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PubMed2006

A direct interaction between actin and vimentin filaments mediated by the tail domain of vimentin.

The assembly and organization of the three major eukaryotic cytoskeleton proteins, actin, microtubules, and intermediate filaments, are highly interdependent. Through evolution, cells have developed specialized multifunctional proteins that mediate the cross-linking of these cytoskeleton filament networks. Here we test the hypothesis that two of these filamentous proteins, F-actin and vimentin filament, can interact directly, i.e. in the absence of auxiliary proteins. Through quantitative rheological studies, we find that a mixture of vimentin/actin filament network features a significantly higher stiffness than that of networks containing only actin filaments or only vimentin filaments. Maximum inter-filament interaction occurs at a vimentin/actin molar ratio of 3 to 1. Mixed networks of actin and tailless vimentin filaments show low mechanical stiffness and much weaker inter-filament interactions. Together with the fact that cells featuring prominent vimentin and actin networks are much stiffer than their counterparts lacking an organized actin or vimentin network, these results suggest that actin and vimentin filaments can interact directly through the tail domain of vimentin and that these inter-filament interactions may contribute to the overall mechanical integrity of cells and mediate cytoskeletal cross-talk.

Actins↗
PubMed1990

Identification of vimentin and novel vimentin-related proteins in Xenopus oocytes and early embryos.

We have made antibodies against fusion proteins of Xenopus vimentin. We show for the first time the distribution of vimentin in larval stages, where it is found in cells of mesenchymal origin, and in radial glial cells. In sections of Xenopus oocytes and early embryos, immunocytochemistry reveals the presence of an extensive cytoplasmic network, distributed in an animal-vegetal gradient. Germ plasm stains particularly strongly. The form of the IF proteins in this network is unusual. In immunoblot experiments the anti-vimentin antibodies detect a number of distinct proteins. We have identified those that are the products of the two known vimentin genes, by injection of synthetic mRNA transcribed from cloned vimentin cDNAs into oocytes, followed by two-dimensional Western blotting. This has demonstrated unambiguously that one Xenopus vimentin, Vim1, is present in oocytes and early embryos. However, two other immunoreactive proteins detected in Triton extracts of oocytes and early embryos are not the products of Vim1, since depletion of vimentin mRNA by antisense oligonucleotide injection has no effect on the synthesis of these proteins. These results suggest that novel IF-like proteins are expressed in Xenopus oocytes and early embryos.

Animals↗
PubMed1992

Biochemical and structural aspects of transiently and stably expressed mutant desmin in vimentin-free and vimentin-containing cells.

Using immunoelectron microscopy it is demonstrated that desmin subunits missing their complete carboxy-terminal domain are incapable of homopolymeric filament formation in vivo. Furthermore it is shown that, in vimentin-containing cells, desmin integrates into preexisting vimentin filaments resulting in desmin/vimentin heteropolymers. Removal of the amino-terminal or both nonhelical end domains of desmin increases Triton X-100 solubility of the mutant desmin subunits. Expression of desmin mutants containing deletions in the C-terminal part of the rod in vimentin-free cells results in an increase of the Triton X-100 solubility too. In contrast, if expressed in vimentin-containing cells, these mutant subunits remain in the Triton X-100 insoluble fraction. Deletion of the nonhelical carboxy-terminal domain only has no effect on solubility. In vimentin-free cells, stably expressed desmin subunits missing their amino-terminal domains display a slightly higher turnover rate compared to wild-type desmin. Transiently expressed desmin subunits missing 18 or more carboxy-terminal residues of the rod domain are rapidly degraded in vimentin-free cells. In vimentin-containing cells, turnover rates were much less pronounced. Finally, by using site-directed mutagenesis, we were able to map specific residues important for de novo filament assembly within the amino-terminal domain and in the conserved part at the C-terminus of the alpha-helical domain.

Amino Acid Sequence↗
PubMed1992

Interference in vimentin assembly in vitro by synthetic peptides derived from the vimentin head domain.

