Decoupling cell size homeostasis in diatoms from the geometrical constraints of the silica cell wall.

Unicellular organisms are known to exert tight control over their cell size. In the case of diatoms, abundant eukaryotic microalgae, two opposing notions are widely accepted. On the one hand, the rigid silica cell wall that forms inside the parental cell is thought to enforce geometrical reduction of the cell size. On the other hand, numerous exceptions cast doubt on the generality of this model. Here, we monitored clonal cultures of the diatom Stephanopyxis turris for up to 2 yr, recording the sizes of thousands of cells, in order to follow the distribution of cell sizes in the population. Our results show that S. turris cultures above a certain size threshold undergo a gradual size reduction, in accordance with the postulated geometrical driving force. However, once the cell size reaches a lower threshold, it fluctuates around a constant size using the inherent elasticity of cell wall elements. These results reconcile the disparate observations on cell size regulation in diatoms by showing two distinct behaviors, reduction and homeostasis. The geometrical size reduction is the dominant driving force for large cells, but smaller cells have the flexibility to re-adjust the size of their new cell walls.

Transport-Limited Growth of Coccolith Crystals.

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.

Polymer-Rich Dense Phase Can Concentrate Metastable Silica Precursors and Regulate Their Mineralization.

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.

Exocytosis of the silicified cell wall of diatoms involves extensive membrane disintegration.

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.

Efficient gene replacement by CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana.

CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism. Homologous recombination was induced in T. pseudonana using sequence-specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT). Up to c. 85% of NAT-resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms.

Periplasmic Bacterial Biomineralization of Copper Sulfide Nanoparticles.

Metal sulfides are a common group of extracellular bacterial biominerals. However, only a few cases of intracellular biomineralization are reported in this group, mostly limited to greigite (Fe3 S4 ) in magnetotactic bacteria. Here, a previously unknown periplasmic biomineralization of copper sulfide produced by the magnetotactic bacterium Desulfamplus magnetovallimortis strain BW-1, a species known to mineralize greigite (Fe3 S4 ) and magnetite (Fe3 O4 ) in the cytoplasm is reported. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide structure that remains in a thermodynamically unstable state. The particles appear to be surrounded by an organic matrix as found from staining and electron microscopy inspection. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this biomineralization process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization that involves intriguing biological control and holds promise for biological metal recovery in times of copper shortage.

The roles of intracellular and extracellular calcium in Bacillus subtilis biofilms.

In nature, bacteria reside in biofilms- multicellular differentiated communities held together by an extracellular matrix. This work identified a novel subpopulation-mineral-forming cells-that 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.

The variability in the structural and functional properties of coccolith base plates.

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 it 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 developed 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. STATEMENT OF SIGNIFICANCE: Biological organic scaffolds can serve as functional surfaces that guide the formation of inorganic materials. However, in many cases the specific interactions that facilitate such tight regulation are complex and not fully understood. In this work, we elucidate the architecture of such amodel biological template, an organic scale that directs the assembly of exquisite crystalline arrays of marine microalgae. By using cryo electron microscopy, we reveal the native state organization of these scales from several species. The observed similarities and differences allow us to propose that the chemical microenvironment, rather than stereochemical matching, is the pivotal regulator of the process.

Complex morphologies of biogenic crystals emerge from anisotropic growth of symmetry-related facets.

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.

Phase Separation of Oppositely Charged Polymers Regulates Bioinspired Silicification.

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.

Ion Pathways in Biomineralization: Perspectives on Uptake, Transport, and Deposition of Calcium, Carbonate, and Phosphate.

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.

Dense intracellular ion pools in unicellular organisms.

Uptake and concentration of inorganic ions are part of the complex cellular processes required for cell homeostasis, as well as for mineral formation by organisms. These ion transport mechanisms include distinct cellular compartments and chemical phases that play various roles in the physiology of organisms. Here, the prominent cases of dense ion pools in unicellular organisms are briefly reviewed. The specific observations that were reported for different organisms are consolidated into a wide perspective that emphasizes general traits. It is suggested that the intracellular ion pools can be divided into three types: a high cytoplasmic concentration, a labile storage compartment that hosts dense ion-rich phases, and a mineral-forming compartment in which a stable long-lived structure is formed. Recently, many labile pools were identified in various organisms using advanced techniques, bringing many new questions about their possible roles in the formation of the stable mineralized structures.

