Composition, in vivo dynamics, and regulation of the exocyst in plant cells

项目摘要 外囊复合物对于真核细胞的极化分泌和生长是必需的，并且已经被广泛地研究。 研究了各个王国。尽管有这些研究，但其功能和调节机制仍不完全清楚。 理解。没有这种理解，就不可能操纵和解决与疾病有关的疾病。 这个进化保守的复合体的缺陷和失调。长期目标是建立一个 了解真核细胞中极化胞吐作用的调节。本申请的总体目标 目的是利用生物化学纯化技术研究植物细胞外孢囊的动态组成和调控， 阳离子、蛋白质-蛋白质相互作用测定以及定位和动力学的体内分析。中央hypoth- 植物胞外泌体的调节依赖于亚细胞定位及其与细胞外泌体的关系。 膜蛋白和磷酸肌醇，并不严重依赖于亚复合物的协会和dissoci， 行动。这一假设是基于Sec 6在苔藓细胞中的定位分析和现有的 在其他工厂工作。提出这项研究的理由是，有了这些新知识， 阐明极化分泌调节的关键方面，以及自分歧以来它是如何演变的 最后的真核生物共同祖先苔藓小立碗藓，由于其遗传，细胞生物学- 和显微镜工具，提供了一个强大的和独特的模型系统，以调查这一假设在植物中。 本研究将从以下两个方面对这一假说进行验证：1）从植物细胞中分离出胞外复合体 并通过结合相互作用确定其结构的调节;和2）确定 外囊，并建立其组装和相互作用动力学的计算机模拟。根据第一项 目的，一种基于亲和纯化技术，蛋白质组学和纯化的玉米体外相互作用的方法， 将使用ponents。所有这些活动将由来自生物学和 生物化学专业。在第二个目标下，外囊亚基的内源基因座将被荧光标记， 蛋白融合，并通过高分辨率多色成像，定量显微镜和荧光分析 光漂白后恢复。为了促进对外囊功能和调节的机制性理解， 计算机模拟方法将被使用的基础上的工作假设，扩散，组装动力学， 和定位都参与了外囊功能的调节。这些实验和分析将是 由来自生物学、生物信息学和计算生物学的本科生团队完成， 物理专业。提出的方法是创新的，因为它使用了模式植物，P. patens，和一个com-marticles。 显微镜、结构生物化学和模拟的结合，使理解向前迈进了一大步。 外囊是如何被调节的这项拟议中的研究意义重大，因为它将提供证据证明 或不存在亚复合体以及外囊在植物细胞中的动态定位。它还将提供一个 理论框架来解释显微镜观察，并得出现实模型的外囊调节。

PROJECT SUMMARY The exocyst complex is essential for polarized secretion and growth in eukaryotic cells and has been extensively studied across kingdoms. Despite these studies, its mechanism of function and regulation are still not fully un- derstood. Without this understanding, it will not be possible to manipulate, and address diseases associated with defects and deregulation of this evolutionarily conserved complex. The long-term goal is to have a mechanistic understanding of the regulation of polarized exocytosis in eukaryotic cells. The overall objective of this application is to determine the dynamic composition and regulation of the exocyst in plant cells by using biochemical purifi- cation, protein-protein interaction assays, and in vivo analyses of localization and dynamics. The central hypoth- esis is that the regulation of exocyst in plants is dependent on subcellular localization and its association with membrane proteins and phosphoinositides, and not heavily dependent on subcomplex association and dissoci- ation. This hypothesis was formulated based on localization analysis of Sec6 in moss cells and from existing work in other plants. The rationale for the proposed research is that, with this new knowledge, it will be possible to elucidate critical facets of the regulation of polarized secretion, and how it has evolved since the divergence from the last eukaryotic common ancestor. The moss Physcomitrella patens, because of its genetic, cell biolog- ical and microscopy tools, offers a powerful and unique model system to investigate this hypothesis in plants. The hypothesis will be tested by the following two specific aims: 1) Isolate the exocyst complex from plant cells and determine the regulation of its structure by binding interactions; and 2) Determine the in vivo dynamics of the exocyst and establish computational simulations of its assembly and interaction dynamics. Under the first aim, an approach based on affinity purification techniques, proteomics, and in vitro interactions of purified com- ponents will be used. All these activities will be performed by teams of undergraduate students from Biology and Biochemistry majors. Under the second aim, endogenous loci of exocyst subunits will be tagged with fluorescent protein fusions and analyzed by high-resolution multi-color imaging, quantitative microscopy, and fluorescence recovery after photobleaching. To advance a mechanistic understanding of exocyst function and regulation, a computer simulation approach will be used based on the working hypothesis that diffusion, assembly dynamics, and localization all participate in the regulation of exocyst function. These experiments and analyses will be completed by teams of undergraduate students from Biology, Bioinformatics and Computational Biology, and Physics majors. The approach proposed is innovative, because it uses the model plant, P. patens, and a com- bination of microscopy, structural biochemistry, and simulations to make major steps forward in understanding how exocyst is regulated. The proposed research is significant, because it will provide evidence for the presence or absence of subcomplexes and the dynamic localization of the exocyst in plants cells. It will also provide a theoretical framework to interpret microscopy observations and derive realistic models of exocyst regulation.