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PubMed2002

Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process.

Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at approximately 2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.

Amino Acid Sequence↗
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↗
PubMed2002

Vimentin intermediate filament reorganization by Cdc42: involvement of PAK and p70 S6 kinase.

Rho family GTPases play a major role in actin cytoskeleton reorganization. Recent studies have shown that the activation of Rho family GTPases also induces collapse of the vimentin intermediate filament (IF) network in fibroblasts. Here, we report that Cdc42V12 induces the reorganization of vimentin IFs in Hela cells, and such reorganization is independent of actin and microtubule status. We analyzed the involvement of three serine/threonine kinase effectors, MRCK, PAK and p70 S6K in the Cdc42-induced vimentin reorganization. Surprisingly, the ROK-related MRCK is not involved in this IF reorganization. We detected phosphorylation of vimentin Ser72, a site phosphorylated by PAK, after Cdc42 activation. PAK inhibition partially blocked Cdc42-induced vimentin IF collapse suggesting the involvement of other effectors. We report that p70 S6 kinase (S6K)1 participates in this IF rearrangement since the inhibitor rapamycin or a dominant inhibitory S6K could reduce the Cdc42V12 or bradykinin-induced vimentin collapse. Further, inhibition of PAK and S6K in combination very effectively prevents Cdc42-induced vimentin IF collapse. Conversely, only in combination active PAK and S6K could induce a vimentin IF rearrangement that mimics the Cdc42 effect. Thus, Cdc42-induced vimentin reorganization involves PAK and, in a novel cytoskeletal role, p70 S6K.

3T3 Cells↗
PubMed1983

Epinemin: a new protein associated with vimentin filaments in non-neural cells.

In this report I describe a new protein, defined by a monoclonal antibody, which is associated with vimentin filaments in a variety of cultured cells and in skeletal muscle. By immunofluorescence it is absent in smooth muscle, in cells without vimentin, and in neural vimentin containing cells. This protein has a molecular weight of 44,500, a pl of 5, a two-dimensional tryptic peptide fingerprint pattern different from vimentin, is unrelated to actin by Cleveland peptide analysis and by light and electron microscopy, and is not recognized by either a polyclonal antivimentin antibody (Frank, E.D., and L. Warren, 1981, Proc. Natl. Acad. Sci. USA, 78:3020-3024) or a monoclonal antibody against all classes of intermediate filaments (Pruss, R.M., R. Mirsky, M.C. Raff, R. Thorpe, A.J. Dowding, and B.H. Anderton, 1981, Cell, 27:419-428). The protein is resistant to nonionic detergent extraction, is soluble in high salt and can thus be removed from vimentin filaments, but fragments with vimentin in either low salt or anionic detergent and collapses with vimentin in colchicine-treated cells. By light microscopy, the distribution of the protein is indistinguishable from vimentin filaments and appears uniform along them. In contrast, immunoferritin electron microscopy reveals that the molecule is distributed in an intermittent pattern on vimentin filaments. Adopting the terminology of Granger and Lazarides (1980, Cell, 30:263-275), the molecule is called epinemin, meaning "upon filaments."

Animals↗
PubMed1984

Differentiation of murine erythroleukemia cells results in the rapid repression of vimentin gene expression.

We show that vimentin filaments are present in undifferentiated Friend murine erythroleukemia cells, but are lost progressively to undetectable levels by 96 h of dimethyl sulfoxide-mediated differentiation. The amount of newly synthesized cytoskeletal vimentin is decreased dramatically by 24 h of induction, and is paralleled by a rapid loss of vimentin mRNA (approximately 25-fold reduction at 96 h). Hence, disappearance of vimentin filaments in these cells appears to be regulated at the level of vimentin mRNA abundance. On the other hand, the levels of actin synthesis and actin mRNA remain essentially unchanged. The kinetics of vimentin mRNA reduction during dimethyl sulfoxide-mediated differentiation, and the levels of vimentin mRNA observed in the presence of hexamethylene-bisacetamide or hemin as inducers suggest that the cessation of vimentin expression precedes, but may be associated with commitment to terminal differentiation. Our results demonstrate the dynamic regulation of vimentin expression in mammalian erythropoiesis.

