In vivo mechanisms of epithelial cell polarization and junction formation

描述（由申请人提供）：上皮细胞排列在我们器官的表面，对它们的形成和功能至关重要。在胚胎发生过程中，大多数上皮细胞发育时，间充质细胞沿其顶基轴极化并组装细胞-细胞连接（间充质-上皮转化，MET）。极性或连接的丧失可导致毁灭性疾病，包括肾脏疾病、由细胞增殖和侵袭增加引起的癌症，以及由形态发生受损引起的出生缺陷。我们对MET的大部分理解来自体外培养细胞的研究，这些细胞在e -钙粘蛋白介导的相互接触中发生极化。然而，MET的机制在体内可能存在差异，因为在果蝇、秀丽隐杆线虫和一些哺乳动物的上皮细胞中，e -钙粘蛋白并不需要启动极性，而且细胞接触并不总是足以诱导极化。这个项目的长期目标是确定细胞和分子机制用于极化上皮细胞和组装连接在一个发育中的有机体。我们已经开发了实时成像和遗传工具来研究秀丽隐杆线虫活体胚胎中的MET机制。使用这些工具，我们已经表明，保守的极性调节器诱导结形成的顺序步骤。首先，PAR-3通过将连接蛋白和其他极性调节因子聚集到皮质病灶中来建立极性，然后将其迁移到根尖表面。然后，与激酶PKC-3一起起作用的PAR-6将顶端连接蛋白聚集成环绕细胞的带。我们还表明，在管状上皮细胞和片状上皮细胞之间，极化机制可能不同，其中顶端的碎屑蛋白EAT-20与PAR-3一起冗余地发挥极化细胞的作用。尽管PAR-3、par -20、PAR-6和PKC-3同源物在上皮细胞中具有保守的作用，但这些蛋白如何在体内使细胞极化和组装连接在很大程度上是未知的。本研究的具体目标是确定PAR-3和EAT-20用于上皮细胞极化的分子机制，并确定PAR-6和PKC-3如何组装连接。通过实时成像和遗传分析，我们将确定PAR-3病灶是如何形成的，连接蛋白和极性蛋白是如何装载到病灶上的，并测试病灶沿着微管移动到根尖表面的假设。我们将验证EAT-20通过向根尖表面招募蛋白质来建立极性的假设，确定EAT-20下游使细胞极化的途径，并确定EAT-20根尖定位是由细胞接触还是胚胎外配体指导的。最后，我们将使用我们已经进行过的大规模遗传选择筛选来鉴定调节或发挥PAR-6和PKC-3下游组装连接功能的基因。这一建议将通过建立上皮细胞在体内极化和组装连接的机制来推进这一领域，增加我们对包括肾脏疾病、癌症和出生缺陷在内的上皮疾病的理解。

DESCRIPTION (provided by applicant): Epithelial cells line the surfaces of our organs and are critical for their formation and function. During embryogenesis most epithelial cells develop when mesenchymal cells polarize along their apicobasal axis and assemble cell-cell junctions (mesenchymal-to-epithelial transformation, MET). Loss of polarity or junctions can lead to devastating diseases, including kidney disease, cancer caused by increased cell proliferation and invasion, and birth defects arising from impaired morphogenesis. Much of our understanding of MET comes from in vitro studies of cultured cells, which polarize as they make E-cadherin-mediated contact with one another. However, mechanisms of MET likely differ in vivo since E-cadherin is not needed to initiate polarity in Drosophila, C. elegans, and some mammalian epithelia, and because cell contact is not always sufficient to induce polarization. The long-term goal of this project is to determine the cellular and molecular mechanisms used to polarize epithelial cells and assemble junctions in a developing organism. We have developed live-imaging and genetic tools to investigate mechanisms of MET in living C. elegans embryos. Using these tools we have shown that conserved polarity regulators induce junction formation in sequential steps. First, PAR-3 establishes polarity by aggregating junction proteins and other polarity regulators into cortical foci, which then travel to the apical surface. Then PAR-6, which functions with the kinase PKC-3, condenses clusters of apical junction proteins into belts that encircle the cell. We have also shown that polarization mechanisms can differ between tube-forming epithelial cells and sheet-forming epithelial cells, where the apical Crumbs protein EAT-20 functions redundantly with PAR-3 to polarize cells. Although PAR-3, EAT-20, PAR-6, and PKC-3 homologues have conserved roles in epithelial cells, how these proteins function to polarize cells and assemble junctions in vivo is largely unknown. The specific objectives of the proposed research are to define the molecular mechanisms that PAR-3 and EAT-20 use to polarize epithelial cells, and to determine how PAR-6 and PKC-3 assemble junctions. Using live imaging and genetic analysis, we will determine how PAR-3 foci form, how junction and polarity proteins load onto foci, and test the hypothesis that foci move to the apical surface along microtubules. We will test the hypothesis that EAT-20 establishes polarity by recruiting proteins to the apical surface, define the pathways downstream of EAT-20 that polarize cells, and determine if EAT-20 apical localization is directed by cell contacts or extra- embryonic ligands. Finally, we will use large-scale genetic selection screens we have already performed to identify genes that regulate or function downstream of PAR-6 and PKC-3 to assemble junctions. This proposal will advance the field by establishing mechanisms epithelial cells use to polarize and assemble junctions in vivo, increasing our understanding of epithelial diseases including kidney disease, cancer, and birth defects. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to determine how epithelial cells form and adhere to each other in developing organisms. Epithelial cells line the surfaces of our organs and are essential for organ formation and function. The proposed research will provide new insights into the basis of epithelial-based diseases such as kidney disease and cancer, as well as human birth defects.