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

项目总结 大多数内脏由围绕中央管腔的单层极化上皮细胞组成，该管腔 建立一道屏障，将内部媒介与外部环境隔离。关键决定因素之一 在上皮细胞中建立顶基底极性的关键是Scribble复合体。Scribble建筑群是一个多- 蛋白质支架平台，通过招募其他结合伙伴来构建不同的空间 信号复合体。理解上皮形成的一个主要障碍是我们对 控制Scribble复合体功能的机制。二十多年来，斯克里布尔建筑群 已知作为遗传学研究的一个模块发挥作用，但尚不清楚该复合体的成员是如何 相互作用，并将其与粘附性和极性的调节联系起来。为了缩小这一差距，我的实验室 重点定义了Scribble复合体的确切组成、形成机制和调控。我们 最近的研究表明，特定于RhoG的全球环境基金是Scribble复合体的一个新组件，该复合体作用于 作为支架直接与Scribble和Dlg1相互作用形成三元络合物，并在 连接形成、心尖收缩、E-钙粘附素稳定性和管腔形成。基于这些发现， 这项建议的目的是确定调节Scribble复合体功能的分子机制 在细胞-细胞连接的组装和维持以及管腔形成的调节中。我们将测试中央 假设Sgef加入到Scribble复合体中会刺激其催化活性，这反过来又会 激活RhoG来调节细胞-细胞连接的稳定性，以及管腔的开度和数量。我们建议 下面的具体内容旨在检验这一假设。目标1.界定SEF的时空调节和 Scribble复合体的RhoG活动。在这里，我们将测试我们的工作假设，即绑定到Scribble和 Dlg1靶向和/或激活促进RhoG局部激活的细胞-细胞连接；目标2。 确定Scribble/Sgef/Dlg1复合体调节E-钙粘蛋白稳定性的机制。在这里我们 将检验我们的工作假设，即RhoG下游SGF的激活使E-钙粘蛋白稳定在 通过调节膜的循环和/或降解；目标3.确定Scribble/Sgef/Dlg1 复合体调节管腔的开度和数量。在这里，我们将检验我们的工作假设，即 Scribble/Sgef/Dlg1是在3D囊中形成具有单一中央开放管腔的囊腔所必需的。 这种方法是创新的，因为它提供了对Scribble建筑群功能的机械性洞察。 这一贡献将是重大的，因为它将阐明控制细胞-细胞的基本机制 黏附及其在上皮细胞和组织中的作用。了解细胞黏附和 在正常细胞中建立和维持极性对于粘附性丧失和 极性是一个问题，例如占人类所有癌症85%的上皮性癌症。

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.