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PubMed1993

Separation and characterization of homo and hetero-oligomers of the intermediate filament proteins desmin and vimentin.

Affinity chromatography on single-stranded (ss)DNA-cellulose in conjunction with gel permeation chromatography in the presence of urea was employed to separate the intermediate filament (IF) protein complement of catalytically oxidized BHK-21 cell Triton cytoskeletons into disulfide-cross-linked homo- and heterodimers of desmin and vimentin and uncross-linked homodimers. The same separation was performed on a desmin-vimentin mixture under autoxidizing conditions in 6 M-urea to obtain the respective cross-linked collision complexes of both proteins. In 5 M-urea, the oxidation products were identified as dimers that were physically indistinguishable from uncross-linked homodimers, suggesting that they were in the form of partially denatured face-to face pairs. Heterodimers derived from intact IFs were identical to those derived from collision complexes. In the presence of 2-mercaptoethanol, heterodimers were unstable and transformed spontaneously into homodimers. After removal of urea, all cross-linked dimers were totally unable to polymerize into filaments; however, in the presence of 2-mercaptoethanol they showed a normal assembly competence. This inability of oxidized homo- and heterodimers to polymerize, together with the relatively low yield of cross-linked dimers obtained from cytoskeletons, is probably due to the introduction of steric strain into the dimers by disulfide bond formation. Substantial amounts of cross-linked heterodimers could also be isolated from IFs reconstituted from mixtures of desmin and vimentin in their homodimeric or tetrameric forms. Taken together, these results suggest that the cross-linked dimers isolated from cytoskeletons arise from a reaction between subfilament strands of IFs rather than from disulfide bond formation within pre-existing dimers and that the heterotypic IFs of BHK-21 cells are largely formed from homodimers and tetramers, respectively, rather than from heterodimers. The differential capacity of desmin and vimentin to interact with ssDNA has also been exploited to distinguish between homotypic and heterotypic protofilaments, the latter consisting of one homodimer of each protein species. This distinction could be made on the basis of characteristic differences in the sedimentation behavior of the respective protein-DNA complexes.

Animals↗
PubMed1988

Paragangliomas of the head and neck: immunohistochemical neuroendocrine and intermediate filament typing.

Twenty-nine paragangliomas of the head and neck region including 20 glomus jugulare (GJ) and nine carotid body (CB) tumors were evaluated for the presence of neuroendocrine and intermediate filament antigens. Immunohistochemistry on formalin-fixed, paraffin-embedded tissue was used to identify: S-100 protein (S-100); neuron-specific enolase (NSE); chromogranin A (CHA); serotonin (SER); synaptophysin (SYN); cytokeratin (CK); neurofilament (NF); desmin (DES); vimentin (VIM); and glial fibrillary acidic protein (GFAP). S-100 protein staining of sustentacular cell nuclei and cytoplasm was found in all tumors and was present in chief cells in 4 of 20 GJ and 3 of 9 CB tumors. All tumors stained with at least three neuroendocrine markers (29 of 29 NSE, 28 of 29 SYN, 26 of 29 CHA, 25 of 29 SER). CK was detected in 2 GJ and 1 CB tumor using anticytokeratins AE 1/3 and CAM 5.2. Neurofilament protein could not be demonstrated in fixed material, and all tumors were negative for GFAP and desmin. Vimentin was inconsistently detected in chief and sustentacular cells. We conclude that, in formalin-fixed material, paragangliomas have S-100 protein staining of sustentacular cells with chief cells containing antigens associated with neuroendocrine differentiation. The presence of CK in some paragangliomas is consistent with recent tissue culture studies demonstrating immunoblot confirmation of CK in pheochromocytomas and represents a potential source of immunohistologic misinterpretation in diagnosis, unless a panel of markers is utilized.

Antibodies↗
PubMed1984

Typing of intermediate filaments in malignant fibrous pleural tumors.

