Antibody staining of 10-nm (100-A) filaments in cultured smooth, cardiac and skeletal muscle cells

Abstract

Antibodies were prepared against the SDS-denatured 10-nm filament protein 'skeletin' extracted from chicken gizzard. The specificity of the antibody to the 10-nm filament protein was shown by immunodiffusion before and after purification of the protein on SDS gels by the enzyme-linked immunoabsorbent assay (ELISA) and by its specific absorption with purified skeletin. In immunofluorescence (where preimmune sera and antigen-absorbed antisera gave negative results), cultured cardiac, skeletal and smooth muscle cells and endothelial cells stained intensely. No staining was observed in fibroblasts present in these cultures, nor was there staining in glial cells or nerve cell bodies and fibres from sympathetic ganglion and Auerbach's plexus cultures. Smooth muscle cells (regardless of their source and phenotypic state) and endothelial cells stained intensely in the perinuclear region and in a fine filamentous network that existed throughout the cytoplasm. In both chick and rat skeletal and cardiac muscle (cultures and frozen sections) filamentous network staining was observed, while in rat muscle the antibody was additionally localized in a regular pattern in the region of the Z-disk, and in the case of cardiac muscle associated with the intercalated disk. The addition of 10(-6) M colchicine to the culture medium of smooth and striated muscle and endothelial cells resulted in an aggregation of the filaments in the nuclear region. Cultured smooth and striated muscle and endothelial cells and freshly isolated smooth muscle cells extracted of actomyosin and tubulin by high and low ionic strength solutions gave a staining pattern similar to non-extracted cells and in the electron microscope, exhibited filaments of predominantly 10 nm diameter.