Mechanisms of vinculin activation and force transmission

纽蛋白激活和力传递机制

• 批准号: 9107123
• 负责人: Sharon L Campbell
• 金额: $ 38.04万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9107123
• 关键词: Actins; Adherens Junction; Adhesions; Affect; Apoptotic; Binding; Binding Sites; Biochemical; Biological Assay; Biosensor; Bundling; Cell Survival; Cell-Cell Adhesion; Cells; Cellular Morphology; Collaborations; Complex; Cryoelectron Microscopy; Cytoskeletal Proteins; Cytoskeleton; Data; Defect; Dimerization; Disease; Docking; Embryonic Development; Essential Genes; Event; F-Actin; Focal Adhesions; Head; Heart Diseases; In Vitro; Knock-out; Length; Ligand Binding; Ligands; Link; Mediating; Membrane; Microfilaments; Modeling; Molecular; Molecular Conformation; Monitor; Mutation; Nature; Null Lymphocytes; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Phosphorylation; Play; Property; Proteins; Publishing; RNA Splicing; Regulatory Element; Resistance; Role; Scaffolding Protein; Structure; Tail; Testing; United States National Institutes of Health; Variant; Vinculin; base; biophysical properties; cell motility; combinatorial; design; dimer; gene function; human disease; migration; molecular dynamics; muscular structure; mutant; novel therapeutic intervention; public health relevance; receptor; response; scaffold; simulation; transmission process; tumorigenic; Actins; Adherens Junction; Adhesions; Affect; Apoptotic; Binding; Binding Sites; Biochemical; Biological Assay; Biosensor; Bundling; Cell Survival; Cell-Cell Adhesion; Cells; Cellular Morphology; Collaborations; Complex; Cryoelectron Microscopy; Cytoskeletal Proteins; Cytoskeleton; Data; Defect; Dimerization; Disease; Docking; Embryonic Development; Essential Genes; Event; F-Actin; Focal Adhesions; Head; Heart Diseases; In Vitro; Knock-out; Length; Ligand Binding; Ligands; Link; Mediating; Membrane; Microfilaments; Modeling; Molecular; Molecular Conformation; Monitor; Mutation; Nature; Null Lymphocytes; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Phosphorylation; Play; Property; Proteins; Publishing; RNA Splicing; Regulatory Element; Resistance; Role; Scaffolding Protein; Structure; Tail; Testing; United States National Institutes of Health; Variant; Vinculin; base; biophysical properties; cell motility; combinatorial; design; dimer; gene function; human disease; migration; molecular dynamics; muscular structure; mutant; novel therapeutic intervention; public health relevance; receptor; response; scaffold; simulation; transmission process; tumorigenic

 DESCRIPTION (provided by applicant): This proposal is focused on vinculin, a cytoskeletal protein that is a prominent component of focal adhesions and adherens junctions. Vinculin exists in an autoinhibited conformation and upon activation, functions as a scaffold to regulate cellular events resulting in cell migration, cell survival and embryogenesis. Vinculin null cells display tumorigenic properties and mutation or loss of vinculin is associated with cardiac disease. Although vinculin binds actin and phosphatidylinositol 4,5- bisphosphate (PIP2), we do not understand the nature of these interactions or their precise role in regulating vinculin function. In particular, the interaction between vinculin and actin plays a pivotal role in linking transmembrane receptors to the cytoskeleton, which, in turn, is important for controlling cellular cell morphology, force transmission and motility. Vinculin binds to F-actin and undergoes a conformational change that induces formation of a cryptic dimer necessary for actin filament bundling, but the conformation change that occurs and dimer that is formed is unknown. It is also unclear how vinculin recognizes PIP2, inserts into membranes and is regulated by this interaction. We propose highly integrated computational and experimental approaches to generate and test models for these important interactions and assess their significance in vinculin function both in vitro and in cells. This will be accomplished by generating and characterizing vinculin variants with specific defects in actin binding, actin-induced vinculin dimer formation and PIP2 association in vitro, and then expressing the full length wild type protein and mutants in vinculin null cells. The role of these interactions in regulating the activation state of vinculin as well as vinculin's force response and transmission properties will be probed at both the sub-cellular and whole cell level.

 描述（由适用提供）：该建议集中于Vinculin，这是一种细胞骨架蛋白，是焦点广告和依从性连接的重要组成部分。 Vinculin存在于自身抑制的会议中并激活后，作为调节细胞迁移，细胞存活和胚胎发生的细胞事件的脚手架。 Vinculin null细胞表现出结核菌特性，突变或Vinculin的突变与心脏病有关。尽管Vinculin结合肌动蛋白和磷脂酰肌醇4,5-双磷酸（PIP2），但我们不了解这些相互作用的性质或它们在调节Vinculin功能中的精确作用。特别是，vinculin和肌动蛋白之间的相互作用在连接中起关键作用 跨膜接收器到细胞骨架，这反过来对于控制细胞的形态，力传递和运动性很重要。 Vinculin与F-肌动蛋白结合，并经历会议变化，从而导致肌动蛋白细丝束必需的加密二聚体的形成，但是发生的会议变化和形成的二聚体是未知的。尚不清楚Vinculin如何识别PIP2，插入膜并受到这种相互作用的调节。我们提出了高度整合的计算方法和实验方法，以生成这些重要相互作用的模型和测试模型，并评估其在体外和细胞中的杂质蛋白功能中的重要性。这将通过在肌动蛋白结合，肌动蛋白诱导的Vinculin二聚体形成和PIP2的体外产生和表征具有特定缺陷的质子蛋白变异来实现，然后在体外表达全长野生型蛋白质和杂种蛋白null细胞中的突变体。在确定Vinculin的激活状态以及Vinculin的力反应和透射特性方面的相互作用将在亚细胞和全细胞水平上进行探测。