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)：Arg和Cortactin Abl家族激酶控制肌动蛋白的稳定性和分支，作用于细胞表面受体的下游，协调肌动蛋白细胞骨架结构的变化。这些信号转导作用使得Abl激酶在依赖肌动蛋白调控的基本过程中必不可少，如细胞迁移和细胞和组织的形态形成。Abl家族的异常激活也会导致白血病，并导致其他癌症的进展。这些生物学过程在很大程度上被归因于激酶的激活，但我们的实验室已经证明，ABL2/Arg激酶也可以直接结合和稳定肌动蛋白细丝。此外，Arg与其底物Cortactin协同激活Arp/3复合体介导的肌动蛋白分支。我们的实验室已经确定Arg调节富含肌动蛋白的结构，这些结构对于成纤维细胞迁移、乳腺癌侵袭和神经元形态发生是必不可少的。阐明Arg-肌动蛋白结合的直接作用对于了解Abl家族激酶如何调节这些不同类型细胞的运动和形态发生至关重要。在这项提议中，我将检验Arg介导的肌动蛋白细丝稳定和刺激的假设 细丝分支对肌动蛋白细胞边缘突起的形成和周转至关重要。我的第一个目标是确定Arg和Cortactin的哪些结构域介导了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Cortactin突变体对肌动蛋白稳定性和分支形成的影响。Arg使皮质肌动蛋白磷酸化，但我们的实验室已经证明，这对于Arg和Cortactin在肌动蛋白调控中的协同作用不是必需的。我将利用一组Arg和Cortactin片段和缺失突变体来确定Arg和Cortactin在稳定肌动蛋白和促进Arp2/3复合体介导的分支中协同所需的最小功能域。我的第二个目标是阐明Arg-肌动蛋白相互作用如何激活皮质肌动蛋白和Arp2/3复合体介导的细丝分支。Arg协同结合肌动蛋白，改变微丝的螺旋缠绕，从而增加皮质肌动蛋白与肌动蛋白的结合。我将使用共构筑试验来检验Arg通过将Arp2/3复合体招募到微丝而增加肌动蛋白分支的假设。我还将使用双色TIRF显微镜来确定相对于裸露的肌动蛋白细丝，精氨酸-肌动蛋白细丝上的分支是否增加。我的第三个目标是确定精氨酸-肌动蛋白相互作用如何驱动体内动态的细胞边缘突起。这两个Arg肌动蛋白结合域(ABD)对肌动蛋白的稳定性和分支有不同的影响，然而，Arg-肌动蛋白结合在调控细胞内肌动蛋白结构形成中的作用尚不清楚。精氨酸是成纤维细胞黏附依赖性细胞边缘突起所必需的。为了确定哪些Arg残基是突起所必需的，我将在Arg-/-细胞中的每个ABD中表达缺失和突变的突变体。Arg可能对其ABD之一AB2调节的肌动蛋白分支具有不依赖于皮质肌动蛋白的作用。我将在Cortactin基因敲除的成纤维细胞中表达Arg突变体，以阐明Arg在细胞中对Cortactin非依赖性的作用。