Intracellular nanoscale architecture as a master regulator of calcium carbonate crystallization in marine microalgae.

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.

Structural evidence for extracellular silica formation by diatoms.

The silica cell wall of diatoms, a widespread group of unicellular microalgae, is an exquisite example for the ability of organisms to finely sculpt minerals under strict biological control. The prevailing paradigm for diatom silicification is that this is invariably an intracellular process, occurring inside specialized silica deposition vesicles that are responsible for silica precipitation and morphogenesis. Here, we study the formation of long silicified extensions that characterize many diatom species. We use cryo-electron tomography to image silica formation in situ, in 3D, and at a nanometer-scale resolution. Remarkably, our data suggest that, contradictory to the ruling paradigm, these intricate structures form outside the cytoplasm. In addition, the formation of these silica extensions is halted at low silicon concentrations that still support the formation of other cell wall elements, further alluding to a different silicification mechanism. The identification of this unconventional strategy expands the suite of mechanisms that diatoms use for silicification.

Exploring Intracellular Ion Pools in Coccolithophores Using Live-Cell Imaging.

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.

Role of silicon in the development of complex crystal shapes in coccolithophores.

The development of calcification by the coccolithophores had a profound impact on ocean carbon cycling, but the evolutionary steps leading to the formation of these complex biomineralized structures are not clear. Heterococcoliths consisting of intricately shaped calcite crystals are formed intracellularly by the diploid life cycle phase. Holococcoliths consisting of simple rhombic crystals can be produced by the haploid life cycle stage but are thought to be formed extracellularly, representing an independent evolutionary origin of calcification. We use advanced microscopy techniques to determine the nature of coccolith formation and complex crystal formation in coccolithophore life cycle stages. We find that holococcoliths are formed in intracellular compartments in a similar manner to heterococcoliths. However, we show that silicon is not required for holococcolith formation and that the requirement for silicon in certain coccolithophore species relates specifically to the process of crystal morphogenesis in heterococcoliths. We therefore propose an evolutionary scheme in which the lower complexity holococcoliths represent an ancestral form of calcification in coccolithophores. The subsequent recruitment of a silicon-dependent mechanism for crystal morphogenesis in the diploid life cycle stage led to the emergence of the intricately shaped heterococcoliths, enabling the formation of the elaborate coccospheres that underpin the ecological success of coccolithophores.

Imaging and quantifying homeostatic levels of intracellular silicon in diatoms.

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 cryo-electron 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.

In situ electron microscopy characterization of intracellular ion pools in mineral forming microalgae.

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.

Native-state imaging of calcifying and noncalcifying microalgae reveals similarities in their calcium storage organelles.

Calcium storage organelles are common to all eukaryotic organisms and play a pivotal role in calcium signaling and cellular calcium homeostasis. In most organelles, the intraorganellar calcium concentrations rarely exceed micromolar levels. Acidic organelles called acidocalcisomes, which concentrate calcium into dense phases together with polyphosphates, are an exception. These organelles have been identified in diverse organisms, but, to date, only in cells that do not form calcium biominerals. Recently, a compartment storing molar levels of calcium together with phosphorous was discovered in an intracellularly calcifying alga, the coccolithophore Emiliania huxleyi, raising a possible connection between calcium storage organelles and calcite biomineralization. Here we used cryoimaging and cryospectroscopy techniques to investigate the anatomy and chemical composition of calcium storage organelles in their native state and at nanometer-scale resolution. We show that the dense calcium phase inside the calcium storage compartment of the calcifying coccolithophore Pleurochrysis carterae and the calcium phase stored in acidocalcisomes of the noncalcifying alga Chlamydomonas reinhardtii have common features. Our observations suggest that this strategy for concentrating calcium is a widespread trait and has been adapted for coccolith formation. The link we describe between acidocalcisomal calcium storage and calcium storage in coccolithophores implies that our physiological and molecular genetic understanding of acidocalcisomes could have relevance to the calcium pathway underlying coccolithophore calcification, offering a fresh entry point for mechanistic investigations on the adaptability of this process to changing oceanic conditions.