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

PAR 蛋白在细胞内管管腔扩张过程中外囊招募和囊泡膜融合中的作用

• 批准号: 10265314
• 负责人: Joshua Michael Abrams
• 金额: $ 1.84万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-10 至 2020-11-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10265314
• 关键词: Acute; Affect; Alleles; Apical; Biological; Biological Assay; Blood Vessels; Blood capillaries; Caenorhabditis elegans; Candidate Disease Gene; Cardiovascular Diseases; Cardiovascular Physiology; Cardiovascular system; Cell Polarity; Cell physiology; Cells; Centers for Disease Control and Prevention (U.S.); Complex; Cues; Data; Defect; Development; Disease; Embryo; Endothelial Cells; Ensure; Genes; Genetic; Genetic Epistasis; Genetic Screening; Goals; Growth; Health; Human; Intervention; Invaded; Ischemia; Lead; Maintenance; Mediating; Membrane; Membrane Fusion; Methods; Microcirculation; Molecular; Morphology; Mutation; Myocardial Infarction; Myocardial Ischemia; Organ; Pathway interactions; Process; Proteins; Recovery; Reporter; Research; Role; Signal Transduction; Site; Stroke; Surface; System; Testing; Therapeutic; Tissues; Transmission Electron Microscopy; Tube; Vascular Diseases; Vesicle; angiogenesis; apical membrane; base; blastomere structure; capillary bed; cardiovascular disorder therapy; cardiovascular injury; effective therapy; improved; in vivo; insight; loss of function; luminal membrane; myocardial injury; neovascularization; new therapeutic target; novel; recruit; response; rho GTP-Binding Proteins; tool; trafficking; vascular bed; vascular injury

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.

项目总结/摘要 在单细胞无缝管中的管腔形成在细胞的发育和功能中是必不可少的。 心血管系统小管，如微循环内的终末血管床毛细血管，通常 通过胞内囊泡聚结和膜融合与侵入的细胞的前缘形成腔 顶端域我的长期目标是确定如何调节极化囊泡的运输，以确保 在体内适当的细胞中空和管形成。我将使用单细胞C。elegans排泄道作为一个 简单的系统来研究这一过程，因为它提供了强大的遗传和细胞生物学工具，并已被证明， 利用在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，包括心肌梗死和中风，以及分子 对管腔在血管发育和疾病期间如何扩张的理解仍然是难以捉摸的。我预计 我的研究结果将提供重要的见解，以更好地了解无缝毛细血管内， 血管形成，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剂，有助于不同的细胞过程。在 在排泄管中，PAR定位于腔膜，在那里它们与囊泡栓系共同定位 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， PARs可诱导胚胎外囊蛋白的不对称性。基于这些发现，我假设 PAR通过募集外囊并引导囊泡融合到这些部位来确定腔的形成位置。 我将使用我们实验室开发的一种方法来急性消耗特定细胞中的蛋白质， 体内通过去除PAR和外囊功能在运河。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 外囊复合体在PAR复合体下游发挥功能，介导腔囊泡募集; 3） 确定区分无缝管中管腔表面和非管腔表面所需的新基因。第一、 我将产生PAR的条件性功能丧失等位基因，以耗尽其在运河中的功能，并确定 它们在腔形成和外囊募集过程中的作用。我还将使用一种条件性功能丧失策略 从髓管中消除核心外囊组分SEC-5，以确定其与PAR的上位性 通过评估PAR定位。最后，我将测试候选基因，并进行基因筛查， 新的基因所需的管腔PAR和外囊定位。我的发现将大大扩展我们的 理解极性线索和囊泡运输在细胞中空化过程中的作用。理解这一 这一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， 缺血性疾病因此，我的研究结果将提供新的见解，如何新生血管生长， 恢复作为一种手段，改善目前的治疗心血管疾病。