Anchorage-independent muscle cell differentiation

Open Access
Publisher Website
Google Scholar
Abstract
Cells from embryonic chicken muscle were cultivated in serum-free medium. After 2 days the suspended cells (almost all of which were nondividing myoblasts) were subcultured in serum-containing medium, in gelatin-coated tissue culture dishes (to promote reattachment) or in bacteriological dishes (to prevent reattachment). The extent of fusion was high in suspended and reattached cultures. Newly synthesized proteins from day 5 cultures were resolved by 2-dimensional electrophoresis and detected by autoradiography. The same protein species were synthesized and the relative intensities of the spots corresponding to known muscle-specific proteins and the patterns of the many unidentified spots were similar. Synthesis of creatine kinase subunits B and M at different times was determined. In 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 [relative MW] 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. Proper alignment of parallel arrays of myofibrils in register apparently requires cell attachment to substrate.