The basal apparatus sub-proteome of Chlamydomonas reinhardtii - composition, gene expression and functional analysis

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Chlamydomonas reinhardtii has become the experimental model system for the functional and developmental analysis of the flagellar apparatus in eukaryotic cells. The basal apparatus anchors the flagella inside the cell and represents the functional equivalent of the mammalian centrosome. Several basal apparatus proteins have been first identified in green flagellates such as centrin and SF-assemblin. They are now known to occur in many other eukaryotic organisms performing essential functions in flagellate/ciliate cells. Whereas the protein composition and function of the axoneme are relatively well understood, comparable studies on the basal apparatus are still lacking. The basal apparatus is not only involved in the formation of the flagella, but also plays important roles in miosis, cytokinesis, and in maintaining cell polarity. It is likely that basal apparatus proteins involved in these essential cellular functions were conserved throughout evolution and that analysis of the basal apparatus sub-proteome of C. reinhardtii may help to identify novel disease-related genes in mammalian and human cells. The project aims to identify most or all of the proteins in the basal apparatus sub-proteome of C. reinhardtii. Basal apparatuses will be isolated, and purified and proteins will be identified following high-resolution 2D-PAGE by mass spectrometry. Functional analyses of selected novel basal apparatus proteins will involve gene silencing by RNAi, and functional rescue by protein targeting using an established electroporation protocol. Finally, global gene expression of a selection of basal apparatus proteins using DNA-microarrays will be analyzed with emphasis on changes in the basal apparatus sub-proteome during cell cycle, sexual differentiation and under circadian control.