Publications

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.

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.

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.

Biomineralization processes exert varying levels of control over crystallization, ranging from poorly ordered polycrystalline arrays to intricately shaped single crystals. Coccoliths, calcified scales formed by unicellular algae, are a model for a highly controlled crystallization process. The coccolith crystals nucleate next to an organic oval structure that was termed the base plate, leading to the assumption that the base plate is responsible for the oriented nucleation of the crystals via stereochemical interactions. In recent years, several works focusing on a well-characterized model species demonstrated a fundamental role for indirect interactions that facilitate coccolith crystallization. Here, we develop the tools to extract the base plates from five different species, giving the opportunity to systematically explore the relations between base plate and coccolith properties. We used multiple imaging techniques to evaluate the structural and chemical features of the base plates under native hydrated conditions. The results show a wide range of properties, overlaid on a common rudimentary scaffold that lacks any detectable structural or chemical motifs that can explain direct nucleation control. This work emphasizes that it is the combination between the base plate and the chemical environment inside the cell that cooperatively facilitate the exquisite control over the crystallization process.

In nature, simple organisms evolved mechanisms to form intricate biosilica nanostructures, far exceeding current synthetic manufacturing. Based on the properties of extracted biomacromolecules, polycation-polyanion pairs were suggested as moderators of biosilica formation. However, the chemical principles of this polymer-induced silicification remain unclear. Here, we used a biomimetic polycation-polyanion system to study polymer-induced silicification. We demonstrate that it is the polymer phase separation process, rather than silica-polymer interactions, which controls silica precipitation. Since ionic strength controls this electrostatic phase separation, it can be used to tune the morphology and structure of the precipitates. In situ cryo electron microscopy highlights the pivotal role of the hydrated polymer condensates in this process. These results pave the road for developing nanoscale morphologies of bioinspired silica based on the chemistry of liquid-liquid phase separation.

Minerals are formed by organisms in all of the kingdoms of life. Mineral formation pathways all involve uptake of ions from the environment, transport of ions by cells, sometimes temporary storage, and ultimately deposition in or outside of the cells. Even though the details of how all this is achieved vary enormously, all pathways need to respect both the chemical limitations of ion manipulation, as well as the many "housekeeping"roles of ions in cell functioning. Here we provide a chemical perspective on the biological pathways of biomineralization. Our approach is to compare and contrast the ion pathways involving calcium, phosphate, and carbonate in three very different organisms: the enormously abundant unicellular marine coccolithophores, the well investigated sea urchin larval model for single crystal formation, and the complex pathways used by vertebrates to form their bones. The comparison highlights both common and unique processes. Significantly, phosphate is involved in regulating calcium carbonate deposition and carbonate is involved in regulating calcium phosphate deposition. One often overlooked commonality is that, from uptake to deposition, the solutions involved are usually supersaturated. This therefore requires not only avoiding mineral deposition where it is not needed but also exploiting this saturated state to produce unstable mineral precursors that can be conveniently stored, redissolved, and manipulated into diverse shapes and upon deposition transformed into more ordered and hence often functional final deposits.

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.

Some microorganisms, such as coccolithophores, produce an intricate exoskeleton made of inorganic solids. Coccoliths, the calcium carbonate scales of coccolithophores, are examples of the precise bioproduction of such complex 3D structures. However, the understanding of the cellular mechanisms that control mineral formation inside the cell, specifically the ability of these microalgae to transport high fluxes of inorganic building blocks, is still limited. Recently, using cryo-electron and X-ray microscopy, several intracellular compartments are shown to store high concentrations of calcium and phosphorous and are suggested to have a dominant role in the intracellular mineralization pathway. Here, live-cell confocal microscopy and fluorescent markers are used to examine the dynamics of ion stores in coccolithophores. Using calcein and 4',6-diamidino-2-phenylindole (DAPI) as fluorescent proxies for calcium and polyphosphates, the experiments reveal an unexpected plethora of organelles with distinct fluorescent signatures over a wide range of strains and conditions. Surprisingly, the fluorescent labeling does not show changes along the calcification process and is similar between calcifying and noncalcifying cells, suggesting that these ion pools may not be a dynamic avenue for calcium transport. In such a case, the enigma behind the ability of coccolithophores to sustain intracellular calcification still awaits comprehensive elucidation.

