our method for quantum imaging in life science microscopy.
Ben Yaakov L., Mutsafi Y., Porat Z., Dadosh T. & Minsky A. (2019) Cytometry. Part A : the journal of the International Society for Analytical Cytology. 95, 5, p. 534-548
Due to the heterogeneity of viruses and their hosts, a comprehensive view of viral infection is best achieved by analyzing large populations of infected cells. However, information regarding variation in infected cell populations is lost in bulk measurements. Motivated by an interest in the temporal progression of events in virally infected cells, we used image flow cytometry (IFC) to monitor changes in Acanthamoeba polyphaga cells infected with Mimivirus. This first use of IFC to study viral infection required the development of methods to preserve morphological features of adherent amoeba cells prior to detachment and analysis in suspension. It also required the identification of IFC parameters that best report on key events in the Mimivirus infection cycle. The optimized IFC protocol enabled the simultaneous monitoring of diverse processes including generation of viral factories, transport, and fusion of replication centers within the cell, accumulation of viral progeny, and changes in cell morphology for tens of thousands of cells. After obtaining the time windows for these processes, we used IFC to evaluate the effects of perturbations such as oxidative stress and cytoskeletal disruptors on viral infection. Accurate dose-response curves could be generated, and we found that mild oxidative stress delayed multiple stages of virus production, but eventually infection processes occurred with approximately the same amplitudes. We also found that functional actin cytoskeleton is required for fusion of viral replication centers and later for the production of viral progeny. Through this report, we demonstrate that IFC offers a quantitative, high-throughput, and highly robust approach to study viral infection cycles and virus-host interactions. (C) 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Koganitsky A., Tworowski D., Dadosh T., Cecchini G. & Eisenbach M. (2019) Journal of Molecular Biology. 431, 19, p. 3662-3676
Fumarate, an electron acceptor in anaerobic respiration of Escherichia coli, has an additional function of assisting the flagellar motor to shift from counterclockwise to clockwise rotation, with a consequent modulation of the bacterial swimming behavior. Fumarate transmits its effect to the motor via the fumarate reductase complex (FrdABCD), shown to bind to FliGone of the motor's switch proteins. How binding of the FrdABCD respiratory enzyme to FliG enhances clockwise rotation and how fumarate is involved in this activity have remained puzzling. Here we show that the FrdA subunit in the presence of fumarate is sufficient for binding to FliG and for clockwise enhancement. We further demonstrate by in vitro binding assays and super-resolution microscopy in vivo that the mechanism by which fumarate-occupied FrdA enhances clockwise rotation involves its preferential binding to the clockwise state of FliG (FliG
<sub>cw</sub>). Continuum electrostatics combined with docking analysis and conformational sampling endorsed the experimental conclusions and suggested that the FrdAFliG
<sub>cw</sub> interaction is driven by the positive electrostatic potential generated by FrdA and the negatively charged areas of FliG. They further demonstrated that fumarate changes FrdA's conformation to one that can bind to FliG
<sub>cw</sub>. These findings also show that the reason for the failure of the succinate dehydrogenase flavoprotein SdhA (an almost-identical analog of FrdA shown to bind to FliG equally well) to enhance clockwise rotation is that it has no binding preference for FliG
<sub>cw</sub>. We suggest that this mechanism is physiologically important as it can modulate the magnitude of ΔG
<sup>0</sup> between the clockwise and counterclockwise states of the motor to tune the motor to the growth conditions of the bacteria.
Anbalagan S., Blechman J., Gliksberg M., Gordon L., Rotkopf R., Dadosh T., Shimoni E. & Levkowitz G. (2019) eLife. 8, e45650
The regulation of neuropeptide level at the site of release is essential for proper neurophysiological functions. We focused on a prominent neuropeptide, oxytocin (OXT) in the zebrafish as an in vivo model to visualize and quantify OXT content at the resolution of a single synapse. We found that OXT-loaded synapses were enriched with polymerized actin. Perturbation of actin filaments by either cytochalasin-D or conditional Cofilin expression resulted in decreased synaptic OXT levels. Genetic loss of robo2 or slit3 displayed decreased synaptic OXT content and robo2 mutants displayed reduced mobility of the actin probe Lifeact-EGFP in OXT synapses. Using a novel transgenic reporter allowing real-time monitoring of OXT-loaded vesicles, we show that robo2 mutants display slower rate of vesicles accumulation. OXT-specific expression of dominant-negative Cdc42, which is a key regulator of actin dynamics and a downstream effector of Robo2, led to a dose-dependent increase in OXT content in WT, and a dampened effect in robo2 mutants. Our results link Slit3-Robo2-Cdc42, which controls local actin dynamics, with the maintenance of synaptic neuropeptide levels.
Farack L., Golan M., Egozi A., Dezorella N., Halpern K. B., Ben-Moshe S., Garzilli I., Toth B., Roitman L., Krizhanovsky V. & Itzkovitz S. (2019) Developmental Cell. 48, 1, p. 115-125
Pancreatic beta cells have been shown to be heterogeneous at multiple levels. However, spatially interrogating transcriptional heterogeneity in the intact tissue has been challenging. Here, we developed an optimized protocol for single-molecule transcript imaging in the intact pancreas and used it to identify a sub-population of "extreme'' beta cells with elevated mRNA levels of insulin and other secretory genes. Extreme beta cells contain higher ribosomal and proinsulin content but lower levels of insulin protein in fasted states, suggesting they may be tuned for basal insulin secretion. They exhibit a distinctive intra-cellular polarization pattern, with elevated mRNA concentrations in an apical ER-enriched compartment, distinct from the localization of nascent and mature proteins. The proportion of extreme cells increases in db/db diabetic mice, potentially facilitating the required increase in basal insulin. Our results thus highlight a sub-population of beta cells that may carry distinct functional roles along physiological and pathological timescales.
Lonetto G., Koifman G., Silberman A., Attery A., Solomon H., Levin-Zaidman S., Goldfinger N., Porat Z., Erez A. & Rotter V. (2019) Cell Death and Differentiation. 26, 9, p. 1566-1581
It is well accepted that malignant transformation is associated with unique metabolism. Malignant transformation involves a variety of cellular pathways that are associated with initiation and progression of the malignant process that remain to be deciphered still. Here we used a mouse model of mutant p53 that presents a stepwise progressive transformation of adult Mesenchymal Stem Cells (MSCs). While the established parental p53Mut-MSCs induce tumors, the parental p53WT-MSCs that were established in parallel, did not. Furthermore, tumor lines derived from the parental p53Mut-MSCs (p53Mut-MSC-TLs), exhibited yet a more aggressive transformed phenotype, suggesting exacerbation in tumorigenesis. Metabolic tracing of these various cell types, indicated that while malignant transformation is echoed by a direct augmentation in glycolysis, the more aggressive p53Mut-MSC-TLs demonstrate increased mitochondrial oxidation that correlates with morphological changes in mitochondria mass and function. Finally, we show that these changes are p53Mut-dependent. Computational transcriptional analysis identified a mitochondrial gene signature specifically downregulated upon knock/out of p53Mut in MSC-TLs. Our results suggest that stem cells exhibiting different state of malignancy are also associated with a different quantitative and qualitative metabolic profile in a p53Mut-dependent manner. This may provide important insights for cancer prognosis and the use of specific metabolic inhibitors in a personalized designed cancer therapy.
Tamary E., Nevo R., Naveh L., Levin-Zaidman S., Kiss V., Savidor A., Levin Y., Eyal Y., Reich Z. & Adam Z. (2019) Plant Direct. 3, 3, 127
The earliest visual changes of leaf senescence occur in the chloroplast as chlorophyll is degraded and photosynthesis declines. Yet, a comprehensive understanding of the sequence of catabolic events occurring in chloroplasts during natural leaf senescence is still missing. Here, we combined confocal and electron microscopy together with proteomics and biochemistry to follow structural and molecular changes during Arabidopsis leaf senescence. We observed that initiation of chlorophyll catabolism precedes other breakdown processes. Chloroplast size, stacking of thylakoids, and efficiency of PSII remain stable until late stages of senescence, whereas the number and size of plastoglobules increase. Unlike catabolic enzymes, whose level increase, the level of most proteins decreases during senescence, and chloroplast proteins are overrepresented among these. However, the rate of their disappearance is variable, mostly uncoordinated and independent of their inherent stability during earlier developmental stages. Unexpectedly, degradation of chlorophyll-binding proteins lags behind chlorophyll catabolism. Autophagy and vacuole proteins are retained at relatively high levels, highlighting the role of extra-plastidic degradation processes especially in late stages of senescence. The observation that chlorophyll catabolism precedes all other catabolic events may suggest that this process enables or signals further catabolic processes in chloroplasts.
Balgley R., Rechav K., Lahav M. & van der Boom M. E. (2019) ChemistrySelect. 4, 41, p. 12104-12110
Flower-like gold microstructures are directly formed from solution on the surface of nanometric molecular assemblies. We show that the size and morphology of these microstructures are controlled by the nanoscale thickness of the assemblies, which consist of ruthenium polypyridyl complexes crosslinked with a palladium salt. Gold electrodeposition on these ultrathin (3-15 nm) molecular assemblies, bound to a conductive substrate, follows an instantaneous nucleation regime that results in multiple small clusters. On thicker assemblies (15-55 nm) a progressive nucleation mode is dominant, which leads to the formation of larger (up to 50 times) and highly branched microstructures. The ability to control the characteristics of these microstructures by nanoscale assemblies is based on the mechanistic insights of the nucleation and growth processes obtained by electrochemical means and scanning electron microscopy (SEM) measurement.
Elazar N., Vainshtein A., Rechav K., Tsoory M., Eshed-Eisenbach Y. & Peles E. (2019) Journal of Cell Biology. 218, 9, p. 2887-2895
Oligodendrocyte-axon contact is mediated by several cell adhesion molecules (CAMs) that are positioned at distinct sites along the myelin unit, yet their role during myelination remains unclear. Cadm4 and its axonal receptors, Cadm2 and Cadm3, as well as myelin-associated glycoprotein (MAG), are enriched at the internodes below the compact myelin, whereas NF155, which binds the axonal Caspr/contactin complex, is located at the paranodal junction that is formed between the axon and the terminal loops of the myelin sheath. Here we report that Cadm4-, MAG-, and Caspr-mediated adhesion cooperate during myelin membrane ensheathment. Genetic deletion of either Cadm4 and MAG or Cadm4 and Caspr resulted in the formation of multimyelinated axons due to overgrowth of the myelin away from the axon and the forming paranodal junction. Consequently, these mice displayed paranodal loops either above or underneath compact myelin. Our results demonstrate that accurate placement of the myelin sheath by oligodendrocytes requires the coordinated action of internodal and paranodal CAMs.
Maria R., Ben-Zvi Y., Rechav K., Klein E., Shahar R. & Weiner S. (2019) Journal of Structural Biology. 206, 1, p. 128-137
Teeth are subjected to compressive loads during mastication. Under small loads the soft tissue periodontal ligament (PDL) deforms most. However when the loads increase and the PDL is highly compressed, the tooth and the alveolar bone supporting the tooth, begin to deform. Here we report on the structure of this alveolar bone in the upper furcation region of the first molars of mature minipigs. Using light microscopy and scanning electron microscopy (SEM) of bone cross-sections, we show that this bone is hypermineralized, containing abundant small pores around 1-5 mu m in diameter, lacunae around 10-20 mu m as well as larger spaces. This bone does not possess the typical lamellar motif or other repeating structures normally found in cortical or trabecular mammalian bone. We also use high resolution focused ion beam scanning electron microscopy (FIB-SEM) in the serial surface mode to image the 3D organization of the demineralized bone matrix. We show that the upper furcation bone matrix has a disordered isotropic structure composed mainly of individual collagen fibrils with no preferred orientation, as well as highly staining material that is probably proteoglycans. Much larger aligned arrays of collagen fibers - presumably Sharpey's fibers - are embedded in this material. This unusual furcation bone material is similar to the disordered material found in human lamellar bone. In the upper furcation region this disordered bone comprises almost all the volume excluding Sharpey's fibers. We surmise that this most unusual bone type functions to resist the repeating compressive loads incurred by molars during mastication.
Weiner A., Orange F., Lacas-Gervais S., Rechav K., Ghugtyal V., Bassilana M. & Arkowitz R. A. (2019) Cellular Microbiology. 21, 1, 12963
Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip-growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long-range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three-dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub-apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkorper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short-range "on-site" secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.
Nissan H., Blum S., Shimoni E. & Elbaum R. (2019) Silicon. 11, 5, p. 2377-2383
Purpose - Silicon (Si) is an abundant element in the earth's crust and is available to plants as silicic acid. Silicon uptake by plants is correlated with increased tolerance to various biotic and abiotic stresses. However, cellular mechanisms responsible for its beneficial effects are still unknown. Even its cellular import mechanisms are not well understood. We thus aimed to characterize silicon localization within minimally differentiated Zea mays (Black Mexican Sweet) cells in suspension. Methods - Cells were grown in a medium containing silicon, and the mRNA levels of silicon transporters were measured by real-time PCR. Cells were separated into an insoluble (mainly walls and starch) and a cytoplasmic fraction. Soluble and total silicon was measured by inductively-coupled-plasma - atomic-emission-spectroscopy. Silicon distribution was assessed by transmission electron microscopy. The cell walls were analyzed chemically, and by Raman micro-spectroscopy and thermal gravimetric analysis. Results - Silicon treatment reduced the levels of silicon transporters transcripts, without affecting cell proliferation. About 70 % of the silicon was localized in the cytoplasm, mostly in vesicles. We found indications that silicon affected the secondary structure of proteins and thermally stabilized starch. Silicon was loosely bound, and diffused out of the cells within 24 hours. Conclusions - Our results show that silicon binds spontaneously to cell walls/starch and accumulates in cytoplasm vesicles. These processes allow the cells to accumulate silicon against its concentration gradient in solution. However, cellular intake acts against reversible diffusion processes, probably through the aquaporin silicon channels (Lsi1, Lsi6) that exchange the cellular silicon with the surrounding medium.
Gordon L., Blechman J., Shimoni E., Gur D., Anand-Apte B. & Levkowitz G. (2019) Development. 146, 23, dev177790
To maintain body homeostasis, endocrine systems must detect and integrate blood-borne peripheral signals. This is mediated by fenestrae, specialized permeable pores in the endothelial membrane. Plasmalemma vesicle-associated protein (Plvap) is located in the fenestral diaphragm and is thought to play a role in the passage of proteins through the fenestrae. However, this suggested function has yet to be demonstrated directly. We studied the development of fenestrated capillaries in the hypophysis, a major neuroendocrine interface between the blood and brain. Using a transgenic biosensor to visualize the vascular excretion of the genetically tagged plasma protein DBP-EGFP, we show that the developmental acquisition of vascular permeability coincides with differential expression of zebrafish plvap orthologs in the hypophysis versus brain. Ultrastructural analysis revealed that plvapb mutants display deficiencies in fenestral diaphragms and increased density of hypophyseal fenestrae. Measurements of DBP-EGFP extravasation in plvapb mutants provided direct proof that Plvap limits the rate of blood-borne protein passage through fenestrated endothelia. We present the regulatory role of Plvap in the development of blood-borne protein detection machinery at a neuroendocrine interface through which hormones are released to the general circulation.
Bussi Y., Shimoni E., Weiner A., Kapon R., Charuvi D., Nevo R., Efrati E. & Reich Z. (2019) Proceedings of the National Academy of Sciences of the United States of America. 116, 44, p. 22366-22375
Plant photosynthetic (thylakoid) membranes are organized into complex networks that are differentiated into 2 distinct morphological and functional domains called grana and stroma lamellae. How the 2 domains join to form a continuous lamellar system has been the subject of numerous studies since the mid-1950s. Using different electron tomography techniques, we found that the grana and stroma lamellae are connected by an array of pitch-balanced right- and left-handed helical membrane surfaces of different radii and pitch. Consistent with theoretical predictions, this arrangement is shown to minimize the surface and bending energies of the membranes. Related configurations were proposed to be present in the rough endoplasmic reticulum and in dense nuclear matter phases theorized to exist in neutron star crusts, where the right- and left-handed helical elements differ only in their handedness. Pitch-balanced helical elements of alternating handedness may thus constitute a fundamental geometry for the efficient packing of connected layers or sheets.
Charuvi D., Nevo R., Aviv-Sharon E., Gal A., Kiss V., Shimoni E., Farrant J. M., Kirchhoff H. & Reich Z. (2019) Environmental and Experimental Botany. 157, p. 100-111
The vegetative tissues of resurrection plants are able to withstand severe protoplasmic dehydration and revive quickly upon rehydration. Resurrection species defined as homoiochlorophyllous retain most or part of their chlorophyll and photosynthetic complement in the dry state, and rely on various mechanisms to protect themselves against photo-damage. In this study, we investigated the changes in chlorophyll distribution, light absorption gradients as well as the alterations in ultrastructure that take place during dehydration of the homoiochlorophyllous species Craterostigma pumilum. Chlorophyll fluorescence profiles show that light absorption is attenuated in dry leaves, likely minimizing generation of reactive oxygen species. These are accompanied by changes that take place in the supramolecular organization of the photosynthetic protein complexes, and ordered functional adjustments of the photosynthetic apparatus, further lessening the excitation and electron pressures. Albeit these, the ultrastructural studies reveal that chloroplasts in dehydrated leaf tissues exhibit features indicative of oxidative stress, which are also reminiscent of senescing chloroplasts. These include mass proliferation of plastoglobules, variable degrees of thylakoid dismantling, as well as chloroplast fragmentation and seemingly vacuolar degradation of such fragments. In addition, unique vesicular structures between the two chloroplast envelope membranes were visualized, some of which appeared to detach from chloroplasts, likely en route to degradation. Together, the data indicate that homoiochlorophyllous resurrection species handle photo-induced damage during dehydration on two levels. Minimization of photo-damage is achieved by attenuation of light absorption and other photo-protective mechanisms. When this is insufficient and significant damage does occur, elimination of damaged components takes place via processes resembling senescence. Nevertheless, these processes are reversible, enabling the plants to avoid the terminal steps of senescence and, hence, to recover.
Toffolo M. B., Ricci G., Caneve L. & Kaplan-Ashiri I. (2019) Scientific Reports. 9, 16170
In nature, calcium carbonate (CaCO3) in the form of calcite and aragonite nucleates through different pathways including geogenic and biogenic processes. It may also occur as pyrogenic lime plaster and laboratory-precipitated crystals. All of these formation processes are conducive to different degrees of local structural order in CaCO3 crystals, with the pyrogenic and precipitated forms being the least ordered. These variations affect the manner in which crystals interact with electromagnetic radiation, and thus formation processes may be tracked using methods such as X-ray diffraction and infrared spectroscopy. Here we show that defects in the crystal structure of CaCO3 may be detected by looking at the luminescence of crystals. Using cathodoluminescence by scanning electron microscopy (SEM-CL) and laser-induced fluorescence (LIF), it is possible to discern different polymorphs and their mechanism of formation. We were thus able to determine that pyrogenic calcite and aragonite exhibit blue luminescence due to the incorporation of distortions in the crystal lattice caused by heat and rapid precipitation, in agreement with infrared spectroscopy assessments of local structural order. These results provide the first detailed reference database of SEM-CL and LIF spectra of CaCO3 standards, and find application in the characterization of optical, archaeological and construction materials.
Niazov-Elkan A., Sui X., Kaplan-Ashiri I., Shimon L. J. W., Leitus G., Cohen E., Weissman H., Wagner H. D. & Rybtchinski B. (2019) ACS Nano. 13, 10, p. 11097-11106
In view of their facile fabrication and recycling, functional materials that are built from small molecules ("molecular plastics") may represent a cost-efficient and sustainable alternative to conventional covalent materials. We show how molecular plastics can be made robust and how their (nano)structure can be tuned via modular construction. For this purpose, we employed binary composites of organic nanocrystals based on a perylene diimide derivative, with graphene oxide (GO), bentonite nanoclay (NC), or hydroxyethyl cellulose (HEC), that both reinforce and enable tailoring the properties of the membranes. The hybrids are prepared via a simple aqueous deposition method, exhibit enhanced mechanical robustness, and can be recycled. We utilized these properties to create separation membranes with tunable porosity that are easy to fabricate and recycle. Hybrids 1/HEC and 1/NC are capable of ultrafiltration, and 1/NC removes heavy metals from water with high efficiency. Hybrid 1/GO shows mechanical properties akin to covalent materials with just 2-10% (by weight) of GO. This hybrid was used as a membrane for immobilizing β-galactosidase that demonstrated long and stable biocatalytic activity. Our findings demonstrate the utility of modular molecular nanoplastics as robust and sustainable materials that enable efficient tuning of structure and function and are based on self-assembly of readily available inexpensive components.
Zhang Q., Pardo M., Rudich Y., Kaplan-Ashiri I., Wong J. P. S., Davis A. Y., Black M. S. & Weber R. J. (2019) Environmental Science & Technology. 53, 20, p. 12054-12061
Consumer-level 3D printers emit ultrafine and fine particles, though little is known about their chemical composition or potential toxicity. We report chemical characteristics of the particles in comparison to raw filaments and assessments of particle toxicity. Particles emitted from polylactic acid (PLA) appeared to be largely composed of the bulk filament material with mass spectra similar to the PLA monomer spectra. Acrylonitrile butadiene styrene (ABS), extruded at a higher temperature than PLA, emitted vastly more particles and their composition differed from that of the bulk filament, suggesting that trace additives may control particle formation. In vitro cellular assays and in vivo mice exposure all showed toxic responses when exposed to PLA and ABS-emitted particles, where PLA-emitted particles elicited higher response levels than ABS-emitted particles at comparable mass doses. A chemical assay widely used in ambient air-quality studies showed that particles from various filament materials had comparable particle oxidative potentials, slightly lower than those of ambient particulate matter (PM2.5). However, particle emissions from ABS filaments are likely more detrimental when considering overall exposure due to much higher emissions. Our results suggest that 3D printer particle emissions are not benign and exposures should be minimized.
Reicher N., Budke C., Eickhoff L., Raveh-Rubin S., Kaplan-Ashiri I., Koop T. & Rudich Y. (2019) Atmospheric Chemistry and Physics. 19, 17, p. 11143-11158
The prediction of cloud ice formation in climate models remains a challenge, partly due to the complexity of ice-related processes. Mineral dust is a prominent aerosol in the troposphere and is an important contributor to ice nucleation in mixed-phase clouds, as dust can initiate ice heterogeneously at relatively low supercooling conditions. We characterized the ice nucleation properties of size-segregated mineral dust sampled during dust events in the eastern Mediterranean. The sampling site allowed us to compare the properties of airborne dust from several sources with diverse mineralogy that passed over different atmospheric paths. We focused on particles with six size classes determined by the Micro-Orifice Uniform Deposit Impactor (MOUDI) cutoff sizes: 5.6, 3.2, 1.8, 1.0, 0.6 and 0.3 μm. Ice nucleation experiments were conducted in the Weizmann Supercooled Droplets Observation on a Microarray (WISDOM) setup, whereby the particles are immersed in nanoliter droplets using a microfluidics technique. We observed that the activity of airborne particles depended on their size class; supermicron and submicron particles had different activities, possibly due to different composition. The concentrations of ice-nucleating particles and the density of active sites (n<sub>s</sub>) increased with the particle size and particle concentration. The supermicron particles in different dust events showed similar activity, which may indicate that freezing was dominated by common mineralogical components. Combining recent data of airborne mineral dust, we show that current predictions, which are based on surface-sampled natural dust or standard mineral dust, overestimate the activity of airborne dust, especially for the submicron class. Therefore, we suggest including information on particle size in order to increase the accuracy of ice formation modeling and thus weather and climate predictions.
Bronshtein I. A., Weissman H., Kaplan-Ashiri I. & Rybtchinski B. (2019) Small. 15, 38, 1902936
The widely employed crystallization of organic molecules in solution is not well understood and is difficult to control. Employing polymers as crystallization media may allow enhanced control via temperature-induced regulation of polymer dynamics. Crystallization of a small organic molecule (perylene diimide) is investigated in polymer matrices (polystyrene) that enable the mechanistic study and control over order evolution. The crystallization is induced by heating above the glass transition temperature of the polymer, and quenched by cooling, leading to stabilization of crystallization intermediates. The mechanistic studies include direct imaging by electron microscopy, revealing a complex self-assembly process starting from amorphous aggregates that densify and transform into an unstable crystalline phase of N,N '-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide (DMP-PDI), followed by a conversion into a more stable crystalline form. Stabilization of crystallization intermediates at room temperature provides diverse structures based on a single molecular component. These findings have implications for the rational design of organic crystalline materials.
Electron cryo-tomography using the scanning transmission modality (STEM)enables 3D reconstruction of unstained, vitrified specimens as thick as 1 μm or more. Contrast is related to mass/thickness and atomic number, providing quantifiable chemical characterization and mass mapping of intact prokaryotic and eukaryotic cells. Energy dispersive X-ray spectroscopy by STEM provides a simple, on-the-spot chemical identification of the elemental composition in sub-cellular organic bodies or mineral deposits. This chapter provides basic background and practical information for performing cryo-STEM tomography on vitrified biological cells.
Baron S., Peleg Y., Grunwald J., Morgenstern D., Elad N., Peretz M., Albeck S., Levin Y., Welch J. T., DeWeerd K. A., Schwarz A., Burstein Y., Diskin R., Shakked Z. & Zimhony O. (2018) PLoS ONE. 9, e0204457
Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.
Tuberculosis (TB) is a devastating and rapidly spreading disease caused by Mycobacterium tuberculosis (Mtb). Therapy requires prolonged treatment with a combination of multiple agents and interruptions in the treatment regimen result in emergence and spread of multidrug resistant (MDR) Mtb strains. MDR Mtb poses a significant global health problem, calling for urgent development of novel drugs to combat TB. Here, we report the 3.3 angstrom resolution structure of the similar to 2 MDa type-I fatty acid synthase (FAS-I) from Mtb, determined by single particle cryo-EM. Mtb FAS-I is an essential enzymatic complex that contributes to the virulence of Mtb, and thus a prime target for anti-TB drugs. The structural information for Mtb FAS-I we have obtained enables computer-based drug discovery approaches, and the resolution achieved by cryo-EM is sufficient for elucidating inhibition mechanisms by putative small molecular weight inhibitors.
Varsano N., Beghi F., Elad N., Pereiro E., Dadosh T., Pinkas I., Perez-Berna A. J., Jin X., Kruth H. S., Leiserowitz L. & Addadi L. (2018) Proceedings Of The National Academy Of Sciences Of The United States Of America-Biological Sciences. 115, 30, p. 7662-7669
The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rodlike crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.
