Modifications in the interactions of the muscle cytoskeleton with the cell membrane occur during cell growth and adaptation, although the mechanisms regulating these interactions are unknown. We have observed that myotendinous junctions (MTJs), which are the primary sites of turnover of the thin filament-membrane associations in skeletal muscle, are greatly enriched in receptors for PDGF. The high concentration of PDGF receptors at MTJs suggested to us that receptor binding may initiate cytoskeletal remodeling in skeletal muscle. We tested this possibility by examining the organization and phosphorylation of cytoskeletal components of L6 myocytes after PDGF stimulation. We have found that 10 min after PDGF stimulation, L6 myoblasts exhibit no stress fibers discernible by phalloidin binding, and that vinculin relocates from focal contacts into a diffuse cytoplasmic distribution. After 60 min of incubation, these changes are largely reversed. Indirect immunofluorescence shows that at 10-min PDGF stimulation, there are no changes in the distribution of talin, the beta 1 subunit of integrin, pp125FAK or desmin. Phosphotyrosine distribution changes upon stimulation from focal contacts to being located both in focal contacts and granules concentrated in perinuclear regions. These granules also immunolabel with anti-PDGF receptor Immunoprecipitations with anti-phosphotyrosine show that polypeptides at 180 and 230 kD show the greatest increase in tyrosine phosphorylation after PDGF stimulation. Immunoblots of anti-phosphotyrosine precipitates show that these polypeptides are the PDGF receptor and talin. We also examined the possibility that the cytoskeletal reorganization observed may result from calpain activation caused by elevated intracellular calcium induced by PDGF stimulation. However, immunoblots of control and stimulated cells show no decrease in the inactive calpain proenzyme or increase in the proteolytic, autolyzed forms of calpain pursuant to stimulation. Furthermore, stimulation produces no increase in the proportion of the 190-kD talin fragment characteristic of calpain-mediated cleavage. The retention of talin and integrin at focal contacts after talin phosphorylation, while vinculin is redistributed, indicate that phosphorylation of talin in PDGF-stimulated cells leads to separation of talin-vinculin associations but not talin-integrin associations. We propose that PDGF binding to PDGF receptors at MTJs may provide one means of regulating myofibril associations with the muscle cell membrane.

Junctions formed by skeletal muscles where they adhere to tendons, called myotendinous junctions, are sites of tight adhesion and where forces generated by the cell are placed on the substratum. In this regard, myotendinous junctions and focal contacts of fibroblasts in vitro are analogues. Talin is a protein located at focal contacts that may be involved in force transmission from actin filaments to the plasma membrane. This study investigates whether talin is also found at myotendinous junctions. Protein separations on SDS polyacrylamide gels and immunolabeling procedures show that talin is present in skeletal muscle. Immunofluorescence microscopy using anti-talin indicates that talin is found concentrated at myotendinous junctions and in lesser amounts in periodic bands over nonjunctional regions. Electron microscopic immunolabeling shows talin is a component of the digitlike processes of muscle cells that extend into tendons at myotendinous junctions. These findings indicate that there may be similarities in the molecular composition of focal contacts and myotendinous junctions in addition to functional analogies.