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

Abstract

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