Regulation of epithelial junctions and lumen morphogenesis by the Scribble/SGEF/Dlg1 complex

项目摘要 大多数内脏器官由单层极化上皮细胞组成， 建立隔离内部介质与外部环境的屏障。一个关键的决定因素 在上皮细胞中建立顶基极性的关键是Scribble复合物。Scribble复合体是一个多- 蛋白质支架平台，其通过招募其他结合伴侣来发挥功能，以构建空间上不同的 信号复合物理解上皮形成的一个主要障碍是我们对 控制涂鸦复合体功能的机制二十多年来，涂鸦情结 在遗传学研究中被认为是一个模块，但目前还不清楚该复合体的成员是如何 相互作用，以及它们与粘附力和极性调节器的联系。为了缩小这一差距，我的实验室 专注于定义Scribble复合物的精确组成，形成机制和调节。我们 最近表明，SGEF，一种RhoG特异性GEF，是Scribble复合物的一种新成分， 作为支架，通过直接与Scribble和Dlg 1相互作用形成三元复合物，并在 连接形成、顶端收缩性、E-钙粘蛋白稳定性和管腔形成。根据这些发现， 本提案的目的是确定调节Scribble复合物功能的分子机制 在调节细胞-细胞连接的组装和维持以及管腔形成中起重要作用。我们将测试中央 假设SGEF掺入Scribble复合物刺激其催化活性，这反过来 激活RhoG以调节细胞-细胞连接的稳定性，以及管腔开放和数量。我们提出 以下具体目的是检验这一假设。目标1.定义SGEF的时空调节， 通过Scribble复合物的RhoG活性。在这里，我们将测试我们的工作假设，绑定到Scribble和 Dlgl靶向和/或激活SGEF至细胞-细胞连接，其促进RhoG的局部激活; Aim 2. 确定Scribble/SGEF/Dlg 1复合物调节E-钙粘蛋白稳定性的机制。这里我们 将测试我们的工作假设，即RhoG下游SGEF的激活使E-钙粘蛋白稳定在 膜通过调节其回收和/或降解;目的3.确定Scribble/SGEF/Dlg 1 复合体调节管腔开口和数量。在这里，我们将测试我们的工作假设， Scribble/SGEF/Dlg 1是形成具有3D囊肿中的单个中心开放腔的囊肿所必需的。 这种方法是创新的，因为它提供了关于Scribble复合体功能的机械见解。 这一贡献将是重要的，因为它将阐明控制细胞-细胞的基本机制。 粘附及其在上皮细胞和组织中的建立中的作用。了解细胞粘附和 在正常细胞中建立和维持极性对于其中丧失粘附和 极性代表一个问题，例如上皮癌，其占所有人类癌症的85%。

PROJECT SUMMARY Most internal organs consist of a monolayer of polarized epithelial cells surrounding a central lumen that establish a barrier that segregate the internal medium from the outside environment. One of the key determinants of establishment of apicobasal polarity in epithelial cells is the Scribble complex. The Scribble complex is a multi- protein scaffolding platform which functions by recruiting other binding partners, to build spatially distinct signaling complexes. A major barrier to understand the formation of the epithelium is our poor understanding of the mechanisms that control the function of the Scribble complex. For over two decades, the Scribble complex was known to function as a module from genetic studies, but it was unclear how the members of the complex interacted with each other, and their link to the regulators of adhesion and polarity. To close this gap, my lab is focused in defining the precise composition, mechanism of formation and regulation of the Scribble complex. We have recently shown that SGEF, a RhoG-specific GEF, is a new component of the Scribble complex that acts as a scaffold to form a ternary complex by interacting directly with Scribble and Dlg1, and plays a role during junction formation, apical contractility, E-cadherin stability and lumen formation. Based on these findings, the objective of this proposal is to define the molecular mechanisms that regulate the function of the Scribble complex in the regulation of cell-cell junctions’ assembly and maintenance, and lumen formation. We will test the central hypothesis that the incorporation of SGEF into the Scribble complex stimulates its catalytic activity, which in turn activates RhoG to regulate the stability of cell-cell junctions, as well as lumen opening and number. We propose the following specific aims to test this hypothesis. Aim 1. Define the spatiotemporal regulation of SGEF and RhoG activity by the Scribble complex. Here we will test our working hypothesis that binding to Scribble and Dlg1 targets and/or activates SGEF to cell-cell junctions which promote the localized activation of RhoG; Aim 2. Determine the mechanism by which the Scribble/SGEF/Dlg1 complex regulates E-cadherin stability. Here we will test our working hypothesis that the activation of RhoG downstream SGEF stabilizes E-cadherin at the membrane by regulating its recycling and/or degradation; Aim 3. Determine how the Scribble/SGEF/Dlg1 complex regulates lumen opening and number. Here, we will test our working hypothesis that, the formation of the Scribble/SGEF/Dlg1 is required the formation of cysts with a single, central open lumen in 3D cysts. This approach is innovative, as it provides mechanistic insight on the function of the Scribble complex. This contribution will be significant because it will shed light on the fundamental mechanisms controlling cell-cell adhesion and its role in the establishment of in epithelial cells and tissues. Understanding how cell adhesion and polarity are established and maintained in normal cells is important for situations in which loss of adhesion and polarity represent a problem, such as epithelial cancers, which comprise 85% of all human cancers.