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PubMed1996

Immunocytochemical colocalization of desmin and vimentin in human fetal skeletal muscle cells.

BACKGROUND: Desmin and vimentin are the major intermediate filaments in muscle. They have been extensively studied in animal experiments. This study is the first to identify the distribution and to analyse the correlation of desmin and vimentin in human fetal skeletal muscle. Vimentin might be replaced by or transformed into desmin during myogenesis in chick embryo, although the precise process remains to be elucidated. The aim of this report is to evaluate the ratio of desmin to vimentin in human fetal muscle. METHODS: By double-labeling immunoelectron microscopy, desmin and vimentin intermediate filaments were localized in developing skeletal muscles of 20-29-week-old human fetuses. The ratio of desmin and vimentin was analyzed statistically. RESULTS: Two sizes of colloidal gold particles, 5 nm (vimentin) and 10 nm (desmin), were distributed along the intermediate filaments. The commonest distance between gold particles was approximately 40-50 nm. Desmin and vimentin labeled with gold particles were arranged very close together in the same intermediate filament. The ratio of vimentin to desmin varied but the amount of vimentin decreased progressively from the undifferentiated myoblast to the differentiated myocytes. As the fetuses developed, desmin increased and vimentin decreased. Desmin and vimentin intermediate filaments were identified in the intermyofibrils of differentiated myocytes, in subsarcolemmal space, and in myoblast. CONCLUSIONS: Desmin and vimentin were colocalized in the same intermediate filaments. More vimentin existed in the less differentiated myocytes, although a small number of desmin filaments were already found in undifferentiated myoblasts. These intermediate filaments may not only connect myofibril bundles, cell organelles, and cell membrane but also provide a basis for myofibrillogenesis that is similar to relation between connective fibers and parenchymal cells.

Desmin↗
PubMed1989

Vimentin: changes in distribution during brain development.

This paper examines both the anatomical changes in the distribution of vimentin intermediate filament protein and the biochemical changes in vimentin and its degradative enzyme during postnatal brain development in the tree shrew (Tupaia belangeri). A pattern of multiple immunoreactive bands at birth (postnatal day 0, or P0) was revealed in nitrocellulose blots of polyacrylamide gels ("Western blots"). These multiple bands gradually disappear during development, and in the adult a single band at the published molecular weight for vimentin (57 kD) is seen. This pattern of bands probably reflects shifts in the activity of a calcium-activated vimentin protease. The changes in the anatomical distribution of vimentin-immunoreactive (vimentin+) cells and their fine processes parallel the biochemical shifts seen in immunoblots. We have examined the neocortex, lateral geniculate nucleus (LGN), and hippocampus in detail. During the first postnatal week, vimentin+ glia, especially radial glia, are prominent in both neocortex and hippocampus. In contrast, only a few vimentin+ radial glia remain in the thalamus at this age. Vimentin+ glia appear to coincide with bundles of axons and often seem to outline subdivisions of thalamic nuclei. Additionally, cellular layers of the lateral geniculate nucleus (LGN) appear to stain with antibodies to vimentin several days before the characteristic neuronal cell layers appear in this area. During the second postnatal week, vimentin+ cells appear in "patches" throughout the cortex. Some subdivisions of the thalamus and hippocampus (as defined by cytoarchitectonic differences in the adult) are distinct when the tissue is stained with an antibody to vimentin, even though a conventional Nissl stain at this age shows no apparent delineation in these same regions. Finally, in the adult, only a few vimentin+ cells remain, primarily in the white matter. Taken together, these results suggest that the remodeling of vimentin+ intermediate filaments in immature glial cells (including radial glia) is paralleled by the action of the enzyme which breaks down these filaments. The apparent activity of this enzyme is high early in development as radial and other glia are rapidly dividing and undergoing morphological changes, with a decrease in activity in the juvenile and adult brain, as immature glial cells are supplanted by mature forms.

Aging↗
PubMed2006

Role of vimentin in smooth muscle force development.

