Topogenesis of Na/K-ATPase in Polarized MDCK Epithelial Cells

描述(申请人提供)：我们工作的长期目标是通过定义细胞-细胞黏附如何启动肌动蛋白重塑和极化细胞组织来深入了解简单上皮形成的基本过程。我们的理论基础是，简单的上皮细胞是后生动物和一些非后生动物的基本构件。它包括一层封闭的静止的、功能极化的细胞，通过细胞-细胞黏附复合体结合在一起，并被细胞外基质(ECM)包围。我们在当前阶段的研究结果表明，细胞间黏附和上皮细胞极性之间的机制联系是古老的，也是理解简单上皮如何形成的基础。然而，目前涉及钙粘附素介导的黏附的蛋白质和通路的库存可能是不完整的，并且由于许多这些蛋白质/通路在其他黏附系统和细胞迁移中的重叠作用而变得复杂。我们的策略是利用一种新的全基因组RNAi筛查果蝇S2细胞中依赖钙和E-钙粘附素的细胞-细胞黏附的结果，该筛查排除了钙非依赖性的细胞-细胞黏附、基于整合素的ECM黏附和扩散以及细胞迁移。基于严格的筛选和验证方案，我们暂时将~100个确认的“命中”分配给6个交叉的调控“枢纽”，其中包含许多以前没有直接与钙粘素功能相关的蛋白质。我们下一阶段的目标是：1)。系统地定义参与钙粘附素介导的细胞-细胞黏附的信号通路的功能，以及在一系列尺度上上皮极性的组织。我们将使用DE-cad/S2屏幕上蛋白质的优先列表，以及我们先前工作中定义的细胞-细胞黏附过程中蛋白质重组的空间地图和时间线；和2)。研究细胞-细胞黏附复合体对机械应变引起的上皮内稳态扰动的反应中涉及的信号通路，这导致静止的细胞通过依赖于E-钙粘附素、β-连环素和α-连环素的不同途径重新进入细胞周期。我们预计，我们的结果将为调节上皮形态发生和动态平衡的途径提供新的机制理解。

DESCRIPTION (provided by applicant): The long-term OBJECTIVE of our work is to gain a deep mechanistic understanding of the fundamental process of how a simple epithelium forms by defining how cell-cell adhesion initiates actin remodeling and polarized cell organization. Our RATIONALE is that a simple epithelium is the fundamental building block of metazoans and some non-metazoans. It comprises a closed monolayer of quiescent, functionally polarized cells held together by cell-cell adhesion complexes and surrounded by the extracellular matrix (ECM). Our results from the current period showed that the mechanistic link between cell-cell adhesion and epithelial polarity is ancient and fundamental to understanding how a simple epithelium forms. However, the current inventory of proteins and pathways involved in cadherin-mediated adhesion is likely incomplete, and is complicated by the overlapping roles of many of those proteins/pathways in other adhesion systems and cell migration. Our STRATEGY is to exploit the results of a novel genome-wide RNAi screen for Ca++- and E-cadherin-dependent cell-cell adhesion in Drosophila S2 cells that excluded Ca++-independent cell-cell adhesion, integrin-based ECM adhesion and spreading, and cell migration. Based on a stringent screen and validation protocol, we have provisionally assigned ~100 confirmed "hits" to 6 intersecting regulatory "hubs" that contain many proteins not previously associated directly with cadherin function. OUR GOALS in the next period are: 1). systematically define the functions of signaling pathways involved in cadherin-mediated cell-cell adhesion and the organization of epithelial polarity across a range of scales. We will use a prioritized list of proteins from the DE-cad/S2 screen, together with the spatial map and time line of protein reorganization during cell-cell adhesion defined in our previous work; and 2). Examine signaling pathways involved in the response of cell-cell adhesion complexes to perturbation of epithelial homeostasis upon mechanical strain, which results in the re-entry of quiescent cells into the cell cycle through different pathways that are dependent on E-cadherin, beta-catenin and alpha-catenin. We anticipate that our results will provide new a mechanistic understanding of pathways that regulate epithelial morphogenesis and homeostasis.