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PubMed1989

Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities.

The phosphorylation of the intermediate filament protein vimentin was examined under in vitro conditions. Cell cytosol and Triton-insoluble cytoskeleton preparations from nonmitotic and mitotically selected mouse L-929 cells exhibited vimentin kinase activity that is apparently cAMP and Ca2+ independent. The level of vimentin kinase activity was greater in preparations from mitotically selected cells than nonmitotic cells. Addition of Ca2+ to mitotic cytosol decreased net vimentin phosphorylation. Dephosphorylation experiments indicated that there is phosphatase activity in these preparations which is stimulated by addition of Ca2+. Fractionation of cytosol from nonmitotic cells on DEAE-Sephacel and phosphocellulose revealed a single major vimentin kinase activity (peak I). Fractionation of cytosol from mitotically selected cells yielded a similar activity (peak I) and an additional vimentin kinase activity (peak II) that was not found in nonmitotic preparations. Based on substrate specificity and lack of inhibition to characteristic inhibitors, the semipurified peak I and II vimentin kinase activities appear to be cAMP-independent enzymes that are distinct from casein kinases I and II. Phosphopeptide mapping studies indicated that both peak I and peak II vimentin kinases phosphorylate tryptic peptides in the NH2-terminal region of vimentin that are phosphorylated in intact cells. Electron microscopic examination of reconstituted vimentin filaments phosphorylated with both semipurified kinases indicated that phosphorylation induced filament disassembly. These experiments indicate that the increased phosphorylation of vimentin during mitosis may be catalyzed by a discrete cAMP-independent protein kinase. In addition, preparations from mitotic cells exhibited a Ca2+-stimulated phosphatase activity, suggesting that Ca2+ may play a regulatory role in vimentin dephosphorylation during mitosis.

Animals↗
PubMed1990

Regulation of the expression of vimentin gene during the differentiation of mouse myeloid leukemia cells.

We have examined the expression of vimentin during the differentiation of mouse myeloid leukemia cells (M1), which were induced to differentiate into macrophages by exposure to conditioned medium (CM) obtained from rat embryo fibroblasts. The synthesis of vimentin, which was examined by two-dimensional gel electrophoresis, increased after 12-24 h of incubation of M1 cells in CM and the elevated level of synthesis continued up to 96 h. A macrophage cell line (Mm1) that was derived from spontaneously differentiated M1 cells constantly synthesized much higher levels of vimentin. The amount of vimentin, which was revealed by immunoblot analysis using an mAb against human vimentin, also increased after differentiation by a factor of 7 when compared on the basis of constant protein and by a factor of 17 on the basis of constant cell numbers. Mm1 cells contained greater than 12- and 45-fold more vimentin compared with undifferentiated M1 cells on the bases of constant protein and constant cell numbers, respectively. Northern blot analysis using vimentin cDNA as a probe revealed increases in vimentin mRNA in the differentiated M1 cells and Mm1 cells. Nuclear run-on assay showed that the expression of vimentin gene during the differentiation of M1 cells was transcriptionally regulated. Observations in indirect immunofluorescence microscopy and EM clearly showed that vimentin bundles were rarely observed in undifferentiated M1 cells, and increased amounts of and large-size vimentin bundles were easily observed in differentiated M1 and Mm1 cells. These results suggest the participation of increased amounts of vimentin filaments in the maldistribution of nuclei in M1 cells during differentiation.

Animals↗
PubMed1992

Regulation of vimentin expression in cultured epithelial cells.

Most cell types start expressing vimentin when brought into tissue culture. Using both vimentin-expressing (HeLa) and vimentin-negative (MCF-7) epithelial cell lines, we have identified the cis-regulatory DNA elements involved in this process. Sequences located 1.1-0.6 kb upstream of the vimentin transcription-initiation site strongly enhance expression in HeLa cells, but are silenced in MCF-7 cells. Other regulatory elements in the vimentin promoter (an enhancer 3.2-2.6 kb upstream and a minimal promoter region including the CAAT-box) are potentially active in both cell types, but are silenced by the 0.5-kb fragment in MCF-7 cells. Deletion of this fragment restores transcriptional activity of a transfected vimentin promoter. Our data indicate that a double AP 1/jun-binding site present in the 0.5-kb fragment mediates the induction of vimentin expression in cultured epithelial cells, while silencing sequences located within the same fragment are responsible for the absence of vimentin expression in MCF-7 cells. In contrast to MCF-7 cells, a transfected vimentin promoter and gene are transcriptionally active in the vimentin-negative epithelial cell line T24. Transfection studies show that type-III-intermediate-filament expression is not impaired at any level in these cells. Upon transfection and expression of a desmin construct in T24 cells not only desmin, but also vimentin was detected. Both proteins assembled into intermediate filaments. This induction of vimentin expression appeared to be regulated at the post-transcriptional level.

