Kinesin like proteins in the parasite, Giardia

寄生虫贾第鞭毛虫中的驱动蛋白样蛋白质

• 批准号: 6701821
• 负责人: William Zacheus Cande
• 金额: $ 30.4万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6701821
• 关键词: Giardia; cell components; cell cycle; chromosome movement; cytoskeleton; electron microscopy; epitope mapping; green fluorescent proteins; kinesin; laboratory rabbit; light microscopy; microtubules; molecular assembly /self assembly; molecular dynamics; polymerase chain reaction; protein structure function; transmission electron microscopy; video microscopy

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.

描述（由申请人提供）：贾第鞭毛虫是人类和动物的广泛且进化上的肠道寄生虫，也是最早分歧的真核生物谱系之一。通常，几乎没有工作来开发细胞骨架介导的染色体隔离或寄生虫附着在宿主肠道上皮上的分子模型，这是通过专用的腹盘结构。该提案的目标是确定示威动机在有丝分裂以及贾第二亚腹盘的组装和功能中的功能。驱动蛋白样蛋白（KLP）分布在大约十个系统发育亚类中，并且是许多细胞机制的重要组成部分，包括有丝分裂，鞭毛运动，细胞内转运和细胞极性。我们已经确定并克隆了总共二十四个贾第鞭毛虫KLP，将每个主要的驱动蛋白子类别以及多个新型的贾第鞭毛虫动力蛋白谱系分组。我们预测了贾第鞭毛虫中KLP的功能保守性，因为贾第氏菌具有至少一个有丝分裂亚类的KLP。相反，我们建议新型的贾第鞭毛虫KLP有助于腹盘的独特收缩功能和组装，腹盘是一种新型的细胞骨架结构，负责遵守宿主上皮（或实验室底物）。为了检验这些假设，我们将首先使用GFP和表位蛋白标记方法将GKLP定位在细胞内。基于有丝分裂或椎间盘定位，我们将使用几种功能抑制方法，然后使用有丝分裂和椎间盘功能的测定方法来监测细胞行为，并评估精选贾第鞭毛虫KLP对有丝分裂以及贾第氏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和电子显微镜来表征贾第鞭毛虫和腹盘功能/组装，以提供结构基线，以分析这些过程中的驱动蛋白功能。这些细胞生物学分析WILT使我们能够定义驱动蛋白在纺锤体组装和功能以及染色体分离中的作用，并开发出贾第藻菌的分子模型。此外，我们将评估驱动蛋白在腹盘组件和附着/脱离中的功能。这些对贾第鞭毛虫动力功能的分析应为抗甘迪亚抗运动蛋白化合物提供新的治疗靶标。