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囊泡招募和组装参与囊泡系结、对接和融合的机制。胞囊是一种八聚体蛋白复合物（Sec3/5/6/8/10/15/Exo70/84），它在空间上将囊泡定位于质膜上的特定位置，例如酵母的芽尖，在一系列的种子研究中发现了它。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的形成。尽管囊泡的作用无处不在且至关重要，但在了解囊泡“系绳”复合物和其他假定的系绳如何锚定囊泡并与下游snare介导的融合机制协调方面仍存在关键空白。虽然系索被认为涉及远程相互作用，是可逆的，并调节囊泡融合的保真度，但这些概念通常未经验证，并且很少有监测囊泡离散生物物理和分子状态动态变化的分析。解决这些问题将包括解决哺乳动物复合体的化学计量学，外囊整体复合体的组装和拆卸，以及它与融合机制的协调，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)批判性地验证长期存在的“外囊作为系绳”假说，并研究其向囊泡募集的动力学；2)研究囊泡如何从生物物理角度促进对接和融合；3)研究胞囊的翻译后修饰如何影响其稳定性和调节其功能。这些研究将提供关于系绳功能的新知识，并建立一个新的平台来监测它们的离散状态。了解胞囊功能的基本机制与人类疾病密切相关，因为其功能障碍与癌细胞、糖尿病和纤毛病的转移有关，并且是炭疽毒素的病理靶点。更深入地了解外囊作为系索的功能以及它是如何被调节的，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.