Acetamides↗
PubMed2001

Role of the intermediate filament protein vimentin in delaying senescence and in the spontaneous immortalization of mouse embryo fibroblasts.

Because knockout of the vimentin gene in mice did not produce an immediately obvious, overt, or lethal specific phenotype, the conjecture was made that the mutation affects some subtle cellular functions whose loss manifests itself only when the mutant animals are exposed to stress. In order to substantiate this idea in a tractable in vitro system, primary embryo fibroblasts from wildtype (V(+/+)) and vimentin-knockout (V(-/-)) mice were compared with regard to their growth behavior under the pseudophysiologic conditions of conventional cell culture. Whereas in the course of serial transfer, the V(+/+) fibroblasts progressively reduced their growth potential, passed through a growth minimum around passage 12 (crisis), and, as immortalized cells, resumed faster growth, the V(-/-) fibroblasts also cut down their growth rate but much earlier, and they either did not immortalize or did so at an almost undetectable rate. Cells withdrawing from the cell cycle showed increased concentrations of reactive oxygen species and signs of oxidative damage: enlarged and flattened morphology, large nuclear volume, reinforced stress fiber system as a result of increased contents of actin and associated proteins, prominent extracellular matrix, and perinuclear masses of pathological forms of mitochondria with low membrane potential. The differences in the cell cycle behavior of the V(+/+) and V(-/-) cells in conjunction with the morphologic changes observed in mitotically arrested cells suggests a protective function of vimentin against oxidative cell damage. Because vimentin exhibits affinity for and forms crosslinkage products with recombinogenic nuclear as well as mitochondrial DNA in intact cells, it is credible to postulate that vimentin plays a role in the recombinogenic repair of oxidative damage inflicted on the nuclear and mitochondrial genome throughout the cells' replicative lifespan. Recombinational events mediated by vimentin also appear to take place when the cells pass through the genetically unstable state of crisis to attain immortality. The residual immortalization potential of V(-/-) fibroblasts might be attributable to their capacity to synthesize, in place of vimentin, the tetrameric form of a lacZ fusion protein carrying, in addition to a nuclear localization signal, the N-terminal 59 amino acids of vimentin and thus its DNA-binding site. On the basis of these results and considerations, a major biologic role of vimentin may be to protect animals during development and postnatal life against genetic damage and, because of its contribution to the plasticity of the genome, to allow them to respond to environmental challenges.

Animals↗
PubMed2001

Isolation of SDS-stable complexes of the intermediate filament protein vimentin with repetitive, mobile, nuclear matrix attachment region, and mitochondrial DNA sequence elements from cultured mouse and human fibroblasts.

