The membrane skeleton of costameres

Abstract

Ultrastructural studies of mammalian skeletal muscle cells have demonstrated that myofibrils are attached laterally to the sarcolemma. The light microscopic correlates of these attachments are believed to be sub-sarcolemmal structures called costameres to which a number of cytoskeletal and integral membrane proteins have been localized. In indirect immunofluorescence studies, antibodies to dystrophin (the protein missing in patients with duchenne muscular dystrophy (DMD)), a 58 kilodalton, dystrophin-associated protein (the 58K protein), and both muscle {dollar}\beta{dollar}-spectrin and {dollar}\beta{dollar}-fodrin labeled the cortical cytoplasm of mammalian skeletal myofibers in a costameric pattern with enrichment over I bands, but not Z lines, and M lines. When the distribution of dystrophin was compared by confocal scanning laser microscopy (CSLM) to that of the 58K protein and of vinculin, the prototypic costameric protein, the patterns were identical. Thus, the five proteins studied were present in costameres. Costameres do not, however, require the presence of dystrophin as they were present in muscles from humans with DMD and mdx mice, which lack dystrophin. My results further suggest that costameres may contain sub-domains, as CSLM showed that labeling for {dollar}\alpha{dollar}-fodrin, although present in costameres, was sparse over M lines, thinner over I bands, and not decreased over Z lines. Mammalian skeletal muscle contained {dollar}\alpha{dollar}- and {dollar}\beta{dollar}-fodrin and muscle {dollar}\beta{dollar}-spectrin, but not erythrocyte {dollar}\alpha{dollar}- and {dollar}\beta{dollar}-spectrin. Immunoprecipitation followed by immunoblotting showed that two populations of these three spectrin subunits were present at the sarcolemma. One population contained {dollar}\alpha{dollar}- and {dollar}\beta{dollar}-fodrin and muscle {dollar}\beta{dollar}-spectrin; the other contained only muscle {dollar}\beta{dollar}-spectrin. Further investigations into the physical association of the five costameric proteins studied showed that anti-muscle {dollar}\beta{dollar}-spectrin also precipitated some 58K protein but no dystrophin or vinculin. In contrast, anti-58K protein antibody precipitated the 58K protein and dystrophin but no spectrin and vinculin. These results demonstrate the presence in costameres of five proteins that may participate in a sub-sarcolemmal cytoskeleton like that of the erythrocyte. Such a cytoskeleton may help to connect the contractile apparatus to the sarcolemma. This structure may also stabilize the sarecolemma by providing a flexible, but strong, sub-membrane scaffold at sites where stress is applied during the contractile cycle. These regular connections would also permit ordered folding of the sarcolemma during contraction.