In vivo mechanisms of epithelial cell polarization and junction formation

描述(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。在胚胎发育过程中，当间充质细胞沿其顶端基底轴极化并组装细胞-细胞连接(间充质-上皮转化，MET)时，大多数上皮细胞都会发育。极性或连接的丧失可能导致毁灭性的疾病，包括肾脏疾病、由于细胞增殖和侵袭增加而导致的癌症，以及由于形态发生障碍而引起的出生缺陷。我们对MET的大部分了解来自于对培养细胞的体外研究，培养细胞在E-钙粘素介导的相互接触时会两极分化。然而，MET的机制在体内可能不同，因为E-钙粘附素不需要在果蝇、线虫和一些哺乳动物的上皮细胞中启动极性，而且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在发育中的生物体中用于极化上皮细胞和组装连接的细胞和分子机制。我们已经开发了活的成像和遗传工具来研究活的线虫胚胎中MET的机制。使用这些工具，我们已经证明了守恒的极性调节器在连续的步骤中诱导结的形成。首先，PAR-3通过聚集连接蛋白和其他极性调节剂到皮质病灶，然后到达顶端表面来建立极性。然后，与PKC-3一起发挥作用的PAR-6将一簇簇的顶端连接蛋白凝聚成带，环绕细胞。我们还表明，管状上皮细胞和片状上皮细胞之间的极化机制可能不同，在这些细胞中，顶端Crumbs蛋白Eat-20与PAR-3冗余地发挥功能，使细胞极化。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，但这些蛋白如何在体内极化细胞和组装连接在很大程度上是未知的。这项研究的具体目标是确定PAR-3和EAT-20用来极化上皮细胞的分子机制，并确定PAR-6和PKC-3如何组装连接。利用实时成像和遗传分析，我们将确定PAR-3焦点是如何形成的，连接蛋白和极性蛋白是如何加载到焦点上的，并检验焦点沿着微管向根尖表面移动的假设。我们将检验EAT-20通过将蛋白质招募到顶端表面来建立极性的假设，定义EAT-20下游使细胞极化的路径，并确定EAT-20的顶端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的大规模基因选择筛选来识别调节或作用于PAR-6和PKC-3下游的基因，以组装连接。这项提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动该领域的发展，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了解。 公共卫生相关性：这项拟议研究的目标是确定在发育中的生物体中，上皮细胞是如何形成和相互黏附的。上皮细胞排列在我们器官的表面，是器官形成和功能所必需的。这项拟议的研究将为以上皮为基础的疾病，如肾脏疾病和癌症，以及人类出生缺陷的基础提供新的见解。

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.