Elemental compositions of sea urchin larval cell vesicles evaluated by cryo-STEM-EDS and cryo-SEM-EDS.

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. STATEMENT OF SIGNIFICANCE: The 'inorganic side of life', encompassing ion trafficking and ion storage in soft tissues of organisms, is a generally overlooked problem. Addressing such a problem becomes possible through the application of innovative techniques, performed in cryogenic conditions, which preserve the tissues in quasi-physiological state. We developed here a set of analytical tools, cryo-SEM-EDS, and cryo-STEM-EDS, which allow reconstructing the ion composition inside vesicles in sea urchin larval cells, on their way to deposit mineral in the skeletons. The techniques are complex, and we evaluate here the advantages and disadvantages of each technique. The methodologies that we are developing here can be applied to other cells and other pathways as well, eventually leading to quantitative elemental analyzes of tissues under cryogenic conditions.

Calculations of the Evolution of the Ca L23 Fine Structure in Amorphous Calcium Carbonate.

Amorphous calcium carbonate (ACC) has been found in many different organisms. Biogenic ACC is frequently a precursor in the formation of calcite and aragonite. The process of structural transformation is therefore of great interest in the study of crystallization pathways in biomineralization. Changes in the prepeak/main peak (L2'/L2) intensity ratio of the Ca L23-edge X-ray absorption spectroscopy (XAS) of Ca-rich particles in skeleton-building cells of sea urchin larva revealed that ACC precipitates through a continuum of states rather than through abrupt phase transitions involving two distinct phases as formerly believed. Using an atomic multiplet code, we show that only a tetragonal or "umbrella-like" distortion of the Ca coordination polyhedron can give rise to the observed continuum of states. We also show on the basis of the structures obtained from previous molecular dynamics simulations of hydrated nanoparticles that the Ca L23-edge is not sensitive to atomic arrangements in the early stages of the transformation process.

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.

Characterization of the growth plate-bone interphase region using cryo-FIB SEM 3D volume imaging.

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.

Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae.

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L2'/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L2'/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

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.

Characterization of unusual MgCa particles involved in the formation of foraminifera shells using a novel quantitative cryo SEM/EDS protocol.

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 SIGNIFICANCE: Ions 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.

Minerals in the pre-settled coral Stylophora pistillata crystallize via protein and ion changes.

Aragonite skeletons in corals are key contributors to the storage of atmospheric CO2 worldwide. Hence, understanding coral biomineralization/calcification processes is crucial for evaluating and predicting the effect of environmental factors on this process. While coral biomineralization studies have focused on adult corals, the exact stage at which corals initiate mineralization remains enigmatic. Here, we show that minerals are first precipitated as amorphous calcium carbonate and small aragonite crystallites, in the pre-settled larva, which then evolve into the more mature aragonitic fibers characteristic of the stony coral skeleton. The process is accompanied by modulation of proteins and ions within these minerals. These findings may indicate an underlying bimodal regulation tactic adopted by the animal, with important ramification to its resilience or vulnerability toward a changing environment.

Phosphate-water interplay tunes amorphous calcium carbonate metastability: spontaneous phase separation and crystallization vs stabilization viewed by solid state NMR.

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.