Isolation and characterization of a temperature-sensitive rat myoblast cell line
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A temperature-sensitive (ts) variant of the established rat myoblast line L6 has isolated and partially characterized. At the permissive temperature (37 [degrees]) the variant cells proceed through the normal sequence of differentiation characterized by growth, alignment, and subsequent fusion to form myotubes. At the nonpermissive temperature (40 [degrees]) neither alignment nor fusion take place. A temperature-shift experiment performed at 3 hour intervals after seeding demonstrated that the temperature-sensitive event occurs 1-5 days after initial plating, while alignment and fusion take place at day 4 and day 5 respectively. Experiments utilizing mixed cultures of L6D0 and tsA1 cells provide evidence for interaction between the two cell types while conditioned medium experiments provide no evidence for the participation of a diffusible extracellular factor in alignment and fusion, and therefore support the hypothesis of surface protein involvement. Extrinsic surface proteins of parental cells (L6D0) and the ts variant were labeled via lactoperoxidase catalyzed iodination and analyzed by SDS-polyacrylamide gel electrophoresis. This experiment revealed substantial differences between the two cell types. L6D0 cultures incubated at both 37 [degrees] and 40 [degrees] and the ts variant grown at 37 [degrees] exhibit similar developmental alterations in surface proteins during myogenesis. Proteins with apparent molecular weights of 98.000, 66.000, 60.000, and 47.000 are associated with alignment. The onset of fusion is associated with the appearance of proteins which migrate at 74.000, 56.000, 40.000, 12.000, and 11.000 D. The temperature-sensitive variant possesses all of the proteins associated with alignment; however the appearance of the 98.000 D protein on day 4 is accompanied by a peptide migrating at 96.000 D. In addition, the non-fusing variant possesses only two of the five proteins associated with fusion.
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