In vivo mechanisms for epithelial cell polarization

上皮细胞极化的体内机制

• 批准号: 8389805
• 负责人: Stephen Thomas Armenti
• 金额: $ 4.72万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-10 至 2015-06-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389805
• 关键词: Address; Alleles; Apical; Binding; Biological Models; Caenorhabditis elegans; Cell Adhesion; Cell Polarity; Cell-Cell Adhesion; Cells; Chronic; Chronic Kidney Failure; Complex; Cues; Cultured Cells; Defect; Development; Drosophila genus; E-Cadherin; Eating; Embryo; Epidermis; Epithelial; Epithelial Cell Junction; Epithelial Cells; Epithelium; Event; Exclusion; Fibrosis; Foundations; Future; Genetic; Goals; Homologous Gene; Human; Image; Injury; Intercellular Junctions; Kidney; Kidney Diseases; Knowledge; Lead; Learning; Life; MDCK cell; Maintenance; Mediating; Membrane; Molecular; Morphogenesis; Organ; PARD6A gene; Pathologic; Play; Process; Protein Kinase; Proteins; RNA Interference; Recruitment Activity; Renal carcinoma; Renal function; Research; Role; Scaffolding Protein; Secondary to; Signal Transduction; Site; Sterility; Structure; Surface; System; Testing; Tissues; Tube; Vitelline Membrane; base; design; epithelial to mesenchymal transition; extracellular; gene function; genome sequencing; human JTB protein; in vivo; insight; mutant; nephrogenesis; novel; prevent; research study

DESCRIPTION (provided by applicant): Establishing epithelial cell polarity and assembling cell-cell junctions are critical steps in tissue morphogenesis. One of the earliest events in epithelial differentiation is the formation of intercellular junctions required for cell-cell adhesion, barrier function and apicobasal polarity. In order to form mature junctions, epithelial cells must (1) respond to polarity cues and localize junction proteins to the site of future junction formation and (2) coalesce these proteins into fully formed junctions. This process is essential for the development of organs such as the kidney, which features a complex epithelial tubule system that relies on the establishment of polarity and maintenance of complex intercellular junctions to regionally segregate functional membrane domains. When renal epithelial cells lose polarity and junctions (for instance, following chronic injury), epithelial cells delaminate and undergo an epithelial-to-mesenchymal transition that leads to tissue fibrosis and impaired kidney function. The proposed research will further our understanding of how epithelial cells polarize and form junctions in vivo by determining the molecular mechanisms of localizing proteins to junctions and coalescing junction proteins into fully-formed junctions. This information will be instrumental in learning how to prevent pathologic EMTs that occur in kidney disease. Mechanisms of epithelial polarization have been studied predominantly in cultured MDCK cells, where the initial polarity cue is generated by E-cadherin-mediated cell adhesion. However, studies examining epithelial polarization in Drosophila, C. elegans and some types of human cultured cells have shown that polarization can occur in the absence of E-cadherin, indicating that epithelial polarization may occur through different mechanisms in vivo. C. elegans is an excellent model system for studying in vivo mechanisms of epithelial polarization and junction assembly during MET as it offers the possibility to watch polarization as it occurs in wild-type and mutant embryos. The specific goal of the proposed research is to determine how homologues of two conserved polarity regulators (Crumbs/EAT-20 and aPKC/PKC-3) regulate C. elegans epithelial cell polarization and junction assembly, respectively. We have shown that EAT-20 functions redundantly with the scaffolding protein PAR-3 to polarize the C. elegans epidermis. In my first aim, I will determine how EAT-20 localizes and functions using genetics and live imaging. To address mechanisms of junction assembly by aPKC/PKC-3, we have performed a genetic suppressor screen to identify genes that function with pkc-3 to regulate epithelial junctions. In my second aim, I will clone two pkc-3 suppressors and determine how they regulate epithelial junctions. Together, I anticipate that my findings will provide new insights into the basic mechanisms of epithelial polarization and junction formation as they occur in vivo, providing a foundation for understanding the molecular mechanisms responsible for junction loss in kidney disease and cancer.

描述（由申请人提供）：建立上皮细胞极性和组装细胞-细胞连接是组织形态发生的关键步骤。上皮分化的最早期事件之一是细胞间连接的形成，这是细胞-细胞粘附、屏障功能和顶基极性所必需的。为了形成成熟的连接，上皮细胞必须（1）对极性线索做出反应，并将连接蛋白定位于未来连接形成的位点，以及（2）将这些蛋白结合成完全形成的连接。这一过程对于器官如肾脏的发育至关重要，肾脏具有复杂的上皮小管系统，该系统依赖于极性的建立和复杂细胞间连接的维持，以区域性地分离功能性膜结构域。当肾上皮细胞失去极性和连接时（例如，在慢性损伤之后），上皮细胞分层并经历上皮至间充质转化，导致组织纤维化和肾功能受损。拟议的研究将通过确定蛋白质定位于接头和将接头蛋白结合成完全形成的接头的分子机制，进一步了解上皮细胞如何在体内形成和形成接头。这些信息将有助于学习如何预防肾脏疾病中发生的病理性EMT。 上皮极化的机制主要在培养的MDCK细胞中进行了研究，其中初始极性线索由E-钙粘蛋白介导的细胞粘附产生。然而，在果蝇，C。elegans和某些类型的人类培养细胞已经表明，极化可以在缺乏E-钙粘蛋白的情况下发生，这表明上皮极化可以通过体内不同的机制发生。C. elegans是研究MET期间上皮极化和连接组装的体内机制的极好模型系统，因为它提供了观察在野生型和突变胚胎中发生的极化的可能性。这项研究的具体目标是确定两个保守的极性调节因子（Crumbs/EAT-20和aPKC/PKC-3）的同源物如何调节C。elegans上皮细胞极化和连接组装。我们已经证明EAT-20与支架蛋白PAR-3冗余地起作用，以使C.秀丽隐翅虫表皮在我的第一个目标中，我将确定EAT-20如何使用遗传学和实时成像定位和发挥作用。为了阐明aPKC/PKC-3的连接组装机制，我们进行了遗传抑制筛选，以鉴定与PKC-3一起调节上皮连接的基因。在我的第二个目标中，我将克隆两个pkc-3抑制因子，并确定它们如何调节上皮连接。总之，我预计我的研究结果将提供新的见解上皮极化和连接形成的基本机制，因为它们发生在体内，为理解肾脏疾病和癌症的连接损失的分子机制提供了基础。