Stolovas D., Serra M., Popovitz-Biro R., Pinkas I., Houben L., Calvino J. J., Joselevich E., Tenne R., Arenal R. & Lajaunie L. (2018) Chemistry of Materials. 30, 24, p. 8829-8842
Misfit layered compounds (MLC) with the composition (LaS)(1.15)TaS2 (for simplicity denoted as LaS-TaS2) and LaS-NbS2 were prepared and studied in the past. Nanotubes of LaS-TaS2 could be easily synthesized, while tubular structure of the LaS-NbS2 were found to be rather rare in the product. To understand this riddle, quaternary alloys of LaS-NbxTa(1-x)S2 with ascending Nb concentration were prepared herein in the form of nanotubes (and platelets). Not surprisingly, the concentration of these quaternary nanotubes shrank (and the relative density of platelets increased) with increasing Nb content in the precursor. The structure and chemical composition of such nanotubes was elucidated by electron microscopy. Conceivably, the TaS2 in the MLC compounds LnS-TaS2 (Ln = lanthanide atom) crystallizes in the 2H polytype. High resolution transmission electron microscopy showed however that, invariably, MLC nanotubes prepared from 80 at% Nb content in the precursor belonged to the 1T polytype. Raman spectroscopy of individual tubes revealed that up to 60 at% Nb, they obey the standard model of MLC, while higher Nb lead to large deviations, which are discussed in brief. The analysis indicated also that such nanotubes do not exhibit the pattern assigned to charge density wave transition so typical for binary 1T-TaS2. The prospect for revealing interesting quasi-1D behavior of such quaternary nanotubes is also briefly discussed.
Stern C., Grinvald S., Kirshner M., Sinai O., Oksman M., Alon H., Meiron O. E., Bar-Sadan M., Houben L. & Naveh D. (2018) Scientific Reports. 8, 16480
Thin films of layered semiconductors emerge as highly promising materials for energy harvesting and storage, optoelectronics and catalysis. Their natural propensity to grow as oriented crystals and films is one of their distinct properties under recent focal interest. Specifically, the reaction of transition metal films with chalcogen vapor can result in films of vertically aligned (VA) layers, while metal-oxides react with chalcogens in vapor phase to produce horizontally aligned crystals and films. The growth mechanisms of vertically oriented films are not yet fully understood, as well as their dependence on the initial metal film thickness and growth conditions. Moreover, the resulting electronic properties and the role of defects and disorder had not yet been studied, despite their critical influence on catalytic and device performance. In this work, we study the details of oriented growth of MoS2 with complementary theoretical and experimental approaches. We present a general theoretical model of diffusion-reaction growth that can be applied to a large variety of layered materials synthesized by solid-vapor reaction. Moreover, we inspect the relation of electronic properties to the structure of vertically aligned MoS2 and shed light on the density and character of defects in this material. Our measurements on Si-MoS2 p-n hetero-junction devices point to the existence of polarizable defects that impact applications of vertical transition-metal dichalcogenide materials.
Chakrabarty T., Goldin N., Feintuch A., Houben L. & Leskes M. (2018) ChemPhysChem. 19, 17, p. 2139-2142
Dynamic nuclear polarization (DNP), a technique in which the high electron spin polarization is transferred to surrounding nuclei via microwave irradiation, equips solid-state NMR spectroscopy with unprecedented sensitivity. The most commonly used polarization agents for DNP are nitroxide radicals. However, their applicability to inorganic materials is mostly limited to surface detection. Paramagnetic metal ions were recently introduced as alternatives for nitroxides. Doping inorganic solids with paramagnetic ions can be used to tune material properties and introduces endogenous DNP agents that can potentially provide sensitivity in the particles' bulk and surface. Here we demonstrate the approach by doping Li4Ti5O12 (LTO), an anode material for lithium ion batteries, with paramagnetic ions. By incorporating Gd(III) and Mn(II) in LTO we gain up to 14 fold increase in signal intensity in static Li-7 DNP-NMR experiments. These results suggest that doping with paramagnetic ions provides an efficient route for sensitivity enhancement in the bulk of micron size particles.
di Gregorio M. C., Ranjan P., Houben L., Shimon L. J. W., Rechav K., Lahav M. & van der Boom M. E. (2018) Journal of the American Chemical Society. 140, 29, p. 9132-9139
In this work, we report the formation of superstructures assembled from organic tubular crystals mediated by metal-coordination chemistry. This template-free process involves the crystallization of molecules into crystals having a rectangular and uniform morphology, which then go on to fuse together into multibranched superstructures. The initially hollow and organic crystals are obtained under solvothermal conditions in the presence of a copper salt, whereas the superstructures are subsequently formed by aging the reaction mixture at room temperature. The mild thermodynamic conditions and the favorable kinetics of this unique self-assembly process allowed us to ex-situ monitor the superstructure formation by electron microscopy, highlighting a pivotal and unusual role for copper ions in their formation and stabilization.
Serra M., Stolovas D., Houben L., Popovitz-Biro R., Pinkas I., Kampmann F., Maultzsch J., Joselevich E. & Tenne R. (2018) Chemistry-A European Journal. 24, 44, p. 11354-11363
The synthesis and characterization of nanotubes from misfit layered compounds (MLCs) of the type (LnS)(1+y)TaS2 (denoted here as LnS-TaS2; Ln=Pr, Sm, Gd, and Yb), not reported before, are described (the bulk compound YbS-LaS2 was not previously documented). Transmission electron microscopy and selected area electron diffraction showed that the interlayer spacing along the c axis decreased with an increase in the atomic number of the lanthanide atom, which suggested tighter interaction between the LnS layer and TaS2 for the late lanthanides. The Raman spectra of the tubules were studied and compared to those of the bulk MLC compounds. Similar to the bulk MLCs, the Raman spectra could be divided into the low-frequency modes (110-150cm(-1)) of the LnS lattice and the high-frequency modes (250-400cm(-1)) of the TaS2 lattice. The Raman spectra indicated that the vibrational lattice modes of the strained layers in the tubes were stiffer than those in the bulk compounds. Furthermore, the modes of the late lanthanides were higher in energy than those of the earlier lanthanides, which suggested larger charge transfer between the LnS and TaS2 layers for the late lanthanides. Polarized Raman measurements showed the expected binodal intensity profile (antenna effect). The intensity ratio of the Raman signal showed that the E-2g mode of TaS2 was more sensitive to the light-polarization effect than its A(1g) mode. These nanotubes are expected to reveal interesting low-temperature quasi-1D transport behavior.
Ranjan P., Kaplan-Ashiri I., Popovitz-Biro R., Cohen S. R., Houben L., Tenne R., Lahav M. & van der Boom M. E. (2018) ACS Omega. 3, 6, p. 6533-6539
In this paper, we demonstrate the formation of hybrid nanostructures consisting of two distinctive components mainly in a one-to-one ratio. Thermolysis of inorganic nanotubes (INT) and closed-cage, inorganic fullerene-like (IF) nanoparticles decorated with a dense coating of metallic nanoparticles (M = Au, Ag, Pd) results in migration of relatively small NPs or surface-enhanced diffusion of atoms or clusters, generating larger particles (ripening). AuNP growth on the surface of INTs has been captured in real time using in situ electron microscopy measurements. Reaction of the AuNP-decorated INTs with an alkylthiol results in a chemically induced NP fusion process at room temperature. The NPs do not dissociate from the surfaces of the INTs and IFs, but for proximate IFs we observed fusion between AuNPs originating from different IFs.
Rothman A., Forsht T., Danieli Y., Popovitz-Biro R., Rechav K., Houben L. & Joselevich E. (2018) Journal of Physical Chemistry C. 122, 23, p. 12413-12420
The surface-guided growth of horizontal nanowires (NWs) allows assembly and alignment of the NWs on the substrate during the synthesis, thus eliminating the need for additional processes after growth. One of the major advantages of guided growth over postgrowth assembly is the control on the NWs direction, crystallographic orientation, and position. In this study, we use the guided growth approach to synthesize high-quality, single-crystal, aligned horizontal ZnS NWs on flat and faceted sapphire surfaces, and show how the crystal planes of the different substrates affects the crystal structure and orientation of the NWs. We also show initial results of the effect of Cu doping on their photoluminescence. Such high-quality aligned ZnS NWs can potentially be assembled as key components in phosphorescent displays and markers due to their unique optical properties. The ZnS NWs have either wurtzite or zinc-blende structure depending on the substrate orientations and contain intrinsic point defects such as sulfur vacancies, which are common in this material. The crystallographic orientations are consistent with those of guided NWs from other semiconductor materials, demonstrating the generality of the guided growth phenomenon. The successfully grown ZnS NWs and the Cu doping are the first step toward the fabrication of optoelectronic devices based on ZnS nanostructures.
Chen X., Chen C., Levi A., Houben L., Deng B., Yuan S., Ma C., Watanabe K., Taniguchi T., Naveh D., Du X. & Xia F. (2018) ACS Nano. 12, 5, p. 5003-5010
A high saturation velocity semiconductor is appealing for applications in electronics and optoelectronics. Thin-film black phosphorus (BP), an emerging layered semiconductor, shows a high carrier mobility and strong mid-infrared photoresponse at room temperature. Here, we report the observation of high intrinsic saturation velocity in 7 to 11 rim thick BP for both electrons and holes as a function of charge-carrier density, temperature, and crystalline direction. We distinguish a drift velocity transition point due to the competition between the electron-impurity and electron phonon scatterings. We further achieve a room-temperature saturation velocity of 1.2 (1.0) X 10(7) cm s(-1) for hole (electron) carriers at a critical electric field of 14 (13) kV cm(-1), indicating current-gain cutoff frequency similar to 20 GHz center dot mu m for radio frequency applications. Moreover, the current density is as high as 580 mu A mu m(-1) at a low electric field of 10 kV cm(-1). Our studies demonstrate that thin-film BP outperforms silicon in terms of saturation velocity and critical field, revealing its great potential in radio-frequency electronics, high-speed mid-infrared photodetectors, and optical modulators.
Udayabhaskararao T., Houben L., Cohen H., Menahem M., Pinkas I., Avram L., Wolf T., Teitelboim A., Leskes M., Yaffe O., Oron D. & Kazes M. (2018) Chemistry of Materials. 30, 1, p. 84-93
Active control over the shape, composition, and crystalline habit of nanocrystals has long been a goal. Various methods have been shown to enable postsynthesis modification of nanoparticles, including the use of the Kirkendall effect, galvanic replacement, and cation or anion exchange, all taking advantage of enhanced solid-state diffusion on the nanoscale. In all these processes, however, alteration of the nanoparticles requires introduction of new precursor materials. Here we show that for cesium lead halide perovskite nanoparticles, a reversible structural and compositional change can be induced at room temperature solely by modification of the ligand shell composition in solution. The reversible transformation of cubic CsPbX3 nanocrystals to rhombohedral Cs4PbX6 nanocrystals is achieved by controlling the ratio of oleylamine to oleic acid capping molecules. High-resolution transmission electron microscopy investigation of Cs4PbX6 reveals the growth habit of the rhombohedral crystal structure is composed of a zero-dimensional layered network of isolated PbX6 octahedra separated by Cs cation planes. The reversible transformation between the two phases involves an exfoliation and recrystalliztion process. This scheme enables fabrication of high-purity monodispersed Cs4PbX6 nanoparticles with controlled sizes. Also, depending on the final size of the Cs4PbX6 nanoparticles as tuned by the reaction time, the back reaction yields CsPbX3 nanoplatelets with a controlled thickness. In addition, detailed surface analysis provides insight into the impact of the ligand composition on surface stabilization that, consecutively, acts as the driving force in phase and shape transformations in cesium lead halide perovskites.
Oksenberg E., Sanders E., Popovitz-Biro R., Houben L. & Joselevich E. (2018) Nano Letters. 18, 1, p. 424-433
All-inorganic lead halide perovskite nanowires have been the focus of increasing interest since they exhibit improved stability compared to their hybrid organic inorganic counterparts, while retaining their interesting optical and optoelectronic properties. Arrays of surface-guided nanowires with controlled orientations and morphology are promising as building blocks for various applications and for systematic research. We report the horizontal and aligned growth of CsPbBr3 nanowires with a uniform crystallographic orientation on flat and faceted sapphire surfaces to form arrays with 6-fold and 2-fold symmetries, respectively, along specific directions of the sapphire substrate. We observed waveguiding behavior and diameter-dependent photoluminescence emission well beyond the quantum confinement regime. The arrays were easily integrated into multiple devices, displaying p-type behavior and photoconductivity. Photodetectors based on those nanowires exhibit the fastest rise and decay times for any CsPbBr3-based photodetectors reported so far. One-dimensional arrays of halide perovskite nanowires are a promising platform for investigating the intriguing properties and potential applications of these unique materials.
Wolf S. G., Shimoni E., Elbaum M. & Houben L. (2018) . p. 33-60
STEM modality provides major advantages for electron tomography of thicker (>300 nm) biological specimens, both for plastic-embedded, heavy-metal stained samples, and for vitrified, unstained cells. With the proliferation of modern TEM microscopes that allow for switching between TEM and STEM modes with relative ease, we expect the use of STEM tomography to increase. The concepts for STEM imaging are significantly different than for TEM, and therefore we will describe in detail the STEM imaging modality, followed by STEM tomography concepts and applications.
Winer H., Fraiberg M., Abada A., Dadosh T., Tamim-Yecheskel B. & Elazar Z. (2018) Nature Communications. 9, 3744
Autophagy, a conserved membrane trafficking process, sequesters cytoplasmic components into autophagosomes and targets them for lysosomal degradation. The TNF receptor Fn14 participates in multiple intracellular signaling pathways and is strongly induced upon tissue injury and solid tumorigenesis. While Fn14 is a short-lived protein, the regulation of its levels is largely obscure. Here we uncover a role for autophagy in Fn14 turnover, wherein specific core autophagy Atg8 proteins play distinct roles: Fn14 accumulates in the ERGIC in absence of GABARAP but within endosomes in the vicinity of autophagic membranes in absence of GATE-16. Moreover, GABARAP regulates overall cellular levels of Fn14, whereas GATE-16 regulates TWEAK signaling by Fn14 and thereby NF-κB activity. These findings not only implicate different Atg8 proteins in distinct roles within the mechanism of selective autophagic regulation of Fn14, but may also provide a more general view of their role in mediating autophagosome biogenesis from different membrane sources.
Goliand I., Adar-Levor S., Segal I., Nachmias D., Dadosh T., Kozlov M. M. & Elia N. (2018) Cell Reports. 24, 7, p. 1756-1764
The ESCRT machinery mediates membrane fission in a variety of processes in cells. According to current models, ESCRT-III proteins drive membrane fission by assembling into helical filaments on membranes. Here, we used 3D STORM imaging of endogenous ESCRT-III component IST1 to reveal the evolution of the structural organization of ESCRT-III in mammalian cytokinetic abscission. Using this approach, ESCRT-III ring and spiral assemblies were resolved and characterized at different stages of abscission. Visualization of IST1 structures in cells lacking the microtubule-severing enzyme spastin and in cells depleted of specific ESCRT-III components or the ATPase VPS4 demonstrated the contribution of these components to the organization and function of ESCRTs in cells. This work provides direct evidence that ESCRT-III proteins form helical filaments to mediate their function in cells and raises new mechanistic scenarios for ESCRT-driven cytokinetic abscission.
Ranjan P., Shankar S., Popovitz-Biro R., Cohen S. R., Kaplan-Ashiri I., Dadosh T., Shimon L. J. W., Visic B., Tenne R., Lahav M. & van der Boom M. E. (2018) Journal of Physical Chemistry C. 122, 12, p. 6748-6759
We report here a unique and efficient methodology for the surface functionalization of closed-cage inorganic fullerene-like (IF) nanoparticles and inorganic nanotubes (INTs) composed of two-dimensional nanomaterials of transition-metal chalcogenides (MS
<sub>2</sub>; M = W or Mo). The first step is the physical coverage of these robust inorganic materials with monodispersed and dense monolayers of gold, silver, and palladium nanoparticles. The structural continuity at the interface between the IF/INT and the metallic nanoparticles is investigated. Lattice matching between these nanocrystalline materials and strong chemical affinity lead to efficient binding of the metallic nanoparticles onto the outer sulfide layer of the MS
<sub>2</sub>-based structures. It is shown that this functionalization results in narrowing of the IF/INT optical band gap, increased work function, and improved surface-enhanced Raman scattering. In the second step, functionalization of the surface-bound nanoparticles is carried out by a ligand-exchange reaction. This ligand exchange involving the tetraoctylammonium bromide capping layer and an alkyl thiol enhances the solubility (∼10×) of the otherwise nearly insoluble materials in organic solvents. The scope of this method is further demonstrated by introducing a ruthenium(II) polypyridyl complex on the surface of the surface-bound AuNPs to generate fluorescent multicomponent materials.
Artzi L., Dadosh T., Milrot E., Morais S., Levin-Zaidman S., Morag E. & Bayer E. A. (2018) mBio. 9, 1, 00012-18
Cellulosomes are multienzyme complexes produced by anaerobic, cellulolytic bacteria for highly efficient breakdown of plant cell wall polysaccharides. Clostridium clariflavum is an anaerobic, thermophilic bacterium that produces the largest assembled cellulosome complex in nature to date, comprising three types of scaffoldins: a primary scaffoldin, ScaA; an adaptor scaffoldin, ScaB; and a cell surface anchoring scaffoldin, ScaC. This complex can contain 160 polysaccharide-degrading enzymes. In previous studies, we proposed potential types of cellulosome assemblies in C. clariflavum and demonstrated that these complexes are released into the extracellular medium. In the present study, we explored the disposition of the highly structured, four-tiered cell-anchored cellulosome complex of this bacterium. Four separate, integral cellulosome components were subjected to immunolabeling: ScaA, ScaB, ScaC, and the cellulosome's most prominent enzyme, GH48. Imaging of the cells by correlating scanning electron microscopy and three-dimensional (3D) super-resolution fluorescence microscopy revealed that some of the protuberance-like structures on the cell surface represent cellulosomes and that the components are highly colocalized and organized by a defined hierarchy on the cell surface. The display of the cellulosome on the cell surface was found to differ between cells grown on soluble or insoluble substrates. Cell growth on microcrystalline cellulose and wheat straw exhibited dramatic enhancement in the amount of cellulosomes displayed on the bacterial cell surface.IMPORTANCE Conversion of plant biomass into soluble sugars is of high interest for production of fermentable industrial materials, such as biofuels. Biofuels are a very attractive alternative to fossil fuels, both for recycling of agricultural wastes and as a source of sustainable energy. Cellulosomes are among the most efficient enzymatic degraders of biomass known to date, due to the incorporation of a multiplicity of enzymes into a potent, multifunctional nanomachine. The intimate association with the bacterial cell surface is inherent in its efficient action on lignocellulosic substrates, although this property has not been properly addressed experimentally. The dramatic increase in cellulosome performance on recalcitrant feedstocks is critical for the design of cost-effective processes for efficient biomass degradation.
Lewinsky H., Barak A. F., Huber V., Kramer M. P., Radomir L., Sever L., Orr I., Mirkin V., Dezorella N., Shapiro M., Cohen Y., Shvidel L., Seiffert M., Herishanu Y., Becker-Herman S. & Shachar I. (2018) Journal of Clinical Investigation. 128, 12, p. 5465-5478
Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and progressive accumulation of mature B lymphocytes in the peripheral blood, lymphoid tissues, and bone marrow. CLL is characterized by profound immune defects leading to severe infectious complications. T cells are numerically, phenotypically, and functionally highly abnormal in CLL, with only limited ability to exert antitumor immune responses. Exhaustion of T cells has also been suggested to play an important role in antitumor responses. CLL-mediated T cell exhaustion is achieved by the aberrant expression of several inhibitory molecules on CLL cells and their microenvironment, prominently the programmed cell death ligand 1/programmed cell death 1 (PD-L1/PD-1) receptors. Previously, we showed that CD84, a member of the SLAM family of receptors, bridges between CLL cells and their microenvironment. In the current study, we followed CD84 regulation of T cell function. We showed that cell-cell interaction mediated through human and mouse CD84 upregulates PD-L1 expression on CLL cells and in their microenvironment and PD-1 expression on T cells. This resulted in suppression of T cell responses and activity in vitro and in vivo. Thus, our results demonstrate a role for CD84 in the regulation of immune checkpoints by leukemia cells and identify CD84 blockade as a therapeutic strategy to reverse tumor-induced immune suppression.
Fredlund J., Santos J. C., Stevenin V., Weiner A., Latour-Lambert P., Rechav K., Mallet A., Krijnse-Locker J., Elbaum M. & Enninga J. (2018) Cellular Microbiology. 20, 4, e12816
Salmonella enterica induces membrane ruffling and genesis of macropinosomes during its interactions with epithelial cells. This is achieved through the type three secretion system-1, which first mediates bacterial attachment to host cells and then injects bacterial effector proteins to alter host behaviour. Next, Salmonella enters into the targeted cell within an early membrane-bound compartment that matures into a slow growing, replicative niche called the Salmonella Containing Vacuole (SCV). Alternatively, the pathogen disrupts the membrane of the early compartment and replicate at high rate in the cytosol. Here, we show that the in situ formed macropinosomes, which have been previously postulated to be relevant for the step of Salmonella entry, are key contributors for the formation of the mature intracellular niche of Salmonella. We first clarify the primary mode of type three secretion system-1 induced Salmonella entry into epithelial cells by combining classical fluorescent microscopy with cutting edge large volume electron microscopy. We observed that Salmonella, similarly to Shigella, enters epithelial cells inside tight vacuoles rather than in large macropinosomes. We next apply this technology to visualise rupturing Salmonella containing compartments, and we use extended time-lapse microscopy to establish early markers that define which Salmonella will eventually hyper replicate. We show that at later infection stages, SCVs harbouring replicating Salmonella have previously fused with the in situ formed macropinosomes. In contrast, such fusion events could not be observed for hyper-replicating Salmonella, suggesting that fusion of the Salmonella entry compartment with macropinosomes is the first committed step of SCV formation.
Scheunert G., McCarron R., Kullock R., Cohen S. R., Rechav K., Kaplan-Ashiri I., Bitton O., Hecht B. & Oron D. (2018) Journal of Applied Physics. 123, 14, 143102
Creating sub-micron hotspots for applications such as heat-assisted magnetic recording (HAMR) is a challenging task. The most common approach relies on a surface-plasmon resonator (SPR), whose design dictates the size of the hotspot to always be larger than its critical dimension. Here, we present an approach which circumvents known geometrical restrictions by resorting to electric field confinement via excitation of a gap-mode (GM) between a comparatively large Gold (Au) nano-sphere (radius of 100 nm) and the magnetic medium in a grazing-incidence configuration. Operating a lambda = 785 nm laser, sub-200 nm hot spots have been generated and successfully used for GM-assisted magnetic switching on commercial CoCrPt perpendicular magnetic recording media at laser powers and pulse durations comparable to SPR-based HAMR. Lumerical electric field modelling confirmed that operating in the near-infrared regime presents a suitable working point where most of the light's energy is deposited in the magnetic layer, rather than in the nano-particle. Further, modelling is used for predicting the limits of our method which, in theory, can yield sub-30 nm hotspots for Au nano-sphere radii of 25-50 nm for efficient heating of FePt recording media with a gap of 5 nm. Published by AIP Publishing.
Xu J., Rechav K., Popovitz-Biro R., Nevo I., Feldman Y. & Joselevich E. (2018) Advanced Materials. 30, 20, 1800413
1D core-shell heterojunction nanostructures have great potential for high-performance, compact optoelectronic devices owing to their high interface area to volume ratio, yet their bottom-up assembly toward scalable fabrication remains a challenge. Here the site-controlled growth of aligned CdS-CdSe core-shell nanowalls is reported by a combination of surface-guided vaporliquid-solid horizontal growth and selective-area vapor-solid epitaxial growth, and their integration into photodetectors at wafer-scale without postgrowth transfer, alignment, or selective shell-etching steps. The photocurrent response of these nanowalls is reduced to 200 ns with a gain of up to 3.8 x 10(3) and a photoresponsivity of 1.2 x 10(3) A W-1, the fastest response at such a high gain ever reported for photodetectors based on compound semiconductor nanostructures. The simultaneous achievement of sub-microsecond response and high-gain photocurrent is attributed to the virtues of both the epitaxial CdS-CdSe heterojunction and the enhanced charge-separation efficiency of the core-shell nanowall geometry. Surface-guided nanostructures are promising templates for wafer-scale fabrication of self-aligned core-shell nanostructures toward scalable fabrication of high-performance compact photodetectors from the bottom-up.
Khalifa G. M., Kahil K., Erez J., Ashiri I. K., Shimoni E., Pinkas I., Addadi L. & Weiner S. (2018) Acta Biomaterialia. 77, p. 342-351
Quantifying ion concentrations and mapping their intracellular distributions at high resolution can provide much insight into the formation of biomaterials. The key to achieving this goal is cryo-fixation, where the biological materials, tissues and associated solutions are rapidly frozen and preserved in a vitreous state. We developed a correlative cryo-Scanning Electron Microscopy (SEM)/Energy Dispersive Spectroscopy (EDS) protocol that provides quantitative elemental analysis correlated with spatial imaging of cryo-immobilized specimens. We report the accuracy and sensitivity of the cryo-EDS method, as well as insights we derive on biomineralization pathways in a foraminifer. Foraminifera are marine protozoans that produce Mg-containing calcitic shells and are major calcifying organisms in the oceans. We use the cryo-SEM/EDS correlative method to characterize unusual Mg and Ca-rich particles in the cytoplasm of a benthic foraminifer. The Mg/Ca ratio of these particles is consistently lower than that of seawater, the source solution for these ions. We infer that these particles are involved in Ca ion supply to the shell. We document the internal structure of the MgCa particles, which in some cases include a separate Si rich core phase. This approach to mapping ion distribution in cryo-preserved specimens may have broad applications to other mineralized biomaterials.Statement of significanceIons are an integral part of life, and some ions play fundamental roles in cell metabolism. Determining the concentrations of ions in cells and between cells, as well as their distributions at high resolution can provide valuable insights into ion uptake, storage, functions and the formation of biomaterials. Here we present a new cryo-SEM/EDS protocol that allows the mapping of different ion distributions in solutions and biological samples that have been cryo-preserved. We demonstrate the value of this novel approach by characterizing a novel biogenic mineral phase rich in Mg found in foraminifera, single celled marine organisms. This method has wide applicability in biology, and especially in understanding the formation and function of mineral-containing hard tissues. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Cohen E., Weissman H., Pinkas I., Shimoni E., Rehak P., Kral P. & Rybtchinski B. (2018) ACS Nano. 12, 1, p. 317-326
Designing supramolecular nanotubes (SNTs) with distinct dimensions and properties is highly desirable, yet challenging, since structural control strategies are lacking. Furthermore, relatively complex building blocks are often employed in SNT self-assembly. Here, we demonstrate that symmetric bolaamphiphiles having a hydrophobic core comprised of two perylene diimide moieties connected via a bipyridine linker and bearing polyethylene glycol (PEG) side chains can self-assemble into diverse molecular nanotubes. The structure of the nanotubes can be controlled by assembly conditions (solvent composition and temperature) and a PEG chain length. The resulting nanotubes differ both in diameter and cross section geometry, having widths of 3 nm (triangular-like cross-section), 4 nm (rectangular), and 5 nm (hexagonal). Molecular dynamics simulations provide insights into the stability of the tubular superstructures and their initial stages of self-assembly, revealing a key role of oligomerization via side-by-side aromatic interactions between bis-aromatic cores. Probing electronic and photonic properties of the nanotubes revealed extended electron delocalization and photoinduced charge separation that proceeds via symmetry breaking, a photofunction distinctly different from that of the fibers assembled from the same molecules. A high degree of structural control and insights into SNT self-assembly advance design approaches toward functional organic nanomaterials.
