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

项目摘要/摘要 单细胞无缝管内管腔的形成对血管的发育和功能至关重要。 心血管系统。微循环中的小管，如末梢血管床毛细血管，通常 通过细胞内小泡合并和膜融合与入侵的前沿形成管腔 顶端区域。我的长期目标是确定极化囊泡贩运是如何受到监管的，以确保 体内适当的细胞空化和管状形成。我会用单细胞线虫排泄道作为 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，并已证明 利用血管发育和疾病期间保存的途径。新生血管管腔缺陷 扩张导致许多血管疾病，包括心肌梗死和中风，以及一种分子 对血管发育和疾病过程中管腔如何扩张的理解仍然难以捉摸。我期待着 我的发现将提供重要的见解，以更好地理解 血管系统的形成，希望改善心血管疾病的干预。 PAR蛋白是细胞极性的保守调节者，对不同的细胞过程有贡献。在……里面 排泄管，部分定位于腔膜，在那里它们与囊泡拴系在一起。 胞囊复合体。我们的实验室最近发现，在管腔形成过程中，囊泡融合需要外囊， 在胚胎中，PARS可以诱导外囊蛋白的不对称。基于这些发现，我假设 这些解析器通过招募外囊并将囊泡融合引导到这些位置来定义管腔的形成位置。 使用我们实验室开发的一种方法来急剧耗尽特定细胞中的蛋白质，我将在 体内通过清除PAR和管内排囊的功能。我的提议的具体目的是：1)测试 假设PAR蛋白是管腔形成和/或维持所必需的；2)确定 外囊复合体在PAR复合体下游发挥作用，以调节腔泡的募集；3) 识别无缝管中区分管腔和非管腔表面所需的新基因。第一, 我将产生有条件的功能丧失等位基因，以使PAL在管内耗尽其功能，并确定 它们在管腔形成和外囊招募中的作用。我还将使用有条件的功能丧失策略 从管内清除核心外囊成分SEC-5，以确定其相对于PARS的上位性 通过评估PAR本地化。最后，我将测试候选基因，并进行基因筛查以发现 腔内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 了解极性信号和囊泡运输在细胞空心化过程中的作用。理解这一点 这一过程直接关系到人类健康的许多方面，包括从心血管损伤中恢复和 缺血性疾病。因此，我的研究结果将为新生的血管生长如何 将其恢复为改善目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.