We report two cases of a malignant fibrous pleural tumor, one of the localized type and one of the diffuse type. In both cases, typing of intermediate filaments by immunofluorescence microscopy showed that the tumor cells were positive for vimentin and negative for (cyto)keratin and desmin. This result supports the concept that malignant fibrous pleural tumors do not arise from the (cyto)keratin-positive pleural mesothelium but from the submesothelial fibrous tissue. Thus, instead of the usual term "malignant fibrous mesothelioma", the term "malignant submesothelial fibrosarcoma" should be preferred.

Adolescent↗
PubMed1986

Reorganization of keratin intermediate filaments by the drug-induced disruption of microfilaments in cultured human keratinocytes.

It has been shown to date that a combined treatment with microtubule and microfilament inhibitors alters the cytoskeletal organization of keratin intermediate filaments in cultured HeLa, fetal mouse epidermal, and epithelial PtK2 cells, although neither of these inhibitors alone is able to do so. In the present study, we found that disruption of microfilaments with cytochalasin B induced a remarkable reorganization of keratin filaments in cultured human keratinocytes, while disruption of microtubules with colchicine did not affect keratin filaments. Keratin filament organization in the presence of cytochalasin B demonstrated a network of connecting star-like knots or foci. These foci coincided with actin aggregates that were formed by depolymerization of actin filaments as studied by double immunofluorescence using antiactin and antikeratin antibodies. Under these conditions, no change in microtubule arrangement was observed. Our observations suggest that the stability and architecture of keratin filament organization may be supported with the microfilament rather than the microtubule cytoskeleton in cultured human keratinocytes.

Actin Cytoskeleton↗
PubMed1995

Distribution of the intermediate filament proteins vimentin, keratin, and desmin in the bovine ovary.

The distribution of the intermediate filament (IF) proteins desmin, keratin, and vimentin was studied immunohistochemically in bovine ovaries. Special attention was paid to granulosa cells to examine possible marked changes of IF distribution in relation to folliculogenesis during ovarian development. Therefore, ovaries were used from fetuses from 3 months of gestation onward, calves, heifers, and cows. In all ovaries, desmin immunoreactivity was restricted to smooth muscle cells in blood vessel walls. Keratin appeared a characteristic of the ovarian surface epithelium. Co-localization of keratin and vimentin was observed in the epithelium of rete ovarii tubules in fetuses and calves, and in cortical cord epithelium and pregranulosa cells of primordial follicles in fetuses at 3-7 months of gestation. Vimentin was demonstrated in endothelium and in fibroblasts. In addition, vimentin immunoreactivity was present in granulosa cells of primary, secondary, and antral follicles. In antral follicles, these granulosa cells mainly had an elongated appearance and either contained an oblong or a round nucleus. Those with an oblong nucleus were characteristic for atretic antral follicles. In nonatretic follicles, numerous vimentin immunoreactive, elongated granulosa cells with a round nucleus were observed, especially in the peripheral granulosa layer and in small ( < 3 mm in diameter) antral follicles. Additionally, in antral follicles, protrusions of vimentin-positive corona radiata cells were observed, that penetrated the zona pellucida to contact the oocyte. The data show that the distribution of vimentin containing IFs is associated with various aspects of granulosa cell activity, as mitosis, atresia, and intercellular transport.

Animals↗
PubMed1982

In vitro induction of anti-intermediate filament antibody in lymphocyte cultures by Epstein-Barr virus.

Serum antibodies reactive with intermediate filaments of the cytoskeleton (anti-IF antibodies) are often present in infectious mononucleosis, some other viral diseases, and rheumatoid arthritis. The mechanism of their production is not known, but it is possible that the formation of this and other autoantibodies result from polyclonal activation of B-cells. Peripheral blood mononuclear cells from subjects with or without serum anti-IF antibody were therefore cultured in the presence or absence of Epstein-Barr virus (EBV). IgM anti-IF antibody was produced in both unfractionated and T-cell-depleted cultures, but not in the supernatants of the same cells cultured without added EBV.

Antibodies↗
PubMedDate not supplied

The cytoskeletal intermediate filaments of canine retinal pigment epithelial cells in vivo and in vitro.