The importance of the amino-terminal domain ("head") of type III intermediate filament (IF) proteins in IF assembly has been examined by testing the influence of synthetic peptides representing a highly conserved decameric motif, KSSSYRRIMFGG, located near the amino terminus of vimentin. When added to soluble vimentin subunits this peptide induces, at fourfold molar excess or slightly above, the appearance of short, regular rod-like structures as determined by electron microscopy of negatively stained and rotary-shadowed preparations as well as by viscometry. At higher peptide concentrations large, irregularly shaped aggregates of mostly non-IF structures formed, but this aggregation was reversible by prolonged dialysis against low ionic strength buffer. The aggregating effect of this peptide was highly sequence-specific and was not seen with point-mutated sequences such as RR----TR or with unrelated peptides containing a central diarginine, indicating that it is not simply ionic. When different hexapeptides representing different "head" positions were compared, only the central sequence, SYRRXF, was as effective as the decamer. The addition of peptide during IF assembly did not prevent filament formation, although 50-fold molar excess of peptide resulted in a drastic increase (up to 40 nm) in the width of the filaments, which also appeared less regular, thus reflecting some interference with assembly. In contrast to the effects on soluble vimentin, the decameric peptide did not disturb IFs, indicating that the binding domain is "masked" or stabilized in the filaments. To identify the domain to which the peptide binds, three different binding assays using vimentin fragments and genetically engineered vimentin deletion mutants were employed. The results indicate that the binding domain of the near-amino-terminal peptide is located at the start of the alpha-helical "rod" domain of the protein. Possible mechanisms of interaction of these two portions of vimentin during IF assembly are discussed.

Amino Acid Sequence↗
PubMed1994

Transient increase in vimentin phosphorylation and vimentin-HSC70 association in 9L rat brain tumor cells experiencing heat-shock.

Characteristic changes in vimentin were studied in 9L rat brain tumor cells treated at 45 degrees C. During heat-shock treatment, vimentin molecules were rapidly phosphorylated and reorganized from a filamentous form into a perinuclear higher-order structure that was less extractable by nonionic detergent. These effects were found to be highly transient, peaked at 30 min after the onset of heat-shock treatment, and subsided thereafter. Simultaneously, the solubility of the constitutively expressed heat-shock protein 70 (HSC70) was also temporarily decreased and the kinetics was identical to that of vimentin. The results indicated that HSC70 and vimentin were co-insolubilized during the heat-shock treatment. We propose that the reorganization of the intermediate filaments resulted from enhanced phosphorylation of vimentin leads to the concurrent association of HSC70 to the intermediate filaments. This process may play an essential role in regulating heat-shock genes.

Animals↗
PubMed1996

Modifications of vimentin filament architecture and vimentin-nuclear interactions by cholesterol oxides in 73/73 endothelial cells.

Among the different targets of the cytodamaging effects of cholesterol oxides in endothelial cells, cytoskeleton is one of the most relevant, due to the large variety of biological events controlled by this subcellular structure. The modifications of the intermediate filament network caused by three cholesterol oxides (cholestane-3beta,5alpha,6beta-triol, CH, 7-keto-cholesterol, KC, and 25-OH-cholesterol, COH) was investigated in the endothelial cell line 73/73 using immunofluorescence and laser scanner confocal microscopy. All three cholesterol oxides promoted a redistribution of vimentin filaments that took place well before cell detachment and the occurrence of any detectable sign of cell death. CH-induced alterations were characterized by the polarization of vimentin to the edges of the cell and a concomitant destruction of its interaction with the nucleus. In KC-treated cells, vimentin filaments appeared cross-linked and formed a sort of circular network ring between the nucleus and the cell periphery. COH promoted the aggregation of vimentin filaments in thick and irregular bundles that delimited apparently empty regions. All these changes occurred independently of gross modifications in microtubule organization, which was generally retained except for the appearance of immunoreactive tubulin spots throughout the cytoplasm. These results indicate that the organization of the intermediate-size filament protein vimentin is markedly affected by cholesterol oxides. The different rearrangements caused by CH, KC, and COH may derive from different pathobiochemical processes triggered by these compounds.

Animals↗
PubMed1986

Immunocytochemical studies of endothelial cells in vivo. I. The presence of desmin only, or of desmin plus vimentin, or vimentin only, in the endothelial cells of different capillaries of the adult chicken.

It is currently believed that the intermediate filaments of endothelial cells contain vimentin subunits exclusively. This inference, however, is derived from studies of only a few types of endothelial cells. By double indirect immunofluorescence and immunoelectron microscopy, we have now examined the endothelial cells of the micro- and macrovasculature of a variety of tissues and organs of adult chicken in vivo for their content of desmin and vimentin. Endothelial cells of the peritubular capillary in the renal cortex, the hepatic sinusoid, and the splenic sinusoid were found to contain only desmin; those of the exocrine pancreas capillary contained both desmin and vimentin; and the endothelial cells of the macrovasculatures and of all the other microvasculatures examined, including the vasa recta of the renal medulla, contained only vimentin. Such heterogeneity suggests that different types of adult chicken endothelial cells may have different embryological origins. To the extent that desmin and vimentin intermediate filaments may be functionally distinct, these results also suggest that different capillary endothelial cells may have different functional properties.

