Morris G E, Head L P
Exp Cell Res. 1985 May;158(1):177-91. doi: 10.1016/0014-4827(85)90442-2.
Two monoclonal antibodies, FIFI and PHIL, have been prepared using detergent-washed myogenic cells as immunogen. On Western blots of total protein extracts of muscle cells, both antibodies bind to vimentin (52 kD) and its degradation products (major band at 42 kD), but do not bind to mouse proteins or to actin (42 kD). Specificity for a determinant common to vimentin and desmin was confirmed by 2-D gel electrophoresis of muscle cell extracts and purified desmin. Western blots with FIFI reveal particularly well the extreme sensitivity of intermediate filaments (IFs) to proteolysis, which was preventable in brain tissue only by boiling in 1% SDS, although it could be reduced in both brain and muscle by less extreme methods. Western blots suggest a large increase in IF content of differentiating myoblast cell cultures at the time of cell fusion and an increase of at least 4-fold is confirmed by a quantitative immunoassay using a direct ELISA method. Immunofluorescence microscopy shows that this increase is due to the appearance of high concentrations of the intermediate filament antigen at the ends of early myotubes, preceding the appearance of cross-striations in myofibrils. Furthermore, whereas the polar filaments detected by FIFI run right to the ends of the early myotubes and only sparingly penetrate the central area, cross-striated myofibrils (as detected by the monoclonal antibody, SAM) run the length of the myotube but do not reach the ends. Colcemid and colchicine cause the vimentin filaments in fibroblasts to collapse into perinuclear rings or caps, but do not have this effect on the polar fluorescence in early myotubes. Heat shock (2 h at 45 degrees C) has a similar differential effect. The results suggest that early in muscle differentiation intermediate filament proteins accumulate rapidly at myotube ends, where they are organized differently from those in fibroblasts.
已使用经去污剂洗涤的成肌细胞作为免疫原制备了两种单克隆抗体，即FIFI和PHIL。在肌肉细胞总蛋白提取物的蛋白质印迹上，两种抗体均与波形蛋白（52kD）及其降解产物（主要条带在42kD）结合，但不与小鼠蛋白或肌动蛋白（42kD）结合。通过肌肉细胞提取物和纯化的结蛋白的二维凝胶电泳证实了对波形蛋白和结蛋白共有的一个决定簇的特异性。用FIFI进行的蛋白质印迹特别好地揭示了中间丝（IFs）对蛋白水解的极端敏感性，这种敏感性在脑组织中只有通过在1% SDS中煮沸才能防止，尽管通过不太极端的方法在脑和肌肉中都可以降低。蛋白质印迹表明，在细胞融合时，分化的成肌细胞培养物中的IF含量大幅增加，并且使用直接ELISA方法的定量免疫测定证实增加了至少4倍。免疫荧光显微镜显示，这种增加是由于在肌原纤维出现横纹之前，早期肌管末端出现了高浓度的中间丝抗原。此外，虽然FIFI检测到的极性丝一直延伸到早期肌管的末端，并且只是很少地穿透中心区域，但横纹肌原纤维（由单克隆抗体SAM检测）贯穿肌管的长度但没有到达末端。秋水仙酰胺和秋水仙碱会使成纤维细胞中的波形蛋白丝塌陷成核周环或帽，但对早期肌管中的极性荧光没有这种作用。热休克（45℃ 2小时）有类似的差异效应。结果表明，在肌肉分化早期，中间丝蛋白在肌管末端迅速积累，在那里它们的组织方式与成纤维细胞中的不同。