Animals↗
PubMed1994

Coexpression of cytokeratin and vimentin intermediate filaments in benign and malignant sweat gland tumors.

The coexpression of cytokeratin and vimentin intermediate filaments has been immunohistochemically evaluated in 124 benign and malignant sweat gland tumors of various types in comparison to normal sweat glands. In addition, all neoplasms have been stained by an antibody to alpha-smooth muscle actin. Epithelial cells reacted with the pan-cytokeratin antibody lu-5. In normal sweat glands, vimentin immunoreactivity was restricted to myoepithelial cells and to some cells of the coiled duct. In benign sweat gland tumors (n = 88), coexpression of vimentin and alpha-smooth muscle actin was frequently found in basal cells of neoplasms considered to differentiate towards the secretory coil of the eccrine or apocrine gland. These included eccrine spiradenoma, apocrine cystadenoma, hidradenoma papilliferum, syringocystadenoma papilliferum, and cylindroma. Thus, in these tumors, vimentin-reactive cells corresponded to myoepithelial cells. Vimentin-positive cells were also found in 14 of 36 sweat gland carcinomas, including 1 case of sclerosing sweat duct carcinoma, 1 case of porocarcinoma, 4 cases of eccrine adenocarcinoma, 1 case of mucinous eccrine carcinoma, and 5 cases of apocrine adenocarcinoma. Co-expression of vimentin and alpha-smooth muscle actin was observed in some cells of eccrine and apocrine adenocarcinomas. Therefore, in these neoplasms, some vimentin-positive cells appear to represent myoepithelial cells. In contrast, vimentin-positive cells in all other malignant tumors did not express alpha-smooth muscle actin. Our results indicate that coexpression of cytokeratin and vimentin may be frequently found in a variety of benign and malignant sweat gland tumors. In the majority of these neoplasms, vimentin-positive cells correspond to myoepithelial cells. Because vimentin is not specific for myoepithelial cells, additional stains for alpha-smooth muscle actin should be performed to prove the myoepithelial nature of vimentin-positive cells.

Humans↗
PubMed1983

The interaction in vitro of the intermediate filament protein vimentin with synthetic polyribo- and polydeoxyribonucleotides.

In a continuation of our studies on the association of the intermediate filament protein vimentin with unfolded ribosomal subunits, rRNA and various naturally occurring RNAs and DNAs, we have investigated the binding of vimentin to a variety of synthetic polyribo- and polydeoxyribonucleotides. The vimentin-binding potentials of the different nucleic acids were determined in competition with 28S rRNA from Ehrlich ascites tumor cells. The reaction products were analysed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Among the homopolynucleotides tested, poly(rG), poly(rX), poly(rI), poly(rs4U) and poly(dG) were very good competitors, whereas poly(dC) was moderately so. Poly(rA), poly(rU), poly(rC), poly(dA), poly(dU), poly(dT) and poly(dI) were very weak competitors. Annealing of the active homopolymers to their complementary homopolymer strands resulted in a reduction of the vimentin-binding potential; however, the resulting duplexes still had substantial vimentin-binding capacity. A similar reduction was observed when the mononucleotides of the active homopolymers were randomly copolymerized with their complementary nucleotides or incorporated into strictly alternating, double-stranded DNAs. DNA-type copolymers, duplexes and alternating copolymers containing the nuclear bases adenine, thymine and uracil were moderate competitors, but, in general, they were significantly more active than the corresponding homopolymers. This result, together with those of the analysis of a series of random heteropolyribonucleotides, indicates that there is some influence of the base sequence and/or secondary structure of the nucleic acids on their vimentin-binding capacities. Whereas the minimum length of oligo(dG) for efficient vimentin binding was between 11 and 18 nucleotides, (dT)18 was still incapable of interacting with vimentin. In order to obtain information on the salt stability of vimentin-nucleic acid adducts, we have also performed affinity chromatographies on single-stranded calf thymus DNA-cellulose and on rRNA and homopolyribonucleotides covalently coupled to agarose. Although because of unspecific secondary interaction of vimentin with the matrix, these nucleic acid-substituted absorbents could not be used for this purpose under non-denaturing conditions, in the presence of 6M urea a relationship between the nucleic acid substitution and the salt stability of vimentin binding was observed. Finally, the nucleic acid-binding properties of vimentin are compared with those of cytosolic steroid hormone receptors, particularly with those of the androgen receptor.

