Expression of intermediate filaments in cultured cells.

Abstract

ABSTRACT The occurrence of different types of intermediate filaments in primary cultures of cells and in cultured cell lines was studied by the indirect immunofluorescence (IFL) technique. The antibodies used were spontaneous monoclonal human antibodies of immunoglobulin M (IgM) class against vimentin-type intermediate filaments (fibroblast 58 × 103 mol. wt subunit protein), and experimental rabbit antibodies of IgG class against vimentin, desmin (muscle 55 × 103 mol. wt subunit protein), glial fibrillary acidic protein (GFA), 68 × 103 mol. wt neurofilament polypeptide and human keratin polypeptides. Cultured fibroblasts from different species, and both aortic and venous endothelial cells, showed a fibrillar cytoplasmic fluorescence when stained with antibodies against vimentin. On the other hand, in cultures of chicken embryonal fibroblasts, cells showing a bright desminspecific fluorescence, but lacking vimentin-βpecific staining, were seen even after several subcultivations. The presence of both desmin and vimentin polypeptides in these cultures was also confirmed by polyacrylamide gel electrophoresis. In chicken fibroblast cultures the 2 types of intermediate filaments were not expressed simultaneously in individual cells, whereas in baby hamster kidney (BHK-21) cells and human rhabdomyosarcoma cells a variable co-staining with anti-vimentin and anti-desmin antibodies could be seen. In contrast, cultured human fibrosarcoma cells and simian virus 40-transformed human fibroblasts showed only vimentin-specific fibrillar fluorescence. Glial cells from mouse embryonic spinal cord appeared to express only GFA-containing intermediate filaments in a primary culture, whereas both subcultured mouse glial cells and a cultured glioma cell-line also showed vimentin-specific staining. On the other hand, neuronelike cells in the primary cultures could only be stained with the antibodies to 68 × 103 mol. wt neurofilament polypeptide. Interestingly, clones of mouse neuroblastoma (C1 300) cells contained only vimentin-type intermediate filaments, whereas rat pheochromocytoma (PC 12) cells contained both vimentin- and neurofilament-specific fluorescence. Two types of intermediate filaments were also seen in cultured epithelial cells. In primary cultures of human amnion epithelial cells a fibrillar keratin-specific fluorescence was seen in all cells but only a few of the cells also showed vimentin-specific fluorescence as distinct juxta-nuclear aggregates. On the other hand, subcultured amnion epithelial cells and various epithelial cell lines contained both keratin and vimentin fibrils. Our results show that cultured fibroblasts contain only vimentin-type intermediate filaments and that differentiated cells in primary culture contain primarily tissue-specific intermediate filaments. On the other hand, all proliferating cultured cells appeared to contain vimentin-type filaments in addition to tissue-specific intermediate filaments. This suggests that vimentin expression is connected with the adaptation of cells to culture conditions.