Immunolocalization of a novel, cytoskeleton-associated polypeptide of Mr 230,000 daltons (p230).
Antibodies were raised against a cytoskeleton-associated, nonphosphorylated, 230,000-dalton bovine lens polypeptide (designated p230), and rendered monospecific by using a novel immunoaffinity technique. In immunofluorescence and electron microscopy of cultured fibroblasts, as well as of various other cells (endothelial, epithelial, lenticular, monocytes, neuroblastoma cells) and tissues (human kidney and liver), p230 was localized as a distinct subplasmalemmal layer in the peripheral cytoplasm of the cells. It constituted less than 0.3% of the total cellular protein in cultured fibroblasts and was not extractable with Triton X-100. In detergent-extracted cytoskeletal preparations of cultured fibroblasts, p230 remained as an elaborate peripheral network that showed a distribution distinctly different from that of the major cytoskeletal structures, stress fibers, cortical myosin, vinculin, and intermediate filaments (IF). The distribution was not dependent on the presence of intact stress fibers or microtubules, as shown by double-fluorescence microscopy of cells exposed to cytochalasin B or cultured in the presence of monensin and of cold-treated cells. Upon demecolcine-induced reorganization of intermediate filaments, however, the localization of p230 was rapidly altered to a dense plaque underneath the perinuclear aggregate of intermediate filaments. On the other hand, p230 seemed to colocalize with the detergent-resistant cell surface lamina, visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin-lectin. The results suggest that p230 is part of a cell surface- and cytoskeleton-associated subplasmalemmal structure that may play an important role in cell surface-cytoskeleton interaction in various cells both in vitro and in vivo.
D000818:Animals; D000918:Antibody Specificity; D002462:Cell Membrane / Q000032:analysis; D002478:Cells, Cultured; D003593:Cytoplasm / Q000032:analysis*; D004727:Endothelium / Q000032:analysis; D005347:Fibroblasts / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D007106:Immune Sera; D007668:Kidney / Q000032:analysis; D007908:Lens, Crystalline / Q000032:analysis; D008099:Liver / Q000032:analysis; D008264:Macrophages / Q000032:analysis; D008854:Microscopy, Electron; D008970:Molecular Weight; D009474:Neurons / Q000032:analysis; D017830:Octoxynol; D011092:Polyethylene Glycols; D011506:Proteins / Q000032:analysis* / Q000276:immunology
D007106:Immune Sera; D011506:Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol