Occurrence and immunolocalization of plectin in tissues

Abstract

Various tissues from rat were examined for the occurrence and cellular localization of plectin, a 300,000-dalton polypeptide component present in intermediate filament-enriched cytoskeletons prepared from cultured cells by treatment with nonionic detergent and high salt solution. The extraction of liver, heart, skeletal muscle, tongue, and urinary bladder with 1% Triton/0.6 M KCl yielded insoluble cell residues that contained polypeptides of Mr 300,000 in variable amounts. These high Mr polypeptide species and a few bands of slightly lower Mr (most likely proteolytic breakdown products) were shown to react with antibodies to rat glioma C6 cell plectin using immunoautoradiography and/or immunoprecipitation. By indirect immunofluorescence microscopy using frozen sections (4 micron) of stomach, kidney, small intestine, liver, uterus, urinary bladder, and heart, antigens reacting with antibodies to plectin were found in fibroblast, endothelial, smooth, skeletal, and cardiac muscle, nerve, and epithelial cells of various types. Depending on the cell type, staining was observed either throughout the cytoplasm, or primarily at the periphery of cells, or in both locations. In hepatocytes, besides granular staining at the cell periphery, conspicuous staining of junctions sealing bile canaliculi was seen. In cardiac muscle strong staining was seen at intercalated disks and, as in skeletal muscle, at Z-lines. In cross sections through smooth muscle, most strikingly of urinary bladder, antibodies to plectin specifically decorated regularly spaced, spot-like structures at the cell periphery. By immunoelectron microscopy using the peroxidase technique, antiplectin-reactive material was found along cell junctions of hepatocytes and was particularly enriched at desmosomal plaques and structures associated with their cytoplasmic surfaces. A specific immunoreaction with desmosomes was also evident in sections through tongue. In cardiac muscle, besides Z-lines, intercalated disks were reactive along almost their entire surface, suggesting that plectin was associated with the fascia adherens, desmosomes, and probably gap junctions. In smooth muscle cells, regularly spaced lateral densities probably representing myofilament attachment sites were immunoreactive with plectin antibodies. The results show that plectin is of widespread occurrence with regard to tissues and cell types. Furthermore, immunolocalization by light and electron microscopy at junctional sites of various cell types and at attachment sites of cytoplasmic filaments in epithelial and muscle cells suggests that plectin possibly plays a universal role in the formation of cell junctions and the anchorage of cytoplasmic filaments.

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