Animals↗
PubMed1991

Expression of vimentin and glial fibrillary acidic protein in the developing rat spinal cord: an immunocytochemical study of the spinal cord glial system.

The glial system in the developing rat spinal cord was studied using immunocytochemistry. Antibodies to vimentin and glial fibrillary acidic protein (GFAP) were used. At E11, vimentin was first found in the membrana limitans externa. In the matrix layer, short vimentin protrusions were found near the membrana limitans externa at E12. In addition, vimentin was scattered throughout the matrix layer, where it was also present as vimentin-positive tangles. Later in development, vimentin immunoreactivity was distributed in a distinct radial pattern in the matrix layer. During the first postnatal weeks, vimentin was replaced by GFAP which is therefore expressed in a similar radial pattern. This orderly structural organisation of vimentin and GFAP in the matrix layer could indicate the involvement of both proteins in morphogenetic processes such as neuron migration and cell organisation. In the mantle layer, a distinct radial vimentin immunoreactivity was replaced by GFAP immunoreactivity during the first 2 postnatal weeks. In addition, GFAP fibres appeared first, at E18, in the ventral mantle layer associated with the motor neuron columns. These glial fibres originated from a local source. In the dorsal mantle layer, GFAP-positive fibres were oriented tangentially, which is different from the overall radial arrangement. This expression pattern may be related to the ingrowth of primary afferents. In the ventral and dorsal raphe, a major vimentin expression was replaced by a minor presence of GFAP. Within the white matter, a vimentin-positive radial pattern was demonstrated which, after birth, was replaced by GFAP. This palisading pattern suggested an involvement of both proteins in the development and guidance of the ascending and descending spinal cord fibre systems. The general transition from the expression of vimentin to the expression of GFAP in the rat spinal cord takes place during the first 3 postnatal weeks.

Animals↗
PubMed1985

Co-expression of cytokeratin and vimentin filaments in mesothelial, granulosa and rete ovarii cells of the human ovary.

The intermediate filament (IF) system of the various cells of human, pig and rat ovaries was studied by electron microscopy, by immunolocalization using antibodies to cytokeratins, vimentin, desmin and desmoplakin, and by two-dimensional gel electrophoresis of cytoskeletal proteins from microdissected tissue samples. In human ovaries, surface epithelial cells (mesothelium) were stained by antibodies against cytokeratins, desmoplakins and vimentin. Biochemical analysis revealed cytokeratins Nos. 8, 18 and 19, together with variable amounts of No. 7. Granulosa cells of follicles of all stages were also positive for cytokeratins, desmoplakins and vimentin, in agreement with the electron microscopic finding of desmosomes in these cells. As the follicle matured, the cytokeratin content usually appeared to decrease, whereas vimentin remained unchanged. On gel electrophoresis, granulosa cells presented cytokeratins Nos. 8 and 18 and vimentin. Rete ovarii cells were also positive for both cytokeratins, desmoplakins and vimentin, and the electron microscopy revealed numerous desmosome-tonofilament complexes. Oocytes appeared to be devoid of IFs. Corpus luteum cells were rich in vimentin but biochemical analysis also revealed small amounts of cytokeratins Nos. 8 and 18. In contrast, cells of the ovarian stroma and luteinized stromal nodules were positive for vimentin only. A certain type of scattered stromal cells, especially around tertiary follicles and corpora lutea, and also desmin-positive. Pig and rat ovaries differed from human ones in that vimentin was not detected in ovarian mesothelium and cytokeratins were not seen in granulosa cells. The latter, however, contained significant amounts of vimentin. These results indicate that three cell types of human ovary, i.e. surface epithelial, granulosa and rete ovarii cells, can be regarded as true epithelial cells which, however, simultaneously express vimentin, a phenomenon frequently seen in cultured epithelial cells but uncommon in epithelial tissues. The presence of cytokeratins in granulosa cells in all types of human follicles is discussed with regard to the development of these cells. In contrast, granulosa cells of the other two mammalian species only display vimentin IF. Such differences between different mammalian species in IF composition of ovarian components present an example which precludes extrapolation of data from one species to another. The results are discussed in relation to current views of the histogenesis of various ovarian tumors.

Animals↗
PubMed1992

Differentiation of bursal secretory-dendritic cells studied with anti-vimentin monoclonal antibody.

Embryonic and posthatched differentiation of bursal secretory dendritic cells, which express vimentin intermediate filaments, were studied with anti-vimentin (clone 3B4) and anti-cytokeratin (clone Lu5) monoclonal antibodies. Anti-cytokeratin staining revealed that medullary reticular epithelial cells formed a continuous network at every age, whereas the vimentin positive cells were single and showed dendritic appearance. On the basis of location, number, shape, polarized appearance, and Ia staining, the vimentin-positive cells and secretory dendritic cells appeared to be the same cell. Secretory dendritic cell precursors entered the bursal epithelium between 11 and 13 days of embryogenesis. The first vimentin positive cell appeared in the bud of 14-day embryos. Bud formation preceded the appearance of vimentin-positive cells. These observations suggested that the secretory dendritic cell precursor did not express vimentin when it entered the epithelium. Between 15 days of embryogenesis and 2 weeks of posthatch development, the changes in vimentin staining pattern revealed a cytological differentiation of the vimentin-positive cell. During rapid bursal growth, the number of secretory dendritic cells (vimentin-positive cells) increased about 18 times possibly by proliferation of vimentin-negative precursors in the epithelial arches of the corticomedullary border.

Animals↗
PubMed1991

Protein kinase C-induced redistribution of the cytoskeleton and phosphorylation of vimentin in cultured brain macrophages.

The phorbol ester 12-O-tetradecanoyl-acetate (TPA) induced prominent and transient changes in the organization of the cytoskeleton in cultured amoeboid microglial cells including redistribution of actin toward the center of the cells and in the subplasmalemmal region, appearance of fine actin filaments, retraction of microtubules (MT), and rearrangement of intermediate filaments (IF) containing vimentin. The possible implication of protein kinase C (PKC) in mediating the effects of TPA was suggested by a parallel shift of PKC activity from the soluble to membrane fractions and phosphorylation of several microglial proteins. The rearrangement of IF closely correlated with increased vimentin phosphorylation, detected by pulse labeling of intact cells. Two monoclonal antivimentin antibodies, B3 and V9, showed different staining patterns. Immunoreactivity with the antibody B3 was more restricted and could be abolished by treatment of fixed, permeabilized cells with alkaline phosphatase, thus suggesting that the antibody reacts with a phosphorylated epitope. Using this antibody, rearrangement of IF involving vimentin phosphorylation was detected within 15 to 60 min of treatment with 50 nM TPA and consisted in the appearance of intense perinuclear fluorescent label. This perinuclear fluorescence persisted up to 24 hr after TPA removal and gradually diminished during the following 2 to 3 days. Immunochemical analysis of nonionic detergent-soluble and -insoluble extracts from untreated and TPA-treated cells revealed no differences in vimentin solubility suggesting that TPA induced vimentin phosphorylation does not result in notable vimentin filament disassembly. However the extent of vimentin degradation was more prominent in TPA-treated cultures indicating a higher sensitivity of vimentin to proteolytic degradation. The data show that PKC-mediated phosphorylation of vimentin results in precise spatial and temporal rearrangement of IF which are not associated with altered vimentin solubility, but possibly changes the mechanical properties and interactions of vimentin filaments.

Actins↗
PubMed2005

The origin of vimentin expression in invasive breast cancer: epithelial-mesenchymal transition, myoepithelial histogenesis or histogenesis from progenitor cells with bilinear differentiation potential?

