Immunolocalization of the costameres in human skeletal muscle fibers: Confocal scanning laser microscope investigations

Background The costameres in skeletal muscle fibers were first described by Pardo et al. (1983a) and have been defined as transverse circumferential elements of the cytoskeleton associated to the sarcolemma. Specific immunostaining for vinculin shows that the costameres overlie I bands. However, an exact correlation between the costameres and the Z line is uncertain, although ∼10 proteins so far have been localized in the costameres. To define the exact localization of costameres in human skeletal muscle fibers, we carried out an immunofluorescence study using confocal scanning laser microscopy on the fascia lata muscle of adult males. Methods Samples were fixed in 3% paraformaldehyde; frozen sections were treated with antivinculin, antitalin, antidesmin, and anti‐α‐actinin, then immunostained with TRITC. For double localization, the TRITC‐streptavidin, as a marker for vinculin and FITC‐streptavidin a marker for desmin, were used. Results The distance between two subsequent transverse lines of actin‐inf indicated that muscle fibers were well stretched. Processing, with different software 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 demonstrate that costameres superimpose underlying I bands without interruption at the Z line. Immunostaining for talin showed that the protein is located in correspondence with the I band and M line. Conclusions We believe that costameres are “proteic machinery.” The findings of the present study suggest that it is possible to determine the width and the period of each proteic component. In addition, we indicate that costameres are present in correspondence with M line. © 1996 Wiley‐Liss, Inc.