Animals↗
PubMed1985

Cell density and cell shape-related regulation of vimentin and cytokeratin synthesis. Inhibition of vimentin synthesis and appearance of a new 45 kD cytokeratin in dense epithelial cell cultures.

The pattern of the intermediate type filament protein synthesis was examined in cultured bovine mammary gland epithelial (BMGE) cells under conditions of varied cell shape and cell-cell contact. In dense monolayer and suspension cultures BMGE cells expressed a new cytokeratin of 45 kD identified as a member of the acidic subfamily of cytokeratins. This polypeptide has a phosphorylated component and is dissociated from the cytokeratins complex in the presence of 6.5 M urea. The mRNA of the new cytokeratin accumulated in dense cell cultures, as revealed by in vitro translation in a cell-free system. In BMGE-H cells that express also vimentin, the synthesis of vimentin decreased dramatically in dense cell cultures, while the synthesis of the 45 kD cytokeratin was maximal under these conditions. The results suggest that the expression of certain cytokeratins and that of vimentin can be coordinately regulated by factors in the cellular environment that effect cell shape and cell surface contacts.

Animals↗
PubMed1983

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis.

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.

Amnion↗
PubMed1989

Coexpression of cytokeratins and vimentin in normal and diseased thyroid glands. Lack of diagnostic utility of vimentin immunostaining.

We documented the coexpression of cytokeratins and vimentin in epithelial cells of the thyroid gland in 100 samples examined from 65 patients. These included normal, inflammatory, and neoplastic tissues that were routinely fixed in formalin and embedded in paraffin. The number of epithelial cells coexpressing the two intermediate filament proteins as well as the subcellular compartmentalization of vimentin immunoreactivity did not correlate with the various conditions of the thyroid gland. Therefore, we conclude that the immunolocalization of vimentin does not represent a useful adjunct tool for the histopathological diagnosis of thyroid diseases.

Humans↗
PubMed2000

Comparative anatomy of the cerebellar cortex in mice lacking vimentin, GFAP, and both vimentin and GFAP.

In the cerebellum of adult mammals, glial fibrillary acidic protein (GFAP) and vimentin (VIM) are coexpressed in Golgi epithelial cells (GEC), also known as Bergmann glia. In this study we used three transgenic knockout mice (GFAP, VIM and double GFAP and VIM) to analyze the involvement of these proteins in the building of glial filaments and in neuron-glia interactions. The cerebella of VIM, GFAP, and GFAP/VIM mutant mice were processed by the rapid Golgi method and also for electron microscopy. In VIM mutant mice, Bergmann fibers are hypertrophic with thickened appendages. In the electron microscope they appear as large glial profiles devoid of glial filaments, with embedded dendritic thorns and parallel fiber boutons. In addition, signs of degeneration are observed in Purkinje cells. In GFAP mutant mice, GEC exhibit fine, delicate processes, as those seen in wild-type animals, however, a large accumulation of lamellae and granular appendages was observed along their surfaces, which came into contact with each other. The electron microscope exhibited fine and scarce astroglial profiles containing some glial filaments, a stunted glia limitans, and the presence of large extracellular spaces. In double mutant mice, the two phenotypes are expressed but appear attenuated, with a total absence of glial filaments and the general appearance of immaturity for GEC. In conclusion, it appears that the absence of each of the proteins yields a specific phenotype and that the defects are not necessarily additive.

Animals↗
PubMed1992

Host cell factors controlling vimentin organization in the Xenopus oocyte.

To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins. Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. Maynell, and A. G. Polson. 1987. Development (Camb.). 100:543-557), which suggests that the same cellular factors are responsible for both. Before germinal vesicle breakdown, in the initial stage of oocyte maturation, large vimentin and keratin filament bundles appear in the animal hemisphere. As maturation proceeds, keratin filaments fragment into soluble oligomers (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787-797), while vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.

Amino Acid Sequence↗
PubMed1995

Association of vimentin intermediate filaments with the centrosome.

