Dynamics of exocyst recruitment and assembly

描述(申请人提供)：这些研究的长期目标是阐明囊泡连接、对接和融合过程中胞囊招募和组装的机制。外囊是一种八聚体蛋白质复合体(Sec3/5/6/8/10/15/Exo70/84)，它将囊泡定位于质膜上的特定位置，如酵母中的芽尖，在那里它被一系列的种子研究所鉴定。在哺乳动物细胞中，它负责一系列极化的细胞过程，如细胞分裂、迁移、连接的稳定和初级纤毛的形成。尽管胞囊具有普遍而重要的作用，但在理解胞囊“系留”复合体和其他假定的系链如何起到锚定囊泡并与下游的圈套介导的融合机制协调的作用方面，仍存在关键的空白。虽然系链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测试，几乎没有检测方法来监测胞囊离散的生物物理和分子状态的动态变化。解决这些主题将包括解决哺乳动物复合体的化学计量学，外囊全复合体的组装和拆解，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。这项研究的具体目的是：1)批判性地检验长期存在的“外囊作为系绳”的假说，并研究其募集到囊泡的动力学；2)研究外囊如何能够在生物物理上促进对接和融合；3)研究外囊的翻译后修饰如何影响其稳定性和调节其功能。这些研究将提供系绳如何工作的新知识，并建立一个新的平台来监测它们的离散状态。了解外囊功能的基本机制与人类疾病高度相关，因为它的功能障碍与癌细胞转移、糖尿病和纤毛疾病有关，而且它是花椒毒素的病理靶点。更深入地了解胞囊是如何发挥系绳作用的，以及它是如何被调控的，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.