Abstract
The control of gene expression during terminal myogenesis was explored in heterokaryons between differentiated and undifferentiated myogenic cells by analyzing the formation of species specific myosin light chains of chick and rat skeletal muscle. Dividing L6 rat myoblasts served as the biochemically undifferentiated parent. The differentiated parental cells were mononucleated muscle cells (myocytes) that were obtained from primary cultures of embryonic chick thigh muscle by blocking myotube formation with EGTA and later incubating the postimitotic cells in cytochalasin B. Heterokaryons were isolated by the selective rescue of fusion products between cells previously treated with lethal doses of different cell poisons. 95-99% pure populations of heterokaryons formed between undifferentiated rat myoblasts and differentiated chick myocytes were obtained. The cells were labeled with [35S]methionine, and whole cell extracts were analyzed on two-dimensional polyacrylamide gels. These heterokaryons synthesize the light chain of chick myosin and both embryonic and adult light chains of rat skeletal myosin. Control homokaryons formed by fusing undifferentiated cells to themselves did not synthesize skeletal myosin light chains. Control heterokaryons formed between undifferentiated rat myoblasts and chick fibroblasts also failed to synthesize myosin light chains. These results indicate that differentiated chick muscle cells provide some factor that induces L6 myoblasts to synthesize rat myosin light chains. This system provides a model for investigating the processes by which differentiated cell functions are induced.
Full Text
The Full Text of this article is available as a PDF (773.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Axelrod D. E., Gopalakrishnan T. V., Willing M., Anderson W. F. Maintenance of hemoglobin inducibility in somatic cell hybrids of tetraploid (2S) mouse erythroleukemia cells with mouse or human fibroblasts. Somatic Cell Genet. 1978 Mar;4(2):157–168. doi: 10.1007/BF01538981. [DOI] [PubMed] [Google Scholar]
- Bischoff R., Holtzer H. Mitosis and the processes of differentiation of myogenic cells in vitro. J Cell Biol. 1969 Apr;41(1):188–200. doi: 10.1083/jcb.41.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Buckley P. A., Konigsberg I. R. Myogenic fusion and the duration of the post-mitotic gap (G1). Dev Biol. 1974 Mar;37(1):193–212. doi: 10.1016/0012-1606(74)90179-1. [DOI] [PubMed] [Google Scholar]
- Carter S. B. Effects of cytochalasins on mammalian cells. Nature. 1967 Jan 21;213(5073):261–264. doi: 10.1038/213261a0. [DOI] [PubMed] [Google Scholar]
- Cohen R., Pacifici M., Rubinstein N., Biehl J., Holtzer H. Effect of a tumour promoter on myogenesis. Nature. 1977 Apr 7;266(5602):538–540. doi: 10.1038/266538a0. [DOI] [PubMed] [Google Scholar]
- Darlington G. J., Bernard H. P., Ruddle F. H. Human serum albumin phenotype activation in mouse hepatoma--human leukocyte cell hybrids. Science. 1974 Sep 6;185(4154):859–862. doi: 10.1126/science.185.4154.859. [DOI] [PubMed] [Google Scholar]
- Davidson R. L., O'Malley K. A., Wheeler T. B. Polyethylene glycol-induced mammalian cell hybridization: effect of polyethylene glycol molecular weight and concentration. Somatic Cell Genet. 1976 May;2(3):271–280. doi: 10.1007/BF01538965. [DOI] [PubMed] [Google Scholar]
- Davidson R. L. Regulation of malanin synthesis in mammalian cells: effect of gene dosage on the expression of differentiation. Proc Natl Acad Sci U S A. 1972 Apr;69(4):951–955. doi: 10.1073/pnas.69.4.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dienstman S. R., Holtzer H. Skeletal myogenesis. Control of proliferation in a normal cell lineage. Exp Cell Res. 1977 Jul;107(2):355–364. doi: 10.1016/0014-4827(77)90357-3. [DOI] [PubMed] [Google Scholar]
- Doering J. L., Fischman D. A. The in vitro cell fusion of embryonic chick muscle without DNA synthesis. Dev Biol. 1974 Feb;36(2):225–235. doi: 10.1016/0012-1606(74)90046-3. [DOI] [PubMed] [Google Scholar]
- Emerson C. P., Jr, Beckner S. K. Activation of myosin synthesis in fusing and mononucleated myoblasts. J Mol Biol. 1975 Apr 25;93(4):431–447. doi: 10.1016/0022-2836(75)90238-7. [DOI] [PubMed] [Google Scholar]
- Fischbach G. D. Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev Biol. 1972 Jun;28(2):407–429. doi: 10.1016/0012-1606(72)90023-1. [DOI] [PubMed] [Google Scholar]
