Intracellular calcium compartmentation in the mineral forming organisms Porcellio scaber (Crustacea), Magellania venosa (Brachiopoda) and Emiliania huxleyi (Haptophyta).

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Formation of biogenic carbonates is one of the most important processes in the biosphere. It has led to the formation of vast limestone deposits representing an important sink for atmospheric CO2. The cellular mechanisms of shell formation involve the transport of calcium through cells to the site of mineralisation. This transport may occur either in the form of ions or as an amorphous mineral precursor phase formed within the cells. In both cases, transport has to take place within cell organelles to avoid high toxic calcium-ion concentrations in the cytosol. However, our knowledge on intracellular calcium compartmentation during biomineralisation is fragmentary. In particular, vesicular structures and the endoplasmatic reticulum have been suggested to contribute to the calcium transport. In addition, the type of organelles involved can vary, depending on the organism and mineralized tissue. Therefore, we plan to investigate the subcellular distribution of calcium within biomineralising cells, with the aim to identify the organelles contributing to calcium transport. To account for possible differences between organisms and tissues, it is planned to investigate five different cell systems. Three of them from the terrestrial crustacean Porcellio scaber: 1) the anterior sternal epithelium, in which calcium transport rates are notably high during formation and resorption of large calcium carbonate reservoirs, 2) the hypodermis of the partes incisivae of the mandibles, as an example of calcium phosphate formation, and 3) the epithelium of the hepatopancreas that, after the animal has ingested its exuviae, transports mineral from the CaCO3-containing exuviae to the hemolymph. Here, it is of particular interest that one cell type accumulates heavy metals within lysosomal granules, raising the possibility that these are also involved in calcium transport. Furthermore, 4) it is planned to investigate the mantle epithelium of the brachiopod Magellania venosa, and 5) the marine coccolithophorid alga Emiliania huxleyi. Brachiopods exist from the early Cambrium with world wide geographic distribution. There fossil shells represent an immense archive for isotope signatures, that are important for reconstruction of paleo-climatic factors and that, however, can be influenced by cellular mechanisms. Emiliania huxleyi is responsible for one third of the marine CaCO3 production, and is an example of calcite formation within an intracellular organelle.For sample preparation high pressure freezing and cryo-ultramicrotomy will be used. Structural aspects and the distribution of calcium will be analysed by cryo-scanning electron microscopy, electron probe microanalysis and energy filtered transmission electron microscopy. The aim of the proposed investigations is to attain new insights in the mechanisms of cellular calcium transport in biomineralisation and its functional diversity and conservation during evolution.