MOLECULAR ANALYSIS OF CYTOKINESIS IN DICTYOSTELIUM

描述（由申请人提供）：尽管最近的进展，胞质分裂仍然是最不了解的方面的细胞周期。在动物细胞中，胞质分裂需要多种细胞组分的协调，包括微管细胞骨架、肌动蛋白细胞骨架和膜运输。目前尚不清楚这些成分是如何调节的，以及它们如何相互作用。这个项目的长期目标是确定参与胞质分裂的元素，并了解这些元素如何整合完成胞质分裂。为了实现这一目标，已经确定了三个蛋白质所需的胞质分裂在网柄藻。这三种蛋白质提供了三种不同途径的手柄，这些途径在胞质分裂期间聚集在一起。这项资助的目的是剖析胞质分裂过程中这三种途径的贡献： 1)将确定LvsA在胞质分裂和骨形成中的作用。LvsA是膜运输蛋白的新型海滩家族的成员，并且是胞质分裂和收缩泡的功能所需的。这提供了在一个简单的模型系统中剖析膜交通对胞质分裂的贡献的机会。将确定每个LvsA结构域对其体内定位和功能的功能贡献。将鉴定LvsA的结合伴侣，并检测其对胞质分裂的要求。将分离LvsA的遗传抑制因子，并确定抑制机制。这些研究将阐明我们对胞质分裂中膜过程的理解，以及LvsA（LYST）的人类同源物突变如何导致人类疾病Chediak-Higashi综合征。 2)将描绘由RacE介导的细胞质分裂所必需的信号通路。小的GTTRY种族是必不可少的皮质张力的发展和卵裂沟的侵入。一种方法的组合将被用来解剖的机制，其中racE控制胞质分裂和皮质张力。racE促进肌动蛋白细胞骨架交联以增强皮质张力的假设将被检验。将确定卵裂沟正确进入所需的皮层张力。结合伴侣racE将被确定，并确定其在皮质张力和胞质分裂的发展中的作用。 3)DdlNCENP在胞质分裂中的作用将被剖析。INCENP是一种微管结合蛋白，已知对所有生物体的有丝分裂和胞质分裂都很重要，但其在胞质分裂中的确切作用尚未确定。我们的小组已经确定了DdlNCENP同源物，并创建了DdlNCENP敲除突变体。这些工具将被用来探索微管细胞骨架与细胞质分裂过程中收缩环组织的关系。由于已知INCENP参与肿瘤发生，这些结果可能提供对肿瘤形成机制的见解。

DESCRIPTION (provided by applicant): Despite recent advances, cytokinesis remains the least understood aspect of the cell cycle. In animal cells, cytokinesis requires the coordination of multiple cellular components including the microtubule cytoskeleton, the actin cytoskeleton and membrane traffic. Today, it is not clear how these components are regulated and how they interact with each other. The long-term goal of this project is to define the elements involved in cytokinesis and to understand how these elements integrate to accomplish cytokinesis. Toward that goal, three proteins required for cytokinesis in Dictyostelium have been identified. These three proteins provide a handle on three different pathways that come together during cytokinesis. The objective of this grant is to dissect the contribution of these three pathways during cytokinesis: 1) The role of LvsA in cytokinesis and osmore, qulation will be determined. LvsA is a member of the novel beach family of membrane trafficking proteins and is required for cytokinesis and for the function of the contractile vacuole. This provides the opportunity to dissect the contribution of membrane traffic to cytokinesis in a simple model system. The functional contribution of each of the LvsA domains to its localization and function in vivo will be ascertained. Binding partners for LvsA will be identified and their requirement for cytokinesis will be tested. Genetic suppressors of LvsA will be isolated and the mechanism of suppression will be determined. These studies will illuminate our understanding of membrane processes in cytokinesis and also how mutations in a human homologue of LvsA (LYST) cause a human disorder, the Chediak-Higashi Syndrome. 2) The signaling pathway mediated by RacE and essential for cytokinesis will be delineated. The small GTPase Race is essential for the development of cortical tension and for the ingression of the cleavage furrow. A combination of approaches will be used to dissect the mechanisms by which racE controls cytokinesis and cortical tension. The hypothesis that racE promotes crosslinking of the actin cytoskeleton to enhance cortical tension will be tested. The requirement of cortical tension for the proper ingression of the cleavage furrow will be determined. Binding partners racE will be identified and their role in the development of cortical tension and cytokinesis will be determined. 3) The role of DdlNCENP in cytokinesis will be dissected. INCENP is a microtubule-binding protein known to be important for mitosis & cytokinesis in all organisms but its exact role in cytokinesis has not been identified. Our group has identified the Dictyostelium INCENP homologue and created a DdlNCENP knockout mutant. These tools will be used to explore the relationship of the microtubule cytoskeleton with the organization of the contractile ring during cytokinesis. Since INCENPs are known to be involved in tumorigenesis these results may provide insights into the mechanisms of tumor formation.