- Fougère C., Ruiz F., Ephrussi B. Gene dosage dependence of pigment synthesis in melanoma x fibroblast hybrids (hamster cells-mouse fibroblast-DOPA-oxidase-irradiation). Proc Natl Acad Sci U S A. 1972 Feb;69(2):330–334. doi: 10.1073/pnas.69.2.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fougère C., Weiss M. C. Phenotypic exclusion in mouse melanoma-rat hepatoma hybrid cells: pigment and albumin production are not reexpressed simultaneously. Cell. 1978 Nov;15(3):843–854. doi: 10.1016/0092-8674(78)90269-6. [DOI] [PubMed] [Google Scholar]
- Garrels J. I. Changes in protein synthesis during myogenesis in a clonal cell line. Dev Biol. 1979 Nov;73(1):134–152. doi: 10.1016/0012-1606(79)90143-x. [DOI] [PubMed] [Google Scholar]
- Keller L. R., Emerson C. P., Jr Synthesis of adult myosin light chains by embryonic muscle cultures. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1020–1024. doi: 10.1073/pnas.77.2.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
- Luzzati D., Drugeon G. The effect of actinomycin D on RNA and protein synthesis during differentiation of myoblasts of line L 6 E: the half-life of functional myosin messenger. Biochimie. 1972;54(9):1157–1167. doi: 10.1016/s0300-9084(72)80020-8. [DOI] [PubMed] [Google Scholar]
- Merlie J. P., Gros F. In vitro myogenesis. Expression of muscle specific function in the absence of cell fusion. Exp Cell Res. 1976 Feb;97(2):406–412. doi: 10.1016/0014-4827(76)90632-7. [DOI] [PubMed] [Google Scholar]
- Moss P. S., Strohman R. C. Myosin synthesis by fusion-arrested chick embryo myoblasts in cell culture. Dev Biol. 1976 Feb;48(2):431–437. doi: 10.1016/0012-1606(76)90104-4. [DOI] [PubMed] [Google Scholar]
- Nadal-Ginard B. Commitment, fusion and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis. Cell. 1978 Nov;15(3):855–864. doi: 10.1016/0092-8674(78)90270-2. [DOI] [PubMed] [Google Scholar]
- Norwood T. H., Zeigler C. J., Martin G. M. Dimethyl sulfoxide enhances polyethylene glycol-mediated somatic cell fusion. Somatic Cell Genet. 1976 May;2(3):263–270. doi: 10.1007/BF01538964. [DOI] [PubMed] [Google Scholar]
- O'Neill M. C., Stockdale F. E. Differentiation without cell division in cultured skeletal muscle. Dev Biol. 1972 Dec;29(4):410–418. doi: 10.1016/0012-1606(72)90081-4. [DOI] [PubMed] [Google Scholar]
- Paterson B., Prives J. Appearance of acetylcholine receptor in differentiating cultures of embryonic chick breast muscle. J Cell Biol. 1973 Oct;59(1):241–245. doi: 10.1083/jcb.59.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peterson J. A., Weiss M. C. Expression of differentiated functions in hepatoma cell hybrids: induction of mouse albumin production in rat hepatoma-mouse fibroblast hybrids. Proc Natl Acad Sci U S A. 1972 Mar;69(3):571–575. doi: 10.1073/pnas.69.3.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rankin J. K., Darlington G. J. Expression of human hepatic genes in mouse hepatoma--human amniocyte hybrids. Somatic Cell Genet. 1979 Jan;5(1):1–10. doi: 10.1007/BF01538781. [DOI] [PubMed] [Google Scholar]
- Rogers J., Ng S. K., Coulter M. B., Sanwal B. D. Inhibition of myogenesis in a rat myoblast line by 5-bromodeoxyuridine. Nature. 1975 Jul 31;256(5516):438–440. doi: 10.1038/256438a0. [DOI] [PubMed] [Google Scholar]
- Sanger J. W. The use of cytochalasin B to distinguish myoblasts from fibroblasts in cultures of developing chick striated muscle. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3621–3625. doi: 10.1073/pnas.71.9.3621. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Trotter J. A., Nameroff M. Myoblast differentiation in vitro: morphological differentiation of mononucleated myoblasts. Dev Biol. 1976 Apr;49(2):548–555. doi: 10.1016/0012-1606(76)90197-4. [DOI] [PubMed] [Google Scholar]
- Whalen R. G., Butler-Browne G. S., Gros F. Identification of a novel form of myosin light chain present in embryonic muscle tissue and cultured muscle cells. J Mol Biol. 1978 Dec 15;126(3):415–431. doi: 10.1016/0022-2836(78)90049-9. [DOI] [PubMed] [Google Scholar]
- Whalen R. G., Butler-Browne G. S., Sell S., Gros F. Transitions in contractile protein isozymes during muscle cell differentiation. Biochimie. 1979;61(5-6):625–632. doi: 10.1016/s0300-9084(79)80160-1. [DOI] [PubMed] [Google Scholar]
- Willing M. C., Nienhuis A. W., Anderson W. F. Selective activation of human beta-but not gamma-globin gene in human fibroblast x mouse erythroleukaemia cell hybrids. Nature. 1979 Feb 15;277(5697):534–538. doi: 10.1038/277534a0. [DOI] [PubMed] [Google Scholar]
- Wright W. E. The isolation of heterokaryons and hybrids by a selective system using irreversible biochemical inhibitors. Exp Cell Res. 1978 Mar 15;112(2):395–407. doi: 10.1016/0014-4827(78)90222-7. [DOI] [PubMed] [Google Scholar]
