Analysis of muscle protein expression in polyethylene glycol-induced chicken: rat myoblast heterokaryons.

Abstract

Heterokaryons derived from polyethylene glycol-mediated fusion of myoblasts at different stages of development were used to investigate the transition of cells in the skeletal muscle lineage from the determined to the differentiated state. Heterokaryons were analyzed by immunofluorescence, using rabbit antibodies against the skeletal muscle isoforms of chicken creatine kinase and myosin, and a mouse monoclonal antibody that cross-reacts with chicken and rat skeletal muscle myosin. When cytochalasin B-treated rat L8(E63) myocytes (Konieczny S.F., J. McKay, and J. R. Coleman, 1982, Dev. Biol., 91:11-26) served as the differentiated parental component and chicken limb myoblasts from stage 23-26 or 10-12-d embryos were used as the determined, undifferentiated parental cell, heterokaryons exhibited a progressive extinction of rat skeletal muscle myosin during a 4-6-d culture period, and no precocious expression of chicken differentiated gene products was detected. In the reciprocal experiment, 85-97% of rat myoblast x chicken myocyte heterokaryons ceased expression of chicken skeletal muscle myosin and the M subunit of chicken creatine kinase within 7 d of culture. Extinction was not observed in heterokaryons produced by fusion of differentiated chicken and differentiated rat myocytes and thus is not due to species incompatibility or to the polyethylene glycol treatment itself. The results suggest that, when confronted in a common cytoplasm, the regulatory factors that maintain myoblasts in a proliferating, undifferentiated state are dominant over those that govern expression of differentiated gene products.

Characterization of Heterokaryons between Skeletal Myoblasts and Somatic Cells Formed by Fusion with HVJ (Sendai Virus): Effects on Myogenic Differentiation.

“…Phenotypic suppression has been reported in various heterokaryons between myogenic and non-myogenic cells, suggesting regulatory factors of differentiation in the cytoplasm (Ringertz and Savage, 1976;Inge et al, 1980;Wright, 1981Wright, , 1984Konieczny et al, 1983;Wright and Aronoff, 1983;Lawrence and Coleman, 1984;Blau et al, 1985). However, from the apparent disturbance of myogenin expression and myofibril formation in our study, it is noteworthy that partial myofibril formation and myogenin expression were frequently seen in the areas far from the nuclei of EAT cells even in heterokaryotic myotubes between QM-RSV and EAT cells (Figs.…”

Limitations of nlsβ-galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer

“…Similarly, when heterokaryons are formed by the fusion of mouse myoblasts and human fibroblasts, muscle genes are activated in the fibroblast nuclei, mediated by trans-acting factors encoded for by the mouse myonucleus. These factors migrate through the cytoplasm and gain access to the fibroblast nuclei (Konieczny et al, 1983;Blau et al, 1985).…”

Do hydrophobic sequences cleaved from cellular polypeptides induce membrane fusion reactions in vivo?

“…There are, furthermore, a number of reports on the fusion of phospholipid vesicles occurring on treatment with peptides and proteins either in the absence of Ca2+ or in the presence of EGTA [51-561. Erythrocytes fuse in the presence of EGTA when they are treated with oleoylglycerol or chlorpromazine as mentioned [ 18,191, or with tetracaine [57], or Sendai virus [58,59] and it has been suggested that the entry of Ca2+ into Sendai virustreated erythrocytes is a consequence and not a cause of the fusion event [60]. Even some fusion of chick myoblasts (but not rat myoblasts) occurs in high-density cultures in 1.75 mM EGTA [61]. Additionally, the toxic effects of PEG are decreased, and the yield of hybrid cells is increased, when PEG is used in a Ca '+-free medium and the cells then maintained in Ca2+-free medium for at least 15 min [62].…”