Crosslinkage of vimentin to DNA in mouse L929 cells by formaldehyde and isolation of SDS-stable DNA-vimentin complexes from normal L929 cells and mouse and human embryo fibroblasts indicated close spatial relations between these components in the intact cell. The adducts, obtained by immunoprecipitation with anti-vimentin antibody, contained substantial quantities, not only of repetitive and mobile sequence elements such as centromeric satellite DNA, telomere DNA, microsatellites and minisatellites, long and short interspersed nucleotide elements, and retroposons, but also of mitochondrial (mt) DNA. Because the SDS-stable complexes could be isolated with distinctly higher yields from oxidatively stressed, senescent fibroblasts and were dissociated by boiling, they possibly arose from accidental condensation reactions mediated by unsaturated and dialdehydes, products of free radical-induced lipid peroxidation. They can therefore be considered vestiges of a general interaction of vimentin with cellular DNA. The sequence patterns of their DNA fragments were similar to those of extrachromosomal circular and linear DNA, including retroviral elements, markers and enhancers of genomic instability that also occur in the cytoplasm and are able to transport vimentin into the nucleus. Many of the fragments were also remarkably similar to AT-rich nuclear matrix attachment regions (MARs) in that they contained, in addition to various mobile elements, a palette of typical MAR motifs. With its tendency to multimerize and to interact with single-stranded and supercoiled DNA, vimentin thus behaves like a nuclear matrix protein and may as such participate in a variety of nuclear matrix-associated processes such as replication, recombination, repair, and transcription of DNA. These activities seem to be extendible to the mitochondrial compartment, as vimentin was also crosslinked to mtDNA, preferentially to its D-loop and hypervariable main control region. These sites are prone to point and deletion mutations and, like nuclear MARs, are associated with the cyto-karyomatrix. Moreover, as a developmentally regulated and tissue-specific cyto-karyomatrix protein, vimentin may contribute to the organization of chromatin, including centromeric and telomeric heterochromatin at the nuclear periphery, with all its consequences for genomic activities during embryogenesis and in adulthood of vertebrates. However, because of its high affinity for hypervariable, recombinogenic DNA sequences, vimentin is proposed to play a major role in both the preservation and the evolution of the nuclear and mitochondrial genome.

Animals↗
PubMed2000

A Stat1alpha factor regulates the expression of the human vimentin gene by IFN-gamma.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. Here, we report an increase in vimentin gene transcription induced by the cytokine interferon-y (IFN-gamma). Northern blot analysis and reporter gene assays reveal that IFN-gamma induces vimentin gene transcription in HeLa cells. However, no increase in vimentin mRNA synthesis was observed de novo in MCF-7 cells, which do not already express vimentin. Band shift analysis shows that the Stat1alpha protein mediates vimentin induction by IFN-gamma. A human mutant fibroblast cell line (U3A), which lacks Stat1alpha but expresses vimentin mRNA, yields no increase in vimentin mRNA levels on the addition of IFN-gamma. These results suggest that the induction of vimentin gene expression might be an important part of a complex cellular response to IFN-gamma.

Adenocarcinoma↗
PubMed1999

Vimentin dephosphorylation by protein phosphatase 2A is modulated by the targeting subunit B55.

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.

Antifungal Agents↗
PubMed2001

Amino-terminal polypeptides of vimentin are responsible for the changes in nuclear architecture associated with human immunodeficiency virus type 1 protease activity in tissue culture cells.

Electron microscopy of human skin fibroblasts syringe-loaded with human immunodeficiency virus type 1 protease (HIV-1 PR) revealed several effects on nuclear architecture. The most dramatic is a change from a spherical nuclear morphology to one with multiple lobes or deep invaginations. The nuclear matrix collapses or remains only as a peripheral rudiment, with individual elements thicker than in control cells. Chromatin organization and distribution is also perturbed. Attempts to identify a major nuclear protein whose cleavage by the protease might be responsible for these alterations were unsuccessful. Similar changes were observed in SW 13 T3 M [vimentin(+)] cells, whereas no changes were observed in SW 13 [vimentin(-)] cells after microinjection of protease. Treatment of SW 13 [vimentin(-)] cells, preinjected with vimentin to establish an intermediate filament network, with HIV-1 PR resulted in alterations in chromatin staining and distribution, but not in nuclear shape. These same changes were produced in SW 13 [vimentin(-)] cells after the injection of a mixture of vimentin peptides, produced by the cleavage of vimentin to completion by HIV-1 PR in vitro. Similar experiments with 16 purified peptides derived from wild-type or mutant vimentin proteins and five synthetic peptides demonstrated that exclusively N-terminal peptides were capable of altering chromatin distribution. Furthermore, two separate regions of the N-terminal head domain are primarily responsible for perturbing nuclear architecture. The ability of HIV-1 to affect nuclear organization via the liberation of vimentin peptides may play an important role in HIV-1-associated cytopathogenesis and carcinogenesis.

