Biomineralisation in Zysten von Flagellaten
Biologische und Biomimetische Chemie
Biomaterialien
Strukturbiologie
Zusammenfassung der Projektergebnisse
Dinoflagellates are ubiquitous freshwater and marine unicellular eukaryotes with great economic and ecological importance. Members of the Thoracosphaeraceae family form extracellular calcitic shells with genetically controlled mineral morphologies. Although dinoflagellates are the second most important calcifying phytoplankton group after coccolithophores, their biomineralization mechanism is still not fully understood and considered as one of the most serious gaps in knowledge. For this reason, the research goal of the Postdoctoral stay was to understand the mineral architecture and stabilization mechanism, to identify subcellular structures that may play a role in biomineralization, as well as to the gain insight into cyst formation mechanism using cryo-electron microscopic techniques (Cryo-SEM and Cryo-FIB-SEM) in combination with various spectroscopic techniques (FT-IR, Raman, Fluorescence, EDX). For the studies, two representative members from two different clades of calcareous dinoflagellates were selected: L. granifera and C. operosum aff. The life cycles of the two clades are significantly different and may also be expressed in different biomineralization modes. In both species, we observed vacuoles containing crystalline inclusions using cryo-SEM. So far, crystalline deposits in the family Thoracosphaeraceae were assigned to calcite and it was assumed that they are involved in the calcitic shell formation. Surprisingly, using in situ Raman spectroscopic imaging we could identify these crystalline inclusions as anhydrous guanine in the biogenic β-form using their low-wavenumber Raman signature. Furthermore, 2D cryo-SEM and 3D cryo-FIB-SEM serial block face imaging of C. operosum aff. show that the deposits of anhydrous guanine crystals are closely associated with the chloroplasts. We suggest that the crystalline deposits scatter light either to enhance light exploitation by the chloroplasts, or possibly for protection from UV radiation. This is consistent with the crystal locations within the cell, their shapes and their sizes. As dinoflagellates are extremely abundant and are a major group of photosynthesizing marine organisms, the presence of guanine crystals in this marine organism may have broad significance. L. granifera, a coccoid-shaped species that produces a new calcite shell during each cell cycle, was established as model system to study dinoflagellate calcification. To study different calcification stages, we performed decalcification with EDTA, followed by re-supplying Ca2+ to the growth medium, which leads to complete re-calcification within 24-72 h. We observe that only calcein-AM enters the cells, indicating active uptake of calcium and other divalent cations. Livecell imaging of cells stained with Calcein-AM, and Cryo-SEM-EDS demonstrate the presence of small MgCaP-rich precursor bodies with a bright ESB-signal in both species. 3D cryo-FIB-SEM serial block face imaging shows a remarkably large number (353) of these bodies distributed in the cell volume. The bodies are seen to be translocated into the outer matrix space where calcite forms. Calcite formation occurs via multiple independent nucleation events and the different crystals grow with preferred orientation into a dense reticular network that forms the calcitic cyst. Based on these observations, we developed a new calcification model in dinoflagellates, which involves four main steps: T (1.) Active uptake of Ca2+ and Mg2+ into the cell by ion pumps and channels; (2.) deposition of an intracellular, disordered Mg-, Ca- and P-rich phase (MgCaP bodies); (3.) Secretion of MgCaP bodies into the outer matrix; (4.) Transformation of the disordered MgCaP-rich phase first into a high Mg-calcite, and then into a low Mg-calcite during structure morphogenesis in the outer matrix.
Projektbezogene Publikationen (Auswahl)
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Anhydrous β-Guanine Crystals in a Marine Dinoflagellate: Structure and suggested function JSB 2019
Jantschke, A.; Pinkas, I.; Hirsch, A.; Elad, N.; Schertel, A.; Addadi, L.; Weiner, S.
