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

Abstract

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.

Keywords: acidocalcisome; biomineralization; coccolithophore; cryo–X-ray tomography; intracellular calcium store.

Fig. 1.

CryoSXT images of vitrified P. carterae cells producing coccoliths. (A) Raw X-ray image of the cell at 0° tilt and 520 eV. (Inset) A light microscopy image of a living cell with a coccolith shell. (B) A slice in the 3D reconstructed volume of the cell showing the chloroplasts (green arrowheads), a large membrane-bound compartment (*), several vesicles, and X-ray−absorbing bodies (dark). (C) Surface rendering of the segmented structures showing the chloroplasts in green, the nucleus in purple, the large vacuole-like compartment in brown, lipid bodies in yellow, several vesicles in gray, and coccoliths in blue. For more details, see Movie S1. (D) Spectromicroscopy at the Ca L-edge, with an image before the edge (342 eV), one at the edge (353.2 eV), and the corresponding difference image showing in dark contrast areas where Ca is abundant. (E) The 3D segmentation of Ca-containing entities. Coccoliths are in blue, Ca-rich bodies are in red, and the compartment they are enclosed in is depicted in brown. A second view at a different angle demonstrates that the Ca-rich bodies are located inside a single compartment. (F and G) CryoSXT Ca maps showing P. carterae cells with high luminal Ca concentrations in the vacuole-like compartment and only a few and small Ca-rich bodies. White ellipses depict direct beam artifacts, which cannot be avoided for such thick samples.

Fig. 2.

CryoSEM micrographs and elemental composition of a high-pressure frozen and freeze-fractured P. carterae cell. (A) A fracture surface showing the inside of a cell. The chloroplast (green arrowheads) was fractured open, while a large organelle in the cell middle remained unfractured (*). (B) Higher magnification of A showing the surface of the organelle with bulging protrusions (red arrowheads) and the cross-section of two extracellular coccoliths (blue arrowheads). (C) Back-scattered electron image of the same area as in B. The coccoliths and the protrusions exhibit bright contrast. (D) EDS maps of the most abundant elements in the cell. (E) EDS spectra taken at the indicated positions. Note that the coccoliths are smaller than the EDS resolution. Therefore, NaCl from the seawater medium (0.5 M NaCl) can be seen in the coccolith spectrum. (F) High-magnification image showing several Ca–P-rich vacuolar protrusions in profile.

Fig. 3.

Ultrastructure of the Ca–P-rich bodies. (A) CryoSEM image of a freeze-fractured P. carterae cell with the Ca storage compartment (*) cut open, showing two Ca–P-rich bodies (in rectangles). (B and C) Higher-magnification images of the framed areas in A showing Ca–P-rich bodies and the nearby compartment membrane (arrowheads). The contrast of the membranes is different from that of the material surrounding them. The boundary of the Ca–P bodies does not show a membrane-characteristic contrast pattern, suggesting the body material to be in direct contact with the compartment solution. (Insets) The corresponding backscattering images in which the brightness of the Ca–P bodies corresponds to their richness in calcium. The membranes are bright due to the steep topography.

Fig. 4.

Calcium pools in noncalcifying haptophytes: (A and B) I. galbana and (C and D) noncalcifying E. huxleyi. (A and C) Calcium distribution maps derived from cryoSXT spectromicroscopy. The circular holes in the support film are indicated with an asterisk (*), and gold nanoparticles that serve as fiducial markers are marked with arrowheads. (Insets) Light microscopy images of the cells before freezing. (B and D) A 3D volume rendering of the intracellular compartments identified with cryoSXT. The chloroplast is shown in green, the nucleus is purple, a large compartment with unknown content is brown, vesicles are gray, a filamentous membrane system is cyan, and lipid bodies are yellow.

Fig. 5.

C. reinhardtii acidocalcisomes analyzed by cryoSXT. (A) A slice in the 3D reconstructed data. Color-coded arrowheads indicate the chloroplast (green) and the nucleus and nucleolus (pink and purple, respectively). (Inset) A light micrograph of a live cell. (B) A 3D volume rendering of an entire cell. The nucleus (pink) and the acidocalcisomes (red) are partially enclosed by the cup-shaped chloroplast (green). (C) Spectromicroscopy at the calcium L-edge revealed the X-ray dense bodies to be Ca-rich and not lipid bodies. (D) A 3D representation of the cell in C showing only the Ca-rich bodies and the enclosing membranes. (E) High magnification showing the organization of a Ca-rich body (red arrowhead) in the acidocalcisome (brown arrows). (F) Ca L-edge XANES spectra extracted from different regions of image stacks traversing the energy range around the Ca L-edge.