RUI: Regulation of Cytoskeletal Linkages by AFAP-110 and Src: Focus on Myofibrils

RUI：AFAP-110 和 Src 对细胞骨架连接的调节：关注肌原纤维

• 批准号: 0212406
• 负责人: Henry Zot
• 金额: $ 26.5万
• 依托单位: Eastern Michigan University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-15 至 2004-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212406&HistoricalAwards=false
• 关键词: RUI; Regulation; Cytoskeletal; Linkages; AFAP

This research project will examine the processes that underlie the assembly and disassembly of myofibrils. Myofibrils are composed of sarcomeres, which are repeating units of thin and thick filaments. Thin filaments composed largely of actin filaments are linked to the Z-line, a structure that resembles cell adhesions in protein composition. Among the proteins found in both focal adhesions and Z-line structures are the barbed ends of the actin filaments, focal adhesion kinase, p130 Cas , alpha-actinin, and AFAP-110. AFAP-110, which is the focus of this project, associates with the nonreceptor tyrosine kinase, Src. Recent evidence demonstrates that AFAP-110 recruits Src to the actin cytoskeleton in response either to elevated protein kinase C (PKC) activity or to mechanical stress. Evidence also suggests that AFAP-110 activates Src directly, resulting in elevated phosphotyrosine levels in the cell. One downstream effect of wide- spread activation of Src substrates is the reorganization of the cytoskeleton brought about by the disassembly of cytoskeletal linkages at focal adhesions and Z-lines. Mature myofibrils disappear from myotubes expressing high Src or PKC activity. Conversely, the generation of force across cell adhesions has been shown to cause the formation of actin filament linkages and to stimulate Src activity restricted to the vicinity of the mechanical strain. The properties that make AFAP-110 a candidate for participating in the formation of cytoskeletal linkages include the ability to bind and crosslink actin filaments, the ability to recruit Src to the cytoskeleton, and the ability to activate Src tyrosine kinase. AFAP-110 could be important in the formation of myofibrils during muscle development. To examine the role of AFAP-110 in muscle development, primary cultures of myotubes will be stained for AFAP-110 and the Z-line marker alpha-actinin. The two proteins should colocalize throughout all developmental stages if AFAP-110 plays a role in the formation of cytoskeletal linkages during myofibrillogenesis. Specific inhibitors of Src will be used to test the hypothesis that the myofibril formation requires activation of Src. To examine the disassembly mechanism, myoblasts will be transfected with a mutant form of AFAP-110, AFAP.lzip , which has been shown to activate Src and disrupt actin cytoskeleton of cultured fibroblasts. Preliminary results show that PKC phosphorylation of AFAP-110 results in the activation of Src tyrosine kinase activity and stress fiber disruption in fibroblasts. Transfection with mutants of AFAP-110 that cannot bind PKC or that cannot interact with Src protect fibroblasts from PKC induced stress fiber disruption. Parallel experiments will be carried out to test whether AFAP-110 serves a similar role in muscle cells. The results of these studies will elucidate the mechanisms by which mature myofibrils turn over and new myofibrils form. The work is to be carried out in large part by undergraduate students, who will carry their training onward to careers in science.

本研究计画将探讨肌原纤维组装与分解的过程。肌原纤维由肌节组成，肌节是薄和厚细丝的重复单元。主要由肌动蛋白丝组成的细丝连接到Z线，这是一种类似于蛋白质组成中的细胞粘附的结构。在粘着斑和Z线结构中发现的蛋白质包括肌动蛋白丝的倒刺末端、粘着斑激酶、p130 Cas、α-辅肌动蛋白和AFAP-110。AFAP-110是本项目的重点，与非受体酪氨酸激酶Src相关。最近的证据表明，AFAP-110招募Src的肌动蛋白细胞骨架的蛋白激酶C（PKC）活性升高或机械应力的反应。 证据还表明，AFAP-110直接激活Src，导致细胞中磷酸酪氨酸水平升高。 Src底物的广泛活化的一个下游效应是由粘着斑和Z线处的细胞骨架连接的分解引起的细胞骨架的重组。成熟的肌原纤维从表达高Src或PKC活性的肌管消失。相反，已显示跨细胞粘附的力的产生导致肌动蛋白丝连接的形成，并刺激限制在机械应变附近的Src活性。使AFAP-110成为参与细胞骨架连接形成的候选物的性质包括结合和交联肌动蛋白丝的能力、将Src募集到细胞骨架的能力以及激活Src酪氨酸激酶的能力。 AFAP-110可能在肌肉发育过程中肌原纤维的形成中起重要作用。 为了检查AFAP-110在肌肉发育中的作用，将对肌管的原代培养物进行AFAP-110和Z线标记α-辅肌动蛋白染色。如果AFAP-110在肌原纤维形成过程中细胞骨架连接的形成中发挥作用，这两种蛋白质应在所有发育阶段共定位。Src的特异性抑制剂将用于检验肌原纤维形成需要Src活化的假设。 为了检查分解机制，将用AFAP-110的突变形式AFAP. lzip转染成肌细胞，AFAP.lzip已显示激活Src并破坏培养的成纤维细胞的肌动蛋白细胞骨架。初步结果表明，AFAP-110的PKC磷酸化导致成纤维细胞中Src酪氨酸激酶活性的激活和应力纤维的破坏。 不能结合PKC或不能与Src相互作用的AFAP-110突变体转染保护成纤维细胞免受PKC诱导的应力纤维破坏。将进行平行实验，以测试AFAP-110是否在肌肉细胞中发挥类似作用。这些研究结果将阐明成熟肌原纤维翻转和新肌原纤维形成的机制。这项工作将在很大程度上由本科生进行，他们将继续他们的培训，以科学的职业生涯。