The cytoskeletal intermediate filament characteristics of normal, freshly isolated and subcultured canine retinal pigment epithelial (RPE) cells were studied using immunocytochemistry and immunoblotting techniques. Commercially available primary antibodies recognising a broad range of cytokeratins and vimentin were selected. Cytokeratin reactivity was a constant feature of all canine RPE cells. The main cytokeratins expressed by cultured RPE cells included 8, 18 and 19. This finding is consistent with the published findings of work carried out in other mammalian species including man. Freshly isolated RPE cells stained positively with broad-spectrum antibodies to cytokeratins but generally did not stain with antibodies specific to cytokeratins 18 or 19 and did not stain with antibodies to vimentin, or stained only very weakly. After a short time in culture however, cells demonstrated intense positive staining for vimentin. This study demonstrated that cytokeratin immunoreactivity (in conjunction with vimentin immunoreactivity in vitro) is a useful and consistent marker for canine RPE cells.

Animals↗
PubMed1995

The 70-kDa heat shock proteins associate with glandular intermediate filaments in an ATP-dependent manner.

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament proteins expressed preferentially in glandular epithelia. We describe the identification, by co-immunoprecipitation from normal human colonic tissues and cultured cell lines, of the 70-kDa heat shock protein (hsp) and its related heat shock cognate protein as K8/18-associated proteins (hsp/c). The association is significant but sub-stoichiometric and occurs preferentially with the soluble rather than the cytoskeletal K8/18 fractions. Heat stress increases the level of soluble K8/18 in association with an increase in hsp70 levels and an increase in the stoichiometry of K8/18-hsp70 association. Identity of the associated proteins was confirmed by microsequencing of a tryptic digest of the purified associated protein and by using anti-hsp/c70-specific antibodies. The K8/18-hsp/c70 complex can be dissociated in a Mg-ATP-dependent manner that requires ATP hydrolysis. Binding of hsp to K8/18 can be reconstituted using purified bovine hsp70 and human K8/18 immunoprecipitates that have been depleted of bound hsp/c70 and increases slightly in the presence of ATP. The reconstituted K8/18-hsp70 complex can be again released in the presence of Mg-ATP. In addition, hsp70 binds to K8/18 without having a significant effect on in vitro filament assembly when added during or after assembly. Using an overlay assay, hsp70 binds exclusively to K8 in the presence of ATP. Our results show direct association of the hsp/c70 proteins with K8/18. This interaction may serve, at least in part, to regulate the function of these two abundant protein groups.

Adenosine Triphosphate↗
PubMed2002

A novel type of regulation of the vimentin intermediate filament cytoskeleton by a Golgi protein.

Whether the highly dynamic structure of the vimentin intermediate filament (IF) cytoskeleton responds to cues from cellular organelles, and what proteins might participate in such events is largely unknown. We have shown previously that the Golgi protein formiminotransferase cyclodeaminase (FTCD) binds to vimentin filaments in vivo and in vitro, and that overexpression of FTCD causes dramatic rearrangements of the vimentin IF cytoskeleton (Gao and Sztul, J. Cell Biol. 152, 877-894, 2001). Using real-time imaging, we now show that FTCD causes bundling of individual thinner vimentin filaments into fibers and that the bundling always originates at the Golgi. FTCD appears to be the molecular "glue" since FTCD cross-links vimentin filaments in vitro. To initiate the analysis of structural determinants required for FTCD function in vimentin dynamics, we used structure-based design to generate individual formiminotransferase (FT) and cyclodeaminase (CD) domains, and to produce an enzymatically inactive FTCD. We show that the intact octameric structure is required for FTCD binding to vimentin filaments and for promoting filament assembly, but that eliminating enzymatic activity does not affect FTCD effects on the vimentin cytoskeleton. Our findings indicate that the Golgi protein FTCD is a potent modulator of the vimentin IF cytoskeleton, and suggest that the Golgi might act as a reservoir for proteins that regulate cytoskeletal dynamics.

Ammonia-Lyases↗
PubMed1989

Intermediate filament expression and the progression of prostatic cancer as studied in the Dunning R-3327 rat prostatic carcinoma system.

