MYOGENIC CELLS EXPRESS MULTIPLE MYOSIN ISOFORMS

In vivo and in vitro, proliferating motile myoblasts form aligned grou ps of cells, with a characteristic bipolar morphology, subsequently be come post-mitotic, begin to express skeletal myosin and fuse. We were interested in whether members of the myosin superfamily were involved in myogenesis. We found that the myoblasts expressed multiple myosin i soforms, from at least five different classes of the myosin superfamil y (classes I, II, V, VII and IX), using RT-PCR and degenerate primers to conserved regions of myosin. All of these myosin isoforms were expr essed most highly in myoblasts and their expression decreased as they differentiated into mature myotubes, by RNAse protection assays, and W estern analysis. However, only myosin I alpha, non-muscle myosin IIA a nd IIB together with actin relocalize in response to the differentiati ve state of the cell. In single cells, myosin I alpha was found at the leading edge, in rear microspikes and had a punctate cytoplasmic stai ning, and non-muscle myosin was associated with actin bundles as previ ously described for fibroblasts. In aligned groups of cells, all these proteins were found at the plasma membrane. Go-staining for skeletal myosin II, and myosin I alpha showed that myosin I alpha also appeared to be expressed at higher levels in post-mitotic myoblasts that had b egun to express skeletal myosin prior to fusion. In early myotubes, ac tin and non-muscle myosin IIA and IIB remained localized at the membra ne. All of the other myosin isoforms we looked at, myosin V, myosin IX and a second isoform of myosin I (mouse homologue to myr2) showed a p unctate cytoplasmic staining which did not change as the myoblasts dif ferentiated. In conclusion, although we found that myoblasts express m any different isoforms of the myosin superfamily, only myosin Iu, non- muscle myosin IIA and IIB appear to play any direct role in myogenesis .