Cell Surface Control of Cellular Physiology

描述（由申请人提供）：本研究的目的是了解黏着斑蛋白如何对细胞运动性发挥肿瘤抑制剂样作用。巨球蛋白是介导细胞内细胞骨架和细胞外基质之间的跨膜连接的细胞和组织结构的主要组分。目前的模型表明，黏着斑蛋白刺激粘附和抑制运动，通过加强这些连接，通过双功能的相互作用之间的塔林整联蛋白复合物黏着斑蛋白的头部域和肌动蛋白丝在其尾部域。由于纯化的黏着斑蛋白是自抑制的，因此假设黏着斑蛋白调节膜蛋白与细胞骨架的附着以控制粘附和运动的机制是调节头/尾相互作用（HTI）以暴露或隐藏配体结合位点。该提案的一个目标是在活细胞中测试该模型。我们开发了两个福斯特共振能量转移（FRET）探针，激活和肌动蛋白结合的构象的黏着斑蛋白，一系列的突变体具有梯度降低的强度的分子内HTI，和塔林结合突变体的报告。我们建议应用这些工具来实现以下具体目标：1）。使用黏着斑蛋白FRET探针测试黏着斑蛋白的组合激活存在冗余机制的假设。Talin和肌动蛋白丝一起可以激活黏着斑蛋白;我们将测试其他黏着斑蛋白配体以及PIP 2的作用，以确定黏着斑蛋白激活的信号和定位线索。2）。使用头/尾相互作用突变体和talin结合突变体来检验以下假设：黏着斑蛋白的激活调节整联蛋白、talin、黏着斑蛋白和肌动蛋白之间的相互作用，这些相互作用控制细胞粘附、运动和力跨细胞膜的转导。3）。使用构象敏感的黏着斑蛋白FRET探针来测试黏着斑蛋白的激活响应于活细胞中的机械力和收缩性的假设。已经与Sharon坎贝尔建立了合作，以促进在黏着斑蛋白（Aim 1的一部分）的组合激活中分析PIP 2，与Andres Garcia建立了合作，以测量细胞中的粘附力，并与Susan Gunst建立了合作，以测量平滑肌组织中的张力发展（Aim 2的一部分）。我们预计，这些研究将提供大量的新信息相关的蛋白质如何建立结构，通过膜传递力的一般性问题，特别是黏着斑蛋白抑制细胞迁移的分子机制。 公共卫生：异常的细胞粘附和迁移是致命癌症的特征。该项目旨在了解细胞迁移和粘附是如何被一种称为黏着斑蛋白的蛋白质调节的。通过了解黏着斑蛋白的工作原理，我们可以更好地了解如何控制癌症的异常细胞行为。

DESCRIPTION (provided by applicant): The goal of this research is to understand how vinculin exerts a tumor-suppressor-like effect on cell motility. Vinculin is a prominent component of cell and tissue structures that mediate transmembrane connections between the intracellular cytoskeleton and the extracellular matrix. Current models suggest that vinculin stimulates adhesion and inhibits motility by strengthening these connections through bifunctional interactions between talin-integrin complexes at vinculin's head domain and actin filaments at its tail domain. Because purified vinculin is autoinhibited, regulation of the head/tail interaction (HTI) to expose or hide ligand binding sites is hypothesized to be the mechanism by which vinculin regulates attachment of membrane proteins to cytoskeleton to control adhesion and motility. A goal of this proposal is to test this model in living cells. We developed two Forster resonance energy transfer (FRET) probes that report on activated and actin-binding conformations of vinculin, a series of mutants having a graded reduction in the strength of the intramolecular HTI, and a talin- binding mutant. We propose to apply these tools to address the following specific aims: 1). Use vinculin FRET probes to test the hypothesis that there are redundant mechanisms for combinatorial activation of vinculin. Talin and actin filaments together can activate vinculin; we will test the roles of other vinculin ligands, as well as PIP2 to define the signaling and localization cues for vinculin activation. 2). Use the head/tail interaction mutants and the talin-binding mutant to test the hypothesis that activation of vinculin regulates interactions between integrin, talin, vinculin, and actin that control cell adhesion, motility, and transduction of force across the cell membrane. 3). Use the conformation-sensitive vinculin FRET probes to test the hypothesis that activation of vinculin responds to mechanical forces and contractility in living cells. Collaborations have been set up with Sharon Campbell to facilitate analyses of PIP2 in combinatorial activation of vinculin (part of Aim1), with Andres Garcia to measure adhesive force in cells, and with Susan Gunst to measure tension development in smooth muscle tissue (parts of Aim2). We anticipate that these studies will provide substantial new information relevant to the general question of how proteins build structures to transmit force across a membrane, and specifically to the molecular mechanism by which vinculin suppresses cell migration. PUBLIC HEALTH REVELANCE: Abnormal cell adhesion and migration are characteristic of cancers that kill people. This project aims to find out how cell migration and adhesion are regulated by a protein called vinculin. By learning how vinculin works, we can better understand how to control the abnormal cell behaviors of cancer.