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，对其形成和功能至关重要。在胚胎发生过程中，当间充质细胞沿其顶基轴沿着伸展并组装细胞-细胞连接时，大多数上皮细胞发育（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的大部分理解来自于对培养细胞的体外研究，这些研究证实了它们之间的E-cadherin介导的接触。然而，MET的机制在体内可能不同，因为E-钙粘蛋白不需要启动果蝇的极性，C。线虫和一些哺乳动物上皮细胞，并且因为细胞接触并不总是足以诱导极化。该项目的长期目标是确定用于在发育中的生物体中形成上皮细胞和组装连接的细胞和分子机制。我们已经开发了实时成像和遗传工具来研究MET在活体C中的机制。线虫胚胎使用这些工具，我们已经表明，保守的极性调节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剂聚集到皮质灶中来建立极性，然后皮质灶行进到顶端表面。然后，与激酶PKC-3一起起作用的PAR-6将顶端连接蛋白簇凝聚成环绕细胞的带。我们还表明，极化机制可以在管形成上皮细胞和片形成上皮细胞之间不同，其中顶端Crumbs蛋白EAT-20与PAR-3到EAT细胞冗余地起作用。虽然PAR-3、EAT-20、PAR-6和PKC-3同源物在上皮细胞中具有保守的作用，但这些蛋白质如何在体内发挥作用以激活细胞并组装连接在很大程度上是未知的。拟议研究的具体目标是确定PAR-3和EAT-20用于上皮细胞增殖的分子机制，并确定PAR-6和PKC-3如何组装连接。使用实时成像和遗传分析，我们将确定PAR-3灶如何形成，连接和极性蛋白如何加载到灶上，并测试灶沿沿着微管移动到顶端表面的假设。我们将测试EAT-20通过将蛋白质募集到顶端表面来建立极性的假设，定义EAT-20下游的使细胞活化的途径，并确定EAT-20顶端定位是否由细胞接触或胚外配体指导。最后，我们将使用我们已经进行的大规模遗传选择筛选来鉴定调节或作用于PAR-6和PKC-3下游以组装连接的基因。该提案将通过建立上皮细胞在体内生长和组装连接的机制来推进该领域，增加我们对上皮疾病，包括肾脏疾病，癌症和出生缺陷的理解。

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.