REMODELING OF CYTOSKELETON AND TRIADS FOLLOWING ACTIVATION OF V-SRC TYROSINE KINASE IN QUAIL MYOTUBES

To study the cellular signals underlying the regulatory mechanisms inv olved in maintenance of sarcomeric integrity, we have used quail skele tal muscle cells that reach a high degree of structural maturation in vitro, and also express a temperature-sensitive mutant of the v-Src ty rosine kinase that allows the control of differentiation in a reversib le manner. By immunofluorescence and electron microscopy we show that v-Src activity in myotubes leads to an extensive cellular remodeling w hich affects components of the sarcomeres, the cytoskeleton network an d the triad junctions. We have previously shown that activation of v-S rc causes a selective dismantling of the I-Z-I segments coupled to the formation of aggregates of sarcomeric actin, alpha-actinin and vincul in, called actin bodies. We now show that intermediate filaments do no t participate in the formation of actin bodies, while talin, a compone nt of costameres, does. The I-Z-I segments are completely dismantled w ithin 24 hours of v-Src activity, but the A-bands persist for a longer time, implying distinct pathways for the turnover of sarcomeric subdo mains. Immunofluorescence labeling of markers of the triad junctions d emonstrates that the localization of the alpha 1 subunit of the dihydr opyridine receptor is disrupted earlier than that of the ryanodine rec eptor after tyrosine kinase activation. Furthermore, the location of j unctional sarcoplasmic reticulum and transverse tubule membranes is ma intained in myotubes in which the I-Z-I have been removed and the regu lar disposition of the intermediate filaments is disrupted, supporting a role for sarcoplasmic reticulum in the proper positioning of triad junctions. Altogether these results point to a tyrosine kinase signali ng cascade as a mechanism for selectively destabilizing sarcomere subd omains and their tethering to the cytoskeleton and the sarcolemma.