IMMUNOLOCALIZATION OF THE COSTAMERES IN HUMAN SKELETAL-MUSCLE FIBERS - CONFOCAL SCANNING LASER MICROSCOPE INVESTIGATIONS

Background: The costameres in skeletal muscle fibers were first descri bed by Pardo et al, (1983a) and have been defined as transverse circum ferential elements of the cytoskeleton associated to the sarcolemma. S pecific immunostaining for vinculin shows that the costameres overlie I bands. However, an exact correlation between the costameres and the Z line is uncertain, although similar to 10 proteins so far have been localized in the costameres. To define the exact localization of costa meres in human skeletal muscle fibers, we carried out an immunofluores cence study using confocal scanning laser microscopy on the fascia lat a muscle of adult males. Methods: Samples were fixed in 3% paraformald ehyde; frozen sections were treated with antivinculin, antitalin, anti desmin, and anti-alpha-actinin, then immunostained with TRITC, For dou ble localization, the TRITC-streptavidin, as a marker for vinculin and FITC-streptavidin a marker for desmin, were used. Results: The distan ce between two subsequent transverse lines of actin-inf indicated that muscle fibers were well stretched, Processing, with different softwar e functions of the images obtained using CLSM, shows that vinculin and talin are only present in the sarcolemmal lattice. Immunostaining for vinculin and double immunostaining for vinculin and desmin demonstrat e that costameres superimpose underlying I bands without interruption at the Z line, Immunostaining for talin showed that the protein is loc ated in correspondence with the I band and M line. Conclusions: We bel ieve that costameres are ''proteic machinery.'' The findings of the pr esent study suggest that it is possible to determine the width and the period of each proteic component, In addition, we indicate that costa meres are present in correspondence with M line. (C) 1996 Wiley-Liss, Inc.