Analysis of repeat motif proteins and cytoskeletal coordination in protozoa

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Single-celled eukaryotes (protists) such as the ciliate Tetrahymena thermophila or the human pathogen Trichomonas vaginalis often have a more complex cytoskeleton than an individual metazoan cell. This proposal targets the analysis of newly discovered cytoskeletal proteins in both of these protists. We will analyse the recruitment, assembly and interaction of these proteins with other cytoskeletal components. In prior work on Tetrahymena, the applicant has found and characterised proteins con-taining charged repeats that make up a large part of the membrane skeleton. Similarly structured pro-teins are found in all eukaryotes and extensive preliminary work shows that some associate with mi-crotubules or basal bodies and some form filaments. The first goal of the present work is to determine if these hitherto unknown proteins function as intermediate filaments and how they are integrated into the Tetrahymena cytoskeleton. The second goal focusses on the actin-based and Ca++-regulated mechanism underlying the amoeboid morphogenesis of Trichomonas cells upon contact with host vaginal epithelia. We have identified a protein of the fimbrin family (TvFim1), which in addition to a Ca++-binding EF-hand contains four calponin homology domains involved in actin bundling. Characterising interactions of actin and TvFim1 using, among other techniques, Trichomonas knock-outs recently established by the applicant will determine if TvFim1 mediates early host parasite interactions.