Animals↗
PubMed1986

Expression of vimentin in surgically resected adenocarcinomas and large cell carcinomas of lung.

The expression of vimentin in pulmonary carcinomas was studied in 285 cases of surgically resected lung cancer from our hospital files. Formalin fixed, paraffin-embedded sections were studied by immunoreactive staining techniques using two monoclonal antibodies against vimentin. Cases demonstrating vimentin positivity by the avidin-biotin-peroxidase method included 11 of 129 adenocarcinomas studied (8.5%), and 15 of 61 large cell carcinomas studied (24.6%). Vimentin expression was not seen in any of the 51 squamous cell carcinomas or 35 small cell carcinomas in our series. The positive cases of adenocarcinoma were in moderately and poorly differentiated cancers. Four of the eight giant cell carcinomas (50%) demonstrated vimentin expression. All cases that exhibited vimentin positivity were studied for cytokeratin expression. Coexpression of vimentin and cytokeratin was demonstrated not only within the same tumor but also within the same cells in some cases stained by double antibody technique, including both adenocarcinomas and large cell carcinomas. Similar immunoreactive methods were also applied to sections from human lung cancer transplants grown in the nude mouse. Of 28 tumors studied, four of 11 adenocarcinomas (36%) and all 4 large cell carcinomas demonstrated coexpression of vimentin and cytokeratin, while none of the five squamous cell carcinomas or eight small cell carcinomas expressed vimentin.

Adenocarcinoma↗
PubMed1983

Synthesis of vimentin in a reticulocyte cell-free system programmed by poly(A)-rich RNA from several cell lines and rat liver.

Poly(A)-rich RNA has been isolated from Ehrlich ascites tumor (EAT) cells and translated in a rabbit reticulocyte cell-free system. The intermediate filament protein, vimentin, was found to be a major translation product. Fractionation of the poly(A)-rich RNA by sucrose gradient centrifugation showed that the vimentin mRNA had a sedimentation coefficient of about 18 S corresponding to a molecular size of about 2000 nucleotides. This means that it must possess significant non-coding regions. Vimentin synthesized in vitro was identical to native vimentin with regard to its precipitability with ammonium sulphate, extent of phosphorylation and susceptibility to digestion by the vimentin-specific, Ca2+-activated proteinase. Poly(A)-rich RNA was also isolated from a number of tissue-culture cells and rat liver, which contain varying amounts of vimentin in situ. It was found that the amount of vimentin synthesized by these RNA preparations in a rabbit reticulocyte cell-free system is proportional to the amount of vimentin detectable in situ, suggesting that the amount of cellular vimentin may be controlled at the level of transcription.

Animals↗
PubMed1994

Unassembled (soluble) vimentin in human myeloid leukemia cell line HL60.

The intermediate filament proteins which include vimentin, desmin, and the keratins are one of three major classes of cytoskeletal proteins in eukaryotic cells. In this study we found that most of the vimentin of undifferentiated HL60 and cells induced to differentiate either along the monocytoid pathway by 12-O-tetradecanoylphorbol-13-acetate (TPA) or along the granulocytic pathway by retinoic acid was soluble in a buffer containing 1% Triton X-100/0.6 mol/l KCl in which the intermediate filament proteins usually are not soluble. HL60 vimentin separated on polyacrylamide gel electrophoresis into two proteins of Mr 55,000 and 54,000 that we detected by immunoblotting. The Mr 55,000 species was the major form in undifferentiated HL60 cells and cells induced by retinoic acid. The distribution of both forms of vimentin changed during induction of differentiation by TPA and after 24 h the Mr 54,000 species was predominant. After an additional 24 h exposure to TPA the relative levels of the two forms of vimentin approached equivalence and a high level of vimentin degradation products was seen. These results suggest that TPA may increase vimentin degradation along a pathway that has a Mr 54,000 intermediate. In addition, the high levels of soluble vimentin in HL60 cells suggests that these cells may be a good model for studying components involved in vimentin assembly.

