Immunoelectron Microscopic Studies of Desmin (Skeletin) Localization and Intermediate Filament Organization in Chicken Skeletal Muscle

We studied the localization of desmin (skeletin), the major subunit of muscle-type intermediate filaments, by high resolution immunoelectron microscopy in adult chicken skeletal muscle. Immunoferritin labeling of ultrathin frozen sections of intact fixed sartorius muscle showed the presence of desmin between adjacent Z-bands and as strands peripheral to Z-bands, forming apparent connections between the Z-bands with adjacent sarcolemma, mitochondria, and nuclei. We observed no desmin labeling, however, in the vicinity of the Ttubules. In addition, intermediate filaments were morphologically discernible at the level of the Z-bands in plastic sections of glycerol-extracted muscle that had been infused with unlabeled antidesmin antibodies. Our results indicate that the desmin present in adult skeletal muscle, that had previously been detected by immunofluorescence light microscopy, is largely if not entirely in the form of intermediate filaments. The results provide evidence that these filaments serve to interconnect myofibrils at the level of their Z-bands, and to connect Z-bands with other specific structures and organelles in the myotube, but not with the T-tubule system. The structure and function of the intermediate filaments of striated muscle have been the subjects of increasing interest in recent years, since it was first shown by Ishikawa et al. (10, 11) that a distinct set of 10-rim filaments different from the actin microfilaments was present in developing skeletal muscle. The main protein subunit of muscle type intermediate fdaments was isolated from smooth muscle independently by Lazarides and Hubbard (16), who called the 55-kdalton protein desmin, and by Small and Sobieszek (21), who called it skeletin. (We use the former designation here.) In adult skeletal muscle, intermediate filaments are less abundant than in embryonic muscle or than in cardiac or smooth muscle. Two approaches have been used to detect their presence in skeletal muscle. One approach has been morphological, by transmission electron microscopy of the intact tissue. It has been difficult, however, to discern 10-rim intermediate filaments in adult skeletal muscle, although Page (19) did describe a network of filaments encircling the myofibrils at the level of the Z-band in chicken anterior latissimus dorsi muscle. The other approach has been immunofiuorescence light microscopy, using antibodies specific for chicken gizzard desmin. With partially extracted and sheared adult skeletal muscle fibers, Lazarides and co-workers (9, 15-17) showed that desmin was present at the periphery of each myofibril at the level of, and surrounding, each Z-band. THE JOURNAL Of CELL BIOLOGY • VOLUME 96 JUNE 1983 1727-1735 © The Rockefeller University Press . 0021-952S/83/06/1727/09 $1.00 The resolution of immunofluorescence microscopy is limited, however, and it is impossible to conclude from these results anything about the filamentous state of the desmin, or the detailed ultrastructural relationship of desmin to the Z-band and to other structures in the muscle fiber. These matters are important to understanding the functions of intermediate filaments in skeletal muscle. It has been shown (2, 6) that during myotube development in culture, there is a drastic reorganization of desmin from fdamentous strands that are longitudinally distributed in the ceil to a highly localized distribution around the Z-bands. Because of this reorganization, and the inability to detect intermediate filaments unequivocally in the mature myotubes, the question has been raised (2) whether in the mature myotube the desmin might be present in a molecular rather than a filamentous form. To investigate these and related problems further, we carried out electron microscopic immunolabeling of desmin in intact adult chicken skeletal muscle. The much higher resolution of immunoelectron microscopy than of immunofluorescence light microscopy has permitted the distribution of desmin to be determined with greater definition than has hitherto been possible. For this purpose, we used the techniques for the immunolabeling of ultrathin frozen sections of lightly fixed intact tissue developed in this laboratory (23, 26). With this 1727 on O cber 9, 2017 jcb.rress.org D ow nladed fom