The Role of PAR Proteins in Exocyst Recruitment and Vesicle Membrane Fusion During Lumen Expansion of Intracellular Tubes

项目概要/摘要 单细胞无缝管内的管腔形成对于细胞的发育和功能至关重要 心血管系统。小管，例如微循环内的终末血管床毛细血管，通常 通过细胞内囊泡聚结和膜与入侵的前缘融合形成管腔 顶端域。我的长期目标是确定如何调控极化囊泡运输以确保 体内适当的细胞空心和管形成。我将使用单细胞线虫排泄管作为 研究这一过程的简单系统，因为它提供了强大的遗传和细胞生物学工具，并且已被证明 利用血管发育和疾病期间保守的途径。新生血管管腔缺陷 扩张导致许多血管疾病，包括心肌梗塞和中风，并且分子 对于血管发育和疾病期间管腔如何扩张的理解仍然难以捉摸。我预计 我的发现将提供重要的见解，以更好地理解体内的无缝毛细血管 血管系统形成，有望改善心血管疾病干预。 PAR 蛋白是细胞极性的保守调节因子，有助于多种细胞过程。在 在排泄道中，PAR 定位于管腔膜，在那里它们与囊泡束缚共定位 外囊复合体。我们的实验室最近表明，在管腔形成过程中，囊泡融合需要外囊， 并且 PAR 可以诱导胚胎中外囊蛋白的不对称性。根据这些发现，我假设 PAR 通过招募外囊并将囊泡融合到这些位点来定义管腔形成的位置。 使用我们实验室开发的一种方法来急剧消耗特定细胞中的蛋白质，我将在 体内通过去除管内的 PAR 和外囊功能。我的建议的具体目标是： 1）测试 假设 PAR 蛋白是管腔形成和/或维持所必需的； 2) 确定是否 外囊复合体在 PAR 复合体下游发挥作用，介导管腔囊泡募集； 3） 识别区分无缝管中的管腔表面和非管腔表面所需的新基因。首先， 我将为 PAR 生成条件功能丧失等位基因，以耗尽它们在根管中的功能并确定 它们在腔发生和外囊招募过程中的作用。我还将使用条件功能丧失策略 从根管中消除核心外囊成分 SEC-5，以确定其相对于 PAR 的上位性 通过评估 PAR 定位。最后，我将测试候选基因并进行基因筛选以发现 管腔 PAR 和外囊定位所需的新基因。我的发现将极大地扩展我们的 了解细胞空心过程中极性线索和囊泡运输的作用。了解这一点 该过程直接关系到人类健康的许多方面，包括心血管损伤的恢复和 缺血性疾病。因此，我的研究结果将为新生血管生长如何发挥作用提供新的见解。 被恢复作为改善当前心血管疾病治疗的一种手段。

Project Summary/Abstract Lumen formation within unicellular, seamless tubes is essential in the development and function of the cardiovascular system. Small tubes, such as terminal vascular bed capillaries within the microcirculation, often form lumens by intracellular vesicle coalescence and membrane fusion with the leading edge of an invading apical domain. My long-term goal is to determine how polarized vesicle trafficking is regulated to ensure proper cell hollowing and tube formation in vivo. I will use the unicellular C. elegans excretory canal as a simple system to study this process, as it offers powerful genetic and cell biological tools, and has proven to utilize pathways conserved during vascular development and disease. Defects in nascent vessel lumen expansion lead to numerous vascular disorders, including myocardial infarction and stroke, and a molecular understanding of how lumens expand during vascular development and disease remains elusive. I anticipate that my findings will provide important insights to better understand how seamless capillaries within the vasculature are formed, with the hope of improving cardiovascular disease intervention. PAR proteins are conserved regulators of cell polarity that contribute to diverse cellular processes. In the excretory canal, PARs localize to the luminal membrane, where they co-localize with the vesicle tethering exocyst complex. Our lab recently showed that exocyst is required for vesicle fusion during lumen formation, and that PARs can induce asymmetry of exocyst proteins in embryos. Based on these findings, I hypothesize that PARs define where the lumen will form by recruiting exocyst and directing vesicle fusion to these sites. Using a method to acutely deplete proteins in specific cells developed in our lab, I will test this hypothesis in vivo by removing PAR and exocyst function in the canal. The specific aims of my proposal are to: 1) Test the hypothesis that PAR proteins are required for lumen formation and/or maintenance; 2) Determine if the exocyst complex functions downstream of the PAR complex to mediate luminal vesicle recruitment; 3) Identify novel genes required to distinguish luminal from non-luminal surfaces in seamless tubes. First, I will generate conditional loss-of-function alleles for PARs to deplete their function in the canal and determine their role during lumenogenesis and exocyst recruitment. I will also use a conditional loss-of-function strategy to eliminate a core exocyst component, SEC-5, from the canal to determine its epistasis with respect to PARs by evaluating PAR localization. Finally, I will test candidate genes and undertake a genetic screen to uncover new genes required for luminal PAR and exocyst localization. My findings will greatly expand our understanding of the role for polarity cues and vesicle trafficking during cell hollowing. Understanding this process directly relates to many aspects of human health, including recovery from cardiovascular injury and ischemic disease. Thus the results of my studies will provide new insights into how nascent vessel growth can be restored as a means of improving current therapies of cardiovascular disease.