Marcovich A. L., Tandogan T., Bareket M., Eting E., Kaplan-Ashiri I., Bukelman A., Auffarth G. U. & Khoramnia R. (2018) BMJ Open Ophthalmology. 3, 1, 000157
Objective To report 11 cases of intraocular lens (IOL) opacification after pars plana vitrectomy (PPV) involving intravitreal gas injection. Methods and analysis Eleven cases of hydrophilic IOLs that opacified following PPV with intravitreal gas injection are described. Eight IOLs were explanted and analysed by light microscopy and scanning electron microscopy. Staining with alizarin red and von Kossa stains, as well as energy dispersive X-ray spectroscopy (EDX) were performed. Three IOLs were not explanted. The surgeons attached the clinical data. Results The IOLs were hydrophilic acrylic produced by six manufacturers. Six patients underwent primarily phacoemulsification with IOL implantation. PPV with intravitreal gas injection was performed 3 months-6 years afterwards. The other five patients underwent combined phacoemulsification with IOL implantation and PPV with intravitreal gas injection. IOL opacification was recorded 1 month-6 years after PPV. In eight patients, the IOLs were explanted 1 month-9 years after opacification was noticed. In three patients, the opacified IOL was not explanted. IOLs had opacified mainly anteriorly at the pupillary entrance or capsulorhexis opening. Light microscopy demonstrated granular surface deposits on the IOLs that stained positive for calcium by alizarin red and von Kossa stains. EDX analysis of the deposits detected calcium and phosphorus. Conclusions Hydrophilic acrylic IOLs can opacify due to calcium deposition after PPV and intravitreal gas injection and may require IOL explantation. A hydrophobic IOL may be preferred when a simultaneous phacoemulsification and vitrectomy with intravitreal gas is performed.
Elianov O., Garusi S., Rosentsveig R., Cohen S. R., Feldman Y., Pinkas B., Bendikov T., Kaplan-Ashiri, Moshkovich A., Perfilyev V., Rapoport L., Moshonov J., Tenne R. & Shay B. (2018) Surface & Coatings Technology. 353, p. 116-125
Self-lubricating films are of immense importance in various tribological applications. Nanoparticles are often used as the lubricating component in such films. Inorganic fullerene-like (IF) nanoparticles of WS2 have been used in the past for variety of tribological applications including for self-lubricating polymer and metallic films. IF nanoparticles from MoS2 with superior tribological behavior were reported in the past. However, such nanoparticles, which are available in minute amounts, were not applied for self-lubricating coatings in the past. In the current work, inorganic fullerene-like nanoparticles of MoS2 (IF-MoS2) were co-evaporated with titanium (cobalt) on metal substrates. The films were characterized by different techniques and were shown to have good adhesion to the underlying substrate. Furthermore, tribological tests indicated that such coatings exhibit improved friction coefficient (roughly 0.1) and very small wear under relatively high load.
Nath J., Dror I., Landa P., Vanek T., Kaplan-Ashiri I. & Berkowitz B. (2018) Environmental Pollution. 242, Part B, p. 1827-1837
In parallel to technological advances and ever-increasing use of nanoparticles in industry, agriculture and consumer products, the potential ecotoxicity of nanoparticles and their potential accumulation in ecosystems is of increasing concern. Because scientific reports raise a concern regarding nanoparticle toxicity to plants, understanding of their bioaccumulation has become critical and demands more research. Here, the synthesis of isotopically-labeled nanoparticles of silver, copper and zinc oxide is reported; it is demonstrated that while maintaining the basic properties of the same unlabeled ("regular") nanoparticles, labeled nanoparticles enable more sensitive tracing of nanoparticles within plants that have background elemental levels. This technique is particularly useful for working with elements that are present in high abundance in natural environments. As a benchmark, labeled and unlabeled metal nanoparticles (Ag-NP, Cu-NP, ZnO-NP) were synthesized and compared, and then exposed in a series of growth experiments to Arabidopsis thaliana; the NPs were traced in different parts of the plant. All of the synthesized nanoparticles were characterized by TEM, EDS, DLS, zeta-potential and single particle ICP-MS, which provided essential information regarding size, composition, morphology and surface charge of nanoparticles, as well as their stability in suspensions. Tracing studies with A. thaliana showed uptake/retention of nanoparticles that is more significant in roots than in shoots. Single particle ICP-MS, and scanning electron micrographs and EDS of plant roots showed presence of Ag-NPs in particular, localized areas, whereas copper and zinc were found to be distributed over the root tissues, but not as nanoparticles. Thus, nanoparticles in any natural matrix can be replaced easily by their labeled counterparts to trace the accumulation or retention of NPs. Isotopically-labeled nanoparticles enable acquisition of specific results, even if there are some concentrations of the same elements that originate from other (natural or anthropogenic) sources. (C) 2018 Elsevier Ltd. All rights reserved.
Shalom H., Feldman Y., Rosentsveig R., Pinkas I., Kaplan-Ashiri I., Moshkovich A., Perfilyev V., Rapoport L. & Tenne R. (2018) International Journal of Molecular Sciences. 19, 3, 657
Films combining hydroxyapatite (HA) with minute amounts (ca. 1 weight %) of (rhenium doped) fullerene-like MoS2 (IF) nanoparticles were deposited onto porous titanium substrate through electrophoretic process (EPD). The films were analyzed by scanning electron microscopy (SEM), X-ray diffraction and Raman spectroscopy. The SEM analysis showed relatively uniform coatings of the HA + IF on the titanium substrate. Chemical composition analysis using energy dispersive X-ray spectroscopy (EDS) of the coatings revealed the presence of calcium phosphate minerals like hydroxyapatite, as a majority phase. Tribological tests were undertaken showing that the IF nanoparticles endow the HA film very low friction and wear characteristics. Such films could be of interest for various medical technologies. Means for improving the adhesion of the film to the underlying substrate and its fracture toughness, without compromising its biocompatibility are discussed at the end.
Garcia-Seisdedos H., Empereur-Mot C., Elad N. & Levy E. D. (2017) Nature. 548, 7666, p. 244-247
The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life(1-6). Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils(7). Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.
Palmer B. A., Taylor G. J., Brumfeld V., Gur D., Shemesh M., Elad N., Osherov A., Oron D., Weiner S. & Addadi L. (2017) Science. 358, 6367, p. 1172-1175
Scallops possess a visual system comprising up to 200 eyes, each containing a concave mirror rather than a lens to focus light. The hierarchical organization of the multilayered mirror is controlled for image formation, from the component guanine crystals at the nanoscale to the complex three-dimensional morphology at the millimeter level. The layered structure of the mirror is tuned to reflect the wavelengths of light penetrating the scallop's habitat and is tiled with a mosaic of square guanine crystals, which reduces optical aberrations. The mirror forms images on a double-layered retina used for separately imaging the peripheral and central fields of view. The tiled, off-axis mirror of the scallop eye bears a striking resemblance to the segmented mirrors of reflecting telescopes.
Hirsch A., Palmer B. A., Elad N., Gur D., Weiner S., Addadi L., Kronik L. & Leiserowitz L. (2017) ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 56, 32, p. 9420-9424
Guanine crystals are widely used in nature as components of multilayer reflectors. Guanine-based reflective systems found in the copepod cuticle and in the mirror of the scallop eye are unique in that the multilayered reflectors are tiled to form a contiguous packed array. In the copepod cuticle, hexagonal crystals are closely packed to produce brilliant colors. In the scallop eye, square crystals are tiled to obtain an image-forming reflecting mirror. The tiles are about 1 mm in size and 70 nm thick. According to analysis of their electron diffraction patterns, the hexagon and square tiles are not single crystals. Rather, each tile type is a composite of what appears to be three crystalline domains differently oriented and stacked onto one another, achieved through a twice-repeated twinning about their and crystal axes, respectively. By these means, the monoclinic guanine crystal mimics higher symmetry hexagonal and tetragonal structures to achieve unique morphologies.
Elad N., Bellapadrona G., Houben L., Sagi I. & Elbaum M. (2017) Proceedings Of The National Academy Of Sciences Of The United States Of America-Physical Sciences. 114, 42, p. 11139-11144
Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.
Luria N., Smith E., Reingold V., Bekelman I., Lapidot M., Levin I., Elad N., Tam Y., Sela N., Abu-Ras A., Ezra N., Haberman A., Yitzhak L., Lachman O. & Dombrovsky A. (2017) PLoS ONE. 12, 1, e0170429
An outbreak of a new disease infecting tomatoes occurred in October-November 2014 at the Ohad village in Southern Israel. Symptomatic plants showed a mosaic pattern on leaves accompanied occasionally by narrowing of leaves and yellow spotted fruit. The disease spread mechanically and rapidly reminiscent of tobamovirus infection. Epidemiological studies showed the spread of the disease in various growing areas, in the South and towards the Southeast and Northern parts of the country within a year. Transmission electron microscope (TEM) analysis showed a single rod-like form characteristic to the Tobamovirus genus. We confirmed Koch's postulates for the disease followed by partial host range determination and revealed that tomato cultivars certified to harbor the Tm-2<sup>2</sup> resistance gene are susceptible to the new viral disease. We further characterized the viral source of the disease using a range of antisera for serological detection and analyzed various virus genera and families for cross-reactivity with the virus. In addition, next generation sequencing of total small RNA was performed on two cultivars grown in two different locations. In samples collected from commercial cultivars across Israel, we found a single virus that caused the disease. The complete genome sequence of the new Israeli tobamovirus showed high sequence identity to the Jordanian isolate of tomato brown rugose fruit virus.
Udayabhaskararao T., Altantzis T., Houben L., Coronado-Puchau M., Langer J., Popovitz-Biro R., Liz-Marzan L. M., Vukovic L., Kral P., Bals S. & Klajn R. (2017) Science. 358, 6362, p. 514-+
Self-assembly of inorganic nanoparticles has been used to prepare hundreds of different colloidal crystals, but almost invariably with the restriction that the particles must be densely packed. Here, we show that non-close-packed nanoparticle arrays can be fabricated through the selective removal of one of two components comprising binary nanoparticle superlattices. First, a variety of binary nanoparticle superlattices were prepared at the liquid-air interface, including several arrangements that were previously unknown. Molecular dynamics simulations revealed the particular role of the liquid in templating the formation of superlattices not achievable through self-assembly in bulk solution. Second, upon stabilization, all of these binary superlattices could be transformed into distinct "nanoallotropes"-nanoporous materials having the same chemical composition but differing in their nanoscale architectures.
Ganai A., Maiti P. S., Houben L., Bar-Ziv R. & Bar Sadan M. (2017) Journal of Physical Chemistry C. 121, 12, p. 7062-7068
Metal semiconductor hybrids are a promising architecture for functional nanostructures because they efficiently promote charge separation. The morphology of the hybrid supports two mechanisms of charge generation and transfer, namely, the excitation of electrons to the conduction band of the semiconductor or the induction of surface plasmon resonance on the metal. Here, we compared the photocatalytic activity of nanoparticles with a core shell or dimer morphology, using Pt, Pd, or Au as the metal and Cu2ZnSnS4 (CZTS), which comprises abundant and environmentally friendly elements, as the semiconductor. Their performance as photocatalysts was evaluated by using Methylene Blue (MB) degradation under light irradiation. We found that although large Au cores improved the photocatalytic activity of the CZTS nanoparticles, the highest catalytic activity was that of Pt CZTS and Pd CZTS dimers. Conversely, using small metal particles as cores degraded the activity of the CZTS due to the formation of an internal boundary and the occupation of potentially optically active volume. In addition, the results point out that depositing multiple metal particles is not beneficial for photocatalysis.
Udayabhaskararao T., Kazes M., Houben L., Lin H. & Oron D. (2017) Chemistry of Materials. 29, 3, p. 1302-1308
Despite the recent surge of interest in lead halide perovskite nanocrystals, there are still significant gaps in the understanding of nucleation and growth processes involved in their formation. Using CsPbX3 as a model system, we systematically study the formation mechanism of cubic CsPbX3 nanocrystals, their growth via oriented attachment into larger nanostructures, and the associated phase transformations. We found evidence to support that the formation of CsPbX3 NCs occurs through the seed-mediated nucleation method, where Pb-o NPs formed during the course of reaction act as seeds. Further growth occurs through self-assembly and oriented attachment. The polar environment is a major factor in determining the structure and shape of the resulting nanoparticles, as confirmed by experiments with aged seed reaction mixtures, and by addition of polar additives. These results provide a fundamental understanding of the influence of the environment polarity on self-assembly, self-healing, and the ability to control the morphology and structure over the perovskite structures. As a result of this understanding, we were able to extend the synthesis to produce other materials such as CsPbBr3 nanowires and orthorhombic CsPbI3 nanowires with tunable length ranging from 200 nm to several microns.
Schreiber R. E., Houben L., Wolf S. G., Leitus G., Lang Z. L., Carbó J. J., Poblet J. M. & Neumann R. (2017) Nature Chemistry. 9, 4, p. 369-373
How molecules in solution form crystal nuclei, which then grow into large crystals, is a poorly understood phenomenon. The classical mechanism of homogeneous crystal nucleation proceeds via the spontaneous random aggregation of species from liquid or solution. However, a non-classical mechanism suggests the formation of an amorphous dense phase that reorders to form stable crystal nuclei. So far it has remained an experimental challenge to observe the formation of crystal nuclei from five to thirty molecules. Here, using polyoxometallates, we show that the formation of small crystal nuclei is observable by cryogenic transmission electron microscopy. We observe both classical and non-classical nucleation processes, depending on the identity of the cation present. The experiments verify theoretical studies that suggest non-classical nucleation is the lower of the two energy pathways. The arrangement in just a seven-molecule proto-crystal matches the order found by X-ray diffraction of a single bulk crystal, which demonstrates that the same structure was formed in each case.
Lavie A., Yadgarov L., Houben L., Popovitz-Biro R., Shaul T. E., Nagler A., Suchowski H. & Tenne R. (2017) Nanotechnology. 28, 24, 24LT03
Nanoparticles, and more specifically gold nanoparticles (AuNPs), have attracted much scientific and technological interest in the last few decades. Their popularity is attributed to their unique optical, catalytic, electrical and magnetic properties when compared with the bulk. However, one of the main problems with AuNPs is their long-term stability. Two-dimensional materials like MoS<sub>2</sub> (WS<sub>2</sub>) are semiconductors that exhibit a combination of properties which make them suitable for electronic, optical and (photo)catalytic devices. Few-layer MoS<sub>2</sub> (WS<sub>2</sub>) nanoparticles (NPs), and in particular single-layer ones, show intriguing optical and electrical properties which are very different from those of the bulk compounds. Here we demonstrate the synthesis of AuNPs sheathed by a single layer of MoS<sub>2</sub> (WS<sub>2</sub>), i.e. a core-shell nanostructure (AuNP@1L-MoS<sub>2</sub>). The hybrid NPs exhibit optical properties that are different from those of either constituent and are amenable for modulation via their chemistry, offering a myriad of applications.
Nerl H. C., Winther K. T., Hage F. S., Thygesen K. S., Houben L., Backes C., Coleman J. N., Ramasse Q. M. & Nicolosi V. (2017) npj 2D Materials and Applications. 1
Excitons and plasmons are the two most fundamental types of collective electronic excitations occurring in solids. Traditionally, they have been studied separately using bulk techniques that probe their average energetic structure over large spatial regions. However, as the dimensions of materials and devices continue to shrink, it becomes crucial to understand how these excitations depend on local variations in the crystal- and chemical structure on the atomic scale. Here, we use monochromated low-loss scanning-transmission-electron-microscopy electron-energy-loss spectroscopy, providing the best simultaneous energy and spatial resolution achieved to-date to unravel the full set of electronic excitations in few-layer MoS2 nanosheets over a wide energy range. Using first-principles, many-body calculations we confirm the excitonic nature of the peaks at similar to 2 and similar to 3 eV in the experimental electron-energy-loss spectrum and the plasmonic nature of higher energy-loss peaks. We also rationalise the non-trivial dependence of the electron-energy-loss spectrum on beam and sample geometry such as the number of atomic layers and distance to steps and edges. Moreover, we show that the excitonic features are dominated by the long wavelength (q = 0) components of the probing field, while the plasmonic features are sensitive to a much broader range of q-vectors, indicating a qualitative difference in the spatial character of the two types of collective excitations. Our work provides a template protocol for mapping the local nature of electronic excitations that open new possibilities for studying photo-absorption and energy transfer processes on a nanometer scale.
Brontvein O., Houben L., Popovitz-Biro R., Levy M., Feuermann D., Tenne R. & Gordon J. M. (2017) Nano. 12, 3, 1750030
New types of core-shell nanoparticles are reported: Pb@GaS fullerene-like and nanotubular structures, achieved via the continuously high reactor temperatures and ultra-hot strong-gradient annealing environments created by highly concentrated sunlight. Structural and chemical characterizations suggest a formation mechanism where vaporized Pb condenses into nanoparticles that are stabilized as they become covered by molten GaS, the ensuing crystallization of which creates the outer layers. Hollow-core GaS fullerene-like nanoparticles and nanotubes were also observed among the products, demonstrating that a single solar procedure can generate a variety of core-shell and hollow nanostructures. The proposed formation mechanisms can account for their relative abundance and the characterization data.
Wolf S. G., Mutsafi Y., Dadosh T., Ilani T., Lansky Z., Horowitz B., Rubin S., Elbaum M. & Fass D. (2017) eLife. 6, e29929
The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.
Rivkin N., Chapnik E., Mildner A., Barshtein G., Porat Z., Kartvelishvily E., Dadosh T., Birger Y., Amir G., Yedgar S., Izraeli S., Jung S. & Hornstein E. (2017) Haematologica. 102, 4, p. 676-685
Hematopoietic-specific microRNA-142 is a critical regulator of various blood cell lineages, but its role in erythrocytes is unexplored. Herein, we characterize the impact of microRNA-142 on erythrocyte physiology and molecular cell biology, using a mouse loss-of-function allele. We report that microRNA-142 is required for maintaining the typical erythrocyte biconcave shape and structural resilience, for the normal metabolism of reactive oxygen species, and for overall lifespan. microRNA-142 further controls ACTIN filament homeostasis and membrane skeleton organization. The analyses presented reveal previously unappreciated functions of microRNA-142 and contribute to an emerging view of small RNAs as key players in erythropoiesis. Finally, the work herein demonstrates how a housekeeping network of cytoskeletal regulators can be reshaped by a single micro-RNA denominator in a cell type specific manner.
Abada A., Levin-Zaidman S., Porat Z., Dadosh T. & Elazar Z. (2017) Proceedings of the National Academy of Sciences of the United States of America. 114, 48, p. 12749-12754
Autophagy, a unique intracellular membrane-trafficking pathway, is initiated by the formation of an isolation membrane (phagophore) that engulfs cytoplasmic constituents, leading to generation of the autophagosome, a double-membrane vesicle, which is targeted to the lysosome. The outer autophagosomal membrane consequently fuses with the lysosomal membrane. Multiple membrane-fusion events mediated by SNARE molecules have been postulated to promote autophagy. alpha SNAP, the adaptor molecule for the SNARE-priming enzyme N-ethylmaleimide-sensitive factor (NSF) is known to be crucial for intracellular membrane fusion processes, but its role in autophagy remains unclear. Here we demonstrated that knockdown of alpha SNAP leads to inhibition of autophagy, manifested by an accumulation of sealed autophagosomes located in close proximity to lysosomes but not fused with them. Under these conditions, moreover, association of both Atg9 and the autophagy-related SNARE protein syntaxin17 with the autophagosome remained unaffected. Finally, our results suggested that under starvation conditions, the levels of alpha SNAP, although low, are nevertheless sufficient to partially promote the SNARE priming required for autophagy. Taken together, these findings indicate that while autophagosomal-lysosomal membrane fusion is sensitive to inhibition of SNARE priming, the initial stages of autophagosome biogenesis and autophagosome expansion remain resistant to its loss.
Lippert L. G., Dadosh T., Hadden J. A., Karnawat V., Diroll B. T., Murray C. B., Holzbaur E. L., Schulten K., Reck-Peterson S. L. & Goldman Y. E. (2017) Proceedings of the National Academy of Sciences of the United States of America. 114, 23, p. E4564-E4573
The force-generating mechanism of dynein differs from the forcegenerating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-μs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.
Banisch T. U., Maimon I., Dadosh T. & Gilboa L. (2017) Development. 144, 11, p. 1937-1947
Two different compartments support germline stem cell (GSC) self-renewal and their timely differentiation: the classical niche provides maintenance cues, while a differentiation compartment, formed by somatic escort cells (ECs), is required for proper GSC differentiation. ECs extend long protrusions that invade between tightly packed germ cells, and alternate between encapsulating and releasing them. How ECs achieve this dynamic balance has not been resolved. By combining live imaging and genetic analyses in Drosophila, wehave characterised EC shapes and their dynamic changes. We show that germ cell encapsulation by ECs is a communal phenomenon, whereby EC-EC contacts stabilise an extensive meshwork of protrusions. We further show that Signal Transducer and Activator of Transcription (Stat) and Epidermal Growth Factor Receptor (Egfr) signalling sustain EC protrusiveness and flexibility by combinatorially affecting the activity of different RhoGTPases. Our results reveal how a complex signalling network can determine the shape of a cell and its dynamic behaviour. It also explains how the differentiation compartment can establish extensive contacts with germ cells, while allowing a continual posterior movement of differentiating GSC daughters.
Milrot E., Shimoni E., Dadosh T., Rechav K., Unger T., Van Etten E. J. L. & Minsky A. (2017) PLoS Pathogens. 13, 8, e1006562
A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic-infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.
Dasari S. K., Schejter E., Bialik S., Shkedy A., Levin-Salomon V., Levin-Zaidman S. & Kimchi A. (2017) Cell Cycle. 16, 21, p. 2003-2010
Autophagy is critical for homeostasis and cell survival during stress, but can also lead to cell death, a little understood process that has been shown to contribute to developmental cell death in lower model organisms, and to human cancer cell death. We recently reported(1) on our thorough molecular and morphologic characterization of an autophagic cell death system involving resveratrol treatment of lung carcinoma cells. To gain mechanistic insight into this death program, we performed a signalome-wide RNAi screen for genes whose functions are necessary for resveratrol-induced death. The screen identified GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase, as an important mediator of autophagic cell death. Here we further show the physiological relevance of GBA1 to developmental cell death in midgut regression during Drosophila metamorphosis. We observed a delay in midgut cell death in two independent Gba1a RNAi lines, indicating the critical importance of Gba1a for midgut development. Interestingly, loss-of-function GBA1 mutations lead to Gaucher Disease and are a significant risk factor for Parkinson Disease, which have been associated with defective autophagy. Thus GBA1 is a conserved element critical for maintaining proper levels of autophagy, with high levels leading to autophagic cell death.
Ruggiero A., Aloni E., Korkotian E., Zaltsman Y., Oni-Biton E., Kuperman Y., Tsoory M., Shachnai L., Levin-Zaidman S., Brenner O., Segal M. & Gross A. (2017) Scientific Reports. 7, 44401
Mitochondrial Carrier Homolog 2 (MTCH2) is a novel regulator of mitochondria metabolism, which was recently associated with Alzheimer's disease. Here we demonstrate that deletion of forebrain MTCH2 increases mitochondria and whole-body energy metabolism, increases locomotor activity, but impairs motor coordination and balance. Importantly, mice deficient in forebrain MTCH2 display a deficit in hippocampus-dependent cognitive functions, including spatial memory, long term potentiation (LTP) and rates of spontaneous excitatory synaptic currents. Moreover, MTCH2-deficient hippocampal neurons display a deficit in mitochondria motility and calcium handling. Thus, MTCH2 is a critical player in neuronal cell biology, controlling mitochondria metabolism, motility and calcium buffering to regulate hippocampal-dependent cognitive functions.
Dasari S. K., Bialik S., Levin-Zaidman S., Levin-Salomon V., Merrill A. H. J., Futerman A. H. & Kimchi A. (2017) Cell Death and Differentiation. 24, p. 1288-1302
Activating alternative cell death pathways, including autophagic cell death, is a promising direction to overcome the apoptosis resistance observed in various cancers. Yet, whether autophagy acts as a death mechanism by over consumption of intracellular components is still controversial and remains undefined at the ultrastructural and the mechanistic levels. Here we identified conditions under which resveratrol-treated A549 lung cancer cells die by a mechanism that fulfills the previous definition of autophagic cell death. The cells displayed a strong and sustained induction of autophagic flux, cell death was prevented by knocking down autophagic genes and death occurred in the absence of apoptotic or necroptotic pathway activation. Detailed ultrastructural characterization revealed additional critical events, including a continuous increase over time in the number of autophagic vacuoles, in particular autolysosomes, occupying most of the cytoplasm at terminal stages. This was followed by loss of organelles, disruption of intracellular membranes including the swelling of perinuclear space and, occasionally, a unique type of nuclear shedding. A signalome-wide shRNA-based viability screen was applied to identify positive mediators of this type of autophagic cell death. One top hit was GBA1, the Gaucher disease-associated gene, which encodes glucocerebrosidase, an enzyme that metabolizes glucosylceramide to ceramide and glucose. Interestingly, glucocerebrosidase expression levels and activity were elevated, concomitantly with increased intracellular ceramide levels, both of which correlated in time with the appearance of the unique death characteristics. Transfection with siGBA1 attenuated the increase in glucocerebrosidase activity and the intracellular ceramide levels. Most importantly, GBA1 knockdown prevented the strong increase in LC3 lipidation, and many of the ultrastructural changes characteristic of this type of autophagic cell death, including a significant decrease in cytoplasmic area occupied by autophagic vacuoles. Together, these findings highlight the critical role of GBA1 in mediating enhanced self-consumption of intracellular components and endomembranes, leading to autophagic cell death.
Silvent J., Akiva A., Brumfeld V., Reznikov N., Rechav K., Yaniv K., Addadi L. & Weiner S. (2017) PLoS ONE. 12, 12, p. e0177731-e0177731, 0177731
Although bone is one of the most studied living materials, many questions about the manner in which bones form remain unresolved, including fine details of the skeletal structure during development. In this study, we monitored skeleton development of zebrafish larvae, using calcein fluorescence, high-resolution micro-CT 3D images and FIB-SEM in the block surface serial imaging mode. We compared calcein staining of the skeletons of the wild type and nacre mutants, which are transparent zebrafish, with micro-CT for the first 30 days post fertilization embryos, and identified significant differences. We quantified the bone volumes and mineral contents of bones, including otoliths,during development, and showed that such developmental differences, including otolith development, could be helpful in identifying phenotypes. In addition, high-resolution imaging revealed the presence of mineralized aggregates in the notochord, before the formation of the first bone in the axial skeleton. These structures might play a role in the storage of the mineral. Our results highlight the potential of these high-resolution 3D approaches to characterize the zebrafish skeleton, which in turn could prove invaluable information for better understanding the development and the characterization of skeletal phenotypes.
