Differential expression and distribution of chicken skeletal- and smooth-muscle-type alpha-actinins during myogenesis in culture

Abstract

Antibodies to chicken fast skeletal muscle (pectoralis) alpha-actinin and to smooth muscle (gizzard) alpha-actinin were absorbed with opposite antigens by affinity chromatography, and four antibody fractions were thus obtained: common antibodies reactive with both pectoralis and gizzard alpha-actinins ([C]anti-P alpha-An and [C]anti-G alpha-An), antibody specifically reactive with pectoralis alpha-actinin ([S]anti-P alpha-An), and antibody specifically reactive with gizzard alpha-actinin ([S]anti-G alpha-An). In indirect immunofluorescence microscopy, (C)anti-P alpha-An, (S)anti-P alpha-An, and (C)anti-G alpha-An stained Z bands of skeletal muscle myofibrils, whereas (S)anti-G alpha-An did not. Although (S)anti-G alpha-An and two common antibodies stained smooth muscle cells, (S)anti-P alpha-An did not. We used (S)anti-P alpha-An and (S)anti-G alpha-An for immunofluorescence microscopy to investigate the expression and distribution of skeletal- and smooth-muscle-type alpha-actinins during myogenesis of cultured skeletal muscle cells. Skeletal-muscle-type alpha-actinin was found to be absent from myogenic cells before fusion but present in them after fusion, restricted to Z bodies or Z bands. Smooth-muscle-type alpha-actinin was present diffusely in the cytoplasm and on membrane-associated structures of mononucleated and fused myoblasts, and then confined to membrane-associated structures of myotubes. Immunoblotting and peptide mapping by limited proteolysis support the above results that skeletal-muscle-type alpha-actinin appears at the onset of fusion and that smooth-muscle-type alpha-actinin persists throughout the myogenesis. These results indicate (a) that the timing of expression of skeletal-muscle-type alpha-actinin is under regulation coordination with other major skeletal muscle proteins; (b) that, with respect to expression and distribution, skeletal-muscle-type alpha-actinin is closely related to alpha-actin, whereas smooth-muscle-type alpha-actinin is to gamma- and beta-actins; and (c) that skeletal- and smooth-muscle-type alpha-actinins have complementary distribution and do not co-exist in situ.