Anchorage-independent muscle cell differentiation

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Cells from embryonic chicken muscle were cultivated in serum-free medium. After two days, the suspended cells (almost all of which were nondividing myoblasts) were subcultured in serum-containing medium, either in gelatincoated tissue culture dishes (to promote reattachment) or in bacteriological dishes (to prevent reattachment). The extent of fusion was high in both suspended and reattached cultures. Newly synthesized proteins from day-5 cultures were resolved by two-dimensional electrophoresis and detected by autoradiography. Not only were the same protein species synthesized, but also the relative intensities of the spots corresponding to known muscle-specific proteins as well as the patterns of the many unidentified spots were similar. Synthesis of creatine kinase subunits B and M at different times was determined. In both suspended and reattached cells there was, as expected for differentiating myogenic cells, a marked increase by day 5 in the ratio of M to B subunits synthesized. Immunofluorescent staining with antibodies against an M-line protein with Mr 165,000 revealed myofibrils partially wound about the nuclei of suspended cells; these became strung out in the axis of the cell as reattached cells elongated. The synthesis of muscle proteins, their assembly into myofilaments, and formation of well-organized myofibrils is evidently not anchorage dependent. However, proper alignment of parallel arrays of myofibrils in register does appear to require cell attachment to substrate.

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