Vimentin intermediate filaments undergo spatial reorganization in cultured smooth muscle cells in response to contractile activation; however, the role of vimentin in the physiological properties of smooth muscle has not been well elucidated. Tracheal smooth muscle strips were loaded with antisense oligonucleotides (ODNs) against vimentin and then cultured for 2 days to allow for protein degradation. Treatment with vimentin antisense, but not sense, ODNs suppressed vimentin protein expression; neither vimentin antisense nor sense ODNs affected protein levels of desmin and actin. Force development in response to ACh stimulation or KCl depolarization was lower in vimentin-deficient tissues than in vimentin sense ODN- or non-ODN-treated muscle strips. Passive tension was also depressed in vimentin-depleted muscle tissues. Vimentin downregulation did not attenuate increases in myosin light chain (MLC) phosphorylation in response to contractile stimulation or basal MLC phosphorylation. In vimentin sense ODN-treated or non-ODN-treated smooth muscle strips, the desmosomal protein plakoglobin was primarily localized in the cell periphery. The membrane-associated localization of plakoglobin was reduced in vimentin-depleted muscle tissues. These studies suggest that vimentin filaments play an important role in mediating active force development and passive tension, which are not regulated by MLC phosphorylation. Vimentin downregulation impairs the structural organization of desmosomes, which may be associated with the decrease in force development.

Acetylcholine↗
PubMed1997

Reorganization of a novel vimentin-associated protein in 3T3-L1 cells during adipose conversion.

We have found that the antibody A2, a marker for the capsule of steroidogenic lipid droplets, reacts with an intermediate filament-associated protein, P200, in 3T3-L1 preadipocytes. Supporting evidence came from the colocalization pattern of P200 with vimentin in double label experiments. The association of P200 with vimentin was further confirmed by its copurification with vimentin after high salt extraction and colocalization of these two proteins in high salt-extracted and vinblastine-treated cells. In preadipocytes this protein was distributed on the vimentin filament network. At the early stage of adipose conversion, this protein was found to encircle nascent lipid droplets ranging from 0.1 to 0.2 micron, accompanied with a decreased distribution on the vimentin filament system. This infers a possible translocation of P200 from the vimentin filaments to the droplet surface. Meanwhile, the vimentin filaments remained in a normal distribution in the cytoplasm and were apparently not associated with the nascent droplet. The association of vimentin filaments to droplet surfaces became prominent in lipid droplets larger than 0.2 micron, forming a typical vimentin cage. Immunogold staining also confirmed the translocation of P200 immunoreactivity from the droplet surface to the vimentin cage. The relocation of P200 from the cytoplasmic vimentin filaments to the droplet surface prior to the formation of the vimentin cage, as well as the reorganization of this protein in the vimentin cage, suggests a stabilizing role in the lipid droplet formation and an inducing function of this protein in the formation of the vimentin cage.

3T3 Cells↗
PubMed1994

Fate of a headless vimentin protein in stable cell cultures: soluble and cytoskeletal forms.

The non-alpha-helical head domains of cytoskeletal intermediate filaments (IFs) are considered to play an important role in IF assembly and stability. We have investigated the fate of a "headless" mutant vimentin protein in cell types that either lack cytoplasmic IFs or contain preexisting IF networks (keratin and vimentin). Stable clones expressing a transfected headless vimentin cDNA were individually analyzed in order to avoid variabilities introduced by transient transfection and to compare the levels and effects of the mutant vimentin protein more accurately. In cells lacking IFs, the mutant protein existed in a diffuse, soluble form as determined by immunofluorescence and biochemical protein fractionation. In cells possessing vimentin IFs, the headless vimentin was highly dispersed throughout the cytoplasm, including lamellopodia. Expression levels in individual clones were as high as sevenfold greater than the endogenous vimentin component. Although the majority of the headless vimentin was highly soluble, a residual portion of the mutant vimentin colocalized with vimentin filaments and consistently comprised about 25% of the cytoskeletal vimentin network. These results demonstrate that the mutant protein can be stably expressed at relatively high levels without deleterious cellular effects or disruption of endogenous vimentin filaments. The observed specific ratio of mutant to wild-type vimentin (1:3) in the cytoskeleton supports IF in vivo assembly via specific hybrid tetramer formation and, further, that at least three intact head domains are required for competent tetramer formation and IF assembly.

