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The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K) . Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm . With maturation, staining of myotubes by anti-58K diminished and eventually disappeared . Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation-from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I-Z region of myofibrils . However, the emergence of a cross-striated anti-55K pattern did not coincide temporally with the emergence of striated myofibrils, but occurred over a period of days thereafter. KEY WORDS intermediate filaments myogenesis " muscle differentiation immunofluorescenceReports from this laboratory and from others have demonstrated that the intermediate or 100-A filament (IF) subunits from different cell types and organisms are not all identical, and can be distinguished immunologically and biochemically (I,2,9, 11a, IIb,21,29) . We demonstrated that antibodies against the 58-kdalton chick fibroblast IF subunit (anti-58K) stained filaments in many different kinds of cultured chick cells, including fibroblasts, chondroblasts, melanocytes, neurons, glia, cardiac and smooth muscle myoblasts, and both replicating presumptive skeletal myoblasts and postmitotic myoblasts and myotubes. Contrastingly, antibodies against the 55-kdalton chick smooth muscle IF subunit (anti-55K) were highly selective and stained only definitive smooth, cardiac, and skeletal muscle cells. Replicating pre-J . CELL BIOLOGY

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The protein subunit of 100-A filaments constitutes approximately 50% of the cytoskeleton protein of chick fibroblasts.

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The synthesis and subsequent modification of neurofilament (NF) polypeptides has been examined in pulse-chase experiments, using cultured chick spinal cord neurons. Fluorography of the [35S]methionine-labeled cytoskeletal proteins, after separation by two-dimensional gel electrophoresis, revealed that (a) the mid-size chicken NF protein, NF-M160, is synthesized as a smaller and more basic precursor, NF-M130; (b) beginning ~8 h after translation, NF-M130 slowly and continuously becomes larger and more acidic, attaining the size and charge of NF-M160 16 or more h later, and undergoing no further change in mobility for many days thereafter; and (c) in contrast, the low molecular weight NF protein, NF-L, is synthesized as such, and undergoes no subsequent change in apparent size or charge.Additional experiments provided evidence that the conversion of NF-M130 to NF-M160 is due, at least in part, to phosphorylation: (a) Incubation of similar cultures in 3~PO4 resulted in incorporation into NF-M160 and transitional forms, but not into NF-M130. (b) An antiserum to NF-M160 was found by immunoblot analysis to bind strongly to untreated NF-M160, but poorly to phosphatase-treated NF-M160, and not at all to NF-M130. It has already been demonstrated (Bennett, G. S., S. J. Tapscott, C. DiLullo, and H. Holtzer, 1984, Brain Res., 304:291-302) that this anti-NF-M160 fails to stain the soma of motor neurons in sections of chick spinal cord, but detects an increasing gradient of immunoreactivity in the proximal axons.These results, together with the known kinetics of axoplasmic transport of NF, suggest that the mid-size chicken NF protein is synthesized as NF-M130 and is extensively modified, at least in part by phosphorylation, to become NF-M160 during transport along proximal neurites. Once maximally modified, NF-M160 undergoes no further net change during transport along distal neurites.