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

 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（由申请人提供）：该提案的重点是纽蛋白，一种细胞骨架蛋白，它是粘着斑和粘附连接的重要组成部分，纽蛋白以自抑制构象存在，并且在激活后充当支架来调节导致细胞发生的细胞事件。纽蛋白缺失细胞表现出致瘤特性，并且纽蛋白的突变或缺失与心脏病相关。磷脂酰肌醇 4,5-二磷酸 (PIP2)，我们不了解这些相互作用的性质或它们在调节纽蛋白功能中的确切作用，特别是纽蛋白和肌动蛋白之间的相互作用在连接中起着关键作用。 纽蛋白与 F-肌动蛋白结合并发生构象变化，诱导形成肌动蛋白丝成束所需的隐秘二聚体。发生的构象变化和形成的二聚体尚不清楚，纽蛋白如何识别 PIP2、插入膜并受这种相互作用的调节。提出高度集成的计算和实验方法来生成和测试这些重要相互作用的模型，并评估它们在体外和细胞中对纽蛋白功能的重要性，这将通过生成和表征具有肌动蛋白结合、肌动蛋白诱导的特定缺陷的纽蛋白变体来实现。纽蛋白二聚体的体外形成和 PIP2 结合，然后在纽蛋白无效细胞中表达全长野生型蛋白和突变体。这些相互作用在调节纽蛋白以及纽蛋白的激活状态中的作用。将在亚细胞和全细胞水平上探测力响应和传递特性。