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

这项研究项目将研究肌原纤维组装和分解的基本过程。肌原纤维由肌节组成，肌节是细丝和粗丝的重复单位。主要由肌动蛋白细丝组成的细丝连接到Z线，这是一种在蛋白质组成上类似于细胞粘连的结构。在局部粘连和Z线结构中发现的蛋白质包括肌动蛋白细丝的带刺末端、粘着斑激酶、p130Cas、α-肌动蛋白和AFAP-110。AFAP-110是这个项目的重点，它与非受体酪氨酸激酶Src相关。最近的证据表明，AFAP-110在蛋白激酶C(PKC)活性升高或机械应激时将Src招募到肌动蛋白细胞骨架上。证据还表明，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在焦点粘连和Z线上的分解所带来的细胞骨架的重组。成熟的肌原纤维从表达高Src或PKC活性的肌管中消失。相反，细胞粘附力的产生已被证明导致肌动蛋白细丝连接的形成，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分子的特性包括结合和交联肌动蛋白细丝的能力、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。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。为了研究AFAP-110在肌肉发育中的作用，将对原代培养的肌管进行AFAP-110和Z线标记α-肌动蛋白的染色。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，这两种蛋白在所有发育阶段都应该是共定位的。将使用Src的特定抑制剂来检验肌原纤维的形成需要激活Src的假设。为了研究分解机制，将AFAP-110的突变形式AFAP.lZIP导入成肌细胞，AFAP-110的突变形式AFAP.lZIP已被证明能激活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激性纤维的断裂。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诱导的应激性纤维断裂。将进行平行实验，以测试AFAP-110是否在肌肉细胞中发挥类似的作用。这些研究的结果将阐明成熟的肌原纤维翻转和新的肌原纤维形成的机制。这项工作将在很大程度上由本科生完成，他们将继续接受科学方面的培训。

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.