Animals↗
PubMed1991

Vimentin mRNA expression increases after corticospinal axotomy in the adult hamster.

We examined changes in vimentin gene expression during Wallerian degeneration after corticospinal axotomy in the adult hamster. Vimentin, which is the product of a type III intermediate filament (IF) gene, is expressed in various cells of mesenchymal origin, including microvascular endothelial cells, microglia and developing astrocytes. While increases in vimentin protein have been observed after various types of central nervous system (CNS) injury, it is not known whether this increase is due to increased vimentin mRNA expression. There is also conflicting evidence as to which cells are expressing increased levels of vimentin. In the present study we used in situ hybridization and double-label immunofluorescence techniques to address these issues. A 35S-labeled vimentin cDNA probe was used for in situ hybridizations of brain stem sections obtained 2, 7 and 14 days after unilateral transection of the corticospinal tract in the caudal medulla of adult hamsters. Autoradiography showed that an increase in vimentin mRNA associated with the degenerating corticospinal tract occurred by 2 days after axotomy and that the levels remained elevated for at least 14 days. Immunoblotting and immunocytochemical studies indicated that vimentin protein levels were increased in the degenerating corticospinal tract. Double-label immunofluorescence revealed many vimentin-positive cells and processes that were also labeled with GFAP antibody. In addition, cells and processes that were vimentin-negative but GFAP-positive were also found in the degenerating tract. We suggest that the reactive cells which possessed both vimentin and GFAP were reactive astrocytes of astroblastic origin while those that expressed only GFAP were derived from mature astrocytes. Other vimentin-positive cells/processes did not label with anti-GFAP and thus were either microglial, endothelial or inflammatory cells. These results demonstrate that an increase in vimentin mRNA occurs during Wallerian degeneration after corticospinal axotomy and that this increase is likely to be due to contributions from more than one cell type.

Animals↗
PubMed1994

Differential organization of desmin and vimentin in muscle is due to differences in their head domains.

In most myogenic systems, synthesis of the intermediate filament (IF) protein vimentin precedes the synthesis of the muscle-specific IF protein desmin. In the dorsal myotome of the Xenopus embryo, however, there is no preexisting vimentin filament system and desmin's initial organization is quite different from that seen in vimentin-containing myocytes (Cary and Klymkowsky, 1994. Differentiation. In press.). To determine whether the organization of IFs in the Xenopus myotome reflects features unique to Xenopus or is due to specific properties of desmin, we used the injection of plasmid DNA to drive the synthesis of vimentin or desmin in myotomal cells. At low levels of accumulation, exogenous vimentin and desmin both enter into the endogenous desmin system of the myotomal cell. At higher levels exogenous vimentin forms longitudinal IF systems similar to those seen in vimentin-expressing myogenic systems and massive IF bundles. Exogenous desmin, on the other hand, formed a reticular IF meshwork and non-filamentous aggregates. In embryonic epithelial cells, both vimentin and desmin formed extended IF networks. Vimentin and desmin differ most dramatically in their NH2-terminal "head" regions. To determine whether the head region was responsible for the differences in the behavior of these two proteins, we constructed plasmids encoding chimeric proteins in which the head of one was attached to the body of the other. In muscle, the vimentin head-desmin body (VDD) polypeptide formed longitudinal IFs and massive IF bundles like vimentin. The desmin head-vimentin body (DVV) polypeptide, on the other hand, formed IF meshworks and non-filamentous structures like desmin. In embryonic epithelial cells DVV formed a discrete filament network while VDD did not. Based on the behavior of these chimeric proteins, we conclude that the head domains of vimentin and desmin are structurally distinct and not interchangeable, and that the head domain of desmin is largely responsible for desmin's muscle-specific behaviors.

