Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry

Distribution of cytoskeletal proteins with emphasis on the membrane-cytoskeleton interface was examined in cultured cardiac myocytes. Using specific antibodies recognizing alpha-sarcomeric actin, desmin, beta-tubulin, spectrin/alpha-fodrin and ankyrin, respectively, the cellular localization of these cytoskeletal proteins was detected by laser scanning confocal microscopy. In addition, the fine filamentous structure of these proteins was identified by combining silver-enhanced immunogold labelling with electron microscopy. The latter technique employed the sequence of quick-freezing, deep-etching and rotary shadowing of the specimens. Conventional transmission electron microscopy of the spherical cardiac myocytes revealed a filamentous submembranous layer, approximately 100 nm thick. Specific immunolabelling of alpha-sarcomeric actin and spectrin/alpha-fodrin as well as ankyrin was seen beneath the plasmalemma. A three-dimensional meshwork of spectrin/alpha-fodrin was shown. Numerous desmin filaments that exhibited a tortuous course throughout the cells were also observed running in parallel with the surface in the submembranous area, whereas beta-tubulin was infrequently detected in these areas. In conclusion, the present study shows that spherical cardiac myocytes contain a distinct and complex three-dimensional membrane skeleton. Major constituents of this distinct submembranous layer were spectrin/alpha-fodrin fibres as well as actin and desmin filaments.