To evaluate if there is any consistent relationship between the expression of intermediate filament proteins (IFP), particularly keratins, and the degree of malignancy of prostatic cancer cells, a series of nine Dunning rat prostatic cancer sublines that span the entire spectrum of progression of prostatic cancer were studied immunocytochemically by the use of a variety of antibodies specific for keratins, vimentin, or desmin. For the keratin studies, monoclonal antibodies with either a general reactivity to several keratins or highly specific for either luminal or basal epithelial cells of the normal rat prostate were used. By use of an antibody specific for luminal cell keratin 18, the luminal tumor cells of the well-differentiated, slow-growing H and HI-S sublines were positively stained. In most of the sublines with a more advanced state of progression (i.e., the moderately differentiated, moderately fast growing HI-M; the poorly differentiated, faster growing HI-F; and the anaplastic, very fast growing AT-1, AT-2, and MAT-Lu tumors), however, no expression of keratin specific for luminal cells was detected. In addition, several of the most advanced sublines (i.e., AT-1, AT-2, and MAT-Lu) were negative using any of the keratin antibodies. In contrast, several of the other sublines with the most advanced degree of progression (i.e., the anaplastic, very fast growing MAT-LyLu tumor derived from the AT-1 subline; and the anaplastic, very fast growing AT-3 tumor, derived from the HI-F subline), however, were positively stained with the keratin antibody specific for the luminal cells. By use of the keratin antibody specific for the basal cells of the normal rat prostate, the basal tumor cells of the well-differentiated slow-growing H and HI-S tumor were positively stained. This positive staining for basal cell keratin was also found in the HI-M and HI-F tumors, while the AT-1, AT-2, MAT-Lu, MAT-LyLu, and AT-3 were negative with this antibody. Thus, a loss in staining for basal cell keratin was consistently associated with the most advanced state of tumor progression. Vimentin-positive staining was demonstrated either alone or with keratin-positive staining in part of the epithelial cancer cells of all the sublines. An increase in the positive staining for vimentin was consistently associated with a more advanced state of tumor progression. Desmin-positive staining was found only in smooth cells present within the various tumor sublines.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗
PubMed1983

Immunocytochemical studies of Alzheimer neuronal perikarya with intermediate filament antisera.

Isolated neuronal perikarya from the brains of patients with Alzheimer's disease were examined in indirect immunofluorescence microscopy with different types of specific antisera against the subunit proteins of cytoskeletal intermediate filaments. From 30 to 50% of the neurofibrillary tangles were stained with antisera against each of the neurofilament triplet proteins, but not with antisera against glial fibrillary acidic protein, vimentin, desmin or cytokeratin. Polyacrylamide gel electrophoresis of Alzheimer perikaryal fractions showed polypeptide patterns markedly similar to control neuronal fractions. Our results thus suggest either that the Alzheimer neurofibrillary tangles and the antigenic neurofilament triplet protein fractions may contain related antigenic determinants or that unaffected perikaryal neurofilament material is associated with the tangles.

Alzheimer Disease↗
PubMed1991

Subclones of C6 rat glioma cells differing in intermediate filament protein expression.

The C6 rat glioma cell line is shown to consist of a mixed population of cells which either contain vimentin (80% of the cells) or completely lack any cytoplasmic intermediate filament (IF) proteins. Subclones could be established with both phenotypes, indicating that these IF protein expression patterns represent stable phenotypic markers. Absence of IF proteins in C6 subclones could consistently be correlated with an altered cell morphology and a pronounced increase in the number of actin stress fibers. In vitro translation and hybridization assays suggest the absence of vimentin to result from a block at the transcriptional level. The data indicate that subcloning of the C6 cell line on the basis of IF protein expression seems to be a reasonable approach for obtaining homogeneous C6 cell populations which may represent suitable experimental models for studies on vimentin expression and glioma cell differentiation.

Animals↗
PubMed2006

Interactions between epiplakin and intermediate filaments.