Antibodies, Monoclonal↗
PubMed1988

Monoclonal antibody-detected vimentin distribution in pleomorphic adenomas of salivary glands.

To evaluate the participation of myoepithelial components in pleomorphic adenomas, an immunohistochemical study was carried out using monoclonal antibodies to vimentin. Of a total of 80 cases, 50 tumors gave positive staining, 5 tumors very slight, and 25 tumors negative staining for vimentin. Localization patterns for vimentin were divided into 3 classes: 1) vimentin staining in fibrous stromal tissue; 2) variable intensities of vimentin staining were found in the outer layers of tumor cells in tubulo-ductal structures (some of which were spindle cells connected to modified myoepithelial cells which also gave variable vimentin staining); and 3) modified myoepithelial cells and chondroidlike cells displayed strongly positive staining for vimentin. Typical histologic features of pleomorphic ademomas, i.e., tubulo-ductal or duct-like structures were characterized by positive vimentin staining in outer tumor cells and by a positive keratin reaction in the luminal tumor cells. In tumors devoid of stromal connective tissues and the near absence of well-developed, or modified myoepithelial cells, vimentin staining was absent.

Adenoma, Pleomorphic↗
PubMed2006

Vimentin and laminin expression is associated with basal-like phenotype in both sporadic and BRCA1-associated breast carcinomas.

AIMS: To determine whether basal-like phenotype and vimentin and/or laminin are related in both sporadic/familial (BRCA1 or BRCA2 mutated) tumours. METHODS: 230 non-familial and 28 hereditary node-negative invasive breast carcinomas were immunohistochemically analysed for oestrogen receptors (ER), progesterone receptors (PR), cytokeratin 5/6 (CK5/6), epidermal growth factor receptors (EGFR), Ki67, p53, vimentin and laminin, using tissue microarrays. Tumours were considered to have basal-like phenotype if they were ER negative and HER2 negative, but positive for CK5/6 and/or EGFR. RESULTS: In sporadic tumours, vimentin expression was found in 77.8% cases with basal-like phenotype and 15.5% of non-basal cases (p<0.001). In familial cases, vimentin was expressed in 83.3% basal-like cancers and 16.7% of non-basal tumours (p<0.001). Vimentin expression was more frequent in BRCA1 than BRCA2 mutation carriers. Vimentin expressing tumours were associated with poor prognosis (p = 0.012) among patients not receiving adjuvant chemotherapy and showed a trend for local recurrence or visceral but not bone metastasis (p = 0.021). Laminin expression was also related to basal-like phenotype in both sporadic/familial cases (p<0.001 and p = 0.007, respectively), but neither with prognosis nor recurrence pattern in sporadic cancers. CONCLUSIONS: Vimentin and laminin expression is associated with basal-like phenotype in breast cancer. Expression of vimentin and laminin is characteristic of BRCA1 associated tumours. Since vimentin and laminin staining is widely used by pathologists for diagnostic purposes, thus demonstrating the robustness of their specific antibodies, the immunohistochemical evaluation of these two molecules could be used in identification of basal-like breast tumours in both sporadic/familial cases.

Biomarkers, Tumor↗
PubMed2000

Vimentin in cultured chromaffin cells: an immunofluorescent, biochemical and functional study.

In tile present study we seek the presence and possible function of the intermediate filament protein vimentin in adrenomedullary chromaffin cells. Vimentin which is not present in the adrenal medulla was clearly showed up after collagenase digestion of the gland in the cultured chromaffin cells by using an immunofluorescent analysis with double cell labeling with monoclonal antibodies against vimentin and dopamine-beta-hydroxylase. Vimentin was also shown to be phosphorylated in a calcium-dependent manner by acetylcholine. The specific protein phosphatase inhibitor calyculin-A, that has been previously shown to increase vimentin phosphorylation, caused a change in the distribution of vimentin which moved from the Triton X-100 insoluble cytoskeletal preparation to the detergent soluble fraction probably as a result of modifications in filament integrity. The possible role of vimentin in secretion was in addition investigated using digitonin-permeabilized cells, in which the specific antibody for vimentin partially inhibited calcium-induced catecholamine release. These results demonstrate the induction of vimentin expression after collagenase digestion in cultured chromaffin cells and suggest that in these conditions this protein is possibly implicated in the regulation of the secretory process through a phosphorylation-dependent mechanism.