The formation of intricately shaped crystalline minerals by organisms is orchestrated by specialized biomacromolecules. The macromolecules associated with coccoliths, nanometer-sized calcite crystal arrays produced by marine microalgae, can form a distinct calcium-rich phase via macromolecular recognition. Here, we show that this calcium-rich phase can be mineralized into a thin film of single-crystalline calcite by the balanced addition of carbonate ions. Such a crystallization process provides a strategy to direct crystalline products via local interactions between soluble macromolecules and compatible templates.

Many organisms build crystals with almost complete control over all aspects of crystal formation, from nucleation to growth, from composition to polymorphic structure, and from morphology to size. In biomineralization, the control is fundamentally always exerted at the level of nanometers, because the building blocks themselves are at the nanoscale. We have chosen to describe in some detail four biological systems that produce nanoscale crystals using different mineralization pathways, different levels of control, and have different functions. These four cases are: bone crystal composites; guanine nanocrystal reflectors; magnetotactic bacteria with single domain magnets; and coccoliths, whose functions have yet to be identified. This is followed by a discussion aimed at identifying possible specific and/or common underlying principles involved in nanocrystal formation in biology.

Organisms tune the metastability of amorphous calcium carbonates (ACC), often by incorporation of additives such as phosphate ions and water molecules, to serve diverse functions, such as modulating the availability of calcium reserves or constructing complex skeletal scaffolds. Although the effect of additive distribution on ACC was noted for several biogenic and synthetic systems, the molecular mechanisms by which additives govern ACC stability are not well understood. By precipitating ACC in the presence of different PO₄^3- concentrations and regulating the initial water content, we identify conditions yielding either kinetically locked or spontaneously transforming coprecipitates. Solid state NMR, supported by FTIR, XRD, and electron microscopy, define the interactions of phosphate and water within the initial amorphous matrix, showing that initially the coprecipitates are homogeneous molecular dispersions of structural water and phosphate in ACC, and a small fraction of P-rich phases. Monitoring the transformations of the homogeneous phase shows that PO₄^3- and waters are extracted first, and they phase separate, leading to solid-solid transformation of ACC to calcite; small part of ACC forms vaterite that subsequently converts to calcite. The simultaneous water-PO₄^3- extraction is the key for the subsequent water-mediated accumulation and crystallization of hydroxyapatite (HAp) and carbonated hydroxyapatite. The thermodynamic driving force for the transformations is calcite crystallization, yet it is gated by specific combinations of water-phosphate levels in the initial amorphous coprecipitates. The molecular details of the spontaneously transforming ACC and of the stabilized ACC modulated by phosphate and water at ambient conditions, provide insight into biogenic and biomimetic pathways.

Many biogenic minerals are composed of aggregated particles at the nanoscale. These minerals usually form through the transformation of amorphous precursors into single crystals inside a privileged space controlled by the organism. Here, in vitro experiments aimed at understanding the factors responsible for producing such single crystals with aggregated particle texture are presented. Crystallization is achieved by a two-step reaction in which amorphous calcium carbonate (ACC) is first precipitated and then transformed into calcite in small volumes of water and in the presence of additives. The additives used are gel-forming molecules, phosphate ions, and the organic extract from sea urchin embryonic spicules - all are present in various biogenic crystals that grow via the transformation of ACC. Remarkably, this procedure yields faceted single-crystals of calcite that maintain the nanoparticle texture. The crystals grow predominantly by the accretion of ACC nanoparticles, which subsequently crystallize. Gels and phosphate ions stabilize ACC via a different mechanism than sea urchin spicule macromolecules. It is concluded that the unique nanoparticle texture of biogenic minerals results from formation pathways that may differ from one another, but given the appropriate precursor and micro-environment, share a common particle accretion mechanism.