Regulation of Epidermal Junctions in C. elegans

描述(申请人提供)：上皮膜的铺展和封闭是动物早期发育过程中的一个基本过程。上皮片封闭的失败对常见的出生缺陷有重要的影响，包括脊柱裂和腭裂，参与这一过程的蛋白质调节不当与许多类型的癌症有关。秀丽线虫表皮是研究上皮片封闭动力学的有力模型系统。我们以前的研究表明，在上皮片封闭过程中，必须动态地招募两个多蛋白复合体：钙粘附素/连环蛋白和SAP97/DLG复合体。有关钙粘附素/连环蛋白功能的一个关键问题是α连环蛋白如何招募并连接到肌动蛋白细胞骨架。关于SAP97家族蛋白，一个需要解决的关键问题是它们如何被招募到膜上，以及一旦进入膜，它们如何形成稳定的复合体。我们将使用遗传学、创新的体内成像和新的体外方法的强大组合来解决这些问题。首先，我们将使用体外分析、遗传学和体内成像来确定HMP-1/α-连环蛋白在形态发生过程中刺激F-肌动蛋白聚合和附着的作用。其次，我们将确定UNC-34/Enabled在刺激F-肌动蛋白在形态发生中的招募中的作用，并寻找新的α-连环蛋白功能的遗传修饰物。第三，我们将确定DLG-1/SAP97结构域在形态发生过程中多蛋白复合体组装中的作用，并将分析候选DLG-1相互作用蛋白。第四，我们将使用生化分析、4D多通道成像和FRET来确定DLG-1结合伙伴AJM-1在形态发生过程中稳定DLG复合体的作用。作为这些研究的结果，我们将阐明钙粘附素/连环蛋白复合体如何招募肌动蛋白细胞骨架，以及SAP97/DLG复合体如何招募并稳定在上皮片封闭过程中新生的细胞-细胞接触处。因为这些研究将在活的有机体中进行，我们将对这些关键的连接复合体如何调节基本的形态发生事件获得基本的新见解。

DESCRIPTION (provided by applicant): The spreading and sealing of epithelial sheets is an essential process during early animal development. Failure of epithelial sheet sealing has important implications for common birth defects, including spina bifida and cleft palate, and misregulation of proteins involved in this process is implicated in many types of cancer. The C. elegans epidermis is a powerful model system in which to study the dynamics of epithelial sheet sealing. Our previous studies have shown that two multiprotein complexes must be dynamically recruited to nascent cell-cell contacts during epithelial sheet sealing: the cadherin/catenin and SAP97/Dlg complexes. A key issue to resolve regarding cadherin/catenin function is how alpha catenin recruits and connects to the actin cytoskeleton. A key issue to resolve regarding SAP97 family proteins is how they are recruited to the membrane and how they form stable complexes once there. We will use a powerful combination of genetics, innovative in vivo imaging, and novel in vitro approaches to address these issues. First, we will determine the role of HMP-1/alpha-catenin in stimulating polymerization of and attachment to F-actin during morphogenesis, using in vitro assays, genetics, and in vivo imaging. Second, we will determine the role of unc-34/Enabled in stimulating recruitment of F-actin during morphogenesis, and we will identify new genetic modifiers of alpha-catenin function. Third, we will determine the role of domains of DLG-1/SAP97 in assembly of a multiprotein complex during morphogenesis, and we will analyze candidate DLG-1 interacting proteins. Fourth, we will determine the role of the DLG-1 binding partner, AJM-1, in stabilizing the DLG complex during morphogenesis, using biochemical assays, 4d multichannel imaging, and FRET. As a result of these studies, we will clarify how the cadherin/catenin complex recruits the actin cytoskeleton, and how the SAP97/Dlg complex is recruited and stabilized at nascent cell-cell contacts during epithelial sheet sealing. Because these studies will be carried out in a living organism, we will gain fundamental new insights into how these key junctional complexes regulate a fundamental morphogenetic event.