Classes Of Intermediate Filaments Research Articles

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

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 alpha-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 (NH4)2SO4 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 Ca2+-activated proteinase specific for intermediate filament proteins in vitro, were identified as products of Ca2+-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 Ca2+-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.

Coexistence of neurofilaments and vimentin in a neurone of adult mouse retina.

Different classes of intermediate filaments are restricted to particular cell types. For example, neurofilaments are found only in neurones, whereas filaments that contain the protein vimentin, which were found in some cells of mesenchymal origin and some forms of glia, are thought to be absent from mature neurones, and present only transiently in early embryonic neurones. However, evidence is presented here of an exception to that rule in the outer plexiform layer of the mouse retina. Double-labelling with antibodies to neurofilaments and vimentin showed that both types of intermediate filaments coexisted in the axonless horizontal cell of that retinal layer, recalling the previous notion that these cells are glial or intermediate between neuronal and glial (reviewed in ref. 10).

Human monoclonal IgM with autoantibody activity against intermediate filaments.

Monoclonal IgMs from two patients with Waldenström macroglobulinemia were found to react with intermediate filaments. This was shown by (a) immunostaining of various tissues and cultured cells and (b) immunological characterization of the reactive antigen after blotting of polypeptides separated from total cell extracts by gel electrophoresis or purified intermediate filaments on nitrocellulose sheets. One monoclonal IgM had an activity directed only against vimentin, whereas the other reacted with four different classes of intermediate filaments--vimentin, desmin, glial fibrillary protein, and keratins. All of the reactivity of the latter IgM was absorbed by purified vimentin, suggesting that different classes of proteins of intermediate filaments share common antigenic determinant(s). The significance of such autoantibody activity of human monoclonal IgM is discussed in the light of the startling frequency of IgM anti-intermediate filaments antibodies in various diseases.

Characterization of cytoskeletal components in epithelial and mesenchymal liver tumors by electron and immunofluorescence microscopy.

Intermediate-sized filaments and microfilaments were studied by electron and immunofluorescence microscopy using antibodies to their constitutive proteins in various rat liver tumors induced by nitrosamines. Hepatocellular carcinomas contained both tonofilaments and microfilaments which could be demonstrated light-microscopically by antibodies to prekeratin and actin, respectively, and were predominantly localized, in a somewhat irregular pattern, in peripheral cytoplasmic regions. The hepatocellular tumors did not show significant amounts of intermediate-sized filaments of the vimentintype. Cholangiofibromas exhibited, within the epithelial duct component, actin-positive microfilaments organized in a terminal web and prekeratinpositive tonofilaments, the latter often being present in large amounts. By contrast, the mesenchymal component of the cholangiofibromas and the cells of angiosarcomas or undifferentiated sarcomas were characterized by intermediate filaments stainable with antibodies against vimentin, but not with those to prekeratin. In addition to vimentin, the mesenchymal tumors contained small amounts of actin. In many cells of the sarcomas a pronounced accumulation of intermediate-sized filaments of the vimentin type, frequently forming whorl-like aggregates, was evident. The characteristic “vimentin storage cells”, many of which were readily identifiable in H&E sections by large fibrillar cytoplasmic inclusions, were pathognomonic for sarcomas, especially for angiosarcomas. The detection of early stages of angiosarcomas by antibodies to vimentin was also facilitated. Our results do not support the concept that presence of an excess of contractile proteins such as actin is an important prerequisite for the metastasic spread of malignant cells. The observations show that antibodies to the different classes of intermediate filaments are a powerful tool for the classification and differential diagnosis of epithelial and mesenchymal tumors of the liver and probably also of other sites. The accumulation of prekeratin in many ductular cells of the cholangiofibromas and the considerable storage of vimentin in sarcoma cells seem to indicate a severe disturbance of the cell metabolism which might be related to the neoplastic transformation.

Purified glial fibrillary acidic protein and desmin are distinct intermediate filament proteins exhibiting similar properties.