Neeman L., Ben-Zvi R., Rechav K., Popovitz-Biro R., Oron D. & Joselevich E. (2017) Nano Letters. 17, 2, p. 842-850
The growth of horizontal nanowires (NWs) guided by epitaxial and graphoepitaxial relations with the substrate is becoming increasingly attractive owing to the possibility of controlling their position, direction, and crystallographic orientation. In guided NWs, as opposed to the extensively characterized vertically grown NWs, there is an increasing need for understanding the relation between structure and properties, specifically the role of the epitaxial relation with the substrate. Furthermore, the uniformity of crystallographic orientation along guided NWs and over the substrate has yet to be checked. Here we perform highly sensitive second harmonic generation (SHG) polarimetry of polar and nonpolar guided ZnO NWs grown on R-plane and M-plane sapphire. We optically map large areas on the substrate in a nondestructive way and find that the crystallographic orientations of the guided NWs are highly selective and specific for each growth direction with respect to the substrate lattice. In addition, we perform SHG polarimetry along individual NWs and find that the crystallographic orientation is preserved along the NW in both polar and nonpolar NWs. While polar NWs show highly uniform SHG along their axis, nonpolar NWs show a significant change in the local nonlinear susceptibility along a few micrometers, reflected in a reduction of 40% in the ratio of the SHG along different crystal axes. We suggest that these differences may be related to strain accumulation along the nonpolar wires. We find SHG polarimetry to be a powerful tool to study both selectivity and uniformity of crystallographic orientations of guided NWs with different epitaxial relations.
Xu J., Oksenberg E., Popovitz-Biro R., Rechav K. & Joselevich E. (2017) J. Am. Chem. Soc. 139, 44, p. 15958-15967
Tri-gate transistors offer better performance than planar transistors by exerting additional gate control over a channel from two lateral sides of semiconductor nanowalls (or ``fins''). Here we report the bottom-up assembly of aligned CdS nanowalls by a simultaneous combination of horizontal catalytic vapor-liquid-solid growth and vertical facet-selective noncatalytic vapor-solid growth and their parallel integration into tri-gate transistors and photodetectors at wafer scale (cm(2)) without postgrowth transfer or alignment steps. These tri-gate transistors act as enhancement-mode transistors with an on/off current ratio on the order of 10(8), 4 orders of magnitude higher than the best results ever reported for planar enhancement-mode CdS transistors. The response time of the photodetector is reduced to the submicrosecond level, 1 order of magnitude shorter than the best results ever reported for photodetectors made of bottom-up semiconductor nanostructures. Guided semiconductor nanowalls open new opportunities for high-performance 3D nanodevices assembled from the bottom up.
Shalev E., Oksenberg E., Rechav K., Popovitz-Biro R. & Joselevich E. (2017) ACS Nano. 11, 1, p. 213-220
One-dimensional semiconductor nanostructures, such as nanowires (NWs), have attracted tremendous attention due to their unique properties and potential applications in nanoelectronics, nano-optoelectronics, and sensors. One of the challenges toward their integration into practical devices is their large-scale controlled assembly. Here, we report the guided growth of horizontal CdSe nanowires on five different planes of sapphire. The growth direction and crystallographic orientation are controlled by the epitaxial relationship with the substrate as well as by a graphoepitaxial effect of surface nanosteps and grooves. CdSe is a promising direct-bandgap II-VI semiconductor active in the visible range, with potential applications in optoelectronics. The guided CdSe nanowires were found to have a wurtzite single-crystal structure. Field-effect transistors and photodetectors were fabricated to examine the nanowire electronic and optoelectronic properties, respectively. The latter exhibited the fastest rise and fall times ever reported for CdSe nanostructures as well as a relatively high gain, both features being essential for optoelectronic applications.
Elool Dov N., Shankar S., Cohen D., Bendikov T., Rechav K., Shimon L. J. W., Lahav M. & van der Boom M. E. (2017) Journal of the American Chemical Society. 139, 33, p. 11471-11481
In this study, we demonstrate a versatile approach for the formation of electrochromic nanoscale assemblies on transparent conductive oxides on both rigid and flexible substrates. Our method is based on the application of alternating spin-coated layers of well-defined metal polypyridyl complexes and a palladium(II) salt to form electrochemically addressable films with a high chromophore density. By varying the central metal ion of the polypyridyl complexes (Os, Ru, and" Fe) and their ligands and by mixing these complexes, coatings with a wide range of colors can be achieved. These coatings cover a large area of RGB color space. The coloration intensities of these nanoscale films can be tuned by "the number of deposition steps. The materials have very attractive ON/OFF ratios, electrochemical stabilities, and coloration efficiencies. Reversible color-to-colorless and color-to-color transitions were demonstrated, and the films were further integrated into sandwich cells.
Herbst R. H., Bar-Zvi D., Reikhav S., Soifer I., Breker M., Jona G., Shimoni E., Schuldiner M., Levy A. A. & Barkai N. (2017) BMC Biology. 15, 38
Background: The merging of genomes in inter-specific hybrids can result in novel phenotypes, including increased growth rate and biomass yield, a phenomenon known as heterosis. Heterosis is typically viewed as the opposite of hybrid incompatibility. In this view, the superior performance of the hybrid is attributed to heterozygote combinations that compensate for deleterious mutations accumulating in each individual genome, or lead to new, over-dominating interactions with improved performance. Still, only fragmented knowledge is available on genes and processes contributing to heterosis.Results: We describe a budding yeast hybrid that grows faster than both its parents under different environments. Phenotypically, the hybrid progresses more rapidly through cell cycle checkpoints, relieves the repression of respiration in fast growing conditions, does not slow down its growth when presented with ethanol stress, and shows increased signs of DNA damage. A systematic genetic screen identified hundreds of S. cerevisiae alleles whose deletion reduced growth of the hybrid. These growth-affecting alleles were condition-dependent, and differed greatly from alleles that reduced the growth of the S. cerevisiae parent.Conclusions: Our results define a budding yeast hybrid that is perturbed in multiple regulatory processes but still shows a clear growth heterosis. We propose that heterosis results from incompatibilities that perturb regulatory mechanisms, which evolved to protect cells against damage or prepare them for future challenges by limiting cell growth.
Cohen E., Weissman H., Shimoni E., Kaplan-Ashiri I., Werle K., Wohlleben W. & Rybtchinski B. (2017) ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 56, 8, p. 2203-2207
Aqua materials that contain water as their major component and are as robust as conventional plastics are highly desirable. Yet, the ability of such systems to withstand harsh conditions, for example, high pressures typical of industrial applications has not been demonstrated. We show that a hydrogel- like membrane self-assembled from an aromatic amphiphile and colloidal Nafion is capable of purifying water from organic molecules, including pharmaceuticals, and heavy metals in a very wide range of concentrations. Remarkably, the membrane can sustain high pressures, retaining its function. The robustness and functionality of the water-based self-assembled array advances the idea that aqua materials can be very strong and suitable for demanding industrial applications.
Nagar E., Zilberman S., Sendersky E., Simkovsky R., Shimoni E., Gershtein D., Herzberg M., Golden S. S. & Schwarz R. (2017) Environmental Microbiology. 19, 7, p. 2862-2872
The hair-like cell appendages denoted as type IV piliare crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II proteinsecretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here, we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo-proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm-promoting function in type IV pili-producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non-piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well-studied type IV pili-producing heterotrophic bacteria.
Scheunert G., Cohen S., Kullock R., McCarron R., Rechev K., Kaplan-Ashiri I., Bitton O., Dawson P., Hecht B. & Oron D. (2017) Beilstein Journal of Nanotechnology. 8, 1, p. 28-37
Heat-assisted magnetic recording (HAMR) is often considered the next major step in the storage industry: it is predicted to increase the storage capacity, the read/write speed and the data lifetime of future hard disk drives. However, despite more than a decade of development work, the reliability is still a prime concern. Featuring an inherently fragile surface-plasmon resonator as a highly localized heat source, as part of a near-field transducer (NFT), the current industry concepts still fail to deliver drives with sufficient lifetime. This study presents a method to aid conventional NFT-designs by additional grazing-incidence laser illumination, which may open an alternative route to high-durability HAMR. Magnetic switching is demonstrated on consumer-grade CoCrPt perpendicular magnetic recording media using a green and a near-infrared diode laser. Sub-500 nm magnetic features are written in the absence of a NFT in a moderate bias field of only μ<sub>0</sub>H = 0.3 T with individual laser pulses of 40 mW power and 50 ns duration with a laser spot size of 3 μm (short axis) at the sample surface - six times larger than the magnetic features. Herein, the presence of a nanoscopic object, i.e., the tip of an atomic force microscope in the focus of the laser at the sample surface, has no impact on the recorded magnetic features - thus suggesting full compatibility with NFT-HAMR.
Schatz D., Rosenwasser S., Malitsky S., Wolf S. G., Feldmesser E. & Vardi A. (2017) Nature Microbiology. 2, 11, p. 1485-1492
Communication between microorganisms in the marine environment has immense ecological impact by mediating trophic-level interactions and thus determining community structure(1). Extracellular vesicles (EVs) are produced by bacteria(2,3), archaea(4), protists(5) and metazoans, and can mediate pathogenicity(6) or act as vectors for intercellular communication. However, little is known about the involvement of EVs in microbial interactions in the marine environment(7). Here we investigated the signalling role of EVs produced during interactions between the cosmopolitan alga Emiliania huxleyi and its specific virus (EhV, Phycodnaviridae)(8), which leads to the demise of these large-scale oceanic blooms(9,10). We found that EVs are highly produced during viral infection or when bystander cells are exposed to infochemicals derived from infected cells. These vesicles have a unique lipid composition that differs from that of viruses and their infected host cells, and their cargo is composed of specific small RNAs that are predicted to target sphingolipid metabolism and cell-cycle pathways. EVs can be internalized by E. huxleyi cells, which consequently leads to a faster viral infection dynamic. EVs can also prolong EhV half-life in the extracellular milieu. We propose that EVs are exploited by viruses to sustain efficient infectivity and propagation across E. huxleyi blooms. As these algal blooms have an immense impact on the cycling of carbon and other nutrients(11,12), this mode of cell-cell communication may influence the fate of the blooms and, consequently, the composition and flow of nutrients in marine microbial food webs.
Falvo E., Tremante E., Arcovito A., Papi M., Elad N., Boffi A., Morea V., Conti G., Toffoli G., Fracasso G., Giacomini P. & Ceci P. (2016) Biomacromolecules. 17, 2, p. 514-522
A novel human ferritin-based nanocarrier, composed of 24 modified monomers able to auto-assemble into a modified protein cage, was produced and used as selective carrier of anti-tumor payloads. Each modified monomer derives from the genetic fusion of two distinct modules, namely the heavy chain of human ferritin (HFt) and a stabilizing/protective PAS polypeptide sequence rich in proline (P), serine (S), and alanine (A) residues. Two genetically fused protein constructs containing PAS polymers with 40- and 75-residue lengths, respectively, were compared. They were produced and purified as recombinant proteins in Escherichia coli at high yields. Both preparations were highly soluble and stable in vitro as well as in mouse plasma. Size-exclusion chromatography, dynamic light scattering, and transmission electron microscopy results indicated that PASylated ferritins are fully assembled and highly monodispersed. In addition, yields and stability of encapsulated doxorubicin were significantly better for both HFt-PAS proteins than for wild-type HFt. Importantly, PAS sequences considerably prolonged the half-life of HFt in the mouse bloodstream. Finally, our doxorubicin-loaded nanocages preserved the pharmacological activity of the drug. Taken together, these results indicate that both of the developed HFt-PAS fusion proteins are promising nanocarriers for future applications in cancer therapy.
Tenne R., Pedetti S., Kazes M., Ithurria S., Houben L., Nadal B., Oron D. & Dubertret B. (2016) Physical Chemistry Chemical Physics. 18, 22, p. 15295-15303
Cadmium chalcogenide nanoplatelet (NPL) synthesis has recently witnessed a significant advance in the production of more elaborate structures such as core/shell and core/crown NPLs. However, controlled doping in these structures has proved difficult because of the restrictive synthetic conditions required for 2D anisotropic growth. Here, we explore the incorporation of tellurium (Te) within CdSe NPLs with Te concentrations ranging from doping to alloying. For Te concentrations higher than similar to 30%, the CdSexTe(1-x) NPLs show emission properties characteristic of an alloyed material with a bowing of the band gap for increased concentrations of Te. This behavior is in line with observations in bulk samples and can be put in the context of the transition from a pure material to an alloy. In the dilute doping regime, CdSe: Te NPLs, in comparison to CdSe NPLs, show a distinct photoluminescence (PL) red shift and prolonged emission lifetimes (LTs) associated with Te hole traps which are much deeper than in bulk samples. Furthermore, single particle spectroscopy reveals dramatic modifications in PL properties. In particular, doped NPLs exhibit photon antibunching and emission dynamics significantly modified compared to undoped or alloyed NPLs.
Aronovitch E., Kalisman P., Houben L., Amirav L. & Bar-Sadan M. (2016) Chemistry of Materials. 28, 5, p. 1546-1552
The displacement of single atoms may have detrimental effects on the functionality of nanoparticle photocatalysts. Understanding the atomic-scale phenomena is the key to the rational design of photocatalysts to prevent undesired events during their synthesis and operation. Here, we used high resolution TEM to study such phenomena within seeded rods of CdSe in CdS, mounted with bimetallic particles of Au and Pd. We show the mobility of Pd atoms during deposition and photocatalysis and relate that to photocatalyst degradation processes that limit overall photocatalyst efficiency. We also show the advantage of the bimetallic composition and the specific internal rearrangement of the two metals for better efficiency and stability to provide a more generalized understanding of prospective design principles.
Elbaum M., Wolf S. G. & Houben L. (2016) MRS Bulletin. 41, 7, p. 542-548
The electron microscope has made paramount contributions to understanding the structure of biological molecules, cells, and tissues. In general, the most faithful preservation of biological specimens and other soft-organic materials is achieved through cryogenic fixation. The embedding medium is the native aqueous environment itself, immobilized in vitrified form by rapid or pressurized cooling. Until recently, imaging of such vitrified thin specimens by electron cryo-microscopy has been nearly synonymous with wide-field transmission electron microscopy (TEM). Several new approaches have entered the cryo-microscopy field, including soft x-ray imaging, serial surface imaging using focused ion beam scanning electron microscopy, phase plates, and scanning TEM (STEM). In this article, we focus on the STEM method and its adaptation to biological cryo-microscopy. Cryogenic imaging of unstained specimens by STEM introduces specific challenges. Difficulties were long considered insurmountable, and the potential advantages were underappreciated. Future developments in experimental setup and detector technologies will allow for extension of the method to thicker specimens with improved resolution and analytic capabilities.
Mangel S., Houben L. & Bar Sadan M. (2016) Nanoscale. 8, 40, p. 17568-17572
On the atomic scale, the exact engineering of interfaces affects the overall properties of functional nanostructures. One factor that is considered both fundamental and practical in determining the structural features of interfaces is the lattice mismatch, but zooming into the atomic scale reveals new data, which suggest that this paradigm should be reconsidered. Here, we used advanced transmission electron microscopy techniques to image, with atomic resolution, the core-shell interfaces of a strain-free system (CdSe@CdSe) and of a strain-induced system (CdSe@CdS). Then, we analyzed the pattern of stacking fault formation in these particles and correlated the location of the stacking faults with the synthetic procedure. We found that, in the strain-free system, the formation of stacking faults is substantial and the faults are located mostly at the core-shell interface, in a pattern that was surprisingly similar to that observed in the strain-induced system. Therefore, we conclude that the formation of faults within the nanoparticles results mainly from the interaction between the last atomic layer and the growth solution, and it is only weakly correlated with lattice mismatch. This finding is important for the design of defect-engineering in multi-step syntheses.
Ben Shoham S. A., Rot C., Stern T., Krief S., Akiva A., Dadosh T., Sabany H., Lu Y., Kadler K. E. & Zelzer E. (2016) Development (Cambridge). 143, 21, p. 3933-3943
Recently, blood vessels have been implicated in the morphogenesis of various organs. The vasculature is also known to be essential for endochondral bone development, yet the underlying mechanism has remained elusive. We show that a unique composition of blood vessels facilitates the role of the endothelium in bone mineralization and morphogenesis. Immunostaining and electron microscopy showed that the endothelium in developing bones lacks basement membrane, which normally isolates the blood vessel from its surroundings. Further analysis revealed the presence of collagen type I on the endothelial wall of these vessels. Because collagen type I is the main component of the osteoid, we hypothesized that the bone vasculature guides the formation of the collagenous template and consequently of the mature bone. Indeed, some of the bone vessels were found to undergo mineralization. Moreover, the vascular pattern at each embryonic stage prefigured the mineral distribution pattern observed one day later. Finally, perturbation of vascular patterning by overexpressing Vegf in osteoblasts resulted in abnormal bone morphology, supporting a role for blood vessels in bone morphogenesis. These data reveal the unique composition of the endothelium in developing bones and indicate that vascular patterning plays a role in determining bone shape by forming a template for deposition of bone matrix.
Addadi L., Varsano N., Kapishnikov S., Pereiro E., Shimoni E., Dadosh T., Jin X., Kruth H. & Leiserowitz L. (2016) Journal of the American Chemical Society. 138, 45, p. 14931-14940
We have developed a high resolution correlative method involving cryo-soft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM), which provides information in three dimensions on large cellular volumes at 70 nm resolution. Cryo-SXT morphologically identified and localized aggregations of carbon-rich materials. STORM identified specific markers on the desired epitopes, enabling colocalization between the identified objects, in this case cholesterol crystals, and the cellular environment. The samples were studied under ambient and cryogenic conditions without dehydration or heavy metal staining. The early events of cholesterol crystal development were investigated in relation to atherosclerosis, using as model macrophage cell cultures enriched with LDL particles. Atherosclerotic plaques build up in arteries in a slow process involving cholesterol crystal accumulation. Cholesterol crystal deposition is a crucial stage in the pathological cascade. Our results show that cholesterol crystals can be identified and imaged at a very early stage on the cell plasma membrane and in intracellular locations. This technique can in principle be applied to other biological samples where specific molecular identification is required in conjunction with high resolution 3D-imaging.
Lippert L., Hallock J., Dadosh T., Diroll B., Murray C. & Goldman Y. (2016) Bioconjugate Chemistry. 27, 3, p. 562-568
We developed methods to solubilize, coat, and functionalize with NeutrAvidin elongated semiconductor nanocrystals (quantum nanorods, QRs) for use in single molecule polarized fluorescence microscopy. Three different ligands were compared with regard to efficacy for attaching NeutrAvidin using the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Biotin-4-fluorescene (B4F), a fluorophore that is quenched when bound to avidin proteins, was used to quantify biotin binding activity of the NeutrAvidin coated QRs and biotin binding activity of commercially available streptavidin coated quantum dots (QDs). All three coating methods produced QRs with NeutrAvidin coating density comparable to the streptavidin coating density of the commercially available quantum dots (QDs) in the B4F assay. One type of QD available from the supplier (ITK QDs) exhibited ∼5-fold higher streptavidin surface density compared to our QRs, whereas the other type of QD (PEG QDs) had 5-fold lower density. The number of streptavidins per QD increased from ∼7 streptavidin tetramers for the smallest QDs emitting fluorescence at 525 nm (QD525) to ∼20 tetramers for larger, longer wavelength QDs (QD655, QD705, and QD800). QRs coated with NeutrAvidin using mercaptoundecanoicacid (MUA) and QDs coated with streptavidin bound to biotinylated cytoplasmic dynein in single molecule TIRF microscopy assays, whereas Poly(maleic anhydride-alt-1-ocatdecene) (PMAOD) or glutathione (GSH) QRs did not bind cytoplasmic dynein. The coating methods require optimization of conditions and concentrations to balance between substantial NeutrAvidin binding vs tendency of QRs to aggregate and degrade over time.
Aram L., Braun T., Braverman C., Kaplan Y., Ravid L., Levin-Zaidman S. & Arama E. (2016) Developmental Cell. 37, 1, p. 15-33
How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. We investigate caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. We show that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase β subunit (A-Sβ), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sβ and the GTP-specific SCSβ (G-Sβ), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sβ in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation.
Buzaglo-Azriel L., Kuperman Y., Tsoory M., Zaltsman Y., Shachnai L., Levin-Zaidman S., Bassat E., Michailovici I., Sarver A., Tzahor E., Haran M., Vernochet C. & Gross A. (2016) Cell Reports. 14, 7, p. 1602-1610
Mitochondrial carrier homolog 2 (MTCH2) is a repressor of mitochondrial oxidative phosphorylation (OXPHOS), and its locus is associated with increased BMI in humans. Here, we demonstrate that mice deficient in muscle MTCH2 are protected from diet-induced obesity and hyperinsulinemia and that they demonstrate increased energy expenditure. Deletion of muscle MTCH2 also increases mitochondrial OXPHOS and mass, triggers conversion from glycolytic to oxidative fibers, increases capacity for endurance exercise, and increases heart function. Moreover, metabolic profiling of mice deficient in muscle MTCH2 reveals a preference for carbohydrate utilization and an increase in mitochondria and glycolytic flux in muscles. Thus, MTCH2 is a critical player in muscle biology, modulating metabolism and mitochondria mass as well as impacting whole-body energy homeostasis.
Nativ-Roth E., Rechav K. & Porat Z. (2016) Thin Solid Films. 603, p. 88-96
Silver and gold were spontaneously deposited on porous silicon (PSi) by immersion-plating. Each metal formed crystallites of typical shapes on top of the PSi layer. Deposition of these metals inside the pores could be achieved by performing the immersion-plating in an ultrasonic bath. Top view and cross-section scanning electron microscope images show slight penetration of silver into the pores but massive filling of gold, to depth of several hundreds of nanometers.
Shadmi N., Kremen A., Frenkel Y., Lapin Z. J., Machado L. D., Legoas S. B., Bitton O., Rechav K., Popovitz-Biro R., Galvao D. S., Jorio A., Novotny L., Kalisky B. & Joselevich E. (2016) Nano Letters. 16, 4, p. 2152-2158
Carbon nanotubes are promising building blocks for various nanoelectronic components. A highly desirable geometry for such applications is a coil. However, coiled nanotube structures reported so far were inherently defective or had no free ends accessible for contacting. Here we demonstrate the spontaneous self-coiling of single-wall carbon nanotubes into defect-free coils of up to more than 70 turns with identical diameter and chirality, and free ends. We characterize the structure, formation mechanism, and electrical properties of these coils by different microscopies, molecular dynamics simulations, Raman spectroscopy, and electrical and magnetic measurements. The coils are highly conductive, as expected for defect-free carbon nanotubes, but adjacent nanotube segments in the coil are more highly coupled than in regular bundles of single-wall carbon nanotubes, owing to their perfect crystal momentum matching, which enables tunneling between the turns. Although this behavior does not yet enable the performance of these nanotube coils as inductive devices, it does point a clear path for their realization. Hence, this study represents a major step toward the production of many different nanotube coil devices, including inductors, electromagnets, transformers, and dynamos.
Reut G., Oksenberg E., Popovitz-Biro R., Rechav K. & Joselevich E. (2016) Journal of Physical Chemistry C. 120, 30, p. 17087-17100
A major challenge toward large-scale integration of nanowires is the control over their alignment and position. A possible solution to this challenge is the guided growth process, which enables the synthesis of well-aligned horizontal nanowires that grow according to specific epitaxial or graphoepitaxial relations with the substrate. However, the guided growth of horizontal nanowires was demonstrated for a limited number of materials, most of which exhibit unintentional n-type behavior. Here we demonstrate the vapor-liquid-solid growth of guided horizontal ZnTe nanowires and nanowalls displaying p-type behavior on four different planes of sapphire. The growth directions of the nanowires are determined by epitaxial relations between the nanowires and the substrate or by a graphoepitaxial effect that guides their growth along nanogrooves or nanosteps along the surface. We characterized the crystallographic orientations and elemental composition of the nanowires using transmission electron microscopy and photoluminescence. The optoelectronic and electronic properties of the nanowires were studied by fabricating photodetectors and top-gate thin film transistors. These measurements showed that the guided ZnTe nanowires are p-type semiconductors and are photoconductive in the visible range. The guided growth of horizontal p-type nanowires opens up the possibility of parallel nanowire integration into functional systems with a variety of potential applications not available by other means.
Kerschnitzki M., Akiva A., Ben Shoham S. A., Koifman N., Shimoni E., Rechav K., Arraf A. A., Schultheiss T. M., Talmon Y., Zelzer E., Weiner S. & Addadi L. (2016) Bone. 83, p. 65-72
During bone formation in embryos, large amounts of calcium and phosphate are taken up and transported to the site where solid mineral is first deposited. The initial mineral forms in vesicles inside osteoblasts and is deposited as a highly disordered calcium phosphate phase. The mineral is then translocated to the extracellular space where it penetrates the collagen matrix and crystallizes. To date little is known about the transport mechanisms of calcium and phosphate in the vascular system, especially when high transport rates are needed and the concentrations of these ions in the blood serum may exceed the solubility product of the mineral phase. Here we used a rapidly growing biological model, the chick embryo, to study the bone mineralization pathway taking advantage of the fact that large amounts of bone mineral constituents are transported. Cryo scanning electron microscopy together with cryo energy dispersive X-ray spectroscopy and focused-ion beam imaging in the serial surface view mode surprisingly reveal the presence of abundant vesicles containing small mineral particles in the lumen of the blood vessels. Morphologically similar vesicles are also found in the cells associated with bone formation. This observation directly implicates the vascular system in solid mineral distribution, as opposed to the transport of ions in solution. Mineral particle transport inside vesicles implies that far larger amounts of the bone mineral constituents can be transported through the vasculature, without the danger of ectopic precipitation. This introduces a new stage into the bone mineral formation pathway, with the first mineral being formed far from the bone itself.
Milrot E., Mutsafi Y., Fridmann Sirkis S. Y., Shimoni E., Rechav K., Gurnon J. R., Van Etten E. J. L. & Minsky A. (2016) Cellular Microbiology. 18, 1, p. 3-16
The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway.
Vidavsky N., Akiva A., Kaplan-Ashiri I., Rechav K., Addadi L., Weiner S. & Schertel A. (2016) Journal of Structural Biology. 196, 3, p. 487-495
Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000 μm<sup>3</sup>) at high resolution (520 nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20 μm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues.
Charuvi D., Nevo R., Kaplan-Ashiri I., Shimoni E. & Reich Z. (2016) Jove-Journal Of Visualized Experiments. 2016, 112, e54066
Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (>100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and lightharvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.
