Analysis of repeat motif proteins and cytoskeletal coordination in protozoa

单细胞真核生物（原生生物），如四膜虫（Tetrahymena thermophila）或人类病原体阴道毛滴虫（Trichomonas vagulosa），通常比单个后生动物细胞具有更复杂的细胞骨架。这一建议的目标是在这两种原生生物中新发现的细胞骨架蛋白的分析。我们将分析这些蛋白质与其他细胞骨架成分的募集、组装和相互作用。在先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序列的蛋白质，所述带电重复序列构成膜骨架的大部分。类似结构的蛋白质在所有真核生物中都有发现，大量的初步工作表明，有些蛋白质与微管或基体结合，有些蛋白质形成细丝。本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发挥作用，以及它们如何整合到四膜虫细胞骨架中。第二个目标集中在肌动蛋白为基础的和Ca++调节机制的变形形态的毛滴虫细胞与宿主阴道上皮细胞接触后。我们已经确定了一个蛋白质的fimplant家庭（TvFim 1），除了钙++结合EF-手包含四个钙调蛋白同源结构域参与肌动蛋白捆绑。使用申请人最近建立的毛滴虫敲除等技术表征肌动蛋白和TvFim 1的相互作用，将确定TvFim 1是否介导早期宿主寄生虫相互作用。

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.