Cytoskeletal properties and endogenous degradation of glial fibrillary acidic protein and vimentin in cultured human glioma cells.
The cytoskeletal properties and endogenous degradation of intermediate filaments in cultured human glioma cells (U-251MG) were studied using monoclonal antibodies in immunohistochemical and immunochemical methods. Both glial fibrillary acidic protein (GFAP)- and vimentin-antibodies gave a fibrillar cytoplasmic staining of the cells, and double immunofluorescence experiments showed the presence of both types of intermediate filaments in the same cells. GFAP and vimentin could also be located to typical coiling perinuclear bundles after vinblastine treatment of the cultures. In the detergent-resistant, adherent cytoskeletons of the glioma cells, both GFAP and vimentin persisted as fibrillar cytoplasmic arrays. Scanning and transmission electron microscopy showed that only intermediate filaments were left in the cytoplasmic domain. Electrophoretic analysis, combined with the immunoblotting method, revealed that the two major detergent-resistant cytoskeletal polypeptides of the cells, with molecular weights of 51 kD and 58 kD, were GFAP and vimentin, respectively. On the other hand, neither GFAP nor vimentin were detected in the detergent extracts of the glioma cells. Detergent-extraction in low ionic strength medium as well as inclusion of Ca2+ into the extraction medium resulted into a rapid degradation of both GFAP and vimentin. These degradation conditions produced different, partially soluble, lower MW immunoreactive polypeptides as detected by the immunoblotting technique. Interestingly, the degradation also produced soluble intact GFAP and vimentin. These results indicate that GFAP and vimentin have closely similar physicochemical properties in the cytoskeletons of human glioma cells including a nearly quantitative localization in filaments, rearrangement upon microtubule disruption, and resistance to extractions by detergents. Proteolytic degradation of both proteins can be induced by a protease activated by both low ionic strength and Ca2+.
D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism*; D005910:Glioma / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D014746:Vimentin / Q000378:metabolism*
D005904:Glial Fibrillary Acidic Protein; D014746:Vimentin