Amino Acid Sequence↗
PubMed2005

Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis.

Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its significance has remained unknown. Here we demonstrated a direct interaction between Plk1 and vimentin-Ser55 phosphorylated by Cdk1, an event that led to Plk1 activation and further vimentin phosphorylation. Plk1 phosphorylated vimentin at approximately 1 mol phosphate/mol substrate, which partly inhibited its filament forming ability, in vitro. Plk1 induced the phosphorylation of vimentin-Ser82, which was elevated from metaphase and maintained until the end of mitosis. This elevation followed the Cdk1-induced vimentin-Ser55 phosphorylation, and was impaired by Plk1 depletion. Mutational analyses revealed that Plk1-induced vimentin-Ser82 phosphorylation plays an important role in vimentin filaments segregation, coordinately with Rho-kinase and Aurora-B. Taken together, these results indicated a novel mechanism that Cdk1 regulated mitotic vimentin phosphorylation via not only a direct enzyme reaction but also Plk1 recruitment to vimentin.

Actin Cytoskeleton↗
PubMed1989

A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus.

We have developed a whole-mount immunocytochemical method for Xenopus and used it to map the expression of the intermediate filament protein vimentin during early embryogenesis. We used two monoclonal antibodies, 14h7 and RV202. Both label vimentin filaments in Xenopus A6 cells, RV202 reacts specifically with vimentin (Mr, 55 x 10(3] on Western blots of A6 cells and embryos. 14h7 reacts with vimentin and a second, insoluble polypeptide of 57 x 10(3) Mr found in A6 cells. The 57 x 10(3) Mr polypeptide appears to be an intermediate filament protein immunochemically related to vimentin. In the whole-mount embryo, we first found vimentin at the time of neural tube closure (stage 19) in cells located at the lateral margins of the neural tube. By stage 26, these cells, which are presumably radial glia, are present along the entire length of the neural tube and in the tail bud. Cells in the optic vesicles express vimentin by stage 24. Vimentin-expressing mesenchymal cells appear on the surface of the somites at stage 22/23; these cells appear first on anterior somites and on progressively more posterior somites as development continues. Beginning at stage 24, vimentin appears in mesenchymal cells located ventral to the somites and associated with the pronephric ducts; these ventral cells first appear below the anterior somites and later appear below more posterior somites. The dorsal fin mesenchyme expresses vimentin at stage 26. In the head, both mesodermally-derived and neural-crest-derived mesenchymal tissues express vimentin by stage 26. These include the mesenchyme of the branchial arches, the mandibular arch, the corneal epithelium, the eye, the meninges and mesenchyme surrounding the otic vesicle. By stage 33, vimentin-expressing mesenchymal cells are present in the pericardial cavity and line the vitelline veins. Vimentin expression appears to be a marker for the differentiation of a subset of central nervous system cells and of head and body mesenchyme in the early Xenopus embryo.

Animals↗
PubMed1998

PDGF induces reorganization of vimentin filaments.

In this study we demonstrate that stimulation with platelet-derived growth factor (PDGF) leads to a marked reorganization of the vimentin filaments in porcine aortic endothelial (PAE) cells ectopically expressing the PDGF beta-receptor. Within 20 minutes after stimulation, the well-spread fine fibrillar vimentin was reorganized as the filaments aggregated into a dense coil around the nucleus. The solubility of vimentin upon Nonidet-P40-extraction of cells decreased considerably after PDGF stimulation, indicating that PDGF caused a redistribution of vimentin to a less soluble compartment. In addition, an increased tyrosine phosphorylation of vimentin was observed. The redistribution of vimentin was not a direct consequence of its tyrosine phosphorylation, since treatment of cells with an inhibitor for the cytoplasmic tyrosine kinase Src, attenuated phosphorylation but not redistribution of vimentin. These changes in the distribution of vimentin occurred in conjunction with reorganization of actin filaments. In PAE cells expressing a Y740/751F mutant receptor that is unable to bind and activate phosphatidylinositol 3'-kinase (PI3-kinase), the distribution of vimentin was virtually unaffected by PDGF stimulation. Thus, PI3-kinase is important for vimentin reorganization, in addition to its previously demonstrated role in actin reorganization. The small GTPase Rac has previously been shown to be involved downstream of PI3-kinase in the reorganization of actin filaments. In PAE cells overexpressing dominant negative Rac1 (N17Rac1), no change in the fine fibrillar vimentin network was seen after PDGF-BB stimulation, whereas in PAE cells overexpressing constitutively active Rac1 (V12Rac1), there was a dramatic change in vimentin filament organization independent of PDGF stimulation. These data indicate that PDGF causes a reorganization of microfilaments as well as intermediate filaments in its target cells and suggest an important role for Rac downstream of PI3-kinase in the PDGF stimulated reorganization of both actin and vimentin filaments.