Vimentin expression is a rather rare finding in invasive breast cancer, and is associated with high tumour invasiveness and chemoresistance. It is currently explained by two different biological theories: direct histogenetic derivation from myoepithelial cells, and epithelial-mesenchymal transition (EMT) reflecting the end-stage of breast cancer dedifferentiation. In this study we aimed to obtain further insights into the biological hallmarks of these vimentin-expressing breast cancers. We applied immunohistochemistry for vimentin and 15 other differentiation markers to a series of 364 invasive breast cancer cases, using tissue microarray technology. 7.7% of all tumours expressed vimentin. Almost all of these cases (19/21) were Grade 3 invasive ductal carcinomas, and the majority (13/21) of these were associated with a ductal in situ component. Vimentin expression was also seen in the respective in situ components and correlated positively with the expression of SMA, CD10, CK 5, p53, Mib-1 and EGFR. A negative correlation was seen for the expression of CK 8/18 and the oestrogen receptor. Vimentin-expressing carcinomas revealed a significantly higher average absolute number of cytogenetic alterations per case, but a significantly lower frequency of chromosome 16q losses compared to vimentin-negative cases. Our present results demonstrate that, despite analogies between vimentin-positive breast cancers and myoepithelial cells in their expression of differentiation-related proteins, neither myoepithelial histogenesis nor EMT can exclusively explain the biology of these distinct tumours. This is mainly supported by the significantly higher incidence of vimentin-expressing breast cancers compared to any other myoepithelial breast tumours and the fact that vimentin is already observed in ductal in situ components. We therefore propose the alternative hypothesis that vimentin-expressing breast carcinomas may derive from breast progenitor cells with bilinear (glandular and myoepithelial) differentiation potential.

Biomarkers, Tumor↗
PubMed1996

Vimentin filaments follow the preexisting cytokeratin network during epithelial-mesenchymal transition of cultured neonatal rat hepatocytes.

Changes in cell cytoskeleton are known to play an important role in differentiation and embryogenesis and also in carcinogenesis. Previous studies indicated that neonatal hepatocytes undergo an epithelial-mesenchymal transition when cultured in a serum-free medium for several days. Here we show by Western blotting of neonatal rat liver cells cultured for 3 days that vimentin and cytokeratin were expressed by these cells. Epidermal growth factor treatment induced high coexpression of vimentin and cytokeratin filaments in hepatocytes from neonatal livers, as detected by double immunofluorescence microscopy. Confocal scanning laser microscopy was used to determine the spatial and cell distribution of cytokeratin and vimentin intermediate filament networks. Vimentin-expressing hepatocytes were mainly located on the periphery of epithelial clusters and presented a migratory morphology, suggesting that vimentin expression was related to the loss of cell-cell contact. Short vimentin filaments were mainly located at the cytoplasmic sites behind the extending lamella. Horizontal and vertical dual imaging of double immunofluorescence with anti-vimentin and anti-cytokeratin antibodies indicated that both filaments colocalize strongly. Three-dimensional reconstruction of serial optical sections revealed that newly synthesized vimentin distributed following the preexisting cytokeratin network and, when present, both filament scaffolds codistributed inside cultured hepatocytes. Immunoelectron microscopy performed in whole-mount-extracted cultured cells revealed that both filaments are closely interrelated but independent. However, a high degree of immunogold colocalization was found in the knots of the filament network. Further experiments with colcemide and cytochalasin treatment indicated that vimentin filament distribution, but not cytokeratin, was dependent on an intact microtubule network. These results are consistent with a mechanism of vimentin assembly, whereby growth of vimentin intermediate filaments is dependent on microtubules in topographically restricted cytoplasmic sites, in close relation to the cytokeratin cytoskeleton and to changes in cell-cell contact and cell shape.

Albumins↗
PubMed2002

Deletion mutagenesis of the amino-terminal head domain of vimentin reveals dispensability of large internal regions for intermediate filament assembly and stability.