Goldbart O., Cohen S. R., Kaplan-Ashiri I., Glazyrina P., Wagner D. H., Enyashin A. & Tenne R. (2016) Proceedings of the National Academy of Sciences of the United States of America. 113, 48, p. 13624-13629
The simple process of a liquid wetting a solid surface is controlled by a plethora of factors-surface texture, liquid droplet size and shape, energetics of both liquid and solid surfaces, as well as their interface. Studying these events at the nanoscale provides insights into the molecular basis of wetting. Nanotube wetting studies are particularly challenging due to their unique shape and small size. Nonetheless, the success of nanotubes, particularly inorganic ones, as fillers in composite materials makes it essential to understand how common liquids wet them. Here, we present a comprehensive wetting study of individual tungsten disulfide nanotubes by water. We reveal the nature of interaction at the inert outer wall and show that remarkably high wetting forces are attained on small, open-ended nanotubes due to capillary aspiration into the hollow core. This study provides a theoretical and experimental paradigm for this intricate problem.
Wolfe S. G., Mutsafi Y., Horowitz B., Elbaum M. & Fass D. (2016) Biophysical Journal. 110, 3, p. 23A
We recently demonstrated that cryo-scanning transmission electron tomography (CSTET) provides tomographic reconstructions of vitrified cells with superior information transfer at high tilts and for thicker specimens than defocus phase contrast (Wolf et al., 2014). In cryoSTEM, there are no image-forming lenses after the electron beam passes through the sample; detection is incoherent and inelastically scattered electrons provide usable contrast information. By obviating the need for zero-loss energy filtration, the STEM modality provides efficient use of electron dose, thereby minimizing specimen damage. Here we demonstrate the use of CSTET for obtaining highly detailed 3D architectures of organelles and macromolecular complexes in unstained, unfixed, and unsectioned cultured fibroblasts while simultaneously collecting analytical information from high-angle, incoherently scattered electrons. As a case in point, cryoSTEM tomograms revealed characteristic patterns of dense deposits sequestered in mitochondria. Energy-dispersive X-ray (EDX) spectroscopy of these deposits revealed calcium and phosphorus. Once the elemental identification was made, the STEM scattering signal could be interpreted quantitatively as a three-dimensional map of mitochondrial calcium deposition. This approach can be extended to identify and map other concentrations of elements in the cell heavier than the pervasive carbon, nitrogen, and oxygen, as we demonstrated for phosphorus in bacterial cells (Wolf et al., 2015). This study provides an example of how imaging with sensitivity to atomic number in whole cells will provide a new dimension in structural cell biology by correlating elemental composition to organelle morphology.
Solomonov I., Zehorai E., Talmi-Frank D., Wolf S. G., Shainskaya A., Zhuravlev A., Kartvelishvily E., Visse R., Levin Y., Kampf N., Jaitin D. A., David E., Amit I., Nagase H. & Sagi I. (2016) Proceedings of the National Academy of Sciences of the United States of America. 113, 39, p. 10884-10889
It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissueremodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.
Rez P., Aoki T., March K., Gur D., Krivanek O. L., Dellby N., Lovejoy T. C., Wolf S. G. & Cohen H. (2016) Nature Communications. 7, 10945
Vibrational spectroscopy in the electron microscope would be transformative in the study of biological samples, provided that radiation damage could be prevented. However, electron beams typically create high-energy excitations that severely accelerate sample degradation. Here this major difficulty is overcome using an 'aloof' electron beam, positioned tens of nanometres away from the sample: high-energy excitations are suppressed, while vibrational modes of energies
Elad N., De Strooper B., Lismont S., Hagen W., Veugelen S., Arimon M., Horre K., Berezovska O., Sachse C. & Chavez-Gutierrez L. (2015) Journal of Cell Science. 128, 3, p. 589-598
The structure and function of the gamma-secretase proteases are of great interest because of their crucial roles in cellular and disease processes. We established a novel purification protocol for the gamma-secretase complex that involves a conformation-and complexspecific nanobody, yielding highly pure and active enzyme. Using single particle electron microscopy, we analyzed the gamma-secretase structure and its conformational variability. Under steady-state conditions, the complex adopts three major conformations, which differ in overall compactness and relative position of the nicastrin ectodomain. Occupancy of the active or substrate-binding sites by inhibitors differentially stabilizes subpopulations of particles with compact conformations, whereas a mutation linked to familial Alzheimer disease results in enrichment of extended-conformation complexes with increased flexibility. Our study presents the gamma-secretase complex as a dynamic population of interconverting conformations, involving rearrangements at the nanometer scale and a high level of structural interdependence between subunits. The fact that protease inhibition or clinical mutations, which affect amyloid beta (A beta) generation, enrich for particular subpopulations of conformers indicates the functional relevance of the observed dynamic changes, which are likely to be instrumental for highly allosteric behavior of the enzyme.
Kalisman P., Houben L., Aronovitch E., Kauffmann Y., Bar Sadan S. M. & Amirav L. (2015) Journal of Materials Chemistry A. 3, 39, p. 19679-19682
We demonstrate improved efficiency for the photocatalytic water splitting reduction half reaction by employing Au-Pt bimetallic cocatalysts. We employed nanoparticle-based photocatalysts consisting of CdSe@CdS rods tipped with Au, Pt, Au-Pt core-shell or Au decorated with Pt islands. By tailoring the composition and morphology of the Au-Pt bimetallic catalysts, we achieved more than a fourfold increase in activity for hydrogen production compared to pure Pt.
Radovsky G., Popovitz-Biro R., Lorenz T., Joswig J. O., Seifert G., Houben L., Dunin-Borkowski R. E. & Tenne R. (2015) Journal of Materials Chemistry C. 4, 1, p. 89-93
Nanotubular structures from a new family of misfit compounds LnS-TaS<sub>2</sub> with (Ln = La, Ce, Nd, Ho, Er) and LaSe-TaSe<sub>2</sub> (some of them not known hitherto) are reported. Stress relaxation originating from the lattice mismatch between the alternating LnS(Se) and TaS<sub>2</sub>(Se) layers, combined with seaming of the dangling bonds in the rim, leads to the formation of a variety of nanotubular structures. Their structures are studied via scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), scanning transmission electron microscopy (STEM) and selected area electron diffraction (SAED). Tubules exhibiting a single folding vector for the LnS(Se) as well as TaS<sub>2</sub>(Se) layers were often found. The small values of the c-axis periodicities are indicative of a strong interaction between the two constituent layers which was also supported by Raman spectroscopy and theoretical calculations.
Meiron O. E., Houben L. & Bar-Sadan M. (2015) RSC Advances. 5, 107, p. 88108-88114
Nanoflowers of layered materials are promising building blocks for photocatalysis due to their unique morphology and exposed edges. Colloidal synthesis of MoS<sub>2</sub>, MoSe<sub>2</sub> and their alloys was used to produce fine nanoflowers with tunable composition. The alloys follow Vegard's law, showing homogeneous composition. They exhibit fully tunable optical properties, where the exciton positions change with alloy composition. Samples annealed under vacuum retain their fine morphology and their composition, which closely follows the feed ratio. Time series analysis was used to investigate the formation mechanism. The results support a growth mechanism of fast-precipitating amorphous material, followed by crystallization of a few layers of small sheets, which curl and tangle around themselves. Electron tomography of the time series reveals a perforated structure without a dense core, supporting the suggested growth mechanism. These structures show multiple exposed edge sites, making them promising materials for applications such as photocatalysis.
Moldavski O., Amen T., Levin-Zaidman S., Eisenstein M., Rogachev I., Brandis A., Kaganovich D. & Schuldiner M. (2015) Developmental Cell. 33, 5, p. 603-610
Exposing cells to folding stress causes a subset of their proteins to misfold and accumulate in inclusion bodies (IBs). IB formation and clearance are both active processes, but little is known about their mechanism. To shed light on this issue, we performed a screen with over 4,000 fluorescently tagged yeast proteins for co-localization with a model misfolded protein that marks IBs during folding stress. We identified 13 proteins that co-localize to IBs. Remarkably, one of these IB proteins, the uncharacterized and conserved protein Iml2, exhibited strong physical interactions with lipid droplet (LD) proteins. Indeed, we here show that IBs and LDs are spatially and functionally linked. We further demonstrate a mechanism for IB clearance via a sterol-based metabolite emanating from LDs. Our findings therefore uncover a function for Iml2 and LDs in regulating a critical stage of cellular proteostasis.
Goldman E. B., Zak A., Tenne R., Kartvelishvily E., Levin-Zaidman S., Neumann Y., Stiubea-Cohen R., Palmon A., Hovav A. H. & Aframian D. J. (2015) Tissue Engineering Part A. 21, 5-6, p. 1013-1023
Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS<sub>2</sub>]) nanotubes (INT-WS<sub>2</sub>) and fullerene-like nanoparticles (IF-WS<sub>2</sub>) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS<sub>2</sub> with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS<sub>2</sub> and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.
Kirchenbuechler D., Mutsafi Y., Horowitz B., Levin-Zaidman S., Fass D., Wolf S. G. & Elbaum M. (2015) AIMS Biophysics. 2, 3, p. 259-273
Cryo-tomography of intact, vitrified cells provides a three dimensional view of their structure and organization in a snapshot of the living state. Lacking heavy metal stains, tilt series images are typically produced by defocus phase contrast. Recently, a number of other methods have been introduced for 3D cryo-imaging. These include phase plate imaging, soft X-ray tomography, serial surface imaging using the focused ion beam-scanning electron microscope, and cryo-STEM tomography (CSTET). Here we explain the basis of the STEM setup and demonstrate the capabilities of CSTET to study unfixed, fully hydrated mammalian cells. Numerous cellular features are recognized in CSTET reconstructions, including membranes, vesicles, cytoskeleton, extracellular matrix, coated pits, and ribosomes. STEM signal acquisition configuration is more flexible than defocus phase contrast, and it imposes a much less severe spatial filter on the original images. Because low spatial frequency information is retained, the STEM tomographic reconstruction more faithfully represents the mass density distribution in the specimen.
Elkis Y., Bel S., Rahimi R., Lerer-Goldstein T., Levin-Zaidman S., Babushkin T., Shpungin S. & Nir U. (2015) PLoS ONE. 10, 12, e0145277
TMF/ARA160 is known to be a TATA element Modulatory Factor (TMF). It was initially identified as a DNA-binding factor and a coactivator of the Androgen receptor. It was also characterized as a Golgi-associated protein, which is essential for acrosome formation during functional sperm development. However, the molecular roles of TMF in this intricate process have not been revealed. Here, we show that during spermiogenesis, TMF undergoes a dynamic change of localization throughout the Golgi apparatus. Specifically, TMF translocates from the cis-Golgi to the trans-Golgi network and to the emerging vesicles surface, as the round spermatids develop. Notably, lack of TMF led to an abnormal spatial orientation of the Golgi and to the deviation of the trans-Golgi surface away from the nucleus of the developing round spermatids. Concomitantly, pro-acrosomal vesicles derived from the TMF-/- Golgi lacked targeting properties and did not tether to the spermatid nuclear membrane thereby failing to form the acrosome anchoring scaffold, the acroplaxome, around the cellnucleus. Absence of TMF also perturbed the positioning of microtubules, which normally lie in proximity to the Golgi and are important for maintaining Golgi spatial orientation and dynamics and for chromatoid body formation, which is impaired in TMF-/- spermatids. In-silico evaluation combined with molecular and electron microscopic analyses revealed the presence of a microtubule interacting domain (MIT) in TMF, and confirmed the association of TMF with microtubules in spermatogenic cells. Furthermore, the MIT domain in TMF, along with microtubules integrity, are required for stable association of TMF with the Golgi apparatus. Collectively, we show here for the first time that a Golgi and microtubules associated protein is crucial for maintaining proper Golgi orientation during a cell developmental process.
Greenman R., Gorelik A., Sapir T., Baumgart J., Zamor V., Segal Salto S. M., Levin-Zaidman S., Aidinis V., Aoki J., Nitsch R., Vogt J. & Reiner O. (2015) Frontiers in Neuroscience. 9, MAR, 53
The intricate formation of the cerebral cortex requires a well-coordinated series of events, which are regulated at the level of cell-autonomous and non-cell autonomous mechanisms. Whereas cell-autonomous mechanisms that regulate cortical development are well-studied, the non-cell autonomous mechanisms remain poorly understood. A non-biased screen allowed us to identify Autotaxin (ATX) as a non-cell autonomous regulator of neural stem cells. ATX (also known as ENPP2) is best known to catalyze lysophosphatidic acid (LPA) production. Our results demonstrate that ATX affects the localization and adhesion of neuronal progenitors in a cell autonomous and non-cell autonomous manner, and strikingly, this activity is independent from its catalytic activity in producing LPA.
Pinnola A., Cazzaniga S., Alboresi A., Nevo R., Levin-Zaidman S., Reich Z. & Bassi R. (2015) Plant Cell. 27, 11, p. 3213-3227
Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.
Revach O., Weiner A., Rechav K., Sabanay I., Livne A. & Geiger B. (2015) Scientific Reports. 5, 9466
Invadopodia are actin-rich membrane protrusions through which cells adhere to the extracellular matrix and degrade it. In this study, we explored the mechanical interactions of invadopodia in melanoma cells, using a combination of correlative light and electron microscopy. We show here that the core actin bundle of most invadopodia interacts with integrin-containing matrix adhesions at its basal end, extends through a microtubule-rich cytoplasm, and at its apical end, interacts with the nuclear envelope and indents it. Abolishment of invadopodia by microtubules or src inhibitors leads to the disappearance of these nuclear indentations. Based on the indentation profile and the viscoelastic properties of the nucleus, the force applied by invadopodia is estimated to be in the nanoNewton range. We further show that knockdown of the LINC complex components nesprin 2 or SUN1 leads to a substantial increase in the prominence of the adhesion domains at the opposite end of the invadopodia. We discuss this unexpected, long-range mechanical interplay between the apical and basal domains of invadopodia, and its possible involvement in the penetration of invadopodia into the matrix.
Oksenberg E., Popovitz-Biro R., Rechav K. & Joselevich E. (2015) Advanced Materials. 27, 27, p. 3999-4005
Perfectly aligned horizontal ZnSe nanowires are obtained by guided growth, and easily integrated into high-performance blue-UV photodetectors. Their crystal phase and crystallographic orientation are controlled by the epitaxial relations with six different sapphire planes. Guided growth paves the way for the large-scale integration of nanowires into optoelectronic devices.
Lashbrooke J., Adato A., Lotan O., Alkan N., Tsimbalist T., Rechav K., Fernandez-Moreno J., Widemann E., Grausem B., Pinot F., Granell A., Costa F. & Aharoni A. (2015) Plant Physiology. 169, 4, p. 2553-2571
The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning.
Rotem O., Pasternak Z., Shimoni E., Belausov E., Porat Z., Pietrokovski S. & Jurkevitch E. (2015) Proceedings of the National Academy of Sciences of the United States of America. 112, 44, p. E6028-E6037
Predators feed on prey to acquire the nutrients necessary to sustain their survival, growth, and replication. In Bdellovibrio bacteriovorus, an obligate predator of Gram-negative bacteria, cell growth and replication are tied to a shift from a motile, free-living phase of search and attack to a sessile, intracellular phase of growth and replication during which a single prey cell is consumed. Engagement and sustenance of growth are achieved through the sensing of two unidentified prey-derived cues. We developed a novel ex vivo cultivation system for B. bacteriovorus composed of prey ghost cells that are recognized and invaded by the predator. By manipulating their content, we demonstrated that an early cue is located in the prey envelope and a late cue is found within the prey soluble fraction. These spatially and temporally separated cues elicit discrete and combinatory regulatory effects on gene transcription. Together, they delimit a poorly characterized transitory phase between the attack phase and the growth phase, during which the bdelloplast (the invaded prey cell) is constructed. This transitory phase constitutes a checkpoint in which the late cue presumably acts as a determinant of the prey's nutritional value before the predator commits. These regulatory adaptations to a unique bacterial lifestyle have not been reported previously.
Tsarfati Y., Strauss V., Kuhri S., Krieg E., Weissman H., Shimoni E., Baram J., Guldi D. & Rybtchinski B. (2015) Journal of the American Chemical Society. 137, 23, p. 7429-7440
The unique properties of carbon nanotubes (CNT) are advantageous for emerging applications. Yet, the CNT insolubility hampers their potential. Approaches based on covalent and noncovalent methodologies have been tested to realize stable dispersions of CNTs. Noncovalent approaches are of particular interest as they preserve the CNTs structures and properties. We report on hybrids, in which perylene diimide (PDI) amphiphiles are noncovalently immobilized onto single wall carbon nanotubes (SWCNT). The resulting hybrids were dispersed and exfoliated both in water and organic solvents in the presence of two different PDI derivatives, PP2b and PP3a. The dispersions were investigated using cryogenic transmission electron microscopy (cryo-TEM), providing unique structural insights into the exfoliation. A helical arrangement of PP2b assemblies on SWCNTs dominates in aqueous dispersions, while a single layer of PP2b and PP3a was found on SWCNTs in organic dispersions. The dispersions were probed by steady-state and time-resolved spectroscopies, revealing appreciable charge redistribution in the ground state, and an efficient electron transfer from SWCNTs to PDIs in the excited state. We also fabricated hybrid materials from the PP2b/SWCNT dispersions. A supramolecular membrane was prepared from aqueous dispersions and used for size-selective separation of gold nanoparticles. Hybrid buckypaper films were prepared from the organic dispersions. In the latter, high conductivity results from enhanced electronic communication and favorable morphology within the hybrid material. Our findings shed light onto SWCNT/dispersant molecular interactions, and introduce a versatile approach toward universal solution processing of SWCNT-based materials.
Elbaz-Alon Y., Eisenberg-Bord M., Shinder V., Stiller S. B., Shimoni E., Wiedemann N., Geiger T. & Schuldiner M. (2015) Cell Reports. 12, 1, p. 7-14
Communication between organelles is crucial for eukaryotic cells to function as one coherent unit. Animportant means of communication is through membrane contact sites, where two organelles come into close proximity allowing the transport of lipids and small solutes between them. Contact sites are dynamic in size and can change in response to environmental or cellular stimuli; however, how this is regulated has been unclear. Here, we show that Saccharomyces cerevisiae Lam6 resides in several central contact sites: ERMES (ER/mitochondria encounter structure), vCLAMP (vacuole and mitochondria patch), and NVJ (nuclear vacuolar junction). Weshow that Lam6 is sufficient for expansion of contact sites under physiological conditions and necessary for coordination of contact site size. Given that Lam6 is part of a large protein family and is conserved in vertebrates, our work opens avenues for investigating the underlying principles of organelle communication.
Dover R. S., Bitler A., Shimoni E., Trieu-Cuot P. & Shai Y. (2015) Nature Communications. 6, 7193
Cell-wall peptidoglycan (PG) of Gram-positive bacteria is a strong and elastic multi-layer designed to resist turgor pressure and determine the cell shape and growth. Despite its crucial role, its architecture remains largely unknown. Here using high-resolution multiparametric atomic force microscopy (AFM), we studied how the structure and elasticity of PG change when subjected to increasing turgor pressure in live Group B Streptococcus. We show a new net-like arrangement of PG, which stretches and stiffens following osmotic challenge. The same structure also exists in isogenic mutants lacking surface appendages. Cell aging does not alter the elasticity of the cell wall, yet destroys the net architecture and exposes single segmented strands with the same circumferential orientation as predicted for intact glycans. Together, we show a new functional PG architecture in live Gram-positive bacteria.
Dhanyasi N., Segal D., Shimoni E., Shinder V., Shilo B., VijayRaghavan K. & Schejter E. D. (2015) The Journal of Cell Biology. 211, 1, p. 191-203
Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell-cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast-myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell-cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process.
Charuvi D., Nevo R., Shimoni E., Naveh L., Zia A., Adam Z., Farrant J. M., Kirchhoff H. & Reich Z. (2015) Plant Physiology. 167, 4, p. 1554-1565
During desiccation, homoiochlorophyllous resurrection plants retain most of their photosynthetic apparatus, allowing them to resume photosynthetic activity quickly upon water availability. These plants rely on various mechanisms to prevent the formation of reactive oxygen species and/or protect their tissues from the damage they inflict. In this work, we addressed the issue of how homoiochlorophyllous resurrection plants deal with the problem of excessive excitation/electron pressures during dehydration using Craterostigma pumilum as a model plant. To investigate the alterations in the supramolecular organization of photosynthetic protein complexes, we examined cryoimmobilized, freeze-fractured leaf tissues using (cryo)scanning electron microscopy. These examinations revealed rearrangements of photosystem II (PSII) complexes, including a lowered density during moderate dehydration, consistent with a lower level of PSII proteins, as shown by biochemical analyses. The latter also showed a considerable decrease in the level of cytochrome f early during dehydration, suggesting that initial regulation of the inhibition of electron transport is achieved via the cytochrome b6f complex. Upon further dehydration, PSII complexes are observed to arrange into rows and semicrystalline arrays, which correlates with the significant accumulation of sucrose and the appearance of inverted hexagonal lipid phases within the membranes. As opposed to PSII and cytochrome f, the light-harvesting antenna complexes of PSII remain stable throughout the course of dehydration. Altogether, these results, along with photosynthetic activity measurements, suggest that the protection of retained photosynthetic components is achieved, at least in part, via the structural rearrangements of PSII and (likely) light-harvesting antenna complexes into a photochemically quenched state.
Sedova A., Bar G., Goldbart O., Ron R., Achrai B., Kaplan-Ashiri I., Brumfeld V., Zak A., Gvishi R., Wagner H. D. & Tenne R. (2015) Journal of Supercritical Fluids. 106, p. 9-15
Silica aerogels are unique solids with extremely high porosity (>99.5% air by volume), transparency and low density. With their high surface area and thermal resistivity they make excellent heat insulators. However, due to their fine structure they are fragile and therefore impractical for structural applications. To improve their mechanical properties we suggest incorporation of minute amounts of tungsten disulfide nanotubes. Nanotubes of tungsten disulfide are known for their high mechanical strength, strain and thermal stability. Adding some 0.11 wt% of tungsten disulfide nanotubes to a variety of polymers clearly lead to substantial improvement in their mechanical properties. The current study reports the preparation of silica aerogels compounded with small amounts of tungsten disulfide nanotubes. Three-point bending and uniaxial compression tests of the composite aerogel revealed 85% and 23% improvement in the composite material toughness, respectively.
Varsano N., Fargion I., Wolf S. G., Leiserowitz L. & Addadi L. (2015) Journal of the American Chemical Society. 137, 4, p. 1601-1607
Atherosclerosis is the major precursor of cardiovascular disease. The formation of cholesterol crystals in atherosclerotic plaques is associated with the onset of acute pathology. The cholesterol crystals induce physical injury in the plaque core, promoting cell apoptosis and triggering an increased inflammatory response. Herein we address the question of how cholesterol crystal formation occurs in atherosclerosis. We demonstrate that three-dimensional (3D) cholesterol crystals can undergo directed nucleation from bilayer membranes containing two-dimensional (2D) cholesterol crystalline domains. We studied crystal formation on supported lipid bilayers loaded with exogenous cholesterol and labeled using a monoclonal antibody that specifically recognizes ordered cholesterol arrays. Our findings show that 3D crystals are formed exclusively on the bilayer regions where there are segregated 2D cholesterol crystalline domains and that they form on the domains. This study has potentially significant implications for our understanding of the crucial step in the mechanism by which atherosclerotic lesions form.
Schreiber R., Cohen H., Leitus G., Wolf S. G., Zhou A., Que L. & Neumann R. (2015) Journal of the American Chemical Society. 137, 27, p. 8738-8748
Manganese(IV,V)-hydroxo and oxo complexes are often implicated in both catalytic oxygenation and water oxidation reactions. Much of the research in this area is designed to structurally and/or functionally mimic enzymes. On the other hand, the tendency of such mimics to decompose under strong oxidizing conditions makes the use of molecular inorganic oxide clusters an enticing alternative for practical applications. In this context it is important to understand the reactivity of conceivable reactive intermediates in such an oxide-based chemical environment. Herein, a polyfluoroxometalate (PFOM) monosubstituted with manganese, [NaH<sub>2</sub>(Mn-L)W<sub>17</sub>F<sub>6</sub>O<sub>55</sub>]<sup>q-</sup>, has allowed the isolation of a series of compounds, Mn(II, III, IV and V), within the PFOM framework. Magnetic susceptibility measurements show that all the compounds are high spin. XPS and XANES measurements confirmed the assigned oxidation states. EXAFS measurements indicate that Mn(II)PFOM and Mn(III)PFOM have terminal aqua ligands and Mn(V)PFOM has a terminal hydroxo ligand. The data are more ambiguous for Mn(IV)PFOM where both terminal aqua and hydroxo ligands can be rationalized, but the reactivity observed more likely supports a formulation of Mn(IV)PFOM as having a terminal hydroxo ligand. Reactivity studies in water showed unexpectedly that both Mn(IV)-OH-PFOM and Mn(V)-OH-PFOM are very poor oxygen-atom donors; however, both are highly reactive in electron transfer oxidations such as the oxidation of 3-mercaptopropionic acid to the corresponding disulfide. The Mn(IV)-OH-PFOM compound reacted in water to form O<sub>2</sub>, while Mn(V)-OH-PFOM was surprisingly indefinitely stable. It was observed that addition of alkali cations (K<sup>+</sup>, Rb<sup>+</sup>, and Cs<sup>+</sup>) led to the aggregation of Mn(IV)-OH-PFOM as analyzed by electron microscopy and DOSY NMR, while addition of Li<sup>+</sup> and Na<sup>+</sup> did not lead to aggregates. Aggregation leads to a lowering of the entropic barrier of the reaction without changing the free energy barrier. The observation that O<sub>2</sub> formation is fastest in the presence of Cs<sup>+</sup> and ∼fourth order in Mn(IV)-OH-PFOM supports a notion of a tetramolecular Mn(IV)-hydroxo intermediate that is viable for O<sub>2</sub> formation in an oxide-based chemical environment. A bimolecular reaction mechanism involving a Mn(IV)-hydroxo based intermediate appears to be slower for O<sub>2</sub> formation.
Wolf S. G., Rez P. & Elbaum M. (2015) Journal of Microscopy. 260, 2, p. 227-233
Bacterial cells often contain dense granules. Among these, polyphosphate bodies (PPBs) store inorganic phosphate for a variety of essential functions. Identification of PPBs has until now been accomplished by analytical methods that required drying or chemically fixing the cells. These methods entail large electron doses that are incompatible with low-dose imaging of cryogenic specimens. We show here that Scanning Transmission Electron Microscopy (STEM) of fully hydrated, intact, vitrified bacteria provides a simple means for mapping of phosphorus-containing dense granules based on quantitative sensitivity of the electron scattering to atomic number. A coarse resolution of the scattering angles distinguishes phosphorus from the abundant lighter atoms: carbon, nitrogen and oxygen. The theoretical basis is similar to Z contrast of materials science. EDX provides a positive identification of phosphorus, but importantly, the method need not involve a more severe electron dose than that required for imaging. The approach should prove useful in general for mapping of heavy elements in cryopreserved specimens when the element identity is known from the biological context.