Actin Cytoskeleton↗
PubMed1998

DNA-mediated transport of the intermediate filament protein vimentin into the nucleus of cultured cells.

A number of characteristic properties of intermediate filament (IF) proteins, such as nucleic acid-binding activity, affinity for histones and structural relatedness to transcription factors and nuclear matrix proteins, in conjunction with the tight association of IFs with the nucleus, suggest that these proteins might also fulfill nuclear functions in addition to their structure-organizing and -stabilizing activities in the cytoplasm. Yet, cytoplasmic IF proteins do not possess nuclear localization signals. In a search for carriers capable of transporting the IF protein vimentin into the nucleus, complexes of FITC-vimentin with various DNAs were microinjected into the cytoplasm of cultured cells and the intracellular distribution of the protein was followed by confocal laser scanning microscopy. The single-stranded oligodeoxyribonucleotides oligo(dG)25, oligo[d(GT)12G] and oligo[d(G3T2A)4G] proved to be excellent nuclear carriers for vimentin. However, in fibroblasts, fluorescence-labeled vimentin taken up by the nuclei remained undetectable with affinity-purified, polyclonal anti-vimentin antibody, whereas it was readily identifiable in the nuclei of microinjected epithelial cells in this way. Moreover, when FITC-vimentin was preinjected into fibroblasts and allowed to assemble into the endogenous vimentin filament system, it was still transferred into the nucleus by post-injected oligo(dG)25, although to a lesser extent. Superhelical circular DNAs, like pBR322, SV40 and mitochondrial DNA, were also characterized by considerable capacities for nuclear vimentin transport; these transport potentials were totally destroyed by relaxation or linearization of the DNA molecules. Nevertheless, certain linear double-stranded DNA molecules with a high affinity for vimentin IFs, such as repetitive telomere and centromere or mobile long interspersed repeat (LINE) DNA, could carry FITC-vimentin into the nucleus. This was also true for a 375 bp extrachromosomal linear DNA fragment which occurs in the cytoplasm of mouse tumor cells and which is capable of immortalizing human lymphocytes. On the basis of these results, it appears very likely that cellular and viral products of reverse transcription as well as other extrachromosomal DNAs, which are circular, superhelical and apparently shuttling between the cytoplasm and the nucleus (eccDNA), are constantly loaded with vimentin in vimentin-positive cells. Since such DNAs are considered as markers of genomic instability, it is conceivable that vimentin directly participates as an architectural, chromatin-modifying protein in recombinatorial processes set off by these DNAs in the nucleus.

Animals↗
PubMedDate not supplied

Expression of vimentin intermediate filament in human odontoblast.