Glial fibrillary acidic (GFA) protein and desmin were purified from bovine brain and large intestine, respectively, and used in a comparison of the major protein components of two classes of intermediate filaments, the glial and smooth muscle filaments. The proteins are similar in size, charge, and amino acid composition, but clearly distinct. By sodium dodecyl sulfate-gel electrophoresis, GFA protein is about 5,000 daltons smaller than desmin. GFA protein is composed of three isoelectric variants which are all slightly more basic than the two variants observed for desmin. One-dimensional peptide mapping following limited proteolysis under denaturing conditions or following cyanogen bromide cleavage demonstrates that the proteins are not closely related in primary structure. Assembly-disassembly experiments reveal that the proteins share solubility properties and that negatively stained preparations of in vitro polymerized filaments are very similar. Limited proteolysis under native conditions demonstrates substructural similarities; comparative peptide mapping following digestion with chymotrypsin and trypsin suggests related core polypeptides of about 37,000 and 21,000 daltons. We conclude that GFA protein and desmin are distinct with respect to primary structure, but probably represent two of the more closely related classes of intermediate filament proteins.

Antibodies to neurofilament, glial filament, and fibroblast intermediate filament proteins bind to different cell types of the nervous system.

Antisera were raised to the 210,000-dalton and the 49,000-dalton proteins of a fraction enriched in intermediate (10 nm) filaments from human brain. Proteins of the filament preparation were separated by SDS-polyacrylamide gel electrophoresis and used for immunization and subsequent analysis of the reactions of the sera by rocket immunoelectrophoresis. Anti-210,000-dalton serum precipitated proteins of molecular weights 210,000, 160,000, and 68,000, and, thus, reacted with all the neurofilament triplet components. Anti-49,000-dalton serum did not react with the triplet proteins but precipitated the 49,000-dalton protein. By immunofluorescence on tissue sections, anti-210,000-dalton serum bound to neuronal axons in sciatic nerve and cerebellum. In dissociated cell cultures, rat dorsal root ganglion cells and their processes bound the serum, whereas nonneuronal cells did not. Some cultured cerebellar neurons were also positive, whereas astrocytes were not. At the ultrastructural level, anti-210,000-dalton serum bound to intermediate filaments inside axonal processes. Anti-49,000-dalton serum bound to astrocytes in sections of the cerebellum, and cultured astrocytes had filaments that stained, whereas other cell types did not. In sciatic nerve sections, elements stained with this serum, but cultured cells from newborn sciatic nerve were negative. An antiserum against the 58,000-dalton protein of the cytoskeleton of NIL-8 fibroblasts strongly stained sciatic nerve sections, binding to Schwann cells but not to axons or to myelin. In cerebellar sections, astrocytes were positive, as were blood vessels and cells in the pia. In cell cultures, anti-58,000-dalton serum stained filaments inside Schwann cells, fibroblasts, and astrocytes, but neurons were negative. Cells in the cultures and tissue sections of the nervous system failed to react with antiserum to the 58,000-dalton protein of skin intermediate filaments. In these studies, astrocytes in vivo and in culture were the only cells which had antigens related to two classes of intermediate filaments.

Antibodies as probes of cellular differentiation and cytoskeletal organization in the mouse blastocyst

Indirect immunofluorescence microscopy has been used to detect cytoskeletal proteins, which allow a distinction between the two cell types present in the mouse blastocyst: i.e. the cells of the inner cell mass (ICM) and the outer trophoblastic cells. Antibodies against three classes of intermediate-sized filaments (cytokeratins, desmin and vimentin), as well as antibodies against actin and tubulin were studied. Antibodies against prekeratin stain the outer trophoblastic cells but not the ICM in agreement with the findings on adult tissues that cytokeratins are a marker for various epithelial cells. Interestingly, vimentin filaments typical of mesenchymal cells as well as of cells growing in culture seem to be absent in both cell types of the blastocyst. Thus, the cytokeratins of the trophoblastic cells seem to be the first intermediate-sized filaments expressed in embryogenesis. Antibodies to tubulin and actin show that microtubules and microfilaments are ubiquitous structures, although microfilaments have a noticeably different organization in the two cell types. In addition, since early embryogenic multipotential cells show close similarities to teratocarcinomic cells, a comparison is made between the cells of the blastocyst, embryonal carcinoma cells (EC cells) and an epithelial endodermal cell line (PYS2 cells) derived from EC cells. EC cells display vimentin filaments whereas PYS2 cells show both vimentin and cytokeratin filaments. The results emphasize the usefulness of antibodies specific for different classes of intermediate filaments in further embryological studies, and suggest that cells of the blastocyst and EC cells differ with respect to vimentin filaments.