Microfilament Organization and Regulation

微丝组织和调控

• 批准号: 9103548
• 负责人: Anthony P. Bretscher
• 金额: $ 51.33万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-04-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9103548
• 关键词: Actins; Address; Adhesions; Affect; Affinity; Animals; Apical; Binding; Binding Proteins; Binding Sites; Bioinformatics; Biological Models; Cell Adhesion Molecules; Cell membrane; Cell physiology; Cells; Cellular biology; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeleton; Defect; Disease; Dissociation; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; F-Actin; Feedback; Functional disorder; Future; Goals; Growth Factor Receptors; Human body; In Vitro; Individual; Lead; Link; Malignant Neoplasms; Mediating; Membrane; Membrane Protein Traffic; Membrane Proteins; Mesenchymal; Microfilaments; Molecular; Morphology; Neoplasm Metastasis; Organ; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Process; Property; Protein Dephosphorylation; Protein Isoforms; Proteins; Proteome; Proteomics; Regulation; Scaffolding Protein; Structure; Surface; System; Tail; Translating; Work; apical membrane; base; cell cortex; cellular microvillus; dynamic system; ezrin; genetic regulatory protein; in vivo; insight; link protein; moesin; novel; public health relevance; radixin protein; trafficking

 DESCRIPTION (provided by applicant): Most cells in the human body are functionally polarized to generate different regions of the plasma membrane having distinct morphologies and protein compositions. Understanding the regulation of this polarity is critical for understanding the cell biology of diseases. For example, cancer metastasis can be initiated when epithelial cells undergo an inappropriate polarity transition to a mesenchymal state. The microfilament-based cell cortex is largely responsible for defining the morphology of plasma membrane domains, as well as modulating their membrane proteome. Our goal is to identify general mechanisms that cells use to generate specific domains. As an accessible model system, we focus on the microfilament-based cytoskeleton of the apical aspect of polarized epithelial cells and how it contributes to the assembly, dynamics and composition of the abundant microvilli that characterize this domain. Ezrin and the ezrin-binding and scaffolding protein EBP50/NHERF1 regulate apical microvilli, as well as many other microfilament-based surface structures, and this proposal is to understand their individual and collective functions. Ezrin is an abundant, conformationally regulated microfilament-membrane linking protein of microvilli that performs an essential function in defining the apical domain. Our recent work has uncovered two unexpected aspects governing the regulation of microvilli on epithelial cells. First, we have shown that restriction of microvilli to the apical domain requires local ezrin phosphocycling mediated by apically localized LOK/SLK kinases and delocalized phosphatases. Second, we have found that the in vivo dynamics of EBP50 is highly regulated by association of proteins with its PDZ domains. This application focuses on how these dynamic systems are mediated and contribute to the structure and composition of the apical membrane. In aim one we build on our preliminary studies with LOK showing that it has unusual properties for a kinase including its association with phosphorylated substrate, suggesting a positive feedback loop for local activation. We propose to define the mechanism by which LOK is regulated to phosphorylate ezrin, and also to identify the mechanism by which LOK is targeted to, and activated at, the apical membrane. In our second aim we will investigate the mechanism underlying the regulation of EBP50 dynamics. Ezrin and EBP50 are known to regulate the plasma membrane abundance of specific membrane proteins, but to date no study has explored the generality of this regulation. Therefore, in our third aim, we use unbiased proteomics to define the apical membrane proteome in cells lacking microvilli as a result of loss or misregulation of ezrin, and in cells in which the dynamics of scaffolding protein EBP50 is altered. Overall, our studies will provide an unprecedented view of how cells regulate the structure and composition of a specific membrane domain. These studies have relevance to many diseases. For example, EBP50 regulates the abundance of apical CFTR and EGFR, and defects in this regulation of these proteins can cause cystic fibrosis or cancer, respectively.

 描述（由适用提供）：人体中的大多数细胞在功能上极化，以产生具有不同形态和蛋白质组成的质膜的不同区域。了解这种极性的调节对于理解疾病的细胞生物学至关重要。例如，当上皮细胞经历不适当的极性过渡到间充质状态时，癌症转移可以开始。基于微丝的细胞皮质在很大程度上负责定义质膜结构域的形态，并调节其膜蛋白质组。我们的目标是确定细胞用于生成特定域的一般机制。作为一个可访问的模型系统，我们专注于极化上皮细胞顶端的基于微丝的细胞骨架，以及它如何贡献表征该域的丰富微壁的组装，动力学和组成。 ezrin和Ezrin结合和脚手架蛋白EBP50/NHERF1调节顶端微绒毛，以及许多其他基于微丝的表面结构，该建议是了解其个体和集体功能。 Ezrin是一种丰富的，构型调节的微丝膜，连接微绒毛的蛋白质，在定义顶端结构域方面起着重要的作用。我们最近的工作发现了两个出乎意料的方面，该方面管理着上皮细胞对微绒毛的调节。首先，我们已经表明，将微绒毛限制到顶端结构域需要由顶端局部局部LOK/SLK激酶和离域磷酸酶介导的局部Ezrin磷酸化。其次，我们发现EBP50的体内动力学受蛋白质与其PDZ结构域的关联高度调节。该应用集中在这些动态系统如何介导，并有助于顶膜的结构和组成。在AIM中，我们以Lok的初步研究为基础，表明它具有与磷酸化底物（包括其与磷酸化的底物相关联）的异常特性，这表明局部激活的阳性反馈回路。我们建议定义LOK被调节以磷酸化的Ezrin的机制，并确定LOK靶向和激活的机制。在第二个目标中，我们将研究EBP50动力学调节的基础机制。已知Ezrin和EBP50可以调节特定膜蛋白的质膜抽象，但迄今为止，尚无研究探讨该调节的一般性。因此，在我们的第三个目标中，我们使用公正的蛋白质来定义缺乏微绒毛的细胞中的根尖膜蛋白，导致ezrin的丧失或不正常，以及在脚手架蛋白EBP50动力学的细胞中改变了细胞。总体而言，我们的研究将为细胞如何调节特定膜结构域的结构和组成提供前所未有的观点。这些研究与许多疾病有关。例如，EBP50调节顶端CFTR和EGFR的抽象，并且这些调节这些蛋白质的缺陷会分别引起囊性纤维化或癌症。