Kinesin like proteins in the parasite, Giardia

描述（由申请人提供）：肠贾第虫是一种广泛存在的、进化上不同的人类和动物肠道寄生虫，是最早分化的真核生物谱系之一的成员。总的来说，很少有工作来开发细胞骨架介导的染色体分离或寄生虫通过专门的腹盘结构附着到宿主肠上皮的分子模型。本提案的目的是确定运动蛋白马达在贾第鞭毛虫有丝分裂和腹侧椎间盘的组装和功能中的功能。运动蛋白样蛋白（klps）分布在大约十个系统发育亚类中，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总共24个贾第鞭毛虫klps，它们属于每个主要的驱动蛋白亚类以及多个新的贾第鞭毛虫驱动蛋白谱系。我们预测在贾第鞭毛虫有丝分裂中klps的功能守恒，因为贾第鞭毛虫拥有来自每个有丝分裂亚类的至少一个klp。相反，我们认为新的贾第鞭毛虫klps有助于独特的收缩功能和腹侧椎间盘的组装，腹侧椎间盘是一种新的细胞骨架结构，负责粘附到宿主上皮（或实验室底物）。为了验证这些假设，我们将首先使用GFP和表位蛋白标记方法来定位细胞内的gklps。基于有丝分裂或盘定位，我们将使用几种功能抑制方法，随后进行有丝分裂和盘功能测定，以监测细胞行为，并评估选定的贾第鞭毛虫klps对有丝分裂和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侧椎间盘功能/组装，为分析这些过程中的驱动蛋白功能提供结构基线。这些细胞生物学分析将使我们能够确定运动蛋白在纺锤体组装和功能以及染色体分离中的作用，并建立贾第鞭毛虫有丝分裂的分子模型。此外，我们将评估运动蛋白在腹盘组装和附着/脱离中的功能。这些贾第鞭毛虫动力蛋白功能的分析将为抗贾第鞭毛虫动力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.