Dynamics of exocyst recruitment and assembly

描述（由申请人提供）：这些研究的长期目标是阐明外囊募集和组装参与囊泡束缚、对接和融合的机制。外囊是一种八聚体蛋白复合物（Sec 3/5/6/8/10/15/Exo 70/84），在空间上将囊泡靶向质膜上的特定位点，例如酵母中的芽尖，在一系列精液研究中鉴定出。在哺乳动物细胞中，它负责一系列极化细胞过程，如细胞分裂、迁移、连接稳定和初级纤毛形成。 尽管外囊的普遍存在和重要的作用，在理解外囊“拴系”复合物和其他假定的拴系物如何发挥锚囊泡的功能并与下游SNARE介导的融合机制协调方面存在关键的差距。虽然栓系被认为涉及长程相互作用，是可逆的，并调节囊泡融合的保真度，这些概念通常是未经测试的，有几个测定来监测外囊的离散的生物物理和分子状态的动态变化。解决这些问题将包括解决哺乳动物复合物的化学计量，外囊holocomplex的组装和拆卸，以及与融合机制的协调，所有这些都是拟议研究的关键目标。本研究的具体目的是：1）严格检验长期存在的“外囊作为系链”假设，并研究其招募到囊泡的动力学; 2）解决外囊如何能够生物促生物学地促进对接和融合; 3）解决外囊的翻译后修饰如何影响其稳定性并调节其功能。这些研究将为系绳的功能提供新的知识，并建立一个新的平台来监测它们的离散状态。 了解外囊如何发挥功能的基本机制与人类疾病高度相关，因为其功能障碍与癌细胞的转移、糖尿病和纤毛病相关，并且其是蒽毒素的病理靶向。更深入地了解外囊如何作为系链发挥作用以及它如何受到调节，可能有助于确定新的干预分子靶点。

DESCRIPTION (provided by applicant): The long-term objective of these studies is to elucidate the mechanisms by which exocyst recruitment and assembly are involved in vesicle tethering, docking and fusion. The exocyst is an octomeric protein complex (Sec3/5/6/8/10/15/Exo70/84) that spatially targets vesicles to specific sites on the plasma membrane, such as the bud tip in yeast, where it was identified in a series of seminal studies. In mammalian cells it is responsible for a range of polarized cellular processes such as cell division, migration, stabilization of junctions and formation of a primary cilium. Despite the ubiquitous and essential role of the exocyst, there are critical gaps in understanding how the exocyst 'tethering' complex and other putative tethers function to anchor vesicles and coordinate with downstream SNARE-mediated fusion machinery. While tethers are believed to involve long-range interactions, be reversible and regulate the fidelity of vesicle fusion, these concepts are generally untested and there are few assays to monitor the dynamic changes in discrete biophysical and molecular states of the exocyst. Resolving these topics will include addressing the stoichiometry of the mammalian complex, assembly and disassembly of the exocyst holocomplex, and its coordination with the fusion machinery, all of which are key goals in the proposed studies. The specific aims of this study are: 1) to critically test the longstanding "exocyst as a tether" hypothesis and to study the dynamics of its recruitment to vesicles; 2) to address how the exocyst is able to biophysically promote docking and fusion; 3) to address how post-translational modifications of the exocyst affects its stability and regulates its function. These studies will provide new knowledge of how tethers function and establish a new platform to monitor their discrete states. Understanding the basic mechanisms of how the exocyst functions is highly relevant to human diseases, since its dysfunction is associated with metastasis of cancer cells, diabetes, and ciliopathies, and it is pathologically targeted by anthra toxin. A deeper understanding of how the exocyst functions as a tether and how it is regulated may help identify new molecular targets of intervention.