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Functional properties of cofilin and C-protein molecules and their roles in formation and maintenance of muscle cellular structure

丝切蛋白和 C 蛋白分子的功能特性及其在肌肉细胞结构形成和维持中的作用

基本信息

批准号：
14340262
负责人：
OBINATA Takashi
金额：
$ 9.28万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

1.Role of cofilin in myofibrillogenesisCofilin expression in cultured muscle cells was suppressed by an anti-sense method. Cofilin expression level was significantly lowered 3 days after injection of antisense-oligo nucleotides. When actin localization was examined with anti-actin antibody, bundles of actin filaments were detected but they were scattered in the cytoplasm without organizing into striated structures. In order to visualize actin dynamics in living muscle cells further, we labeled actin filaments in the cells with fluorescence-labeled C-terminal fragments of moesin, which contains actin-binding site but has no effects on actin dynamics. Actin assembly was traced up to another 3 days in the same cell after injection of anti-sense oligo nucleotides. Again, we observed that actin organization into myofibrils was severely disturbed in the cells with anti-sense oligo nucleotides. Thus, ordered assembly of actin into sarcomeric structures was clearly suppressed by lowering cofil … More in level. On the other hand, we generated chimeric mice that contain normal muscle cells and cofilin-deficient muscle cells in a chimeric pattern. We observed that severe damage emerged in skeletal muscle area, which lacks cofilin, namely irregular assembly of actin and myosin in sarcomere and degeneration of myofibers. These results indicate that cofilin plays a critical role for the regulated assembly of actin in the process of myofibril formation.2.Functional domains in C-protein moleculeN-terminal fragments of chicken cardiac MyBP-C (NC) of different size, namely that contain domain C0-C4,C0-C1,C1-C2,C2-C4,were prepared in an E.coll expression system. Co-sedimentation assay showed that C0-C4 as well as C0-C1 bound to F-actin in a physiological salt solution. C0-C4 showed an inhibitory effect on actin-activated myosin-ATPase but this inhibition was much lowered in the presence of troponin-tropomyosin. Based on these data and together with those previously reported, we conclude that C-protein binds to myosin and connectin mainly at the C-terminal side while it binds to actin at the N-terminal side. Less

1.cofilin在肌纤维形成中的作用用反义法抑制培养肌细胞中cofilin的表达。注射反义寡核苷酸后3 d， Cofilin的表达水平显著降低。当用抗肌动蛋白抗体检测肌动蛋白定位时，检测到成束的肌动蛋白丝，但它们分散在细胞质中，没有组织成条纹结构。为了进一步观察肌动蛋白在活肌细胞中的动态，我们用荧光标记的moesin的c端片段标记细胞中的肌动蛋白丝，该片段含有肌动蛋白结合位点，但对肌动蛋白动力学没有影响。在注射反义寡核苷酸后，在同一细胞中追踪肌动蛋白的组装长达3天。我们再次观察到，在含有反义寡核苷酸的细胞中，肌动蛋白进入肌原纤维的组织受到严重干扰。因此，降低肌动蛋白的水平明显抑制了肌动蛋白在肌肉结构中的有序组装。另一方面，我们以嵌合模式产生了含有正常肌肉细胞和缺乏cofilin肌肉细胞的嵌合小鼠。我们观察到骨骼肌区域出现了严重的损伤，缺乏cofilin，即肌动蛋白和肌球蛋白在肌节中的不规则组装和肌纤维变性。这些结果表明，在肌原纤维形成过程中，cofilin在调节肌动蛋白的组装中起着关键作用。在大肠杆菌表达系统中制备了不同大小的鸡心肌MyBP-C (NC) c蛋白分子末端片段的功能域，即含有结构域C0-C4、C0-C1、C1-C2、C2-C4。共沉降实验表明，C0-C4和C0-C1在生理盐溶液中与f -肌动蛋白结合。C0-C4对肌动蛋白激活的肌球蛋白- atp酶有抑制作用，但在肌钙蛋白-原肌球蛋白存在时，这种抑制作用大大降低。根据这些数据和先前报道的数据，我们得出结论，c蛋白主要在c端与肌凝蛋白和连接蛋白结合，而在n端与肌动蛋白结合。少