Previous studies have shown that the non-alpha-helical head domain of vimentin is required for polymerization of intermediate filaments (IFs) and, furthermore, a nonapeptide highly conserved among type III IF subunit proteins at their extreme amino-terminus is essential for this process. Recombinant DNA technology was employed to produce specific vimentin deletion mutant proteins (for in vitro studies) or vimentin protein expression plasmids (for in vivo studies), which were used to identify other regions of the vimentin head domain important for polymerization. Various vimentin proteins lacking either residues 25-38, 44-95, or 40-95 polymerized into wild-type or largely normal IFs, both in vitro and in vivo. Vimentin proteins lacking residues 44-69 or 25-63 failed to form IFs in vitro, but assembled into IFs in vivo. Vimentin proteins lacking residues 25-68, 44-103, or 88-103 failed to form IFs in vitro or in vivo. Taken together with previous results, these data demonstrate that the middle of the vimentin non-alpha-helical head domain, which is known to be the site of nucleic acid binding, is completely dispensable for IF formation, whereas both ends of the vimentin non-alpha-helical head domain are required for IF formation. The simplest explanation for these results is that the middle of the vimentin non-alpha-helical head domain loops out, thereby permitting the juxtaposition of the ends of the head domain and their productive interaction with other protein domains (probably the C-terminus of the rod domain) during IF polymerization. The ability of some of the mutant proteins to form IFs in vivo, but not in vitro, suggests that as-yet-unknown cellular proteins may interact with and, in some cases, enable polymerization of IFs, even though they are not absolutely required for IF formation by wild-type vimentin.

Adenocarcinoma↗
PubMed1992

Expression of vimentin by rabbit corneal epithelial cells during wound repair.

Intermediate filaments of epithelial cells generally consist of specific combinations of keratins. However, cultured epithelial cells from certain tissues and some epithelial tumors have been shown also to express vimentin. In the present study, the expression of vimentin by epithelial cells in healing corneal wounds (partial thickness penetrating wounds) and in tissue culture was analyzed. Both immunohistochemical and immunotransblot analyses indicated that although vimentin was not detected in the normal rabbit corneal epithelium in vivo, cultured rabbit corneal epithelial cells co-express keratins and vimentin. At 1 day post-wounding, vimentin was not detectable in the epithelial cells that had covered the denuded stroma. However, at 2 days postwounding, the epithelium at the base of the epithelial plug immunoreacted with both anti-vimentin and antikeratin monoclonal antibodies. Immunotransblot analyses of the extracts of the epithelial plugs confirmed the presence of vimentin (Mr = 58k). The 58k band was not detected in the extract of normal rabbit corneal epithelium. At day/5, vimentin was no longer detectable in the epithelium. This study demonstrated that corneal epithelial cells transiently co-express vimentin and keratins in vivo during wound healing and in tissue culture. The time-course of the transient expression of vimentin suggests that the vimentin expression in the epithelial cells during healing is not linked to cell proliferation or to the centripetal migration of the epithelium during early stages (first 24 h) of healing, but may be linked to cell-matrix interactions or the migration of basal cells in the upward direction at the following stage of healing.

Animals↗
PubMed1986

Cleavage of vimentin in dense cell cultures. Inhibition upon transformation by type 5 adenovirus.

The analysis on two-dimensional isoelectric focusing and SDS polyacrylamide gels (2D gels) of the Triton X-100 and high salt-insoluble fraction of fibroblast cell lines, certain epithelial cell lines and granulosa cells revealed various amounts of a vimetin cleavage product, with a more basic pI and with a MW (1,500-2,000) lower than that of intact vimentin. This cleavage product of vimentin which constituted as much as 30% of the total vimentin in an established rat embryo fibroblast cell line (CREF), was detected by a monoclonal antivimentin antibody in whole cell and Triton-insoluble extracts, and it has a phosphorylated variant which can be degraded to form the "staircase pattern" on 2D gels similarly to intact vimentin. This processing of vimentin occurred mainly in dense cell cultures and it could not be induced in sparse cell cultures by inhibiting DNA synthesis with ara C, or by arresting cell growth in medium containing 0.1% serum. Transformation of CREF cells with intact wild-type (H5wt) and host-range cold-sensitive mutants (H5hr1 or H5d1101) of type 5 adenovirus (Ad5), or transformation of CREF cells by Ca2+-mediated DNA transfection with the transforming E1a (0-4.5 map units) or E1a + E1b (0-11.5 map units) region of Ad5 inhibits the cleavage of vimentin in dense cultures only at temperatures which are permissive for expression of the transformed phenotype. The transformation of cells with bovine papilloma virus type 1, with T24 ras oncogene, or with RSV does not interfere with the cleavage of vimentin. The organization of the vimentin network in dense cultures, where the vimentin cleavage occurs, is very different from that of sparse untransformed and sparse or dense Ad5-transformed cells. The possibility that the acidic amino acid-rich C-terminus of vimentin is cleaved in dense cell cultures in conjunction with the reorganization of the vimentin network and the inhibition of this cleavage by transformation with Ad5, are discussed.