Pardo M., Shuster-Meiseles T., Levin-Zaidman S., Rudich A. & Rudich Y. (2014) Environmental Science and Technology. 48, 6, p. 3457-3466
The cytotoxicity of tungsten disulfide nano tubes (INT-WS<sub>2</sub>) and inorganic fullerene-like molybdenum disulfide (IF-MoS<sub>2</sub>) nanoparticles (NPs) used in industrial and medical applications was evaluated in comparison to standard environmental particulate matter. The IF-MoS<sub>2</sub> and INT-WS<sub>2</sub> reside in vesicles/inclusion bodies, suggestive of endocytic vesicles. In cells representing the respiratory, immune and metabolic systems, both IF-MoS<sub>2</sub> and INT-WS<sub>2</sub> NPs remained nontoxic compared to equivalent concentrations (up to 100 μg/mL in the medium) of silica dioxide (SiO<sub>2</sub>), diesel engine-derived and carbon black NPs, which induced cell death. Associating with this biocompatibility of IF-MoS<sub>2</sub>\INT- WS<sub>2</sub>, we demonstrate in nontransformed human bronchial cells (NL-20) relative low induction of the pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α. Moreover, IF-MoS<sub>2</sub> and INT-WS<sub>2</sub> activated antioxidant response as measured by the antioxidant response element (ARE) using a luciferase reporter, and induced Nrf2-mediated Phase II detoxification genes. Collectively, our findings suggest that the lower cytotoxicity of IF-MoS <sub>2</sub> and INT-WS<sub>2</sub> NPs does not reflect general biological inertness. Rather, compared to other NP's, it likely results from decreased pro-inflammatory activation, but a comparable significant capacity to induce protective antioxidant/detoxification defense mechanisms.
From R., Eilam R., Bar-Lev D. D., Levin-Zaidman S., Tsoory M., LoPresti P., Sela M., Arnon R. & Aharoni R. (2014) GLIA. 62, 4, p. 649-665
Myelinogenesis in the mammal nervous system occurs predominantly postnatally. Glatiramer acetate (GA), a drug for the treatment for multiple sclerosis (MS), has been shown to induce immunomodulation and neuroprotection in the inflamed CNS in MS and in experimental autoimmune encephalomyelitis (EAE). Here we investigated whether GA can affect myelinogenesis and oligodendrogenesis in the developing nervous system under nonpathological conditions. Towards this end we studied myelination in mice injected daily by GA, at postnatal Days 7-21. Immunohistological and ultrastructural analyses revealed significant elevation in the number of myelinated axons as well as in the thickness of the myelin encircling them and their resulting g-ratios, in spinal cords of GA-injected mice compared with their PBS-injected littermates, at postnatal Day 14. Elevation in myelinated axons was detected also in the peripheral ventral roots of the motor nerves. GA induced also an increase in axonal diameter, implying an effect on the overall development of the nervous system. A prominent elevation in the amount of progenitor oligodendrocytes and their BrdU incorporation, as well as in mature oligodendrocytes indicated that the effect of GA is linked to increased proliferation and differentiation along the oligodendroglial maturation cascade. In addition, elevation in insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) was found in the white matter of the GA-injected mice. Furthermore, a functional advantage in rotating rod test was exhibited by GA-injected mice over their littermates at postnatal Day 21. These cumulative findings corroborate the beneficial effect of GA on oligodendrogenesis and myelination.
Davidi L., Shimoni E., Khozin-Goldberg I., Zamir A. & Pick U. (2014) Plant Physiology. 164, 4, p. 2139-2156
The halotolerant microalgae Dunaliella bardawil accumulates under nitrogen deprivation two types of lipid droplets: plastoglobuli rich in beta-carotene (beta C-plastoglobuli) and cytoplasmatic lipid droplets (CLDs). We describe the isolation, composition, and origin of these lipid droplets. Plastoglobuli contain beta-carotene, phytoene, and galactolipids missing in CLDs. The two preparations contain different lipid-associated proteins: major lipid droplet protein in CLD and the Prorich carotene globule protein in beta C-plastoglobuli. The compositions of triglyceride (TAG) molecular species, total fatty acids, and sn-1+ 3 and sn-2 positions in the two lipid pools are similar, except for a small increase in palmitic acid in plastoglobuli, suggesting a common origin. The formation of CLD TAG precedes that of beta C-plastoglobuli, reaching a maximum after 48 h of nitrogen deprivation and then decreasing. Palmitic acid incorporation kinetics indicated that, at early stages of nitrogen deprivation, CLD TAG is synthesized mostly from newly formed fatty acids, whereas in beta C-plastoglobuli, a large part of TAG is produced from fatty acids of preformed membrane lipids. Electron microscopic analyses revealed that CLDs adhere to chloroplast envelope membranes concomitant with appearance of small beta C-plastoglobuli within the chloroplast. Based on these results, we propose that CLDs in D. bardawil are produced in the endoplasmatic reticulum, whereas beta C-plastoglobuli are made, in part, from hydrolysis of chloroplast membrane lipids and in part, by a continual transfer of TAG or fatty acids derived from CLD.
Vidavsky N., Addadi S., Mahamid J., Shimoni E., Ben-Ezra D., Shpigel M., Weiner S. & Addadi L. (2014) Proceedings of the National Academy of Sciences of the United States of America. 111, 1, p. 39-44
Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo- SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5-1.5 μm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20-30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule.
Krieg E., Weissman H., Shimoni E., On (Ustinoy) (. A. B. & Rybtchinski B. (2014) Journal of the American Chemical Society. 136, 26, p. 9443-9452
Achieving supramolecular polymerization based on strong yet reversible bonds represents a significant challenge. A solution may be offered by perfluoroalkyl groups, which have remarkable hydrophobicity. We tested the idea that a perfluorooctyl chain attached to a perylene diimide amphiphile can dramatically enhance the strength of supramolecular bonding in aqueous environments. Supramolecular structures and polymerization thermodynamics of this fluorinated compound (1-F) were studied in comparison to its non-fluorinated analogue (1-H). Depending on the amount of organic cosolvent, 1-F undergoes cooperative or isodesmic aggregation. The switching between two polymerization mechanisms results from a change in polymer structure, as observed by cryogenic electron microscopy. 1-F showed exceptionally strong noncovalent binding, with the largest directly measured association constant of 1.7 X 10(9) M-1 in 75:25 water/THF mixture (v/v). In pure water, the association constant of 1-F is estimated to be at least in the order of 10(15) M-1 (based on extrapolation), 3 orders of magnitude larger than that of 1-H. The difference in aggregation strength between 1-F and 1-H can be explained solely on the basis of the larger surface area of the fluorocarbon group, rather than a unique nature of fluorocarbon hydrophobicity. However, differences in aggregation mechanism and cooperativity exhibited by 1-F appear to result from specific fluorocarbon conformational rigidity.
Schatz D., Shemi A., Rosenwasser S., Sabanay H., Wolf S. G., Ben-Dor S. & Vardi A. (2014) New Phytologist. 204, 4, p. 854-863
Marine photosynthetic microorganisms are the basis of marine food webs and are responsible for nearly 50% of the global primary production. Emiliania huxleyi forms massive oceanic blooms that are routinely terminated by large double-stranded DNA coccolithoviruses. The cellular mechanisms that govern the replication cycle of these giant viruses are largely unknown. We used diverse techniques, including fluorescence microscopy, transmission electron microscopy, cryoelectron tomography, immunolabeling and biochemical methodologies to investigate the role of autophagy in host-virus interactions. Hallmarks of autophagy are induced during the lytic phase of E.huxleyi viral infection, concomitant with up-regulation of autophagy-related genes (ATG genes). Pretreatment of the infected cells with an autophagy inhibitor causes a major reduction in the production of extracellular viral particles, without reducing viral DNA replication within the cell. The host-encoded Atg8 protein was detected within purified virions, demonstrating the pivotal role of the autophagy-like process in viral assembly and egress. We show that autophagy, which is classically considered as a defense mechanism, is essential for viral propagation and for facilitating a high burst size. This cellular mechanism may have a major impact on the fate of the viral-infected blooms, and therefore on the cycling of nutrients within the marine ecosystem. 10.1111/(ISSN)1469-8137
Wolf S. G., Houben L. & Elbaum M. (2014) Nature Methods. 11, 4, p. 423-428
Cryo-electron tomography (CET) of fully hydrated, vitrified biological specimens has emerged as a vital tool for biological research. For cellular studies, the conventional imaging modality of transmission electron microscopy places stringent constraints on sample thickness because of its dependence on phase coherence for contrast generation. Here we demonstrate the feasibility of using scanning transmission electron microscopy for cryo-tomography of unstained vitrified specimens (CSTET). We compare CSTET and CET for the imaging of whole bacteria and human tissue culture cells, finding favorable contrast and detail in the CSTET reconstructions. Particularly at high sample tilts, the CSTET signals contain more informative data than energy-filtered CET phase contrast images, resulting in improved depth resolution. Careful control over dose delivery permits relatively high cumulative exposures before the onset of observable beam damage. The increase in acceptable specimen thickness broadens the applicability of electron cryo-tomography.
Ben-Moshe A., Wolf S. G., Sadan M. B., Houben L., Fan Z., Govorov A. O. & Markovich G. (2014) Nature Communications. 5, 4302
A large number of inorganic materials form crystals with chiral symmetry groups. Enantioselectively synthesizing nanostructures of such materials should lead to interesting optical activity effects. Here we report the synthesis of colloidal tellurium and selenium nanostructures using thiolated chiral biomolecules. The synthesis conditions are tuned to obtain tellurium nanostructures with chiral shapes and large optical activity. These nanostructures exhibit visible optical and chiroptical responses that shift with size and are successfully simulated by an electromagnetic model. The model shows that they behave as chiral optical resonators. The chiral tellurium nanostructures are transformed into chiral gold and silver telluride nanostructures with very large chiroptical activity, demonstrating a simple colloidal chemistry path to chiral plasmonic and semiconductor metamaterials. These materials are natural candidates for studies related to interactions of chiral (bio)molecules with chiral inorganic surfaces, with relevance to asymmetric catalysis, chiral crystallization and the evolution of homochirality in biomolecules.
Golan N., Kartvelishvily E., Spiegel I., Salomon D., Sabanay H., Rechav K., Vainshtein A., Frechter S., Maik-Rachline G., Eshed Eisenbach E. Y., Momoi T. & Peles E. (2013) Journal of Neuroscience. 33, 27, p. 10950-10961
The interaction between myelinating Schwann cells and the axons they ensheath is mediated by cell adhesion molecules of the Cadm/Necl/SynCAM family. This family consists of four members: Cadm4/Necl4 and Cadm1/Necl2 are found in both glia and axons, whereas Cadm2/Necl3 and Cadm3/Necl1 are expressed by sensory and motor neurons. By generating mice lacking each of the Cadm genes, we now demonstrate that Cadm4 plays a role in the establishment of the myelin unit in the peripheral nervous system. Mice lacking Cadm4 (PGK-Cre/Cadm4(fl/fl)), but not Cadm1, Cadm2, or Cadm3, develop focal hypermyelination characterized by tomacula and myelin outfoldings, which are the hallmark of several Charcot-Marie-Tooth neuropathies. The absence of Cadm4 also resulted in abnormal axon-glial contact and redistribution of ion channels along the axon. These neuropathological features were also found in transgenic mice expressing a dominant-negative mutant of Cadm4 lacking its cytoplasmic domain in myelinating glia Tg(mbp-Cadm4dCT), as well as in mice lacking Cadm4 specifically in Schwann cells (DHH-Cre/Cadm4(fl/fl)). Consistent with these abnormalities, both PGK-Cre/Cadm4(fl/fl) and Tg(mbp-Cadm4dCT) mice exhibit impaired motor function and slower nerve conduction velocity. These findings indicate that Cadm4 regulates the growth of the myelin unit and the organization of the underlying axonal membrane.
Weiner A., Kapishnikov S., Shimoni E., Cordes S., Guttmann P., Schneider G. & Elbaum M. (2013) Journal of Structural Biology. 181, 1, p. 77-81
Soft X-ray cryo-microscopy (cryo-XT) offers an ideal complement to electron cryo-microscopy (cryo-EM). Cryo-XT is applicable to samples more than an order of magnitude thicker than cryo-EM, albeit at a more modest resolution of tens of nanometers. Furthermore, the natural contrast obtained in the "water-window" by differential absorption by organic matter vs water yields detailed images of organelles, membranes, protein complexes, and other cellular components. Cryo-XT is thus ideally suited for tomography of eukaryotic cells. The increase in sample thickness places more stringent demands on sample preparation, however. The standard method for cryo-EM, i.e., plunging to a cryogenic fluid such as liquid ethane, is no longer ideally suited to obtain vitrification of thick samples for cryo-XT. High pressure freezing is an alternative approach, most closely associated with freeze-substitution and embedding, or with electron cryo-microscopy of vitreous sections (CEMOVIS). We show here that high pressure freezing can be adapted to soft X-ray tomography of whole vitrified samples, yielding a highly reliable method that avoids crystallization artifacts and potentially offers improved imaging conditions in samples not amenable to plunge-freezing.
Kapishnikov S., Weiner A., Shimoni E., Schneider G., Elbaum M. & Leiserowitz L. (2013) Langmuir. 29, 47, p. 14595-14602
Crystallization of the malaria pigment hemozoin sequesters the toxic heme byproduct of hemoglobin digestion in Plasmodium-infected red blood cells (RBCs). Recently, we applied electron and X-ray imaging and diffraction methods to elucidate this process. We observed crystals oriented with their {100} faces at the inner membrane surface of the digestive vacuole (DV) of Plasmodium falciparum in parasitized RBCs. Modeling of the soft X-ray tomographic (SXT) images of a trophozoite-stage parasite indicated a 4-16 nm DV membrane thickness, suggesting a possible role for lipid multilayers. Here, we reanalyzed the trophozoite SXT images quantitatively via X-ray absorption to map the DV membrane thickness. Making use of the chemical structure and crystal density of the lipid, we found, predominantly, a bilayer 4.2 nm thick, and the remainder was interpreted as patches ∼8 nm thick. Image analysis of electron micrographs also yielded a 4-5 nm DV membrane thickness. The DV lipid membrane is thus mainly a bilayer, so induced hemozoin nucleation occurs primarily via the inner of the membrane's two leaflets. We argue that such a leaflet embodying mono- and di-acyl lipids with appropriate OH or NH bearing head groups may catalyse hemozoin nucleation by stereochemical and lattice match to the {100} crystal face, involving a two-dimensional nucleation aggregate of ∼100 molecules.
Shechter N., Zaltzman L., Weiner A., Brumfeld V., Shimoni E., Fridmann-Sirkis Y. & Minsky A. (2013) Journal of Biological Chemistry. 288, 35, p. 25659-25667
Background: Genome-wide homology search is inconsistent with the emerging view of bacterial genome morphology. Results: Stress-induced genome condensation proceeds through nonrandom convergence of sister chromosomes that culminates in spatial proximity of homologous sites. Conclusion: Chromosome convergence enables repair of double strand DNA breaks. Significance: Exposure to diverse stressful conditions primes bacteria to cope with detrimental DNA lesions.
Mutsafi Y., Shimoni E., Shimon A. & Minsky A. (2013) PLoS Pathogens. 9, 5
Although extensively studied, the structure, cellular origin and assembly mechanism of internal membranes during viral infection remain unclear. By combining diverse imaging techniques, including the novel Scanning-Transmission Electron Microscopy tomography, we elucidate the structural stages of membrane biogenesis during the assembly of the giant DNA virus Mimivirus. We show that this elaborate multistage process occurs at a well-defined zone localized at the periphery of large viral factories that are generated in the host cytoplasm. Membrane biogenesis is initiated by fusion of multiple vesicles, similar to 70 nm in diameter, that apparently derive from the host ER network and enable continuous supply of lipid components to the membrane-assembly zone. The resulting multivesicular bodies subsequently rupture to form large open single-layered membrane sheets from which viral membranes are generated. Membrane generation is accompanied by the assembly of icosahedral viral capsids in a process involving the hypothetical major capsid protein L425 that acts as a scaffolding protein. The assembly model proposed here reveals how multiple Mimivirus progeny can be continuously and efficiently generated and underscores the similarity between the infection cycles of Mimivirus and Vaccinia virus. Moreover, the membrane biogenesis process indicated by our findings provides new insights into the pathways that might mediate assembly of internal viral membranes in general.
Sorkin R., Kampf N., Dror Y., Shimoni E. & Klein J. (2013) Biomaterials. 34, 22, p. 5465-5475
Phosphatidylcholine (PC) vesicles have been shown to have remarkable boundary lubricating properties under physiologically-high pressures. Here we carry out a systematic study, using a surface force balance, of the normal and shear (frictional) forces between two opposing surfaces bearing different PC vesicles across water, to elucidate the origin of these properties. Small unilamellar vesicles (SUVs, diameters
Schatz D., Nagar E., Sendersky E., Parnasa R., Zilberman S., Carmeli S., Mastai Y., Shimoni E., Klein E., Yeger O., Reich Z. & Schwarz R. (2013) Environmental Microbiology. 15, 6, p. 1786-1794
Biofilms are consortia of bacteria that are held together by an extracellular matrix. Cyanobacterial biofilms, which are highly ubiquitous and inhabit diverse niches, are often associated with biological fouling and cause severe economic loss. Information on the molecular mechanisms underlying biofilm formation in cyanobacteria is scarce. We identified a mutant of the cyanobacterium Synechococcus elongatus, which unlike the wild type, developed biofilms. This biofilm-forming phenotype is caused by inactivation of homologues of type II secretion /type IV pilus assembly systems and is associated with impairment of protein secretion. The conditioned medium from a wild-type culture represses biofilm formation by the secretion-mutants. This suggested that the planktonic nature of the wild-type strain is a result of a self-suppression mechanism, which depends on the deposition of a factor to the extracellular milieu. We also identified two genes that are essential for biofilm formation. Transcript levels of these genes are elevated in the mutant compared with the wild type, and are initially decreased in mutant cells cultured in conditioned medium of wild-type cells. The particular niche conditions will determine whether the inhibitor will accumulate to effective levels and thus the described mechanism allows switching to a sessile mode of existence.
Krieg E., Albeck S., Weissman H., Shimoni E. & Rybtchinski B. (2013) PLoS ONE. 8, 5, e63188
Membrane separation of biomolecules and their application in biocatalysis is becoming increasingly important for biotechnology, demanding the development of new biocompatible materials with novel properties. In the present study, an entirely noncovalent water-based material is used as a membrane for size-selective separation, immobilization, and biocatalytic utilization of proteins. The membrane shows stable performance under physiological conditions, allowing filtration of protein mixtures with a 150 kDa molecular weight cutoff (∼8 nm hydrodynamic diameter cutoff). Due to the biocompatibility of the membrane, filtered proteins stay functionally active and retained proteins can be partially recovered. Upon filtration, large enzymes become immobilized within the membrane. They exhibit stable activity when subjected to a constant flux of substrates for prolonged periods of time, which can be used to carry out heterogeneous biocatalysis. The noncovalent membrane material can be easily disassembled, purified, reassembled, and reused, showing reproducible performance after recycling. The robustness, recyclability, versatility, and biocompatibility of the supramolecular membrane may open new avenues for manipulating biological systems.
Manukovsky N., Sanders E., Matalon E., Wolf S. G. & Goldfarb D. (2013) Molecular Physics. 111, 18-19, p. 2887-2896
Nitroxide spin-labelled lipid analogues are often used to study model membrane properties using EPR spectroscopy. Whereas in liquid phase membranes the spin label assumes, on average, its putative location, in gel phases and frozen membrane, depending on its position along the acyl chain, it may exhibit a different average location. Here we used <sup>2</sup>H three-pulse Electron Spin Echo Envelope Modulation (ESEEM) of phospholipid spin probes, combined with various deuteration schemes to detect the effect of the model membrane curvature and cholesterol on vertical migrations of the spin label. We compared large and small unilamellar 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles with and without cholesterol (10%). The vertical displacement of the spin label was manifested as an apparently flat trans-membrane profile of water concentration and of label proximity to the head group choline. The spin-label propensity to migrate was found to increase with vesicle curvature and decrease in the presence of cholesterol. This in turn reflects the effect of packing and ordering of the membrane lipids. The results show that in curved vesicles lacking cholesterol, the label attached to carbon 16 may travel as far high along the membrane normal as the location of the label on carbon 5, due to the presence of U-shaped lipid conformations. This phenomenon must be taken into account when using spin-labelled lipids as membrane depth markers or to trace trans-membrane profiles.
Bharat T. A. M., Zbaida D., Eisenstein M., Frankenstein Z., Mehlman T., Weiner L., Sorzano C. O. S., Barak Y., Albeck S., Briggs J. A. G., Wolf S. G. & Elbaum M. (2013) Structure. 21, 7, p. 1158-1167
Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity.
Saar Dover D. R., Bitler A., Nezer R., Shmuel-Galia L., Firon A., Shimoni E., Trieu-Cuot P. & Shai Y. (2012) PLoS Pathogens. 8, 9
Cationic antimicrobial peptides (CAMPs) serve as the first line of defense of the innate immune system against invading microbial pathogens. Gram-positive bacteria can resist CAMPs by modifying their anionic teichoic acids (TAs) with D-alanine, but the exact mechanism of resistance is not fully understood. Here, we utilized various functional and biophysical approaches to investigate the interactions of the human pathogen Group B Streptococcus (GBS) with a series of CAMPs having different properties. The data reveal that: (i) D-alanylation of lipoteichoic acids (LTAs) enhance GBS resistance only to a subset of CAMPs and there is a direct correlation between resistance and CAMPs length and charge density; (ii) resistance due to reduced anionic charge of LTAs is not attributed to decreased amounts of bound peptides to the bacteria; and (iii) Dalanylation most probably alters the conformation of LTAs which results in increasing the cell wall density, as seen by Transmission Electron Microscopy, and reduces the penetration of CAMPs through the cell wall. Furthermore, Atomic Force Microscopy reveals increased surface rigidity of the cell wall of the wild-type GBS strain to more than 20-fold that of the dltA mutant. We propose that D-alanylation of LTAs confers protection against linear CAMPs mainly by decreasing the flexibility and permeability of the cell wall, rather than by reducing the electrostatic interactions of the peptide with the cell surface. Overall, our findings uncover an important protective role of the cell wall against CAMPs and extend our understanding of mechanisms of bacterial resistance.
Kapishnikov S., Weiner A., Shimoni E., Guttmann P., Schneider G., Dahan-Pasternak N., Dzikowski R., Leiserowitz L. & Elbaum M. (2012) Proceedings of the National Academy of Sciences of the United States of America. 109, 28, p. 11188-11193
Heme detoxification is a critical step in the life cycle of malariaca-using parasites, achieved by crystallization into physiologically insoluble hemozoin. The mode of nucleation has profound implications for understanding the mechanism of action of antimalarial drugs that inhibit hemozoin growth. Several lines of evidence point to involvement of acylglycerol lipids in the nucleation process. Hemozoin crystals have been reported to form within lipid nanospheres; alternatively, it has been found in vitro that they are nucleated at an acylglycerol lipid-water interface.We have applied cryogenic soft X-ray tomography and three-dimensional electron microscopy to address the location and orientation of hemozoin crystals within the digestive vacuole (DV), as a signature of their nucleation and growth processes. Cryogenic soft X-ray tomography in the "water window" is particularly advantageous because contrast generation is based inherently on atomic absorption. We find that hemozoin nucleation occurs at the DV inner membrane, with crystallization occurring in the aqueous rather than lipid phase. The crystal morphology indicates a common {100} orientation facing the membrane as expected of templated nucleation. This is consistent with conclusions reached by X-ray fluorescence and diffraction in a companion work. Uniform dark spheres observed in the parasite were identified as hemoglobin transport vesicles. Their analysis supports a model of hemozoin nucleation primarily in the DV. Modeling of the contrast at the DV membrane indicates a 4-nm thickness with patches about three times thicker, possibly implicated in the nucleation.
Peled E., Pick U., Zarka A., Shimoni E., Leu S. & Boussiba S. (2012) Journal of Phycology. 48, 5, p. 1209-1219
Astaxanthin-rich oil globules in Haematococcus pluvialis display rapid light-induced peripheral migration that is unique to this organism and serves to protect the photosynthetic system from excessive light. We observed rapid light-induced peripheral migration that is associated with chlorophyll fluorescence quenching, whereas the recovery was slow. A simple assay to follow globule migration, based on chlorophyll fluorescence level has been developed. Globule migration was induced by high intensity blue light, but not by high intensity red light. The electron transport inhibitor dichlorophenyl-dimethylurea did not inhibit globule migration, whereas the quinone analog (dibromo-methyl-isopropylbenzoquinone), induced globule migration even at low light. Actin microfilament-directed toxins, such as cytochalasin B and latrunculin A, inhibited the light-induced globule migration, whereas toxins against microtubules were ineffective. Electron microscopic (EM) imaging confirmed the cytoplasmic localization and peripheral migration of globules upon exposure to very high light (VHL). Scanning EM of freeze-fractured cells also revealed globules within cytoplasmic bridges traversing the chloroplast, presumably representing the pathway of migration. Close alignments of globules with endoplasmic reticulum (ER) membranes were also observed following VHL illumination. We propose that light-induced globule migration is regulated by the redox state of the photosynthetic electron transport system. Possible mechanisms of actin-based globule migration are discussed.
Goclaw-Binder H., Sendersky E., Shimoni E., Kiss V., Reich Z., Perelman A. & Schwarz R. (2012) Environmental Microbiology. 14, 3, p. 680-690
While tightly regulated, bacterial cell morphology may change substantially in response to environmental cues. Here we describe such changes in the cyanobacterium Synechococcus sp. strain PCC7942. Once maintained in stationary phase, these rod-shaped organisms stop dividing and elongate up to 50-fold. Increase in cell length of a thymidine-auxotroph strain upon thymidine starvation implies that inhibition of DNA replication underlies cell elongation. Elongation occurs under conditions of limiting phosphorus but sufficient nitrogen levels. Once proliferative conditions are restored, elongated cells divide asymmetrically instead of exhibiting the typical fission characterized by mid-cell constriction. The progeny are of length characteristic of exponentially growing cells and are proficient of further proliferation. We propose that the ability to elongate under conditions of cytokinesis arrest together with the rapid induction of cell division upon nutrient repletion represents a beneficial cellular mechanism operating under specific nutritional conditions.
