Analysis of repeat motif proteins and cytoskeletal coordination in protozoa

单细胞真核生物(原生动物)，如纤毛虫四膜虫、嗜热性四膜虫或人类病原体阴道毛滴虫，通常具有比单个后生动物细胞更复杂的细胞骨架。这项建议的目标是对这两个原生生物中新发现的细胞骨架蛋白进行分析。我们将分析这些蛋白质的招募、组装以及与其他细胞骨架成分的相互作用。在之前关于四膜虫的工作中，申请人已经发现并表征了含有带电重复的蛋白质，这些重复组成了膜骨架的很大一部分。在所有真核生物中都发现了结构相似的蛋白，大量的前期工作表明，一些蛋白与微管或基底体有关，一些形成细丝。目前工作的第一个目标是确定这些迄今未知的蛋白质是否作为中间丝起作用，以及它们如何整合到四膜虫细胞骨架中。第二个目标集中在滴虫细胞与宿主阴道上皮接触时基于肌动蛋白和钙离子调节的阿米巴形态发生机制。我们已经鉴定出Fimbrin家族的一个蛋白质(TvFim1)，除了一个与钙离子结合的EF-Hand外，它还包含四个参与肌动蛋白捆绑的Calponin同源结构域。除其他技术外，使用申请人最近建立的滴虫敲除技术来表征肌动蛋白和TvFim1的相互作用将确定TvFim1是否介导早期宿主寄生虫的相互作用。

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.