Control of actin stability and branching by Arg and cortactin

Arg 和 cortactin 控制肌动蛋白稳定性和分支

• 批准号: 9243922
• 负责人: Alexander Scherer
• 金额: $ 3万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-16 至 2018-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9243922
• 关键词: ANGPTL2 gene; Actins; Adhesions; Affect; Binding; Biological Assay; Biological Process; Cell Shape; Cell Surface Receptors; Cells; Color; Complex; Deletion Mutation; Development; Diagnosis; Disease; EMS1 gene; Family; Fibroblasts; Filament; Fluorescence Microscopy; Foundations; Growth Factor Receptors; Imagery; In Vitro; Individual; Lead; Malignant Neoplasms; Measures; Mediating; Methods; Microfilaments; Morphogenesis; Neurodegenerative Disorders; Neurons; Pathologic; Pathway interactions; Phosphorylation; Phosphotransferases; Process; Recruitment Activity; Regulation; Role; Signal Transduction; Structure; Testing; Time; Tissues; Work; base; cell motility; cell type; depolymerization; experimental study; human tissue; in vivo; knock-down; leukemia; malignant breast neoplasm; migration; mutant; polymerization; public health relevance; reconstitution; synergism; tumor progression

 DESCRIPTION (provided by applicant): Control of actin stability and branching by Arg and cortactin Abl family kinases act downstream of cell surface receptors to coordinate changes in actin cytoskeletal structure. These signaling roles make Abl kinases essential in fundamental processes that depend on actin regulation, such as cell migration and cell and tissue morphogenesis. Aberrant Abl family kinase activation also causes leukemia and contributes to the progression of other cancers. These biological processes have been largely ascribed to kinase activation, but our lab has shown that the Abl2/Arg kinase can also directly bind and stabilize actin filaments. In addition, Arg synergizes with its substrate cortactin to activate Arp/3 complex- mediated actin branching. Our lab has established that Arg regulates actin-rich structures that are essential for fibroblast migration, breast cancer invasion, and neuronal morphogenesis. Elucidating the direct effects of Arg- actin binding is critical for understanding how Abl family kinases regulate motility and morphogenesis in these diverse cell types. In this proposal, I will test the hypothesis that Arg-mediated actin filament stabilization and stimulation of filament branching is critical for the formation and turnover of actin-based cell edge protrusions. My first aim is to determine which domains of Arg and cortactin mediate actin stabilization and branching. I will use total internal reflection fluorescence (TIRF) microscopy to measure the effects of Arg and cortactin mutants on actin stability and branch formation. Arg phosphorylates cortactin, but our lab has shown that this is not required for Arg and cortactin synergy in actin regulation. I will utilize a panel of Arg and cortactin fragments and deletion mutants to determine the minimal functional domains of Arg and cortactin necessary for their synergy in stabilizing actin and promoting Arp2/3 complex-mediated branching. My second aim is to elucidate how Arg-actin interactions activate cortactin- and Arp2/3 complex- mediated filament branching. Arg binds actin cooperatively and changes the helical twist of filaments, which increases cortactin binding to actin. I will use cosedimentation assays to test the hypothesis that Arg increases actin branching by recruiting the Arp2/3 complex to filaments. I will also use two-color TIRF microscopy to determine whether branching is increased on Arg-actin filaments relative to naked actin filaments. My third aim is to determine how Arg-actin interactions drive dynamic cell edge protrusions in vivo. The two Arg actin-binding domains (ABDs) have distinct effects on actin stability and branching, however, the role of Arg-actin binding in regulating the formation of actin-based structures in cells is not well understood. Arg is required for adhesion-dependent cell edge protrusions in fibroblasts. To determine which Arg residues are necessary for protrusions, I will express mutants with deletions and mutations in each ABD in arg-/- cells. Arg may have cortactin-independent effects on actin branching modulated by one of its ABDs, AB2. I will express Arg mutants in cortactin knockdown fibroblasts to elucidate the cortactin-independent effects of Arg in cells.

 描述（由申请人提供）：通过Arg和corneumn Abl家族激酶控制肌动蛋白稳定性和分支作用于细胞表面受体下游，以协调肌动蛋白细胞骨架结构的变化。这些信号作用使得Abl激酶在依赖于肌动蛋白调节的基本过程中至关重要，如细胞迁移和细胞及组织形态发生。异常Abl家族激酶激活也会导致白血病，并有助于其他癌症的进展。这些生物学过程在很大程度上归因于激酶激活，但我们的实验室已经表明，β 2/Arg激酶也可以直接结合和稳定肌动蛋白丝。此外，Arg与其底物cornein协同作用，激活阿普/3复合物介导的肌动蛋白分支。我们的实验室已经确定，精氨酸调节肌动蛋白丰富的结构是必不可少的成纤维细胞迁移，乳腺癌的侵袭，神经元形态发生。阐明精氨酸-肌动蛋白结合的直接作用对于理解Abl家族激酶如何调节这些不同细胞类型中的运动性和形态发生是至关重要的。在这个建议中，我将测试的假设，精氨酸介导的肌动蛋白丝的稳定和刺激， 的细丝分支的形成和基于肌动蛋白的细胞边缘突起的周转是至关重要的。我的第一个目标是确定哪些领域的精氨酸和cornein介导肌动蛋白的稳定和分支。我将使用全内反射荧光（TIRF）显微镜， 测量Arg和cornein突变体对肌动蛋白稳定性和分支形成的影响。精氨酸磷酸化coronylates，但我们的实验室已经表明，这是不需要的精氨酸和coronylates在肌动蛋白调节的协同作用。我将利用一个面板的精氨酸和cornein片段和缺失突变体，以确定最小的功能域的精氨酸和cornein必要的协同作用，在稳定肌动蛋白和促进Arp 2/3复合物介导的分支。我的第二个目标是阐明精氨酸-肌动蛋白相互作用如何激活Corp 2/3复合物介导的丝分支。精氨酸协同结合肌动蛋白并改变细丝的螺旋扭曲，从而增加皮质素与肌动蛋白的结合。我将使用共沉淀试验来检验这一假设，即精氨酸增加肌动蛋白的分支招募的Arp 2/3复合物的细丝。我还将使用双色TIRF显微镜，以确定是否增加分支的精氨酸肌动蛋白丝相对于裸露的肌动蛋白丝。我的第三个目标是确定精氨酸-肌动蛋白相互作用如何在体内驱动动态细胞边缘突起。两个精氨酸肌动蛋白结合结构域（ABD）对肌动蛋白的稳定性和分支有不同的影响，然而，精氨酸肌动蛋白结合在调节细胞中肌动蛋白结构形成中的作用还不清楚。成纤维细胞中粘附依赖性细胞边缘突起需要Arg。为了确定哪些Arg残基是突起所必需的，我将在arg-/-细胞中表达每个ABD中具有缺失和突变的突变体。精氨酸可能有皮质素独立的影响肌动蛋白分支调制的ABD之一，AB 2。我将表达精氨酸突变体在cortacin敲低成纤维细胞，阐明皮质素的独立作用的精氨酸在细胞中。