Intermediate filaments in monkey kidney TC7 cells: focal centers and interrelationship with other cytoskeletal...

Two-dimensional gel electrophoresis of intermediate-sized filament-enriched cytoskeletons of epithelial monkey kidney TC7 cells has shown that they are composed of at least two keratins (isoelectric focusing 36, Mr = 48,500; IEF 46, Mr = 43,500; HeLa protein catalogue number) and vimentin. Indirect immunofluorescence as well as immunoelectron microscopy using antibodies directed against specific polypeptides sometimes revealed a discontinuous staining of keratin-containing filaments. Indirect immunofluorescence analysis of cells stained with keratin or vimentin antibodies also revealed a bright perinuclear staining in 58% of the cells in interphase. Of particular interest were focal centers from which filaments radiated. Double-label immunofluorescence using tubulin and keratin antibodies showed that these centers codistributed with focal arrays of microtubules (most likely centrosomes) in interphase cells but were not colocalized with centrioles in mitosis or, in many cases, with the microtubule organizing centers seen after release from nocodazole treatment. Treatment of TC7 cells with demecolcine (10 micrograms/ml, 20 hr) resulted in a drastic rearrangement of the keratin and vimentin filaments. Likewise, treatment with cytochalasin B (10 micrograms/ml, 1 hr) produced a star-like arrangement of the keratin and vimentin filaments and, in most cases, these codistributed with patches of actin. The results provide evidence for the interaction of intermediate filaments (keratins and vimentin) with both microtubules and microfilaments.