Charuvi D., Kiss V., Nevo R., Shimoni E., Adam Z. & Reich Z. (2012) Plant Cell. 24, 3, p. 1143-1157
Chloroplasts of higher plants develop from proplastids, which are undifferentiated plastids that lack photosynthetic (thylakoid) membranes. In flowering plants, the proplastid-chloroplast transition takes place at the shoot apex, which consists of the shoot apical meristem (SAM) and the flanking leaf primordia. It has been believed that the SAM contains only proplastids and that these become chloroplasts only in the primordial leaves. Here, we show that plastids of the SAM are neither homogeneous nor necessarily null. Rather, their developmental state varies with the specific region and/or layer of the SAM in which they are found. Plastids throughout the L1 and L3 layers of the SAM possess fairly developed thylakoid networks. However, many of these plastids eventually lose their thylakoids during leaf maturation. By contrast, plastids at the central, stem cell-harboring region of the L2 layer of the SAM lack thylakoid membranes; these appear only at the periphery, near the leaf primordia. Thus, plastids in the SAM undergo distinct differentiation processes that, depending on their lineage and position, lead to either development or loss of thylakoid membranes. These processes continue along the course of leaf maturation.
Heyman Y., Buxboim A., Wolf S. G., Daube S. S. & Bar-Ziv R. H. (2012) Nature Nanotechnology. 7, 6, p. 374-378
Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.
Ridelman Y., Singh G., Popovitz-Biro R., Wolf S. G., Das S. & Klajn R. (2012) Small. 8, 5, p. 654-660
Well-defined metallic nanobowls can be prepared by extending the concept of a protecting group to colloidal synthesis. Magnetic nanoparticles are employed as "protecting groups" during the galvanic replacement of silver with gold. The replacement reaction is accompanied by spontantous dissociation of the protecting groups, leaving behind metallic nanobowls.
Gordon-Grossman M., Zimmermann H., Wolf S. G., Shai Y. & Goldfarb D. (2012) Journal Of Physical Chemistry B. 116, 1, p. 179-188
Studies of membrane peptide interactions at the molecular level are important for understanding essential processes such as membrane disruption or fusion by membrane active peptides. In a previous study, we combined several electron paramagnetic resonance (EPR) techniques, particularly continuous wave (CW) EPR, electron spin echo envelope modulation (ESEEM), and double electron-electron resonance (DEER) with Monte Carlo (MC) simulations to probe the conformation, insertion depth, and orientation with respect to the membrane of the membrane active peptide melittin. Here, we combined these EPR techniques with cryogenic transmission electron microscopy (cryo-TEM) to examine the effect of the peptide/phospholipid (P/PL) molar ratio, in the range of 1:400 to 1:25, on the membrane shape, lipids packing, and peptide orientation and penetration. Large unilamellar vesicles (LUVs) of DPPC/PG (7:3 dipalmitoylphosphatidylcholine/egg phosphatidylglycerol) were used as model membranes. Spin-labeled peptides were used to probe the peptide behavior whereas spin-labeled phspholipids were used to examine the membrane properties. The cryo-TEM results showed that melittin causes vesicle rupture and fusion into new vesicles with ill-defined structures. This new state was investigated by the EPR methods. In terms of the peptide, CW EPR showed decreased mobility, and ESEEM revealed increased insertion depth as the P/PL ratio was raised. DEER measurements did not reveal specific aggregates of melittin, thus excluding the presence of stable, well-defined pore structures. In terms of membrane properties, the CW EPR reported reduced mobility in both polar head and alkyl chain regions with increasing P/PL. ESEEM measurements showed that, as the P/PL ratio increased, a small increase in water content in the PL headgroup region took place and no change was observed in the alkyl chains part close to the hydrophilic region. In terms of lipid local density, opposite behavior was observed for the polar head and alkyl chain regions with increasing P/PL; while the DPPC density increased in the polar head region, it decreased in the alkyl chain region. These results are consistent with disruption of the lipid order and segregation of the PL constituents of the membrane as a consequence of the melittin binding. This work further demonstrates the applicability and potential of pulse EPR techniques for the study of peptide-membrane interactions.
Orbach M., Lahav M., Milko P., Wolf S. G. & van der Boom M. E. (2012) Angewandte Chemie - International Edition. 51, 29, p. 7142-7145
Nanoparticles (NPs) may be exploited to make practical materials that are capable of the selective detection of (bio)molecules.[17] Sensing with NPs often depends on the ability to selectively form aggregates. For instance, Mirkin et al. introduced a bio-barcode amplification method for ultrasensitive protein detection.[8] Another important study involves the detection of copper ions by hybrid AuNP assemblies in click chemistry.[9, 10] The structures of AuNPbased assemblies can also be controlled electrochemically or by light.[1113] However, despite these successes, controlling the properties and structure of NP-based assemblies with organic cross-linkers (CLs) still remains a challenge.[14] We have previously shown that the molecular geometry of CLs and the number of possible NP binding sites are related to the formation of hybrid AuNP assemblies and their associated optical properties.[15]
Weissman H., Ustinov A., Shimoni E., Cohen S. & Rybtchinski B. (2011) Polymers for Advanced Technologies. 22, 1, p. 133-138
Two-dimensional porous networks self-assembled in solution are rare, while maintaining the solution-phase network structure upon casting on solid supports presents a major challenge. We report on oligoarylacetylene bearing amphiphilic perylene diimide moieties that self-assemble into a two-dimensional porous network in aqueous solution that can be cast on surfaces, while maintaining the porous structure. The networks were characterized by cryogenic electron microscopy (cryo-TEM and cryo-SEM), and by atomic force microscopy, revealing formation of thin porous films (4-nm thick). The network can be cast on various solid surfaces, preserving its solution-phase structure. The design motif utilizing an oligoarylacetylene backbone with interacting amphiphilic pendants appears to be of wide utility for formation of novel assembly patterns. Copyright (C) 2010 John Wiley & Sons, Ltd.
Weiner A., Dahan-Pasternak N., Shimoni E., Shinder V., von Huth P., Elbaum M. & Dzikowski R. (2011) Cellular Microbiology. 13, 7, p. 967-977
The deadliest form of human malaria is caused by the protozoan parasite Plasmodium falciparum. The complex life cycle of this parasite is associated with tight transcriptional regulation of gene expression. Nuclear positioning and chromatin dynamics may play an important role in regulating P. falciparum virulence genes. We have applied an emerging technique of electron microscopy to construct a 3D model of the parasite nucleus at distinct stages of development within the infected red blood cell. We have followed the distribution of nuclear pores and chromatin throughout the intra-erythrocytic cycle, and have found a striking coupling between the distributions of nuclear pores and chromatin organization. Pore dynamics involve clustering, biogenesis, and division among daughter cells, while chromatin undergoes stage-dependent changes in packaging. Dramatic changes in heterochromatin distribution coincide with a previously identified transition in gene expression and nucleosome positioning during the mid-to-late schizont phase. We also found a correlation between euchromatin positioning at the nuclear envelope and the local distribution of nuclear pores, as well as a dynamic nuclear polarity during schizogony. These results suggest that cyclic patterns in gene expression during parasite development correlate with gross changes in cellular and nuclear architecture.
Krieg E., Weissman H., Shirman E., Shimoni E. & Rybtchinski B. (2011) Nature Nanotechnology. 6, 3, p. 141-146
Most practical materials are held together by covalent bonds, which are irreversible. Materials based on noncovalent interactions can undergo reversible self-assembly, which offers advantages in terms of fabrication, processing and recyclability, but the majority of noncovalent systems are too fragile to be competitive with covalent materials for practical applications, despite significant attempts to develop robust noncovalent arrays. Here, we report nanostructured supramolecular membranes prepared from fibrous assemblies in water. The membranes are robust due to strong hydrophobic interactions, allowing their application in the size-selective separation of both metal and semiconductor nanoparticles. A thin (12 μm) membrane is used for filtration (∼5 nm cutoff), and a thicker (45 μm) membrane allows for size-selective chromatography in the sub-5 nm domain. Unlike conventional membranes, our supramolecular membranes can be disassembled using organic solvent, cleaned, reassembled and reused multiple times.
Kirchhoff H., Hall C., Wood M., Herbstova M., Tsabari O., Nevo R., Charuvi D., Shimoni E. & Reich Z. (2011) Proceedings of the National Academy of Sciences of the United States of America. 108, 50, p. 20248-20253
The machinery that conducts the light-driven reactions of oxygenic photosynthesis is hosted within specialized paired membranes called thylakoids. In higher plants, the thylakoids are segregated into two morphological and functional domains called grana and stroma lamellae. A large fraction of the luminal volume of the granal thylakoids is occupied by the oxygen-evolving complex of photosystem II. Electron microscopy data we obtained on dark- and light-adapted Arabidopsis thylakoids indicate that the granal thylakoid lumen significantly expands in the light. Models generated for the organization of the oxygen-evolving complex within the granal lumen predict that the light-induced expansion greatly alleviates restrictions imposed on protein diffusion in this compartment in the dark. Experiments monitoring the redox kinetics of the luminal electron carrier plastocyanin support this prediction. The impact of the increase in protein mobility within the granal luminal compartment in the light on photosynthetic electron transport rates and processes associated with the repair of photodamaged photosystem II complexes is discussed.
Goldschmidt-Arzi M., Shimoni E., Sabanay H., Futerman A. H. & Addadi L. (2011) Journal of Structural Biology. 175, 1, p. 21-30
Lipid microdomains, also called lipid rafts, consisting of sphingolipids and cholesterol, play important roles in membrane trafficking and in signaling. Despite years of study of the composition, size, half-life and dynamic organization of these domains, many open questions remain about their precise characteristics. To address some of these issues, we have developed a new experimental approach involving the use of specific monoclonal antibodies as recognition tools. One such antibody was raised against a homogeneous, mixed, ordered monolayer phase comprised of 60:40. mol% cholesterol:C16-ceramide, and has been used previously to demonstrate the existence of C16-ceramide/cholesterol domains in the membranes of cultured cells. We now use a combination of quantitative fluorescence microscopy, immuno-transmission electron microscopy and immuno-scanning cryo-electron microscopy, optimized for the study of intracellular lipid antigens. In a variety of cultured cells, C16-ceramide/cholesterol structural domains were found at high levels in late endosomes and in the trans-Golgi network, but were not found at statistically significant levels in early endosomes, lysosomes or the endoplasmic reticulum. We discuss the relevance of these results to understanding the role of lipid lateral organization in biological membranes.
Tidhar Y., Weissman H., Wolf S. G., Gulino A. & Rybtchinski B. (2011) Chemistry-A European Journal. 17, 22, p. 6068-6075
Most molecular self-assembly strategies involve equilibrium systems, leading to a single thermodynamic product as a result of weak, reversible non-covalent interactions. Yet, strong non-covalent interactions may result in non-equilibrium self-assembly, in which structural diversity is achieved by forming several kinetic products based on a single covalent building block. We demonstrate that well-defined amphiphilic molecular systems based on perylene diimide/peptide conjugates exhibit kinetically controlled self-assembly in aqueous medium, enabling pathway-dependent assembly sequences, in which different organic nanostructures are evolved in a stepwise manner. The self-assembly process was characterized using UV/Vis circular dichroism (CD) spectroscopy, and cryogenic transmission electron microscopy (cryo-TEM). Our findings show that pathway-controlled self-assembly may significantly broaden the methodology of non-covalent synthesis.
Ziv K., Meir G., Harmelin A., Shimoni E., Klein E. & Neeman M. (2010) NMR in Biomedicine. 23, 5, p. 523-531
The iron storage protein, ferritin, provides an important endogenous MRI contrast that can be used to determine the level of tissue iron. In recent years the impact of modulating ferritin expression on MRI contrast and relaxation rates was evaluated by several groups, using genetically modified cells, viral gene transfer and transgenic animals. This paper reports the follow-up of transgenic mice that chronically over-expressed the heavy chain of ferritin (h-ferritin) in liver hepatocytes (liver-hfer mice) over a period of 2 years, with the aim of investigating the long-term effects of elevated level of h-ferritin on MR signal and on the well-being of the mice. Analysis revealed that aging liver-hfer mice, exposed to chronic elevated expression of h-ferritin, have increased R<sub>2</sub> values compared to WT. As expected for ferritin, R<sub>2</sub> difference was strongly enhanced at high magnetic field. Histological analysis of these mice did not reveal liver changes with prolonged over expression of ferritin, and no differences could be detected in other organs. Furthermore, dietary iron supplementation significantly affected MRI contrast, without affecting animal wellbeing, for both wildtype and ferritin over expressing transgenic mice. These results suggest the safety of ferritin over-expression, and support the use of h-ferritin as a reporter gene for MRI.
Levy-Lior A., Shimoni E., Schwartz O., Gavish-Regev E., Oron D., Oxford G., Weiner S. & Addadi L. (2010) Advanced Functional Materials. 20, 2, p. 320-329
Biological photonic systems composed of anhydrous guanine crystals evolved separately in several taxonomie groups. Here, two such systems found in fish and spiders, both of which make use of anhydrous guanine crystal plates to produce structural colors, are examined. Measurements of the photoniccrystal structures using cryo-SEM show that the crystal plates in both fish skin and spider integument are ∼20-nm thick. The reflective unit in the fish comprises stacks of single plates alternating with ∼ 230-nm-thick cytoplasm layers. In the spiders the plates are formed as doublet crystals, cemented by 30-nm layers of amorphous guanine, and are stacked with ∼200nm of cytoplasm between crystal doublets. They achieve light reflective properties through the control of crystal morphology and stack dimensions, reaching similar efficiencies of light reflectivity in both fish skin and spider integument. The structure of guanine plates in spiders are compared with the more common situation in which guanine occurs in the form of relatively unorganized prismatic crystals, yielding a matt white coloration.
Mahamid J., Aichmayer B., Shimoni E., Ziblat R., Li C., Siegel S., Paris O., Fratzl P., Weiner S. & Addadi L. (2010) Proceedings of the National Academy of Sciences of the United States of America. 107, 14, p. 6316-6321
The continuously forming fin bony rays of zebrafish represent a simple bone model system in which mineralization is temporally and spatially resolved. The mineralized collagen fibrils of the fin bones are identical in structure to those found in all known bone materials. We study the continuous mineralization process within the tissue by using synchrotron microbeam x-ray diffraction and small-angle scattering, combined with cryo-scanning electron microscopy. The former provides information on the mineral phase and the mineral particles size and shape, whereas the latter allows high-resolution imaging of native hydrated tissues. The integration of the two techniques demonstrates that new mineral is delivered and deposited as packages of amorphous calcium phosphate nanospheres, which transform into platelets of crystalline apatite within the collagen matrix.
Santosh G., Shirman E., Weissman H., Shimoni E., Pinkas I., Rudich Y. & Rybtchinski B. (2010) Journal Of Physical Chemistry B. 114, 45, p. 14389-14396
We report on the synthesis of organic dye-metal nanoparticle hybrids from two thiol-derivatized perylenediimide (PDI) ligands and 1.5 nm gold nanoparticles. The hybrids form spherical nanostructures when cast from 40% methanol/chloroform solution and toluene. The spherical aggregates are in the size range 50-230 nm in 40% MeOH/CHCl<sub>3</sub> mixture and 100-400 nm in toluene solution, as evidenced by transmission electron microscopy (TEM). Scanning electron microscopy (SEM) measurements show that these spherical aggregates are vesicles with a hollow interior. The π-π interactions of the perylenediimides are the predominant driving force leading to the aggregation of the hybrids, whereby the sizes of the nanospheres can be regulated via the PDI linker moiety and solvent choice. Femtosecond transient absorption studies of the hybrids reveal complex photophysical behavior involving electron transfer from the gold nanoparticles to the PDI moieties. This study shows that the formation of well-defined hybrid nanostructures as well as tuning their sizes can be achieved through employing a combination of the capping ligand choice and regulating the solvophobic interactions between the ligands.
Rubin N., Perugia E., Wolf S. G., Klein E., Fridkin M. & Addadi L. (2010) Journal of the American Chemical Society. 132, 12, p. 4242-4248
Amyloids are pathological fibrillar aggregates of proteins related to over 20 diseases. Amyloid fibers are characterized by the cross-beta motif, which is minimally defined as a series of beta-strands extended perpendicular to the fiber axis, joined by hydrogen bonds parallel to the fiber direction. Several structures, all in agreement with the cross-beta definition, have been proposed for specific amyloids. We study the correlation among the suprastructural chirality, molecular structure, and molecular chirality of amyloids. Here we investigate the suprastructure chirality of different (all-S) serum amyloid A (SAA) truncated peptides. We found that the suprastructure chirality of amyloid fibers from segments SAA(2-6), SAA(1-11) and the majority of those from SAA(2-9) is left-handed, which is consistent with the beta-sheet protofilament model. In contrast, SAA(1-12) and SAA(2-12) as well as SAA(1-12), where the C-terminal aspartic acid was point mutated to either leucine or alanine, form right-handed helical amyloid fibers. Such a suprastructure switch indicates a molecular change in the protofilament structure. This is supported by the behavior observed in the FTIR spectra, where the amide I peak of all of the right-handed fibers is red shifted relative to the left-handed amyloid fibers. This work is a case study where isolated short fragments of SAA containing the same amyloidogenic core sequence fold into different amyloid structures. We show that core sequences, supposed to start the misfolding aggregation of the full-length amyloid peptides, may have structures different from those assumed by the isolated segments.
Kol N., Tsvitov M., Hevroni L., Wolf S. G., Pang H. B., Kay M. S. & Rousso I. (2010) Journal of Virological Methods. 169, 1, p. 244-247
Elucidating the structure of the immature HIV-1 Gag core is an important aspect of understanding the biology of this virus. In doing so, preservation of the fragile Gag lattice is essential. In this study, the effects of purification methods on the structural and mechanical integrity of immature HIV-1 are examined. The results show that the morphological and mechanical properties of the virion are preserved to a significantly higher degree by Iodixanol (OptiPrep) purification compared to the standard sucrose method. In conclusion, these results indicate that OptiPrep instead of sucrose purification should be employed when conducting structural studies on the HIV-1 virion.
Bar Sadan M., Wolf S. G. & Houben L. (2010) Nanoscale. 2, 3, p. 423-428
Nanotubes and fullerene-like nanoparticles of various inorganic layered compounds have been studied extensively in recent years. Their characterisation on the atomic scale has proven essential for progress in synthesis as well as for the theoretical modelling of their physical properties. We show that with electron tomography it is possible to achieve a reliable reconstruction of the 3D structure of nested WS<sub>2</sub> or MoS<sub>2</sub> fullerene-like and nanotube structures with sub-nanometre resolution using electron microscopes that are not aberration-corrected. Model-based simulations were used to identify imaging parameters, under which structural features such as the shell structure can be retained in the tomogram reconstructed from bright-field micrographs. The isolation of a particle out of an agglomerate for the analysis of a single structure and its interconnection with other particles is facilitated through the tomograms. The internal structure of the layers within the particle alongside the shape and content of its internal void are reconstructed. The tomographic reconstruction yields insights regarding the growth process as well as structural defects, such as non-continuous layers, which relate to the lubrication properties.
Nevo R., Chuartzman S. G., Tsabari O., Reich Z., Charuvi D. & Shimoni E. (2009) . 30, p. 295-327
The primary events of oxygenic photosynthesis are carried out within intricate membrane lamellar systems called thylakoid networks. These networks, which are present in cyanobacteria, algae, and higher plants, accommodate all of the molecular complexes necessary for the light-driven reactions of photosynthesis and provide a medium for energy transduction. Here we describe the ultrastructure of thylakoid membranes and their three-dimensional organization in various organisms along the evolutionary tree. Along the way we discuss issues pertaining to the formation and maintenance of these membranes, the means by which they enable molecular traffic within and across them, and the manner by which they respond to short- and long-term variations in light conditions.
Gladnikoff M., Shimoni E., Gov N. & Rousso I. (2009) Biophysical Journal. 97, 9, p. 2419-2428
The assembly and budding of a new virus is a fundamental step in retroviral replication. Yet, despite substantial progress in the structural and biochemical characterization of retroviral budding, the underlying physical mechanism remains poorly understood, particularly with respect to the mechanism by which the virus overcomes the energy barrier associated with the formation of high membrane curvature during viral budding. Using atomic force, fluorescence, and transmission electron microscopy, we find that both human immunodeficiency virus and Moloney murine leukemia virus remodel the actin cytoskeleton of their host. These actin-filamentous structures assemble simultaneously with or immediately after the beginning of budding, and disappear as soon as the nascent virus is released from the cell membrane. Analysis of sections of cryopreserved virus-infected cells by transmission electron microscopy reveals similar actin filament structures emerging from every nascent virus. Substitution of the nucleocapsid domain implicated in actin binding by a leucine-zipper domain results in the budding of virus-like particles without remodeling of the cell's cytoskeleton. Notably, viruses carrying the modified nucleocapsid domains bud more slowly by an order of magnitude compared to the wild-type. The results of this study show that retroviruses utilize the cell cytoskeleton to expedite their assembly and budding.
Krieg E., Shirman E., Weissman H., Shimoni E., Wolf S. G., Pinkas I. & Rybtchinski B. (2009) Journal of the American Chemical Society. 131, 40, p. 14365-14373
Design of an extensive supramolecular three-dimensional network that is both robust and adaptive represents a significant challenge. The molecular system PP2b based on a perylene diimide chromophore (PDI) decorated with polyethylene glycol groups self-assembles in aqueous media into extended supramolecular fibers that form a robust three-dimensional network resulting in gelation. The self-assembled systems were characterized by cryo-TEM, cryo-SEM, and rheological measurements. The gel possesses exceptional robustness and multiple stimuli-responsiveness. Reversible charging of PP2b allows for switching between the gel state and fluid solution that is accompanied by switching on and off the material's birefringence. Temperature triggered deswelling of the gel leads to the (reversible) expulsion of a large fraction of the aqueous solvent. The dual sensibility toward chemical reduction and temperature with a distinct and interrelated response to each of these stimuli is pertinent to applications in the area of adaptive functional materials. The gel also shows strong absorption of visible light and good exciton mobility (elucidated using femtosecond transient absorption), representing an advantageous light harvesting system.
Zeev-Ben-Mordehai T., Paz A., Peleg Y., Toker L., Wolf S. G., Rydberg E. H., Sussman J. & Silman I. (2009) Protein Expression and Purification. 63, 2, p. 147-157
Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH<sub>2</sub>-terminal His<sub>6</sub>Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high β-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R<sub>H</sub> = 16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.
Golubkov G., Weissman H., Shirman E., Wolf S. G., Pinkas I. & Rybtchinski B. (2009) ANGEWANDTE CHEMIE-INTERNATIONAL EDITION. 48, 5, p. 926-930
(Chemical Equation Presented) Four from one: Nanoscale ribbons, tubes, vesicles, and platelets can be formed from the self-assembly of a single covalent unit, which is based on an amphiphilic perylene diimide functionalized with a terpyridine ligand (see picture). The assembly diversity arises from the encoding of multiple inputs through hydrophobic interactions and metal coordination.
Zauberman N., Mutsafi Y., Ben Halevy H. D., Shimoni E., Klein E., Xiao C., Sun S. & Minsky A. (2008) PLoS Biology. 6, 5, p. 1104-1114
Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the ''stargate'', allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane-containing viruses.
Nudelman F., Shimoni E., Klein E., Rousseau M., Bourrat X., Lopez E., Addadi L. & Weiner S. (2008) Journal of Structural Biology. 162, 2, p. 290-300
A key to understanding control over mineral formation in mollusk shells is the microenvironment inside the pre-formed 3-dimensional organic matrix framework where mineral forms. Much of what is known about nacre formation is from observations of the mature tissue. Although these studies have elucidated several important aspects of this process, the structure of the organic matrix and the microenvironment where the crystal nucleates and grows are very difficult to infer from observations of the mature nacre. Here, we use environmental- and cryo-scanning electron microscopy to investigate the organic matrix structure at the onset of mineralization in the nacre of two mollusk species: the bivalves Atrina rigida and Pinctada margaritifera. These two techniques allow the visualization of hydrated biological materials coupled with the preservation of the organic matrix close to physiological conditions. We identified a hydrated gel-like protein phase filling the space between two interlamellar sheets prior to mineral formation. The results are consistent with this phase being the silk-like proteins, and show that mineral formation does not occur in an aqueous solution, but in a hydrated gel-like medium. As the tablets grow, the silk-fibroin is pushed aside and becomes sandwiched between the mineral and the chitin layer.
Peretti D., Dahan N., Shimoni E., Hirschberg K. & Lev S. (2008) Molecular Biology of the Cell. 19, 9, p. 3871-3884
Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgi-mediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterol-binding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/ phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities.
Cohen-Ben-Lulu G. N., Francis N. R., Shimoni E., Noy D., Davidov Y., Prasad K., Sagi Y., Cecchini G., Johnstone R. M. & Eisenbach M. (2008) EMBO Journal. 27, 7, p. 1134-1144
The mechanism of function of the bacterial flagellar switch, which determines the direction of flagellar rotation and is essential for chemotaxis, has remained an enigma for many years. Here we show that the switch complex associates with the membrane-bound respiratory protein fumarate reductase (FRD). We provide evidence that FRD binds to preparations of isolated switch complexes, forms a 1:1 complex with the switch protein FliG, and that this interaction is required for both flagellar assembly and switching the direction of flagellar rotation. We further show that fumarate, known to be a clockwise/switch factor, affects the direction of flagellar rotation through FRD. These results not only uncover a new component important for switching and flagellar assembly, but they also reveal that FRD, an enzyme known to be primarily expressed and functional under anaerobic conditions in Escherichia coli, nonetheless, has important, unexpected functions under aerobic conditions.
Chuartzman S. G., Nevo R., Shimoni E., Charuvi D., Kiss V., Ohad I., Brumfeld V. & Reich Z. (2008) Plant Cell. 20, 4, p. 1029-1039
Adaptability of oxygenic photosynthetic organisms to fluctuations in light spectral composition and intensity is conferred by state transitions, short-term regulatory processes that enable the photosynthetic apparatus to rapidly adjust to variations in light quality. In green algae and higher plants, these processes are accompanied by reversible structural rearrangements in the thylakoid membranes. We studied these structural changes in the thylakoid membranes of Arabidopsis thaliana chloroplasts using atomic force microscopy, scanning and transmission electron microscopy, and confocal imaging. Based on our results and on the recently determined three-dimensional structure of higher-plant thylakoids trapped in one of the two major light-adapted states, we propose a model for the transitions in membrane architecture. The model suggests that reorganization of the membranes involves fission and fusion events that occur at the interface between the appressed (granal) and nonappressed (stroma lamellar) domains of the thylakoid membranes. Vertical and lateral displacements of the grana layers presumably follow these localized events, eventually leading to macroscopic rearrangements of the entire membrane network.
Medalsy I., Dgany O., Sowwan M., Cohen H., Yukashevska A., Wolf S. G., Wolf A., Koster A., Almog O., Marton I., Pouny Y., Altman A., Hoseyov O. S. & Porath D. (2008) Nano Letters. 8, 2, p. 473-477
Controlled formation of complex nanostructures is one of the main goals of nanoscience and nanotechnology. Stable Protein 1 (SP1) is a boiling-stable ring protein complex, 11 nm in diameter, which self-assembles from 12 identical monomers. SP1 can be utilized to form large ordered arrays; it can be easily modified by genetic engineering to produce various mutants; it is also capable of binding gold nanoparticles (GNPs) and thus forming protein-GNP chains made of alternating SP1s and GNPs. We report the formation and the protocols leading to the formation of those nanostructures and their characterization by transmission electron microscopy, atomic force microscopy, and electrostatic force microscopy. Further control over the GNP interdistances within the protein-GNP chains may lead to the formation of nanowires and structures that may be useful for nanoelectronics.