SW-13 cells that lack cytoplasmic intermediate filaments (IFs) were stably transfected with a human vimentin cDNA expression vector. Isolated subclones displayed two prevalent patterns of vimentin distribution as observed by indirect immuno-localization: (1) cytoplasmic filaments characteristic of a vimentin IF network; and (2) a distinct, juxtanuclear focus with limited filamentous extensions. Comparative analysis of two subclones that uniquely segregated these patterns of vimentin organization indicated that vimentin accumulated as a perinuclear focus in cells that expressed a 4-fold lower level of the protein. The observed variation in cellular organization was not due to detectable differences in vimentin protein modification, as determined by two-dimensional gel analysis. Increasing the amount of vimentin in a low expressing clone by a secondary transfection with human or mouse vimentin cDNA resulted in well-dispersed, cytoplasmic filaments, suggesting that the distinct juxtanuclear organization of vimentin arose due to lower cellular vimentin levels. Employing anti-gamma-tubulin and anti-vimentin antibodies, dual immunofluorescence together with confocal microscopy revealed that the juxtanuclear focus of vimentin was located in the centrosomal region. Electron microscopy showed a spheroidal, filamentous structure with at least some filaments closely associated with the pericentriolar material (PCM). Because vimentin IF organization is at least partially dependent on microtubules, the effects of nocodazole and taxol on perinuclear vimentin foci were examined. Neither drug affected the juxtanuclear localization of foci, although taxol (10 microM, 5 hours) caused a release of pericentriolar gamma-tubulin from the nuclear region in 50-60% of the cells. These studies indicate that lower, in vivo, levels of vimentin fail to form extended IFs but rather are organized as a perinuclear aggregate. Moreover, the PCM of the centrosome appears to possess attachment sites for vimentin IFs.

Animals↗
PubMed1991

Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils.

The effects of cGMP-dependent protein kinase (G-kinase), a major cellular receptor of cGMP, were investigated in activated human neutrophils. Immunocytochemistry demonstrated that G-kinase translocated from a diffuse localization in the cytoplasm to the cytoskeleton and nucleus after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), and transiently co-localized with the intermediate filament protein, vimentin. During this time period, the most remarkable co-localization of G-kinase and vimentin was observed between 1-2.5 min stimulation with fMLP. At that time co-localization of G-kinase and vimentin was predominantly confined to filaments which extended from regions adjacent to the nucleus into the uropod. Distinctive localization for only G-kinase was observed at the microtubule organizing center and euchromatin of the nucleus. The filamentous staining pattern for G-kinase and vimentin was enhanced in the presence of 8-Br-cGMP. Coincident with co-localization of G-kinase and vimentin in adherent neutrophils was a transient increase in cGMP levels and an increase in the phosphorylation of vimentin in fMLP-stimulated cells. The increase in cGMP levels was dependent upon cell adherence, was enhanced by preincubating neutrophils with L-arginine (the precursor for nitric oxide synthesis), and attenuated with the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. Phosphorylation of vimentin in the fMLP-stimulated neutrophil was observed in the presence or absence of exogenous cGMP, although in the presence of low concentrations of 8-Br-cGMP a more rapid phosphorylation of vimentin was observed that correlated with the enhanced co-localization of G-kinase and vimentin. Phosphorylation of vimentin was not observed in non-activated cells treated with 8-Br-cGMP, suggesting that phosphorylation only occurs when G-kinase is co-localized with vimentin. The presence of the protein kinase C inhibitors, staurosporine or H-7, did not inhibit vimentin phosphorylation during fMLP stimulation, while 8-Br-cGMP enhanced phosphorylation in fMLP-treated cells. This suggests that neither protein kinase C nor cAMP-dependent protein kinase catalyze the phosphorylation of vimentin in neutrophils activated by fMLP. These results indicate that vimentin and G-kinase are co-localized in neutrophils and that vimentin is phosphorylated by G-kinase in response to the co-localization of the two proteins. A model for the targeting of G-kinase and vimentin is presented which hypothesizes that the transient redistribution of G-kinase may regulate neutrophil activation.

Calcium↗
PubMed2006

Dissociation of Crk-associated substrate from the vimentin network is regulated by p21-activated kinase on ACh activation of airway smooth muscle.

The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, ACh stimulation of tracheal smooth muscle strips increased the ratio of soluble to insoluble vimentin (an index of vimentin disassembly) in association with force development. ACh activation also induced vimentin phosphorylation at Ser(56) as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips on contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The ACh-elicited decrease in CAS distribution in cytoskeletal vimentin was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser(56) and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during ACh activation of tracheal smooth muscle tissues.

Acetylcholine↗