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在培养的肌肉细胞中的肌原纤维纤维质溶质蛋白表达中的作用被一种抗敏感方法抑制。注射抗oligo核动肽后3天，Cofilin表达水平显着降低。当用抗肌动蛋白抗体检查肌动蛋白定位时，检测到肌动蛋白丝的束，但它们散布在细胞质中，而无需组织成条纹结构。为了进一步可视化活肌细胞中的肌动蛋白动力学，我们标记了带有荧光标记的Moesin的荧光标记的C末端片段的肌动蛋白丝，其中包含肌动蛋白结合位点，但对肌动蛋白动力学没有影响。注射抗敏感性寡核苷酸后，将肌动蛋白组件追溯到同一细胞中的另外3天。同样，我们观察到肌动蛋白在肌纤维中的组织在抗义寡核苷酸的细胞中受到严重干扰。通过降低Cofil的水平，清楚地抑制了将肌动蛋白组装成肌肉结构的序列。另一方面，我们生成了嵌合有正常肌肉细胞和缺陷型肌肉细胞的嵌合小鼠。我们观察到骨骼肌区域中出现了严重损害，缺乏cofilin，即肌动蛋白和肌球蛋白在肌动蛋白和肌纤维变性中的不规则组装。这些结果表明，在肌原纤维形成过程中，Cofilin对于肌动蛋白的调节组装起着至关重要的作用。2。鸡心脏MyBP-C（NC）的C蛋白分子末端片段的功能域，其大小不同，即包含域C0-C4，C0-C1，C1-C2，C1-C2，C2 C2 C2，C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2中。共进行测定测定表明，在物理盐溶液中，C0-C4以及与F-肌动蛋白结合的C0-C1。 C0-C4对肌动蛋白激活的肌球蛋白ATPase具有抑制作用，但在肌钙蛋白 - 肌球蛋白的存在下，这种抑制作用大大降低。基于这些数据以及先前报道的那些数据，我们包括C蛋白与肌球蛋白结合，主要在C末端结合蛋白，同时它与N末端的肌动蛋白结合。较少的