Epiplakin, a cytoskeletal linker protein, was originally identified as an autoantigen in a serum specimen obtained from a patient with subepidermal blistering disease. To examine the binding ability of epiplakin with intermediate filaments (IF), we performed slot-blot assays using fusion proteins that included various domains and subdomains of epiplakin. At least two of the 4.6 copies in the B domains of epiplakin were necessary for the binding of fusion proteins to keratin. The repeated structures of linker domains also played an important role in the binding of epiplakin to keratin in these assays while also increasing the repeated structure in the linker domain of epiplakin which is involved in the increased binding to IF. A similar but weaker binding to vimentin and desmin was also detected. These observations indicated that the highly repeated structures of epiplakin in both the B and the linker domains, which is the unique feature of this molecule in the plakin family, play an essential role in the functioning of this molecule.

Amino Acid Sequence↗
PubMed1987

Immunocytochemical analysis of Ewing's tumors. Patterns of expression of intermediate filaments and desmosomal proteins indicate cell type heterogeneity and pluripotential differentiation.

Examples of classical Ewing's tumors ("Ewing's sarcomas") of both skeletal and extraskeletal locations were analyzed for the expression of intermediate filament (IF) and cell junction proteins, with the use of immunofluorescence and immunoelectron microscopy as well as gel electrophoresis. In all 11 tumors examined vimentin filaments were abundant. A type of plaque-bearing small cell junction, which is common in these tumors but difficult to classify by morphologic criteria, was identified by antibodies to desmoplakins as true desmosomes. These were found in all cases, although in a very variable proportion of cells. Some of these junctions were associated with vimentin IFs. In addition, 9 of the cases examined showed scattered or clustered cells expressing the simple-epithelium type cytokeratins 8 and 18. Moreover, 3 cases displayed dispersed or clustered cells producing neurofilaments. The value of these observations, notably the cell type heterogeneity, for the diagnosis of tumors of this group is discussed. The results further indicate that Ewing's tumors are derived from a primitive, pluripotential cell that may differentiate, in variable proportions, into cells with mesenchymal, epithelial, and, more rarely, even neural features, suggesting that this tumor should be regarded as a blastoma, rather than as a true sarcoma.

Adolescent↗
PubMed1988

Immunocytochemical investigation of intermediate filament proteins and epithelial membrane antigen in spindle cell tumours of the breast.

Seven consecutive cases of primary spindle celled tumours of the breast have been studied immunohistologically using antisera to the intermediate filament proteins (IFP) vimentin, cytokeratin, and desmin, and with an antibody to epithelial membrane antigen. Representative paraffin sections were examined using a peroxidase-antiperoxidase method. In three cases, very occasional foci of epithelial differentiation were apparent by conventional microscopy, and in one case, adjacent ductal carcinoma in situ was present. The remaining three cases were composed of spindle cell elements entirely, with no evidence of epithelial differentiation morphologically. Immunoreactivity of spindle cell elements for vimentin was found in all seven cases, and for cytokeratin in six cases. One case showed immunoreactivity for vimentin, cytokeratin, and desmin, and one case only for vimentin. Epithelial membrane antigen was not identified in the spindle cell elements of any tumour, but was present in the invasive epithelial component of three cases and the in situ component of one case. We conclude that many spindle cell tumours of breast show immunohistological evidence of epithelial differentiation and can be regarded as spindle cell carcinomas. However, in some cases IFP expression may be complex and histogenesis cannot be determined. This technique can aid histological diagnosis in some cases.

Adult↗
PubMed1989

Expression of intermediate filament proteins during development of Xenopus laevis. I. cDNA clones encoding different forms of vimentin.

