A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga

Abstract

Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism.

Figure 1. Speciation of cellular calcium during the early stages of coccolith formation in E. huxleyi.

(a) Time-resolved evolution of the XANES spectra (black) of cells induced to form calcite and of the calculated fits (orange) using linear combinations of three reference standards (coccolith calcite, free calcium ions and amorphous CaCO₃). (b) Ca K-edge XANES spectra of calcium reference standards and of E. huxleyi cells enclosed by a sphere of coccoliths (C cells). Free calcium ions were represented by 10 mM CaCl₂ solution. The spectrum of amorphous calcium phosphate is courtesy of Diane Eichert, Elettra synchrotron, Trieste, Italy. The spectrum of C cells showed the characteristic feature of calcite and was used instead of synthetic calcite for fitting the spectra of induced cells. (c) Relative contribution of the calcium references to the three component fits shown in a.

Figure 2. Cryo-X-ray imaging reveals concentrated calcium pools in E. huxleyi cells.

(a) Two-dimensional slices from a reconstructed X-ray tomogram with (top) an immature coccolith marked by the arrowhead and a calcium-rich body marked by the arrow, and (bottom) with 3D segmentation of the calcium-rich bodies (red) and intracellular coccoliths (blue). (b) X-ray images recorded at an energy below the Ca L_2,3-egde (342 eV), at the edge energy (353.2 eV) and the grey value difference between both images. (c) Averaged XANES spectra of the Ca L_2,3-edge. For each spectrum, data from the relevant pixels of four cells were averaged; the inset shows the exact locations in one of these cells. Notice the difference in the position of the crystal field peak (vertical lines) between coccolith calcite and synthetic calcium phosphate. Figure is accompanied by Supplementary Movie 1.

Figure 3. Cryo-FIB-SEM imaging of vitrified E. huxleyi reveals coccolith calcite and calcium-rich bodies in separate compartments.

(a–d) Two slices from the same cell acquired with in-lens secondary electron detector (a,c) and energy selective backscattered electron detector (b,c) showing a cross-sectioned coccolith in statu nascendi (blue), the Ca-rich body (red), the coccolith vesicle (CV)–reticular body system (RB), the nucleus (N) and the chloroplast (Chl). Additional organelles are visible in the secondary electron images as is shown in Supplementary Fig. 3. (e) Oversampled and contrast-enhanced magnification of the area framed in c, illustrating the membrane that encloses the Ca-rich body (arrows). (f) 3D reconstruction of an E. huxleyi cell from a cryo-FIB-SEM image series, showing the nucleus (violet), chloroplast (dark green), plasma membrane (light green), a coccolith in statu nascendi (blue), Ca-rich bodies (red) and the membranes encompassing Ca-rich bodies (orange). Figure is accompanied by Supplementary Video 2.

Figure 4. Ultrastructural and elemental microanalysis of the calcium-rich body.

(a) HAADF-STEM image of a thin-sectioned cell showing the nucleus (N), the chloroplast (Chl), the coccolith vesicle (CV)–reticular body system (RB) (encircled by the white line), coccolith calcite (blue arrowhead) and the Ca-rich body (red arrow). Additional organelles that are visible in the HAADF-STEM images are shown in Supplementary Fig. 4. (b) High-resolution images and corresponding Fourier-transformed image of coccolith calcite and the Ca-rich body. (c) STEM-EELS spectra measured at the carbon K-edge on the Ca-rich body, coccolith calcite and embedding resin. (d) STEM-EDX spectra of the Ca-rich body and coccolith calcite.

Figure 5. Confocal fluorescence microscopy images of live E. huxleyi.

Cells were dual-stained with DAPI and the membrane-permeable calcium stain calcein-AM. The emitted fluorescence in the wavelength window between 420 and 450 nm originates from DAPI–DNA and DAPI–polyphosphate complexes, whereas the emission between 500 and 550 nm originates from DAPI bound to polyphosphate. The red channel shows the auto-fluorescence of chlorophyll.

Figure 6. Cells without Ca-rich body contained a compartment filled with diluted concentrations of calcium and phosphorus.

(a–e) Slices from cryo-FIB-SEM image series of high-pressure frozen E. huxleyi cells imaged in secondary electron mode (a,c,e) and backscattered electron mode (b,d), showing coccolith calcite (blue arrowhead), the dense Ca–P-rich body (red arrow) and a pool of diluted concentrations of Ca (orange arrow, framed orange in e) in close contact. The white line in e frames the coccolith vesicle–reticular body system. (f) HAADF-STEM image of a thin-sectioned cell showing the nucleus (N), the chloroplast (Chl), coccolith calcite (blue arrowhead), the coccolith vesicle–reticular body system (framed white) and the vacuole-like compartment containing Ca and P (framed orange). (g) EDX spectra taken from inside the compartment framed orange in f and of cytosol. The epoxy resin contributes to the C peak and therefore the C signal does not represent in vivo concentrations.

Figure 7. Conceptual model of the coccolith calcium pathway.

Ca accumulation and calcite precipitation are spatially and temporally separated. Calcium uptake into cells involves Ca transporter. The Ca ions are concentrated by polyphosphates into a disordered phase in a compartment distinct from the coccolith vesicle–reticular body system. The disordered Ca phase is a dynamic reservoir, concentrating and dispatching Ca ions. The released Ca ions are possibly transferred into the coccolith vesicle–reticular body system by Ca transporter and/or passive diffusion driven by a high-concentration gradient in free Ca ions. Inside the coccolith vesicle the calcium is precipitated as calcite.