Adenoviruses, Human↗
PubMed1992

Species-specific recognition patterns of monoclonal antibodies directed against vimentin.

Two commercially available monoclonal antibodies raised against the intermediate filament protein vimentin were characterized concerning their species-specific reaction pattern on vertebrate cells. The antibody V9 exhibited extensive reactivity with vimentin of all mammalian species tested, but specifically did not detect vimentin in mouse cells and chicken fibroblasts. The antibody VIM 3B4 recognized vimentin in cells of chicken and most mammalian species, except for rodent species. Characterization of the binding site of VIM 3B4 on human vimentin by limited proteolysis and immunoblotting as well as by sequence comparison strongly suggested that the epitope is located in the coil 2 part of the vimentin rod domain. Site-directed mutagenesis of a mouse vimentin cDNA clone followed by in vivo expression showed that VIM 3B4 could detect rodent vimentin containing a single amino acid substitution (valine for leucine) at position 353 of the mouse vimentin sequence. Practical application for this finding was demonstrated by the unequivocal identification of a modified murine vimentin protein, distinct from the endogenous vimentin, in a cytoplasmic intermediate filament network in mouse skin fibroblasts transfected with a recombinant plasmid expression vector.

Animals↗
PubMed1992

Cell cycle-dependent vimentin expression in elutriator-synchronized, TPA-treated MPC-11 mouse plasmacytoma cells.

We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).

Animals↗
PubMed1998

Zebrafish vimentin: molecular characterization, assembly properties and developmental expression.

To provide a basis for the investigation of the intermediate filament (IF) protein vimentin in one of the most promising experimental vertebrate systems, the zebrafish (Danio rerio), we have isolated a cDNA clone of high sequence identity to and with the characteristic features of human vimentin. Using this clone we produced recombinant zebrafish vimentin and studied its assembly behaviour. Unlike other vimentins, zebrafish vimentin formed unusually thick filaments when assembled at temperatures below 21 degrees C. At 37 degrees C few filaments were observed, which often also terminated in aggregated masses, indicating that its assembly was severely disturbed at this temperature. Between 21 and 34 degrees C apparently normal IFs were generated. By viscometry, the temperature optimum of assembly was determined to be around 28 degrees C. At this temperature, zebrafish vimentin partially rescued, in mixing experiments, the temperature-dependent assembly defect of trout vimentin. Therefore it is apparently able to "instruct" the misorganized trout vimentin such that it can enter normal IFs. This feature, that assembly is best at the normal body temperature of various species, puts more weight on the assumption that vimentin is vital for some aspects of generating functional adult tissues. Remarkably, like in most other vertebrates, zebrafish vimentin appears to be an abundant factor in the lens and the retina as well as transiently, during development, in various parts of the central and peripheral nervous system. Therefore, promising cell biological investigations may now be performed with cells involved in the generation of the vertebrate eye and brain, and, in particular, the retina. Moreover, the power of genetics of the zebrafish system may be employed to investigate functional properties of vimentin in vivo.

Amino Acid Sequence↗
PubMed1983

Synthesis and post-translational assembly of intermediate filaments in avian erythroid cells: vimentin assembly limits the rate of synemin assembly.

The assembly of vimentin intermediate filaments and the high molecular weight filament crosslinking protein, synemin, was studied in erythroid cells from 10-day chicken embryos. Pulse labeling studies show that newly synthesized vimentin is present both in a Triton X-100-insoluble form and in a soluble form. The incorporation of labeled vimentin into the insoluble fraction increases linearly with time, while the soluble pool of labeled vimentin saturates quickly. In contrast, synemin accumulates rapidly in the Triton X-100-soluble fraction and begins to accumulate in the insoluble fraction only after a considerable lag of time. Pulse-chase studies reveal that the detergent-soluble pools of both vimentin and synemin contain precursors for their post-translational assembly into detergent-insoluble filaments and that the half-life of soluble synemin is about twice as long as that of soluble vimentin. Immunoprecipitation of solubilized filaments with synemin antiserum precipitates vimentin with synemin. On the other hand, soluble vimentin does not coimmunoprecipitate with soluble synemin. These results suggest that, in the assembly of vimentin and synemin into intermediate filaments, vimentin filament elongation generates synemin binding sites, and thus the rate of vimentin filament elongation limits the rate of synemin assembly.

Animals↗