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

1. Cofilin在肌原纤维形成中的作用用反义方法抑制培养的肌细胞中Cofilin的表达。在注射反义寡核苷酸后3天，Cofilin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而没有组织成条纹状结构。为了进一步观察活肌细胞中肌动蛋白的动力学，我们用荧光标记的膜突蛋白C-末端片段标记细胞中的肌动蛋白丝，膜突蛋白C-末端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，在同一细胞中再追踪肌动蛋白组装长达3天。再次，我们观察到，肌动蛋白组织成肌原纤维是严重干扰的细胞与反义寡核苷酸。因此，肌动蛋白有序组装成肌节结构明显受到cofil降低的抑制。 ...更多信息 在水平。另一方面，我们产生了嵌合小鼠，其中含有正常的肌肉细胞和cofilin缺陷的肌肉细胞在嵌合模式。我们观察到在缺乏cofilin的骨骼肌区域出现了严重的损伤，即肌节中肌动蛋白和肌球蛋白的不规则组装和肌纤维的变性。这些结果表明，在肌原纤维形成过程中，cofilin在肌动蛋白的调控组装中起着关键作用。2. C蛋白分子的功能结构域在大肠杆菌科尔表达系统中，制备了鸡心肌MyBP-C（NC）N端不同大小的片段，即含有C 0-C4、C 0-C1、C1-C2、C2-C4结构域的片段。共沉淀实验表明，在生理盐水溶液中，C 0-C4和C 0-C1与F-肌动蛋白结合。C 0-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-原肌球蛋白存在下，这种抑制作用大大降低。根据这些数据，并结合以前的报道，我们得出结论，C-蛋白结合肌球蛋白和连接蛋白主要在C-端侧，而它结合肌动蛋白在N-端侧。少

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