Adrenal Glands↗
PubMed2007

Interaction between Bluetongue virus outer capsid protein VP2 and vimentin is necessary for virus egress.

BACKGROUND: The VP2 outer capsid protein Bluetongue Virus (BTV) is responsible for receptor binding, haemagglutination and eliciting host-specific immunity. However, the assembly of this outer capsid protein on the transcriptionally active viral core would block transcription of the virus. Thus assembly of the outer capsid on the core particle must be a tightly controlled process during virus maturation. Earlier studies have detected mature virus particles associated with intermediate filaments in virus infected cells but the viral determinant for this association and the effect of disrupting intermediate filaments on virus assembly and release are unknown. RESULTS: In this study it is demonstrated that BTV VP2 associates with vimentin in both virus infected cells and in the absence of other viral proteins. Further, the determinants of vimentin localisation are mapped to the N-terminus of the protein and deletions of amino acids between residues 65 and 114 are shown to disrupt VP2-vimentin association. Site directed mutation also reveals that amino acid residues Gly 70 and Val 72 are important in the VP2-vimentin association. Mutation of these amino acids resulted in a soluble VP2 capable of forming trimeric structures similar to unmodified protein that no longer associated with vimentin. Furthermore, pharmacological disruption of intermediate filaments, either directly or indirectly through the disruption of the microtubule network, inhibited virus release from BTV infected cells. CONCLUSION: The principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress. Furthermore, i) the N-terminal 118 amino acids of VP2 are sufficient to confer vimentin interaction. ii) Deletion of amino acids 65-114 or mutation of amino acids 70-72 to DVD abrogates vimentin association. iii) Finally, disruption of vimentin structures results in an increase in cell associated BTV and a reduction in the amount of released virus from infected cells.

Acrylamide↗
PubMed1996

Vimentin in a cold-water fish, the rainbow trout: highly conserved primary structure but unique assembly properties.

We have isolated from a rainbow trout (Oncorhynchus mykiss) spleen cDNA library a clone coding for vimentin. The deduced amino acid sequence reveals a high degree of identity with vimentin from carp (81%), frog (71%), chick and human (73% each). Large stretches in the central alpha-helical rod are identical within all four classes of vertebrates, but in 17 residues spread over the entire rod, the two fish differ distinctly from the tetrapod species. In addition, in the more diverged non-helical head domain, a nonapeptide motif previously shown to be important for regular filament formation is conserved. Recombinant trout vimentin assembles into bona fide filaments in vitro, with a temperature optimum between 18 and 24 degrees C. Above 27 degrees C, however, filament assembly is abruptly abolished and short filaments with thickened ends as well as structures without typical intermediate filament appearance are formed. This distinguishes its assembly properties significantly from amphibian, avian and mammalian vimentin. Also in vivo, after cDNA transfection into vimentin-free mammalian epithelial cells, trout vimentin does not form typical intermediate filament arrays at 37 degrees C. At 28 degrees C, and even more pronounced at 22 degrees C, the vimentin-positive material in the transfected cells is reorganized in the perinuclear region with a partial fibrillar appearance, but typical intermediate filament arrays are not formed. Together with immunoblotting and immunolocalization data from trout tissues, where vimentin is predominantly found in glial and white blood cells, we conclude that vimentin is indeed important in its filamentous form in fish and other vertebrates, possibly fulfilling cellular functions not directly evident in gene targeting experiments carried out in mice.

Amino Acid Sequence↗