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

植物细胞外囊的组成、体内动力学和调控

• 批准号: 9813291
• 负责人: Luis Vidali
• 金额: $ 44.81万
• 依托单位: WORCESTER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813291
• 关键词: Address; Affinity Chromatography; Animals; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biological Assay; Biological Models; Biology; Cell Proliferation; Cell Shape; Cell Size; Cell membrane; Cells; Chimeric Proteins; Color; Complex; Computational Biology; Computer Simulation; Coupled; Data; Defect; Development; Diffusion; Disease; Dissociation; Distant; Eukaryotic Cell; Event; Evolution; Exocytosis; Fluorescence Microscopy; Fluorescence Recovery After Photobleaching; Foundations; Genetic; Goals; Growth; Image; In Vitro; Knowledge; Label; Literature; Mammalian Cell; Mediating; Membrane Fusion; Membrane Proteins; Mentors; Microscopy; Mission; Modeling; Molds; Molecular; Morphogenesis; Mosses; Organism; Outcomes Research; Phosphatidylinositols; Physcomitrella; Physics; Plant Model; Plants; Process; Proteins; Proteomics; Public Health; Quantitative Microscopy; Regulation; Research; Resolution; Secretory Vesicles; Structural Biochemistry; Structure; Supervision; System; Techniques; Testing; Time; Training; United States National Institutes of Health; Work; Yeasts; base; biological research; career; cell growth; experimental study; in vivo; innovation; novel; polarized cell; protein protein interaction; simulation; stoichiometry; tool; undergraduate student

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.

项目概要 外囊复合体对于真核细胞的极化分泌和生长至关重要，并且已被广泛研究 跨王国学习。尽管有这些研究，但其功能和调节机制仍未完全阐明。 明白了。如果没有这种了解，就不可能操纵和解决与以下疾病相关的疾病： 这种进化上保守的复合体的缺陷和放松管制。长远目标是建立机制 了解真核细胞中极化胞吐作用的调节。该应用程序的总体目标 是利用生化纯化技术确定植物细胞胞外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体内定位和动力学分析。中心假设—— esis 是植物中胞囊的调节依​​赖于亚细胞定位及其与 膜蛋白和磷酸肌醇，并不严重依赖于亚复合物的结合和解离 化。这一假设是基于 Sec6 在苔藓细胞中的定位分析和现有的 在其他工厂工作。拟议研究的基本原理是，有了这些新知识，就有可能 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 来自最后一个真核生物的共同祖先。苔藓小立碗藓 (Physcomitrella patens) 因其遗传、细胞生物学特性， 科学和显微镜工具提供了一个强大而独特的模型系统来研究植物中的这一假设。 该假设将通过以下两个具体目标进行检验：1）从植物细胞中分离出胞外囊复合物 并通过结合相互作用确定其结构的调控； 2) 确定体内动力学 外囊并建立其组装和相互作用动力学的计算模拟。在第一个下 目标，一种基于亲和纯化技术、蛋白质组学和纯化化合物的体外相互作用的方法 将使用组件。所有这些活动将由生物学和生物系的本科生团队进行 生物化学专业。在第二个目标下，外囊亚基的内源位点将用荧光标记 蛋白质融合并通过高分辨率多色成像、定量显微镜和荧光进行分析 光漂白后恢复。为了促进对外囊功能和调节的机械理解， 将基于扩散、装配动力学、 和定位均参与外囊功能的调节。这些实验和分析将 由生物学、生物信息学和计算生物学的本科生团队完成，以及 物理专业。所提出的方法是创新的，因为它使用了模型植物 P. patens 和一个组件 显微镜、结构生物化学和模拟的结合在理解方面取得了重大进展 胞外囊是如何调控的。拟议的研究意义重大，因为它将提供存在的证据 或不存在子复合物以及植物细胞中外囊的动态定位。它还将提供一个 解释显微镜观察结果并得出外囊调节的现实模型的理论框架。