Monoclonal antibodies to intermediate filaments in chick muscle cell cultures.
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.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002478:Cells, Cultured; D002642:Chick Embryo; D003599:Cytoskeleton / Q000276:immunology* / Q000648:ultrastructure; D003703:Demecolcine / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006358:Hot Temperature; D007163:Immunosorbent Techniques; D007381:Intermediate Filament Proteins / Q000276:immunology* / Q000378:metabolism; D007526:Isoelectric Point; D008970:Molecular Weight; D009124:Muscle Proteins / Q000276:immunology* / Q000378:metabolism; D009132:Muscles / Q000196:embryology
D000911:Antibodies, Monoclonal; D007381:Intermediate Filament Proteins; D009124:Muscle Proteins; D003703:Demecolcine