BACKGROUND: Vimentin (57 Kda) is a cytoskeletal protein. Odontoblasts contain vimentin and it seems that this protein may function to keep the organelles and the nucleus in a definite place. However little is known about vimentin in the cytoskeleton of odontoblast processes. The purpose of the present study was, therefore to immunolocalize vimentin intermediate filament in odontoblast body and process in order to clarify the distribution of this cytoskeletal element. METHODS: 12 extracted intact premolars, from children, were used in the present study. Each specimen was decalcified in EDTA. Each tissue portion was embedded in paraffin. On sections a monoclonal anti-vimentin antibody was applied. The immunoreaction was visualized by ABC technique. RESULTS: Vimentin was expressed in the cell body and cell process of odontoblasts, however with a different immunolabeling pattern related to the topographical area of observations. In odontoblast cell bodies vimentin showed a perinuclear and cytoplasmatic staining. In the very initial portion of odontoblast process immunoreaction products for vimentin were observed in the core of the process. In the middle zone of dentin vimentin immunoreactions products also showed a granular and cross-bridge arrangements, and also, vimentin was also detected under the plasma membrane, at the periphery of the odontoblast process. Nearby the dentino-enamel junction vimentin immunolabeling was appreciated, mainly under the plasma membrane. CONCLUSIONS: On the basis of vimentin distribution in the odontoblast process it seems plausible to assume that this IF vimentin is important in forming a flexible scaffold essential for structuring cytoplasm.

Child↗
PubMed1992

Vimentin metaplasia in renal cortical tubules of preneoplastic, neoplastic, aging, and regenerative lesions of rats and humans.

Vimentin expression was studied immunohistochemically in renal cortical tubules of untreated male rats of various ages, rats exposed to toxins (barbital sodium, folic acid) and carcinogens (streptozotocin, N-bis(2-hydroxypropyl)nitrosamine, barbital sodium, and in humans of various ages with or without renal epithelial tumors. Fetal, neonatal, and young adult rats did not express vimentin in renal cortical tubules. Regenerative renal tubular lesions from rats with aging nephropathy and from rats with toxic nephropathy both expressed vimentin. Mitogenic lesions induced by folic acid at 24 hours, however, were not immunoreactive for vimentin. Carcinogen-induced preneoplastic renal cortical tubular lesions in rats were most often focally immunoreactive whereas strong vimentin expression was found in almost all induced renal tumors. In kidneys of three children (younger than 2 years of age), vimentin was not found in renal cortical tubular cells except in rare individual cells in one case. Vimentin was abundant in basophilic regenerative tubules in kidneys of aged individuals, however. Most (7/10) human renal carcinomas and latent preneoplastic or neoplastic renal tubular lesions found incidentally at autopsy (2/4) showed vimentin expression. The authors suggest that the switching to vimentin expression in phenotypically normal renal cortical tubular cells in rats and humans, which do not usually express the intermediate filament protein vimentin, should be considered vimentin metaplasia. Vimentin expression is dissociated from increased cell proliferation in hyperplastic and neoplastic lesions, however. Instead the degree of dedifferentiation of the tubule cells and changes in phenotype were associated with vimentin expression.

Age Factors↗
PubMed1996

Expression of intermediate filament desmin and vimentin in the human fetal heart.

BACKGROUND: The intermediate filament (IF) desmin provides support for contractile machinery in muscle cells, and vimentin plays an important role in maintaining the stability of mesenchymal cells and in signal transduction. However, development of IFs in heart tissue during intrauterine life in human is not well established. METHODS: In the present study, development of desmin and vimentin in human fetal hearts aged 9-28 weeks of gestation (n = 41) were investigated by immunohistochemistry with monoclonal antibodies against desmin and vimentin. Relative density of fluorescence of each sample was determined by densitometry. Left ventricle (LV) tissues from a 1-year-old child (n = 1) were examined by immunohistochemistry for postnatal comparison. Western blot analyses were done with only a few randomly selected LV tissues from fetuses of 9, 20, and 28 weeks gestation to assess trends of desmin and vimentin expression. RESULTS: By Western blot analyses, 53-kDa desmin and 54-kDa vimentin were present in all fetal heart tissues examined. Desmin intensity was progressively increased with increasing fetal age, whereas vimentin intensity decreased. Desmin was present only in cardiomyocytes. In the earlier period (10-14 weeks gestation), desmin was localized along the cardiomyocyte membrane and/or Z lines in regular intervals, and later (25-28 weeks gestation) it was structurally well integrated; however, its network was incomplete. Only cardiomyocytes from a 1-year-old child revealed highly developed and integrated desmin lattices. However, vimentin was present in the mesenchymal tissue including fibroblasts and surrounding blood vessels. In part, some cardiomyocytes showed a weakly positive reaction with monoclonal antibody against vimentin in 9-14 weeks gestation. Vimentin-positive areas, however, were progressively diminished with increasing fetal age. Vimentin was present only in the connective tissue and coverings of the 1-year-old child's heart. Relative density of fluorescence of desmin was increased with increasing fetal age, whereas that of vimentin decreased. CONCLUSIONS: These results indicate that there is a fetal age (or gestation)-dependent expression of IFs in human fetal heart: desmin increases with increasing fetal age, whereas vimentin decreases.

