Kinesin like proteins in the parasite, Giardia

描述(由申请人提供)：肠贾第鞭毛虫是一种广泛存在于人类和动物中的进化分化的肠道寄生虫，是最早的分化真核血统之一的成员。总体而言，对于细胞骨架介导的染色体分离或寄生虫通过特殊的腹盘结构附着到宿主肠道上皮的分子模型的研究工作很少。这项建议的目的是确定有丝分裂中动蛋白马达的功能，以及贾第鞭毛虫腹盘的组装和功能。激动素样蛋白(KLP)分布于大约10个系统发育亚类中，是许多细胞机制的重要组成部分，包括有丝分裂、鞭毛运动、细胞内运输和细胞极性。我们已经鉴定并克隆了24个属于每个主要运动蛋白亚类以及多个新的运动蛋白谱系的贾第虫KLP。我们预测在贾第鞭毛虫有丝分裂中KLP的功能是保守的，因为贾第鞭毛虫在每个有丝分裂亚类中至少拥有一个KLP。相反，我们认为新的贾第虫KLP有助于腹盘独特的收缩功能和组装，腹盘是一种新的细胞骨架结构，负责与宿主上皮(或实验室底物)的黏附。为了验证这些假设，我们将首先使用GFP和表位蛋白标签方法在细胞内定位GKLP。基于有丝分裂或圆盘定位，我们将使用几种功能抑制方法，然后进行有丝分裂和圆盘功能的分析，以监测细胞行为，并评估选定的贾第鞭毛虫KLP对有丝分裂和对贾第鞭毛虫特异性细胞骨架结构的组装和功能的贡献。我们还将使用最先进的光学和电子显微镜来表征贾第鞭毛虫有丝分裂和腹盘功能/组装，为分析这些过程中的动蛋白功能提供结构基准。这些细胞生物学分析将使我们能够确定动蛋白在纺锤体组装、功能和染色体分离中的作用，并建立贾第虫有丝分裂的分子模型。此外，我们还将评估动蛋白在腹侧盘装配和附着/脱离中的作用。这些对贾第鞭毛虫运动蛋白功能的分析将为抗贾第鞭毛虫运动蛋白化合物提供新的治疗靶点。

DESCRIPTION (provided by the applicant): Giardia intestinalis is a widespread and evolutionarily divergent intestinal parasite of humans and animals, and a member of one of the earliest diverging eukaryotic lineages. In general, there has been little work to develop molecular models of either cytoskeletal-mediated chromosome segregation or of parasite attachment to the host intestinal epithelium via a specialized ventral disc structure. The goals of this proposal are to establish the function of kinesin motors in mitosis and in the assembly and function of the ventral disc in Giardia. Kinesin-like proteins (klps) are distributed among roughly ten phylogenetic subclasses and are essential components of many cellular mechanisms including mitosis, flagellar motility, intracellular transport, and cell polarity. We have identified and cloned a total of twenty-four Giardia klps that group to each of the major kinesin subclasses as well as multiple novel Giardia kinesin lineages. We predict the functional conservation of klps in Giardia mitosis, as Giardia possesses at least one klp from each mitotic subclass. Conversely, we suggest that novel Giardia klps contribute to the unique contractile functions and assembly of the ventral disc, a novel cytoskeletal structure responsible for adherence to the host epithelium (or laboratory substrates). To test these hypotheses, we will first use GFP and epitope protein tagging approaches to localize gklps within the cell. Based on mitotic or disc-localization, we will use several methods of functional inhibition followed by assays of mitosis and disc function to monitor the cellular behavior and assess the contribution of select Giardia klps to both mitosis and to the assembly and function of Giardia-specific cytoskeletal structures. We will also use state of the art light and electron microscopy to characterize Giardia mitosis and ventral disc function/assembly to provide a structural baseline for the analysis of kinesin function in these processes. These cell biological analyses wilt permit us to define the role of kinesins in spindle assembly and function and chromosome segregation and develop a molecular model of Giardia mitosis. Furthermore, we will assess the functions of kinesins in ventral-disc assembly and attachment/detachment. These analyses of Giardia kinesin functions should provide novel therapeutic targets for anti-Giardia anti-kinesin compounds.