Cell Surface Control of Cellular Physiology

细胞生理学的细胞表面控制

• 批准号: 7931516
• 负责人: SUSAN W CRAIG
• 金额: $ 7.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931516
• 关键词: Abnormal Cell; Actinin; Actins; Address; Adhesions; Adhesives; Binding; Binding Sites; Cell Adhesion; Cell membrane; Cell physiology; Cell surface; Cells; Cellular Structures; Characteristics; Collaborations; Complex; Cues; Cytoskeleton; Development; Energy Transfer; Extracellular Matrix; Focal Adhesions; Generations; Goals; Head; Image; In Vitro; Individual; Integrins; Intercellular Junctions; Learning; Life; Ligand Binding; Ligands; Malignant Neoplasms; Measurement; Measures; Mechanics; Mediating; Membrane; Membrane Proteins; Microfilaments; Modeling; Molecular; Molecular Conformation; Mutation; Phosphatidylinositol 4,5-Diphosphate; Physiological; Process; Proteins; Recruitment Activity; Regulation; Reporting; Research; Role; Series; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Stress; Structure; Tail; Talin; Testing; Time; Tissues; Vinculin; Work; cell behavior; cell motility; combinatorial; controlled release; improved; inhibitor/antagonist; killings; migration; mutant; response; retinal rods; small molecule; tool; transmission process; tumor

The goal of this research is to understand how vinculin exerts a tumor-supressor-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 togetther can activate vinculin; we will test the roles of other vinculin ligands, as well as PIP2 to define the signalling 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. Project Narrative: 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.

本研究的目的是了解黏着斑蛋白如何发挥肿瘤抑制剂样作用， 能动性胰蛋白酶是细胞和组织结构的重要组成部分，介导跨膜 细胞内细胞骨架和细胞外基质之间的连接。现款车型 表明黏着斑蛋白通过加强这些连接来刺激粘附并抑制运动 通过黏着斑蛋白头部结构域的talin-整联蛋白复合物与肌动蛋白之间的双功能相互作用， 在它的尾部区域的细丝。由于纯化的黏着斑蛋白是自抑制的，因此调节头部/尾部黏着斑蛋白的表达是不可能的。 暴露或隐藏配体结合位点的相互作用（HTI）被假设为 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。 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，报告活化和肌动蛋白结合的黏着斑蛋白， 一系列突变体具有分子内HTI强度的分级降低，和talin- 结合突变体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探针来测试存在冗余机制的假设， 黏着斑蛋白的激活。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PIP2，以定义黏着斑蛋白的信号传导和定位线索 activation. 2）。使用头/尾相互作用突变体和talin结合突变体来测试 黏着斑蛋白激活调节整合素、talin、黏着斑蛋白和肌动蛋白之间相互作用的假说 控制细胞粘附、运动和力穿过细胞膜的传递。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 对机械力和活细胞的收缩力作出反应。已与下列机构建立了合作关系： 莎朗·坎贝尔（Sharon Campbell）促进PIP 2在粘着斑蛋白（AIM 1的一部分）组合激活中的分析， Andres Garcia测量细胞的粘附力，Susan Gunst测量张力 平滑肌组织中的发育（Aim2的部分）。我们预计，这些研究将提供 关于蛋白质如何构建结构的一般问题的大量新信息， 通过膜传递力，特别是黏着斑蛋白的分子机制， 抑制细胞迁移。项目叙述： 异常的细胞粘附和迁移是致命癌症的特征。该项目旨在寻找 一种叫做黏着斑蛋白的蛋白质是如何调节细胞迁移和粘附的。通过了解纽蛋白的作用机制， 我们可以更好地了解如何控制癌症的异常细胞行为。