Assaf Gal 1,2 André Scheffel 1 Damien Faivre 2
1Department of Organelle Biology, Max-Planck Institute of Molecular Plant Physiology, Potsdam, Germany
2Department of Biomaterials, Max-Planck Institute of Colloids and Interfaces, Potsdam, Germany

A major part of the global carbon cycle is the burial of calcium carbonates in deep sea sediments. This process is primarily biologically driven, with unicellular calcifying algae being dominant players. We aim to elucidate the underlying mechanisms of intracellular calcification in the most important calcifier in modern oceans, the coccolithophore Emiliania huxleyi. This unicellular alga forms intricate arrays of calcium carbonate crystals, called coccoliths, which are made inside a specialized intracellular compartment. The intracellular pathway of calcium ion accumulation from seawater to the coccolith-forming vesicle has remained elusive. This is mostly due to the experimental difficulties in following mineralization processes in vivo. Such process usually involves metastable and short-lived mineral phases that are difficult to follow at the spatial and temporal resolution characteristic for cellular activity. Therefore, the intracellular pathways responsible for the transport of the constituent ions from seawater to the growing coccolith are mostly unknown.

We used synchrotron soft-X-ray tomography at cryogenic conditions in order to map the intracellular calcium in cells of E. huxleyi. The cells were rapidly frozen and maintained at cryogenic conditions to preserve their intracellular organization. Single cells were imaged with the X-ray microscope at a resolution of 50 nm. Two types of data sets were acquired. The first is a tilt-series at the ‘water window’ energy range. At this X-ray energy the best contrast between carbon-rich intracellular membranes and the water-rich cytoplasm is achieved so the data can be used for 3D reconstruction of cells. The second data set was an energy scan around the Ca L-edge. From these data a complete X-ray Absorption Near-Edge Spectroscopy (XANES) spectrum can be extracted for each pixel in the image, providing information on the concentration of calcium inside intracellular organelles and spectroscopic information on the crystallinity of this Ca-rich phase. In the cell tomograms all major organelles were visible, as well as intracellular membrane-bound coccoliths in status nascendi. To our surprise, the cells contained distinct intracellular compartments packed with highly absorbing material, which the spectroscopic data showed to be rich in calcium. The XANES spectra collected from multiple Ca-rich compartments were clearly different from the spectra of coccolith calcite and exhibited characteristics of disordered local environment around the calcium atoms.

These data provide the first insights on the spatial distribution of calcium in coccolithophorid cells. We discovered high amounts of calcium to be concentrated in membrane-bound compartments that are separate from the coccolith producing compartment and we propose that this calcium pool is used for coccolith calcite formation.

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