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

本研究项目将研究肌原纤维组装和拆卸的过程。肌原纤维由肌节组成，肌节是细纤维和粗纤维的重复单位。主要由肌动蛋白丝组成的细丝与z线相连，这种结构类似于蛋白质组成中的细胞粘附。在局灶黏附和z线结构中发现的蛋白质包括肌动蛋白丝的带刺末端、局灶黏附激酶、p130 Cas、α -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在响应蛋白激酶C （PKC）活性升高或机械应力时，将Src招募到肌动蛋白细胞骨架上。也有证据表明，AFAP-110直接激活Src，导致细胞内磷酸酪氨酸水平升高。Src底物广泛激活的一个下游效应是细胞骨架的重组，这是由聚焦黏附和z线上的细胞骨架连接的解体引起的。表达高Src或PKC活性的成熟肌原纤维从肌管中消失。相反，通过细胞粘附产生的力已被证明会导致肌动蛋白丝连接的形成，并刺激仅限于机械应变附近的Src活性。使AFAP-110成为参与细胞骨架连接形成的候选者的特性包括结合和交联肌动蛋白丝的能力，将Src招募到细胞骨架的能力，以及激活Src酪氨酸激酶的能力。在肌肉发育过程中，AFAP-110可能在肌原纤维的形成中起重要作用。为了研究AFAP-110在肌肉发育中的作用，将对肌管原代培养物进行AFAP-110和z线标记α -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起作用，那么这两种蛋白应该在所有发育阶段都有共定位。Src的特异性抑制剂将用于验证肌原纤维形成需要激活Src的假设。为了研究其分解机制，将用突变形式的AFAP-110 （AFAP）转染成肌细胞。lzip，已被证明可以激活Src并破坏培养成纤维细胞的肌动蛋白细胞骨架。初步结果表明，PKC磷酸化AFAP-110导致成纤维细胞中Src酪氨酸激酶活性激活和应激纤维断裂。转染不能结合PKC或不能与Src相互作用的AFAP-110突变体可以保护成纤维细胞免受PKC诱导的应激纤维破坏。将进行平行实验来测试AFAP-110是否在肌肉细胞中起类似的作用。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。这项工作大部分将由本科生来完成，他们将把他们的训练带到科学事业中去。

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.