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

极化 MDCK 上皮细胞中 Na/K-ATP 酶的拓扑发生

• 批准号: 8503496
• 负责人: W. James Nelson
• 金额: $ 65.83万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8503496
• 关键词: Actins; Adhesions; Affect; B-Lymphocytes; Cadherins; Caenorhabditis elegans; Carcinoma; Cell Cycle; Cell Polarity; Cell-Cell Adhesion; Cells; Complex; Cytoskeleton; Defect; Development; Dictyostelium discoideum; Drosophila genus; E-Cadherin; Epithelial; Epithelial Cells; Epithelium; Equipment and supply inventories; Extracellular Matrix; Genes; Genetic; Goals; Homeostasis; Human; In Situ; In Vitro; Integrins; Intercellular Junctions; Lead; Link; MDCK cell; Malignant Neoplasms; Maps; Mechanics; Mediating; Metabolic Diseases; Morphogenesis; Mutation; Na(+)-K(+)-Exchanging ATPase; Pathway interactions; Process; Property; Protein Biochemistry; Proteins; Protocols documentation; RNA Interference; Role; Signal Pathway; Signaling Protein; Simple Epithelium; Structural Protein; System; Testing; Time; Tumor Suppressor Genes; Validation; Vesicle; Work; Zebrafish; alpha catenin; base; beta catenin; cancer type; cell motility; citrate carrier; design; genetic regulatory protein; genome-wide; in vivo; insight; monolayer; novel; polarized cell; protein complex; protein distribution; protein protein interaction; public health relevance; reconstitution; research study; response; trafficking

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.

描述（由申请人提供）：我们工作的长期目标是通过定义细胞-细胞粘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。我们的理论依据是单层上皮是后生动物和一些非后生动物的基本组成部分。它包括由细胞-细胞粘附复合物保持在一起并被细胞外基质（ECM）包围的静止的功能极化细胞的封闭单层。我们当前的研究结果表明，细胞间粘附和上皮极性之间的机械联系对于理解简单上皮如何形成是古老且基础的。然而，目前的库存涉及钙粘蛋白介导的粘附的蛋白质和途径可能是不完整的，是复杂的许多这些蛋白质/途径在其他粘附系统和细胞迁移的重叠作用。我们的策略是利用一种新的全基因组RNAi筛选的结果在果蝇S2细胞中的Ca++和E-cadherin依赖的细胞-细胞粘附，排除Ca++-非依赖的细胞-细胞粘附，整合素为基础的ECM粘附和扩散，和细胞迁移。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”，这些调控“枢纽”包含许多以前与钙粘蛋白功能不直接相关的蛋白质。我们下一阶段的目标是：1）。系统地定义了参与钙粘蛋白介导的细胞间粘附和上皮极性组织的信号通路的功能。我们将使用来自DE-cad/S2筛选的蛋白质的优先级列表，以及我们先前工作中定义的细胞-细胞粘附期间蛋白质重组的空间图和时间线;和2）。检查细胞-细胞粘附复合物对机械应变后上皮稳态扰动的反应中涉及的信号通路，这导致静止细胞通过依赖于E-钙粘蛋白、β-连环蛋白和α-连环蛋白的不同通路重新进入细胞周期。我们期望我们的研究结果将提供新的机制的理解，调节上皮形态和稳态的途径。