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

Abstract

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-L_2,3 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.

Keywords: ACC; XAS; biomineralization; cryo-SXM; spectromicroscopy.

Fig. 1.

Identification of Ca-rich particles using cryo-SXM in a PMC (A–D) and a non-PMC (E-H). (A and E) Two-dimensional projection transmission images of the PMC and the non-PMC (respectively) taken at the Ca L₂-edge (352.6 eV). (B and F) Two-dimensional projection transmission images of A and E, respectively, taken below the Ca L_2,3-edge (338.3 eV), where Ca is almost transparent. Orange arrowheads point to selected particles that are not visible in B and F but are visible in A and E, respectively. These are Ca-rich particles. (C) and (G) “Ca maps” obtained by subtraction of B from A and F from E. The gray scale is converted to absorbance, so that white areas are locations where Ca is concentrated. (D and H) Superimposition of the 2D Ca-rich particle maps on the 3D segmented and reconstructed tomograms of both cells. Green, Ca-rich particles; blue, nucleus (N); gray, cell membranes, cytoplasm, vesicles, and vacuoles that do not contain Ca.

Fig. 2.

Ca-rich particle distribution patterns. (A–C) Slices through the reconstructed volumes of three PMCs. (Insets) High magnifications of the boxed areas. (A) Ca-rich particles not enveloped by a visible membrane. (B) Clusters of Ca-rich particles within a larger body. (C) Ca-rich particle contained in a larger vesicle. (Scale bars in the insets, 0.5 µm.) (D and E) Cryo-SEM micrographs of vesicles from sea urchin embryos. The vesicles are inside PMCs. (D) Vesicle from high-pressure-frozen and freeze-fractured embryo. The vesicle contains granular, 20-nm particles. (E) Vesicles from a high-pressure-frozen and cryoplaned embryo. The vesicles contain denser particles (arrowheads) surrounded by medium with a texture similar to the cytoplasm.

Fig. 3.

Ca L_2,3 XANES. (A) XANES of four standard samples: calcite in black, anhydrous ACC in green, hydrated ACC (ACC*H₂O) in purple (35), and sea water in blue. (B) XANES spectrum obtained for one particle, from the measurement of the 18 data points, after background subtraction. Green spectrum is the ACC calculated spectrum from ref. . The procedure is explained in detail in SI Appendix, XANES. (C) Superimposition of the 2D Ca-rich particle map on the 3D reconstructed and segmented PMC cell tomogram. The data are overlaid on a slice through the reconstructed volume. Green, Ca-rich particles; gray, cell membranes. Four particles (colored arrows) with their L₂′/L₂ values are shown.

Fig. 4.

Ca-rich particle L₂′/L₂ value distributions. (Ai and Aii) Two segmented reconstructed volumes of PMC tomograms. Ca-rich particles are marked in colors according to their L₂′/L₂ value, as indicated in the color scale on the right side. Blue, nucleus; gray, cytoplasm, membranes, and C-rich vesicles and vacuoles. The average diameter of the Ca-rich particle in the two cells are for cell i 230 ± 50 nm and for cell ii 180 ± 70 nm. (B) L₂′/L₂ values for all of the Ca-rich particles in the two cells shown in A (cell i in orange and cell ii in blue). The values are sorted for convenience in descending order to show the general trend. There is no correlation between the L₂′/L₂ values and the location of a particle within the cell as shown in A. (C) Distribution of the L₂′/L₂ values in B for the cells i and ii.

Fig. 5.

Ca concentration estimation. (Ai and Aii) Superimposition of the cell segmentation on the 3D reconstruction of the PMCs presented in Fig. 4. In green are the Ca-rich particles within 2 µm of the cell closest to the beam entry surface, which were used for the concentration calculations (see the main text). (B) Ca concentrations of the particles in the cells shown in Ai and Aii. The values are sorted in ascending order for convenience. Please note that the concentrations may be slightly underestimated, because the area of the L₂′ peak was not taken into account in the integrated absorption of the L₂ peak. The extent of the underestimation falls, in any case, within the limits of the error. (C) Distribution of the Ca concentration values in B for the cells i and ii.