MOLECULAR ANALYSIS OF CYTOKINESIS IN DICTYOSTELIUM

描述(由申请人提供)：尽管最近取得了进展，但细胞质分裂仍然是细胞周期中最不被了解的方面。在动物细胞中，胞质分裂需要多种细胞成分的协调，包括微管细胞骨架、肌动蛋白细胞骨架和膜运输。如今，尚不清楚这些组成部分是如何受到监管的，以及它们之间是如何相互作用的。这个项目的长期目标是定义参与胞质分裂的元素，并了解这些元素是如何整合在一起完成胞质分裂的。为了实现这一目标，已经确定了网柄蕨胞质分裂所需的三种蛋白质。这三种蛋白质在细胞质分裂过程中连接在一起的三条不同的通路上。这项资助的目的是剖析这三条途径在细胞质分裂过程中的作用： 1)将确定LVSA在胞质分裂和成骨过程中的作用。LVSA是膜转运蛋白BASH家族的一员，是胞质分裂和收缩空泡功能所必需的。这提供了在一个简单的模型系统中剖析膜交通对细胞质分裂的贡献的机会。将确定每个LVSA结构域对其在体内的定位和功能的功能贡献。将确定LVSA的结合伙伴，并测试它们对胞质分裂的要求。我们将分离出LVSA的基因抑制因子，并确定其抑制机制。这些研究将阐明我们对细胞质分裂中的膜过程的理解，以及人类LVSA(Lyst)同源物的突变如何导致人类疾病-切迪亚克-东综合征。 2)RACE介导的胞质分裂所必需的信号转导途径。小的GTP酶竞赛对于皮质张力的发展和裂沟的内向是必不可少的。我们将结合多种方法来剖析RACE控制细胞质分裂和皮质张力的机制。RACE促进肌动蛋白细胞骨架的交联化以增强皮质张力的假设将得到验证。将确定适当地进入乳沟所需的皮质张力。将确定结合伙伴RACE，并确定它们在皮质张力和胞质分裂发展中的作用。 3)DdlNCENP在胞质分裂中的作用。INCENP是一种微管结合蛋白，在所有生物的有丝分裂和胞质分裂中都是重要的，但它在胞质分裂中的确切作用尚未确定。我们的团队已经鉴定了DictyostelialINCENP同源物，并创造了一个DdlNCENP基因敲除突变体。这些工具将被用来探索微管细胞骨架与胞质分裂过程中收缩环的组织之间的关系。由于INCENPs已知与肿瘤的发生有关，这些结果可能为肿瘤形成的机制提供见解。

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.