Kinesin function in mitosis and automixis in the widespread parasite Giardia inte

广泛分布的寄生虫贾第鞭毛虫有丝分裂和自混合中的驱动蛋白功能

• 批准号: 8089321
• 负责人: William Zacheus Cande
• 金额: $ 40.57万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8089321
• 关键词: ATP phosphohydrolase; Animal Model; Animals; Behavior; Biochemical; Biological Assay; Biology; Cell Cycle Stage; Cell Nucleus; Cell division; Cell fusion; Cells; Chromosomes; Cyst; Cytoskeleton; DNA Damage; DNA Repair; Dominant-Negative Mutation; Drug resistance; Electron Microscopy; Eukaryota; Event; Evolution; Family; Gene Expression; Generations; Genetic; Genetic Materials; Genetic Screening; Genome Stability; Giardia; Giardia lamblia; Goals; Health; Homologous Gene; Human; In Vitro; Interphase; Intestines; Investigation; Kinesin; Knowledge; Length; Life; Life Cycle Stages; Location; Maintenance; Meiosis; Metronidazole; Microtubules; Mitosis; Mitotic; Molecular; Motor; Movement; Nuclear; Nuclear Fusion; Organism; Parasites; Pathogenesis; Pharmaceutical Preparations; Phenotype; Play; Process; Reproduction; Research; Role; Testing; Time; chemical genetics; design; drug discovery; homologous recombination; kinase inhibitor; migration; mutant; novel; promoter; small molecule; tool

DESCRIPTION (provided by applicant): The goal of this project is to analyze functions essential to the reproduction and pathogenesis of the intestinal parasite and basal eukaryote, Giardia intestinalis. G. intestinalis belongs to the earliest known diverging lineage of eukaryotes (diplomonads), and it is a widespread intestinal parasite of humans and animals. It is one of the ten major parasites in humans, and in the USA, it is estimated that several million cases occur annually. Further, there is recent in vitro evidence of drug resistance to the widely used antigiardial, Flagyl. These observations underscore the necessity of identifying alternatives to the limited number of known antigiardial compounds, and highlight our incomplete knowledge of Giardia biology. The proposed research will establish fundamental principles governing force generation in the Giardia microtubule cytoskeleton, and will test how selected kinesins (microtubule motors) promote mitosis, ventral disc function and assembly, and karyogamy and automixis during encystation. We hypothesize that the evolutionarily conserved kinesins are involved in conserved processes (for example, mitosis), whereas the novel kinesins are involved in novel functions such askaryogamy during encystation. We have developed new molecular tools for studying kinesin function in Giardia. Analysis of a dominant negative rigor mutant of kin13 demonstrates that it is a master regulator of interphase and mitotic microtubule dynamics, and along with the two kin2 representatives regulates flagellar length. Our approach includes an analysis of kinesin localization in Giardia to prioritize candidates for further study (Aim 1), and an in-depth study of selected kinesin functions using a genetic and biochemical approach. (Aims 1-3). We will continue to investigate the mechanism of mitosis in Giardia, extending our analysis to living cells. The giardial mitotic kinesins under investigation in aim 2 are essential for reproduction and will be prime candidates for chemical genetic screens for small molecule inhibitors of kinase ATPase activity. This analysis will also provide an important evolutionary perspective on kinesin function during mitosis. In specific aims 3 and 4 we will investigate nuclear behavior during encystation that leads to cell and nuclear fusion and automixis. . Automixis may be essential for maintenance of genome stability and evolution of drug resistance, and therefore is a target for drug discovery. We will determine whether kinesins contribute to cell and nuclear movement and fusion (aim 3) and whether processes related to homologous recombination occur after fusion (aim 4). These results will be validated by infecting animal models with cells containing integrated selectable markers. PUBLIC HEALTH RELEVANCE: Giardia intestinalis is one of the ten major parasites in humans, and in the USA, it is estimated that several million cases occur annually. There is evidence of drug resistance to Flagyl, the current drug of choice. These observations underscore the necessity of identifying alternatives to the limited number of known antigiardial compounds, and highlight our incomplete knowledge of Giardia biology. The research proposed here will directly discover as yet unknown essential features of the Giardia life cycle, including cell division and meiosis and offer additional targets for drug discovery.

描述（由申请人提供）：该项目的目的是分析对肠道寄生虫和基础真核生物贾第二硫酸肠的繁殖和发病机理必不可少的功能。 G. intestinalis属于最早已知的真核生物谱系（Eliphomonads），它是人类和动物的广泛肠道寄生虫。它是人类的十个主要寄生虫之一，在美国，据估计每年发生数百万个病例。此外，最近有一些体外证据表明对广泛使用的抗生物鞭毛的耐药性。这些观察结果强调了确定有限数量的已知抗原化合物的替代品的必要性，并强调了我们对贾第鞭毛虫生物学的不完整知识。拟议的研究将在贾第鞭毛虫微管细胞骨架上建立有关力产生的基本原理，并将测试选择的驱动蛋白（微管电动机）如何促进有丝分裂，腹盘功能和组装，以及核果和自闭症。我们假设进化保守的驱动蛋白参与保守的过程（例如有丝分裂），而新型驱动蛋白参与了新型功能，例如在广告过程中。我们已经开发了用于研究贾第烷的动力素功能的新分子工具。对KIN13的主要负严格突变体的分析表明，它是相间和有丝分裂微管动力学的主要调节剂，并且与两个KIN2代表一起调节鞭毛长度。我们的方法包括对贾第藻（Giardia）的动力素定位的分析，以优先考虑候选人进行进一步研究（AIM 1），并使用遗传和生化方法对选定的驱动蛋白功能进行了深入研究。 （目标1-3）。我们将继续研究贾第藻中有丝分裂的机制，将我们的分析扩展到活细胞。在AIM 2中研究的贾dial有丝分裂驱动素对繁殖至关重要，将是用于激酶ATPase活性的小分子抑制剂化学遗传筛选的主要候选者。该分析还将为有丝分裂过程中的驱动蛋白功能提供重要的进化观点。在特定的目标3和4中，我们将研究导致细胞和核融合和汽车的百年核行为。 。汽车可能对于维持基因组稳定性和耐药性的进化至关重要，因此是药物发现的靶标。我们将确定驱动蛋白是否有助于细胞，核运动和融合（AIM 3）以及融合后与同源重组有关的过程是否有助于（AIM 4）。这些结果将通过感染含有综合选择标记的细胞感染动物模型来验证。公共卫生相关性：贾第鞭毛虫是人类的十个主要寄生虫之一，在美国，据估计，每年发生数百万个病例。有证据表明抗鞭毛的耐药性是当前选择的药物。这些观察结果强调了确定有限数量的已知抗原化合物的替代品的必要性，并强调了我们对贾第鞭毛虫生物学的不完整知识。此处提出的研究将直接发现贾第鞭毛虫生命周期的未知基本特征，包括细胞分裂和减数分裂，并为药物发现提供其他靶标。