Blotting, Western↗
PubMed1995

Structural and biological consequences of increased vimentin expression in simple epithelial cell types.

Cytoskeletal intermediate filaments (IFs) constitute a diverse family of proteins whose members are expressed in tissue-specific patterns. Although vimentin IFs are normally restricted to mesenchyme, a variety of cell types express vimentin alone or together with cell-specific IFs during growth, differentiation, and neoplasia. In this study, we have investigated the influence of increased vimentin expression on the simple epithelial cell phenotype. An expression vector encoding a human vimentin cDNA was transfected into murine HR9 endoderm and F9 embryonal carcinoma cell lines, which serve as models for early extraembryonic epithelial differentiation. Stable clones that expressed varying levels of human vimentin were characterized by human vimentin were characterized by immunofluorescence and biochemical analysis. A relatively high level of vimentin expression in HR9 and differentiated F9 epithelial cells resulted in aberrant vimentin structures with co-collapse of keratin K8/K18 filaments and lowered amounts of keratin protein. In F9 epithelial cells, the desmosomal proteins DP I/II did not appear to localize to cell surface desmosome s but rather but rather co-aggregated with the perturbed IFs. Although overall cell morphology was not dramatically altered, individual nuclei were distorted by excess intracellular vimentin. Furthermore, cell proliferation as well as the cell spreading response time were slowed. Ther appears to be a threshold effect regarding overall vimentin levels as cells that expressed lower amounts of the human vimentin exhibited no obvious structural nor biological effects. Our results demonstrate that wild-type vimentin can act as a "mutant" protein when present at high intracellular levels, inducing a variety of phenotypic changes.

Animals↗
PubMed1995

Analysis of the interdependent localization of vimentin and microtubules in neoplastic myoepithelial cells.

Salivary gland neoplastic myoepithelial cells in culture form very thin cytoplasmic processes in which the vimentin network is well dispersed. These vimentin filaments can be individually visualized by immunofluorescence. In this study, we have analyzed the role of microtubules in the distension and organization of the vimentin filament network found in these cells. We find that vimentin filaments colocalize along microtubules; however, a significant number of filaments can also be found in microtubule-free domains. Additionally, vimentin filaments are absent from large domains of microtubule-rich domains. Treatment of neoplastic myepithelial cells with the microtubule inhibitor nocodazole did not cause any retraction of the distended vimentin network. This observation suggests that the structural integrity of microtubules is not important for the stability of the vimentin network. Combining procedures for transient disruption of vimentin filaments and microtubules we observed that, in the absence of microtubules, the vimentin network could reassemble in the perinuclear region but was unable to extend toward the cell periphery. The dispersion of vimentin filaments to the peripheral regions of the cytoplasm could only be observed upon microtubule reassembly. This indicates that microtubules are not required for the stability of the vimentin network, but the dispersion of vimentin filaments to the peripheral cytoplasm depends on active interactions with microtubules.

Adenoma, Pleomorphic↗
PubMed1992

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.

Animals↗
PubMed1988

Acquisition of vimentin in astrocytes cultured from postnatal rat brain.

Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin-GFAP transition which occurs during glial development in situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.

Animals↗