MOLECULAR ANALYSIS OF CYTOKINESIS IN DICTYOSTELIUM

EXCEEDTHESPACE提供。尽管最近取得了进展，但细胞分裂仍然是细胞周期中最不为人所知的方面。在动物细胞中，细胞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。今天，这些成分是如何被调节的以及它们如何相互作用还不清楚。该项目的长期目标是定义细胞分裂所涉及的元素，并了解这些元素如何整合以完成细胞分裂。为了实现这一目标，Dictyostelium细胞分裂所需的两种蛋白质已经被确定。这些蛋白质在细胞分裂过程中提供了两条不同的途径。这项资助的目的是剖析这些途径在细胞分裂过程中的贡献：1)LvsA在渗透调节和细胞分裂中的作用将被定义。LvsA是膜运输蛋白新海滩家族的成员，是细胞分裂和收缩液泡功能所必需的。这为在一个简单的模型系统中剖析膜运输对细胞分裂的贡献提供了机会。每个LvsA结构域对其在体内的定位和功能的功能贡献将被确定。将确定LvsA的结合伙伴，并测试它们对细胞分裂的需求。分离LvsA基因抑制因子，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同源物（LYST）的突变如何导致人类疾病Chediak-Higashi综合征。2)细胞质分裂过程中需要RacE的信号通路将被定义。小的GTPase RacE对于皮质张力的发展和细胞分裂时分裂沟的进入是必不可少的。将使用多种方法来剖析racE控制细胞分裂的机制。基于最近几个实验室关于细胞分裂所需蛋白质的数据，提出了一个解释RacE在细胞分裂过程中作用的模型。该模型的几个预测将得到验证，其中包括：a) RacE通过激活特定的iqgap来控制Cortexillins在卵裂沟处的组织；b) ddraggap1的GAP结构域在体内调节RacE的活性。此外，已经开发了一种纯化RacE的方法，并将用于鉴定RacE的新结合伙伴。这些蛋白在细胞质分裂中的作用将通过生化和遗传方法来确定。PERFORMANCESITE ( ======================================== 节结束 ===========================================

EXCEEDTHESPACE PROVIDED. 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, two proteins required for cytokinesis in Dictyostelium have been identified. These proteins provide a handle on two different pathways that come together during cytokinesis. The objective of this grant is to dissect the contribution of these pathways during cytokinesis: 1) The role of LvsA in osmorequlation and cytokinesis will be defined. 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 that requires RacE durinq cytokinesis will be defined. The small GTPase RacE is essential for the development of cortical tension and for the ingression of the cleavage furrow during cytokinesis. A combination of approaches will be used to dissect the mechanisms by which racE controls cytokinesis. A model that explains the role of RacE during cytokinesis is proposed based on recent data from several laboratories on proteins required in cytokinesis. Several predictions made by this model will be tested, among them: a) that RacE controls the organization of Cortexillins at the cleavage furrow though the activation of specific IQGAPs; b) that the GAP domain of DdRacGAP1 modulates the activity of RacE in vivo. In addition, a method to purify RacE has been developed and will be used to identify novel binding partners for racE. The role of these proteins in cytokinesis will be determined by biochemical and genetic approaches. PERFORMANCESITE( ========================================Section End===========================================