To provide a basis for studies of the expression of genes encoding the diverse kinds of intermediate-filament (IF) proteins during embryogenesis of Xenopus laevis we have isolated and characterized IF protein cDNA clones. Here we report the identification of two types of Xenopus vimentin, Vim1 and Vim4, with their complete amino acid sequences as deduced from the cloned cDNAs, both of which are expressed during early embryogenesis. In addition, we have obtained two further vimentin cDNAs (Vim2 and 3) which are sequence variants of closely related Vim1. The high evolutionary conservation of the amino acid sequences (Vim1: 458 residues; Mr approximately 52,800; Vim4: 463 residues; Mr approximately 53,500) to avian and mammalian vimentin and, to a lesser degree, to desmin from the same and higher vertebrate species, is emphasized, including conserved oligopeptide motifs in their head domains. Using these cDNAs in RNA blot and ribonuclease protection assays of various embryonic stages, we observed a dramatic increase of vimentin RNA at stage 14, in agreement with immunocytochemical results obtained with antibody VIM-3B4. The significance of very weak mRNA signals detected in earlier stages is discussed in relation to negative immunocytochemical results obtained in these stages. The first appearance of vimentin has been localized to a distinct mesenchymal cell layer underlying the neural plate or tube, respectively. The results are discussed in relation to programs of de novo synthesis of other cytoskeletal proteins in amphibian and mammalian development.

Amino Acid Sequence↗
PubMed1991

Glycine loops in proteins: their occurrence in certain intermediate filament chains, loricrins and single-stranded RNA binding proteins.

Quasi-repetitive, glycine-rich peptide sequences are widespread in at least three distinct families of proteins: the keratins and other intermediate filament proteins, including nuclear lamins; loricrins, which are major envelope components of terminally differentiated epithelial cells; and single-stranded RNA binding proteins. We propose that such sequences comprise a new structural motif termed the 'glycine loop'. The defining characteristics of glycine loop sequences are: (1) they have the form x(y)n, where x is usually an aromatic or occasionally a long-chain aliphatic residue; y is usually glycine but may include polar residues such as serine, asparagine, arginine, cysteine, and rarely other residues; and the value of n is highly variable, ranging from 1 to 35 in examples identified to date. (2) Glycine-loop-containing domains are thought to form when at least two and to date, as many as 18, such quasi-repeats are configured in tandem, so that the entire domain in a protein may be 50-150 residues long. (3) The average value of n, the pattern of residues found in the x position and the non-glycine substitutions in the y position appear to be characteristic of a given glycine loop containing domain, whereas the actual number of repeats is less constrained. (4) Glycine loop sequences display a high degree of evolutionary sequence variability and even allelic variations among different individuals of the same vertebrate species. (5) Glycine loop sequences are expected to be highly flexible, but possess little other regular secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMedDate not supplied

Alterations in microtubules, intermediate filaments, and microfilaments induced by microcystin-LR in cultured cells.

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin that inhibits intracellular serine/threonine protein phosphatases causing disruption of actin microfilaments (MFs) and intermediate filaments (IFs) in hepatocytes. This study compared the effects of MCLR on the organization of MFs, IFs, and microtubules (MTs) in hepatocytes and nonhepatocyte cell lines and determined the sequence of toxin-induced changes in these cytoskeletal components. Rat renal epithelial cells and fibroblasts were incubated with MCLR at 100 or 200 microM for 6-18 hr. Rat hepatocytes in primary culture were exposed to the toxin at 1 or 10 microM for 2-64 min. Cells were fixed and incubated with primary antibodies against beta-tubulin, actin, and vimentin or cytokeratin IFs, followed by gold-labeled secondary antibodies with silver enhancement of the gold probe. The fraction of fibroblasts and hepatocytes with altered cytoskeletal morphology was evaluated as a function of MCLR dose and exposure time to assess the sequence of changes in cytoskeletal components. Changes in fibroblasts and some hepatocytes were characterized initially by disorganization of IFs, followed rapidly by disorganization of MTs, with the progressive collapse of both cytoskeletal components around cell nuclei. Many hepatocytes exhibited MT changes prior to effects on IF structure. Alterations in MFs occurred later and included initial aggregation of actin under the plasma membrane, followed by condensation into rosette-like structures and eventual complete collapse into a dense perinuclear bundle. The similarity of effects among different cell types suggests a common mechanism of action, but the independent kinetics of IF and MT disruption in hepatocytes suggests that there may be at least 2 sites of phosphorylation that lead to cytoskeletal alterations.

Actin Cytoskeleton↗