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Polyacrylamide gel electrophoretic screening of mammalian cells cultured in vitro for the presence of the intermediate filament protein vimentin

Traub, Ulrike E. and Nelson, W. James and Traub, Peter (1983) Polyacrylamide gel electrophoretic screening of mammalian cells cultured in vitro for the presence of the intermediate filament protein vimentin. Journal of Cell Science, 62 . pp. 129-147. ISSN 0021-9533.

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A total of 53 different cell lines originating from a variety of mammalian species were cultured in vitro and analysed for the presence of vimentin, employing polyacrylamide gradient slab gel electrophoresis in urea/acetic acid as buffer system. Irrespective of the cell culture conditions, and the growth potential and morphology of the cells, vimentin was expressed in all cell lines examined, with two exceptions: MPC-11 mouse myeloma and MOPC-31C mouse plasmacytoma cells. Immunoblotting with the monoclonal antibody α-IFA, which is directed against an antigenic determinant shared by all classes of intermediate filaments, did not detect any other of the known intermediate filament proteins in MPC-11 and MOPC-31C cells. Vimentin synthesized by various cell lines was characterized by four different criteria: (1) its extractability with Triton X-100 under various ionic conditions; (2) its behaviour in ((NH_4)_2)SO_4 fractionation of cellular extracts; (3) its electrophoretic mobility in polyacrylamide gel electrophoresis in urea/acetic acid; and (4) the co-isolation of polypeptides of higher electrophoretic mobility, which, by comparison with degradation products of vimentin obtained with the Ca^(2+)-activated proteinase specific for intermediate filament proteins in vitro, were identified as products of Ca^(2+)-dependent proteolysis of vimentin. Although the degradation products occurred in different ratios in extracts of different cell lines, they constituted the same characteristic set of proteins whenever degradation of vimentin was observed. The formation of proteolytic breakdown products could be partially to totally suppressed when the cells were harvested, washed and processed in the presence of EGTA and proteinase inhibitors. The experimental data show that: (1) vimentin, as well as the Ca^(2+)-activated proteinase specific for intermediate filament proteins, is highly conserved during the evolution of mammalian species; (2) the proteolytic breakdown products of vimentin, which give rise to a characteristic ‘staircase’ in two-dimensional gel electrophoresis, are probably artefacts of isolation; (3) the expression of vimentin is neither a prerequisite for nor necessarily indicative of rapid cell proliferation in vitro; and (4) the techniques described can be used for the routine identification of vimentin in cells and tissues in case vimentin-specific antibodies are not available.

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Additional Information:© 1983 The Company of Biologists Limited. Received 24 January 1983-Accepted 18 February 1983. We are grateful to Mrs Margot Bialdiga for her invaluable help in growing all the cells used in this investigation and to Miss Brigitte Geisel, Miss Annemarie Scherbarth and Mrs Gabi Shoeman for skillful technical assistance, especially in the performance of polyacrylamide gel electrophoresis. We also thank Dr G. Darai (University of Heidelberg, FRG), Dr M. Schweiger and Dr M. Hirsch-Kaufmann (University of Innsbruck, Austria), Dr J. R. Sheppard (University of Minnesota, Minneapolis, Minn., U.S.A.), Dr A. Shatkin (The Roche Institute, Nutley, N.J., U.S.A.), Dr D. Gallwitz (University of Marburg, FRG), Dr S. H. Revell (Institute for Cancer Research, London, U.K.) and Dr G. Rutter (Pette Institut, Hamburg, FRG) for the provision of various cell lines and Dr A. Dowding and Dr B. Anderton (St George's Hospital Medical School, London, U.K.) for the generous gift of the monoclonal antibody cr-IFA.
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Official Citation:U.E. Traub, W.J. Nelson, and P. Traub Polyacrylamide gel electrophoretic screening of mammalian cells cultured in vitro for the presence of the intermediate filament protein vimentin J Cell Sci 1983 62:129-147
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:32397
Deposited On:13 Jul 2012 14:34
Last Modified:03 Oct 2019 04:00

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