Bar Sadan S. M., Houben L., Wolf S. G., Enyashin A., Seifert G., Tenne R. & Urban K. (2008) Nano Letters. 8, 3, p. 891-896
We present the advancement of electron tomography for three-dimensional structure reconstruction of fullerene-like particles toward atomic-scale resolution. The three-dimensional reconstruction of nested molybdenum disulfide nanooctahedra is achieved by the combination of low voltage operation of the electron microscope with aberration-corrected phase contrast imaging. The method enables the study of defects and irregularities in the three-dimensional structure of individual fullerene-like particles on the scale of 2-3 Å. Control over shape, size, and atomic architecture is a key issue in synthesis and design of functional nanoparticles. Transmission electron microscopy (TEM) is the primary technique to characterize materials down to the atomic level, albeit the images are two-dimensional projections of the studied objects. Recent advancements in aberration-corrected TEM have demonstrated single atom sensitivity for light elements at subångström resolution. Yet, the resolution of tomographic schemes for three-dimensional structure reconstruction has not surpassed 1 nm<sup>3</sup>, preventing it from becoming a powerful tool for characterization in the physical sciences on the atomic scale. Here we demonstrate that negative spherical aberration imaging at low acceleration voltage enables tomography down to the atomic scale at reduced radiation damage. First experimental data on the three-dimensional reconstruction of nested molybdenum disulfide nanooctahedra is presented. The method is applicable to the analysis of the atomic architecture of a wide range of nanostructures where strong electron channeling is absent, in particular to carbon fullerenes and inorganic fullerenes.
Baram J., Shirman E., Ben-Shitrit N., Ustinov A., Weissman H., Pinkas I., Wolf S. G. & Rybtchinski B. (2008) Journal of the American Chemical Society. 130, 45, p. 14966-14967
Self-assembling systems, whose structure and function can be reversibly controlled in situ are of primary importance for creating multifunctional supramolecular arrays and mimicking the complexity of natural systems. Herein we report on photofunctional fibers self-assembled from perylene diimide cromophores, in which interactions between aromatic monomers can be attenuated through their reduction to anionic species that causes fiber fission. Oxidation with air restores the fibers. The sequence represents reversible supramolecular depolymerization-polymerization in situ and is accompanied by a reversible switching of photofunction.
Dym O., Albeck S., Unger T., Jacobovitch J., Branzburg A., Michael Y., Frenkiel-Krispin D., Wolf S. G. & Elbaum M. (2008) Proceedings of the National Academy of Sciences of the United States of America. 105, 32, p. 11170-11175
Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its wide-spread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners.
Paz Y., Shimoni E., Weiss M. & Pick U. (2007) Plant Physiology. 144, 3, p. 1407-1415
Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein ( TTf), which binds and internalizes Fe3(+) ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability.
Nevo R., Charuvi D., Shimoni E., Schwarz R., Kaplan A., Ohad I. & Reich Z. (2007) EMBO Journal. 26, 5, p. 1467-1473
Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher-plant chloroplasts, allowing water-soluble and lipid-soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane-bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes.
Sigal N., Lewinson O., Wolf S. G. & Bibi E. (2007) Biochemistry. 46, 17, p. 5200-5208
MdfA is a 410-residue-long secondary multidrug transporter from E. coli. Cells expressing MdfA from a multicopy plasmid exhibit resistance against a diverse group of toxic compounds, including neutral and cationic ones, because of active multidrug export. As a prerequisite for high-resolution structural studies and a better understanding of the mechanism of substrate recognition and translocation by MdfA, we investigated its biochemical properties and overall structural characteristics. To this end, we purified the β-dodecyl maltopyranoside (DDM)-solubilized protein using a 6-His tag and metal affinity chromatography, and size exclusion chromatography (SE-HPLC). Purified MdfA was analyzed for its DDM and phospholipid (PL) content, and tetraphenylphosphonium (TPP<sup>+</sup>)-binding activity. The results are consistent with MdfA being an active monomer in DDM solution. Furthermore, an investigation of two-dimensional crystals by electron crystallography and 3D reconstruction lent support to the notion that MdfA may also be monomeric in reconstituted proteoliposomes.
Frenkiel-Krispin D., Wolf S. G., Albeck S., Unger T., Peleg Y., Jacobovitch J., Michael Y., Daube S., Sharon M., Robinson C. V., Svergun D. I., Fass D., Tzfira T. & Elbaum M. (2007) Journal of Biological Chemistry. 282, 6, p. 3458-3464
Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss)DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.
Varsano T., Wolf S. G. & Pick U. (2006) Journal of Biological Chemistry. 281, 15, p. 10305-10315
Adaptation of the halotolerant alga Dunaliella salina to iron deprivation involves extensive changes of chloroplast morphology, photosynthetic activities, and induction of a major 45-kDa chloroplast protein termed Tidi. Partial amino acid sequencing of proteolytic peptides suggested that Tidi resembles chlorophyll a/b-binding proteins which compose light-harvesting antenna complexes (LHC) (Varsano, T., Kaftan, D., and Pick, U. (2003) J. Plant Nutr. 26, 2197-2210). Here we show that Tidi shares the highest amino acid sequence similarity with light-harvesting I chlorophyll a/b-binding proteins from higher plants but has an extended proline-rich N-terminal domain. The accumulation of Tidi is reversed by iron supplementation, and its level is inversely correlated with photosystem I (PS-I) reaction center proteins. In native gel electrophoresis, Tidi co-migrates with enlarged PS-I-LHC-I super-complexes. Single particle electron microscopy analysis revealed that PS-I units from iron-deficient cells are larger (31 and 37 nm in diameter) than PS-I units from control cells (22 nm). The 77 K chlorophyll fluorescence emission spectra of isolated complexes suggest that the Tidi-LHC-I antenna are functionally coupled to the reaction centers of PS-I. These findings indicate that Tidi acts as an accessory antenna of PS-I. The enlargement of PS-I antenna in algae and in cyanobacteria under iron deprivation suggests a common limitation that requires rebalancing of the energy distribution between the two photosystems.
Wang W., Dgany O., Wolf S. G., Levy I., Algom R., Pouny Y., Wolf A., Marton I., Altman A. & Shoseyov O. (2006) Biotechnology and Bioengineering. 95, 1, p. 161-168
Stable protein 1 (SP1) is a homo-oligomeric protein isolated from aspen (Populus tremula aspen) plants which forms a ring-shape dodecameric particle with a central cavity. The oligomeric form of SP1 is an exceptionally stable structure that is resistant to proteases (e.g., trypsin, V8, and proteinase K), high temperatures, organic solvents, and high levels of ionic detergent. Analytical ultra-centrifugation, chemical cross-linking, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and transmission electron microscopy were used to further characterize the SP1 dodecamer. Introduction of a single cysteine at the N-terminus of SP1 enabled the formation of disulfide bridges within the SP1 dodecamer, concurrent with increased melting point. A six-histidine tag was introduced at the N-terminus of SP1 to generate 6HSP1, and the ΔNSP1 mutant was generated by a deletion of amino acids 2-6 at the N-terminus. Both 6HSP1 and ΔNSP1 maintained their ability to assemble a stable dodecamer. Remarkably, these SP1 homo-dodecamers were able to re-assemble into stable hetero-dodecamers following co-electro-elution from SDS-PAGE. The exceptional stability of the SP1-nano ring and its ability to self-assemble hetero-complexes paves the way to further research in utilizing this unique protein in nano-biotechnology.
Chapman E., Farr G. W., Usaite R., Furtak K., Fenton W. A., Chaudhuri T. K., Hondorp E. R., Matthews R. G., Wolf S. G., Yates J. R., Pypaert M. & Horwich A. L. (2006) Proceedings of the National Academy of Sciences of the United States of America. 103, 43, p. 15800-15805
In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in ≈2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.
Coquelle F., Levy T., Bergmann S., Wolf S. G., Bar-El D., Sapir T., Brody Y., Orr I., Barkai N., Eichele G. & Reiner O. (2006) Cell Cycle. 5, 9, p. 976-983
The doublecortin-like (DCX) domains serve as protein-interaction platforms. DCX tandem domains appear in the product of the X-linked doublecortin (DCX) gene, in retinitis pigmentosa-1 (RP1), as well as in other gene products. Mutations in the human DCX gene are associated with abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, while DCDC2 has been associated with dyslectic reading disabilities. Motivated by the possible importance of this gene family, a thorough analysis to detect all family members in the mouse was conducted. The DCX-repeat gene superfamily is composed of eleven paralogs, and we cloned the DCX domains from nine different genes. Our study questioned which functions attributed to the DCX domain, are conserved among the different members. Our results suggest that the proteins with the DCX-domain have conserved and unique roles in microtubule regulation and signal transduction. All the tested proteins stimulated microtubule assembly in vitro. Proteins with tandem repeats stabilized the microtubule cytoskeleton in transfected cells, while those with single repeats localized to actin-rich subcellular structures, or the nucleus. All tested proteins interacted with components of the JNK/MAP-kinase pathway, while only a subset interacted with Neurabin 2, and a nonoverlapping group demonstrated actin association. The sub-specialization of some members due to confined intracellular localization, and protein interactions may explain the success of this superfamily.
Shimoni E., Rav-Hon O., Ohad I., Brumfeld V. & Reich Z. (2005) Plant Cell. 17, 9, p. 2580-2586
The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dual-axis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.
Rivenzon-Segal D., Wolf S. G., Shimon L. J. W., Willison K. & Horovitz A. (2005) Nature Structural & Molecular Biology. 12, 3, p. 233-237
The eukaryotic cytoplasmic chaperonin containing TCP-1 (CCT) is a hetero-oligomeric complex that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. To understand the allosteric transitions that occur during the functional cycle of CCT, we imaged the chaperonin complex in the presence of different ATP concentrations. Labeling by monoclonal antibodies that bind specifically to the CCTα and CCTδ subunits enabled alignment of all the CCT subunits of a given type in different particles. The analysis shows that the apo state of CCT has considerable apparent conformational heterogeneity that decreases with increasing ATP concentration. In contrast with the concerted allosteric switch of GroEL, ATP-induced conformational changes in CCT are found to spread around the ring in a sequential fashion that may facilitate domain-by-domain substrate folding. The approach described here can be used to unravel the allosteric mechanisms of other ring-shaped molecular machines.
Li J., Wolf S. G., Elbaum M. & Tzfira T. (2005) Trends in Microbiology. 13, 7, p. 295-298
Type IV secretion systems (T4SSs) are used by various bacteria to deliver protein and DNA molecules to a wide range of target cells. These include systems that are directly involved in pathogenesis, such as the secretion of pertussis toxin by Bordetella pertussis into human cells and the delivery of single-stranded DNA (ssDNA) into plants by Agrobacterium. These complex systems are composed of proteins that span the bacterial cytoplasm. The Agrobacterium T4SS is composed of 12 virulence proteins and delivers its transferred ssDNA and several virulence protein substrates to a variety of eukaryotic cells. Recent studies on the Agrobacterium T4SS have revealed new information on the localization and structure of its proteins in the bacteria, the biochemical properties of its transport signal, the route of a DNA substrate through the secretion system, and the initial point of contact of the system with its host. These findings have expanded our knowledge and understanding of the still mostly obscure structure and function of the T4SSs.
Frenkiel-Krispin D., Ben-Avraham I., Englander J., Shimoni E., Wolf S. G. & Minsky A. (2004) Molecular Microbiology. 51, 2, p. 395-405
The textbook view of the bacterial cytoplasm as an unstructured environment has been overturned recently by studies that highlighted the extent to which non-random organization and coherent motion of intracellular components are central for bacterial life-sustaining activities. Because such a dynamic order critically depends on continuous consumption of energy, it cannot be perpetuated in starved, and hence energy-depleted, stationary-state bacteria. Here, we show that, at the onset of the stationary state, bacterial chromatin undergoes a massive reorganization into ordered toroidal structures through a process that is dictated by the intrinsic properties of DNA and by the ubiquitous starvation-induced DMA-binding protein Dps. As starvation proceeds, the toroidal morphology acts as a structural template that promotes the formation of DNA-Dps crystalline assemblies through epitaxial growth. Within the resulting condensed assemblies, DNA is effectively protected by means of structural sequestration. We thus conclude that the transition from bacterial active growth to stationary phase entails a co-ordinated process, in which the energy-dependent dynamic order of the chromatin is sequentially substituted with an equilibrium crystalline order.
Frenkiel-Krispin D., Sack R., Englander J., Shimoni E., Eisenstein M., Bullitt E., Horowitz-Scherer R., Hayes C., Setlow P., Minsky A. & Wolf S. G. (2004) Journal of Bacteriology. 186, 11, p. 3525-3530
Bacterial spores have long been recognized as the sturdiest known life forms on earth, revealing extraordinary resistance to a broad range of environmental assaults. A family of highly conserved spore-specific DNA-binding proteins, termed α/β-type small, acid-soluble spore proteins (SASP), plays a major role in mediating spore resistance. The mechanism by which these proteins exert their protective activity remains poorly understood, in part due to the lack of structural data on the DNA-SASP complex. By using cryoelectron microscopy, we have determined the structure of the helical complex formed between DNA and SspC, a characteristic member of the α/β-type SASP family. The protein is found to fully coat the DNA, forming distinct protruding domains, and to modify DNA structure such that it adopts a 3.2-nm pitch. The protruding SspC motifs allow for interdigitation of adjacent DNA-SspC filaments into a tightly packed assembly of nucleoprotein helices. By effectively sequestering DNA molecules, this dense assembly of filaments is proposed to enhance and complement DNA protection obtained by DNA saturation with the α/β-type SASP.
Abu-Arish A., Frenkiel-Krispin D., Fricke T., Tzfira T., Citovsky V., Wolf S. G. & Elbaum M. (2004) Journal of Biological Chemistry. 279, 24, p. 25359-25363
Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.
Dgany O., Gonzalez A., Sofer O., Wang W., Zolotnitsky G., Wolf A., Shoham Y., Altman A., Wolf S. G., Shoseyov O. & Almog O. (2004) Journal of Biological Chemistry. 279, 49, p. 51516-51523
We previously reported on a new boiling stable protein isolated from aspen plants (Populus tremula), which we named SP1. SP1 is a stress-related protein with no significant sequence homology to other stress-related proteins. It is a 108-amino-acid hydrophilic polypeptide with a molecular mass of 12.4 kDa (Wang, W. X., Pelah, D., Alergand, T., Shoseyov, O., and Altman, A. (2002) Plant Physiol. 130, 865-875) and is found in an oligomeric form. Preliminary electron microscopy studies and matrix-assisted laser desorption ionization time-of-fliglit mass spectrometry experiments showed that SP1 is a dodecamer composed of two stacking hexamers. We performed a SDS-PAGE analysis, a differential scanning calorimetric study, and crystal structure determination to further characterize SP1. SDS-PAGE indicated a spontaneous assembly of SP1 to one stable oligomeric form, a dodecamer. Differential scanning calorimetric showed that SP1 has high thermostability i.e. T<sub>m</sub> of 107°C (at pH 7.8). The crystal structure of SP1 was initially determined to 2.4 Å resolution by multi-wave-length anomalous dispersion method from a crystal belonging to the space group 1422. The phases were extended to 1.8 Å resolution using data from a different crystal form (P21). The final refined molecule includes 106 of the 108 residues and 132 water molecules (on average for each chain). The R-free is 20.1%. The crystal structure indicated that the SP1 molecule has a ferredoxin-like fold. Strong interactions between each two molecules create a stable dimer. Six dimers associate to form a ring-like-shaped dodecamer strongly resembling the particle visualized in the electron microscopy studies. No structural similarity was found between the crystal structure of SP1 and the crystal structure of other stress-related proteins such as small heat shock proteins, whose structure has been already determined. This structural study further supports our previous report that SP1 may represent a new family of stress-related proteins with high thermostability and oligomerization.
Azubel M., Wolf S. G., Sperling J. & Sperling R. (2004) Molecular Cell. 15, 5, p. 833-839
Splicing of pre-mRNA occurs in a multicomponent macromolecular machine - the spliceosome. The spliceosome can be assembled in vitro by a stepwise assembly of a number of snRNPs and additional proteins on exogenously added pre-mRNA. In contrast, splicing in vivo occurs in preformed particles where endogenous pre-mRNAs are packaged with all five spliceosomal U snRNPs (penta-snRNP) together with other splicing factors. Here we present a three-dimensional image reconstruction by cryo-electron microscopy of native spliceosomes, derived from cell nuclei, at a resolution of 20 Å. The structure revealed an elongated globular particle made up of two distinct subunits connected to each other leaving a tunnel in between. We show here that the larger subunit is a suitable candidate to accommodate the penta-snRNP, and that the tunnel could accommodate the pre-mRNA component of the spliceosome. The features this structure reveals provide new insight into the global architecture of the native splicing machine.
Levin-Zaidman S., Englander J., Shimoni E., Sharma A. K., Minton K. W. & Minsky A. (2003) Science. 299, 5604, p. 254-256
The bacterium Deinococcus radiodurans survives ionizing irradiation and other DNA-damaging assaults at doses that are lethal to all other organisms. How D. radiodurans accurately reconstructs its genome from hundreds of radiationgenerated fragments in the absence of an intact template is unknown. Here we show that the D. radiodurans genome assumes an unusual toroidal morphology that may contribute to its radioresistance. We propose that, because of restricted diffusion within the tightly packed and laterally ordered DNA toroids, radiation-generated free DNA ends are held together, which may facilitate template-independent yet error-free joining of DNA breaks.
Danziger O., Rivenzon-Segal D., Wolf S. G. & Horovitz A. (2003) Proceedings of the National Academy of Sciences of the United States of America. 100, SUPPL. 2, p. 13797-13802
The reaction cycle of the double-ring chaperonin GroEL is driven by ATP binding that takes place with positive cooperativity within each seven-membered ring and negative cooperativity between rings. The positive cooperativity within rings is due to ATP binding-induced conformational changes that are fully concerted. Herein, it is shown that the mutation Asp-155 → Ala leads to an ATP-induced break in intra-ring and inter-ring symmetry. Electron microscopy analysis of single-ring GroEL particles containing the Asp-155 → Ala mutation shows that the break in intra-ring symmetry is due to stabilization of allosteric intermediates such as one in which three subunits have switched their conformation while the other four have not. Our results show that eliminating an intra-subunit interaction between Asp-155 and Arg-395 results in conversion of the allosteric switch of GroEL from concerted to sequential, thus demonstrating that its allosteric behavior arises from coupled tertiary conformational changes.
Minsky A., Shimoni E. & Frenkiel-Krispin D. (2002) Nature Reviews Molecular Cell Biology. 3, 1, p. 50-60
Much of the sophisticated chemistry of life is accomplished by multicomponent complexes, which act as molecular machines. Intrinsic to their accuracy and efficiency is the energy that is supplied by hydrolysis of nucleoside triphosphates. Conditions that deplete energy sources should therefore cause decay and death. But studies on organisms that are exposed to prolonged stress indicate that this fate could be circumvented through the formation of highly ordered intracellular assemblies. In these thermodynamically stable structures, vital components are protected by a physical sequestration that is independent of energy consumption.
Herskovits A. A., Shimoni E., Minsky A. & Bibi E. (2002) Journal of Cell Biology. 159, 3, p. 403-410
In Escherichia coli, ribosomes must interact with translocons on the membrane for the proper integration of newly synthesized membrane proteins, cotranslationally. Previous in vivo studies indicated that unlike the E. coli signal recognition particle (SRP), the SRP receptor FtsY is required for membrane targeting of ribosomes. Accordingly, a putative SRP-independent, FtsY-mediated ribosomal targeting pathway has been suggested (Herskovits, A.A., E.S. Bochkareva, and E. Bibi. 2000. Mol. Microbiol. 38:927-939). However, the nature of the early contact of ribosomes with the membrane, and the involvement of FtsY in this interaction are unknown. Here we show that in cells depleted of the SRP protein, Ffh or the translocon component SecE, the ribosomal targeting pathway is blocked downstream and unprecedented, membrane-bound FtsY-ribosomal complexes are captured. Concurrently, under these conditions, novel, ribosome-loaded intracellular membrane structures are formed. We propose that in the absence of a functional SRP or translocon, ribosomes remain jammed at their primary membrane docking site, whereas FtsY-dependent ribosomal targeting to the membrane continues. The accumulation of FtsY-ribosome complexes induces the formation of intracellular membranes needed for their quantitative accommodation. Our results with E. coli, in conjunction with recent observations made with the yeast Saccharomyces cerevisiae, raise the possibility that the SRP receptor-mediated formation of intracellular membrane networks is governed by evolutionarily conserved principles.
Frenkiel-Krispin D., Levin-Zaidman S., Shimoni E., Wolf S. G., Wachtel E., Arad T., Finkel S., Kolter R. & Minsky A. (2001) EMBO Journal. 20, 5, p. 1184-1191
The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These nonenzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.
Sapir T., Horesh D., Caspi M., Atlas R., Burgess H., Wolf S. G., Francis F., Chelly J., Elbaum M., Pietrokovski S. & Reiner O. (2000) Human Molecular Genetics. 9, 5, p. 703-712
Mutations in the X-linked gene doublecortin (DCX) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified and the sequence differences included nonsense, splice site and missense mutations throughout the gene. Recently, we and others have demonstrated that DCX interacts and stabilizes microtubules. Here, we performed a detailed sequence analysis of DCX and DCX-like proteins from various organisms and defined an evolutionarily conserved Doublecortin (DC) domain. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. We hypothesized that these repeats may be important for microtubule binding. We expressed DCX or DCLK (KIAA0369) repeats in vitro and in vivo. Our results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, we overexpressed seven of the reported mutations in COS7 cells and examined their effect on the microtubule cytoskeleton. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development.
Levin-Zaidman S., Frenkiel-Krispin D., Shimoni E., Sabanay I., Wolf S. G. & Minsky A. (2000) Proceedings of the National Academy of Sciences of the United States of America. 97, 12, p. 6791-6796
The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. Here we present the first study of the morphological aspects that accompany the SOS response in Escherichia coli. We find that induction of the SOS system in wild-type bacteria results in a fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly. The coaggregates comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in vitro RecA-DNA networks, as well as morphological studies of strains carrying RecA mutants, are all consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.
Wolf S. G., Frenkiel D., Arad T., Finkel S., Kolter R. & Minsky A. (1999) Nature. 400, 6739, p. 83-85
The crystalline state is considered to be incompatible with life. However, in living systems exposed to severe environmental assaults, the sequestration of vital macromolecules in intracellular crystalline assemblies may provide an efficient means for protection. Here we report a generic defence strategy found in Escherichia coli, involving co-crystallization of its DNA with the stress-induced protein Dps. We show that when purified Dps and DNA interact, extremely stable crystals form almost instantaneously, within which DNA is sequestered and effectively protected against varied assaults. Crystalline structures with similar lattice spacings are formed in E. coli in which Dps is slightly over expressed, as well as in starved wild- type bacteria. Hence, DNA-Dps co-crystallization is proposed to represent a binding mode that provides wide-range protection of DNA by sequestration. The rapid induction and large-scale production of Dps in response to stress, as well as the presence of Dps homologues in many distantly related bacteria, indicate that DNA protection by biocrystallization may be crucial and widespread in prokaryotes.
Horesh D., Sapir T., Francis F., Wolf S. G., Caspi M., Elbaum M., Chelly J. & Reiner O. (1999) Human Molecular Genetics. 8, 9, p. 1599-1610
X-linked lissencephaly is a severe brain malformation affecting males. Recently it has been demonstrated that the doublecortin gene is implicated in this disorder. In order to study the function of Doublecortin, we analyzed the protein upon transfection of COS cells. Doublecortin was found to bind to the microtubule cytoskeleton. In vitro assays (using biochemical methods, DIC microscopy and electron microscopy) demonstrate that Doublecortin binds microtubules directly, stabilizes them and causes bundling. In vivo assays also show that Doublecortin stabilizes microtubules and causes bundling. Doublecortin is a basic protein with an isoelectric point of 10, typical of microtubule-binding proteins. However, its sequence contains no known microtubule-binding domain(s). The results obtained in this study with Doublecortin and our previous work on another lissencephaly gene (LIS1) emphasize the central role of regulation of microtubule dynamics and stability during neuronal morphogenesis.
Nogales E., Wolf S. G. & Downing K. H. (1998) Nature. 393, 6681, p. 191
In this Letter, the numbers for the secondary structure elements involved in Taxol binding are incorrect (page 202, second-to-last paragraph of main text). The sentences giving the correct numbers are, \u201cIn our model, the C-3 is near the top of helix H1 (that is, between β:1525), and the C2 group near H6 and the H6H7 loop (that is, between β:212222). The main interaction of the taxane ring is at L275, at the beginning of the B7H9 loop.\u201d
Nogales E., Wolf S. G. & Downing K. H. (1998) Nature. 391, 6663, p. 199-203
The alpha beta tubulin heterodimer is the structural subunit of microtubules, which are cytoskeletal elements that are essential for intracellular transport and cell division in all eukaryotes. Each tubulin monomer binds a guanine nucleotide, which is non-exchangeable when it is bound in the alpha subunit, or N site, and exchangeable when bound in the beta subunit, or E site. The alpha- and beta-tubulins share 40% amino-acid sequence identity, both exist in several isotype forms, and both undergo a variety of posttranslational modifications'. Limited sequence homology has been found with the proteins FtsZ(2) and Misato(3), which are involved in cell division in bacteria and Drosophila, respectively. Here we present an atomic model of the alpha beta tubulin dimer fitted to a 3.7-Angstrom density map obtained by electron crystallography of zinc-induced tubulin sheets. The structures of alpha- and beta-tubulin are basically identical: each monomer is formed by a core of two beta-sheets surrounded by alpha-helices. The monomer structure is very compact, but can be divided into three functional domains: the amino-terminal domain containing the nucleotide-binding region, an intermediate domain containing the Taxol-binding site, and the carboxy-terminal domain, which probably constitutes the binding surface for motor proteins.
Nogales E., Wolf S. G. & Downing K. H. (1997) Journal of Structural Biology. 118, 2, p. 119-27
We are in the process of determining the structure of tubulin using electron crystallography of zinc-induced, crystalline sheets. We have now extended the resolution to 4 A, and there are many features in the map that appear to show details of the secondary structure. X-ray crystallographers are well aware of the problems of interpreting maps with such limited resolution, and the additional problem of the missing cone of data inherent in electron crystallography may make interpretation even more difficult. To investigate how reliably these maps can be interpreted, we have calculated density maps of a known structure, actin, under conditions similar to those of the tubulin map. Results of these simulations support the limited interpretations we made previously in the 6.5-A maps and the more extensive interpretations we make here in the 4-A map. Most of the secondary structure of the tubulin dimer can now be identified.
