Intermediate Filament Protein Vimentin Research Articles

Expression of Cytoskeletal Proteins in Hepatic Stellatecells Isolated from Normal and Cirrhotic Rat Liver

Hepatic stellate cells (HSC) are located in Disse spaces of normal rat liver. In their quiescent state they serve as a storage site for vitamin A. In fibrotic liver they become activated, proliferate and they undergo transdifferentiation into myofibroblast-like cells. Changes in the cell phenotype are accompanied by changes in the cellular cytoskeleton. We have studied the expression of alpha-smooth muscle actin and intermediate filament proteins vimentin, desmin and glial fibrillary acidic protein (GFAP) by immunocytochemistry in HSC cultured for 2 or 7 days after isolation. Normal or cirrhotic rat liver was perfused with solutions of pronase and collagenase and HSC were isolated by density gradient centrifugation of the resulting cell suspension. Liver cirrhosis was produced in rats by repeated carbon tetrachloride administration. Vimentin was detected in all cells from normal and cirrhotic liver. The concentration of desmin in the cells from cirrhotic liver was slightly higher than that in normal cells and it increased with time in culture. GFAP could be detected only in normal cells 2 days after their isolation. In contrast, alpha smooth muscle actin (alpha-SMA) was absent from normal cells at this time but its expression was pronouced later. In most cells from cirrhotic liver this antigen was already present on the second day of culture and its expression further increased.

Effect of Vimentin on Reactive Gliosis:In VitroandIn VivoAnalysis

Reactive gliosis is a prominent result of many types of insult to the central nervous system (CNS) and leads to the formation of glial scar that impedes the regeneration of axons. The intermediate filament protein vimentin is found in pathology of the CNS, mainly in the vicinity of injuries to the CNS. In the present study we investigated the role of vimentin in the formation of glial scars in vitro and in vivo by using immunohistochemistry, Western blot analysis, and in situ hybridization. In vitro experiments showed that the intensity of immunofluorescent labeling for vimentin and glial fibrillary acidic protein (GFAP) was consistently decreased in astrocytes after transfection with a retrovirus carrying antisense complementary DNA (cDNA) for vimentin. Transfection also inhibited the growth of astrocytes and decreased the expression of vimentin mRNA. In vivo studies demonstrated that transfection with the retrovirus carrying the antisense cDNA vimentin inhibited the upregulation of vimentin and GFAP in stab wounds in rat cerebrum. These results suggest that vimentin may play a key role in the formation of glial scars in the CNS.

Increased Cell Proliferation and Neurogenesis in the Hippocampal Dentate Gyrus of Old GFAP?/?Vim?/? Mice

In response to central nervous system (CNS) injury, and more discretely so also during aging, astrocytes become reactive and increase their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Studies of mice deficient in astrocytic intermediate filaments have provided insights into the function of reactive gliosis. Recently we demonstrated robust integration of retinal transplants (1) and increased posttraumatic synaptic regeneration (2) in GFAP(-/-)Vim(-/-) mice, suggesting that modulation of astrocyte activity affects the permissiveness of the CNS environment for regeneration. Neurogenesis in the adult mammalian CNS is restricted to essentially two regions, the hippocampus and the subventricular zone. Here, we assessed neurogenesis in the hippocampus of 18-month-old GFAP(-/-)Vim(-/-) mice. In the granular layer of the dentate gyrus, cell proliferation/survival was 34% higher and neurogenesis 36% higher in GFAP(-/-)Vim(-/-) mice than in wildtype controls. These findings suggest that the adult hippocampal neurogenesis in healthy old mice can be increased by modulating astrocyte reactivity.

Astrocytic proliferation in the piriform cortex of amygdala-kindled subjects: a quantitative study in partial versus fully kindled brains

Complex partial epilepsy is a seizure disorder in which attacks frequently arise from foci located in the temporal lobes. The amygdala-kindling model is a widely used model of complex partial epilepsy with secondary generalization. The present study was designed to quantitatively assess astrocytic changes in the rat piriform cortex in the amygdala-kindling model of epilepsy. Bromodeoxyuridine-injected subjects were sacrificed 24 h after the first stage 1 or fifth stage 5 seizure. Brain sections were prepared and examined quantitatively. A significantly higher number of dividing astrocytes (identified by co-labeling with antibodies to bromodeoxyuridine and to one of the astrocytic intermediate filament proteins glial fibrillary acidic protein or vimentin) was found in both partially kindled (stage 1) and fully kindled (stage 5) brains. The partially kindled brains had a significantly higher number of double-labeled cells on the side ipsilateral to stimulation. The opposite trend was observed in the fully kindled brains. Differences between the ipsilateral and contralateral sides of the kindled brain may suggest different role(s) for astrocytes in the development and progression of the seizure-prone state.

Structural proteins during brain development in the preterm and near-term ovine fetus and the effect of intermittent umbilical cord occlusion

ObjectiveThe purpose of this study was to determine the immunoreactivity of selected structural proteins in the preterm and near-term ovine fetal brain and the response to intermittent umbilical cord occlusion as a measure of altered cellular growth. The intermediate filament proteins nestin, vimentin, and glial fibrillary acidic protein was used as markers for astroglial maturation and astrogliosis, and myelin basic protein as a marker for oligodendrocytes and myelin formation. Study designFetal sheep (control and experimental groups at 0.75 and 0.90 of gestation) were studied over 4 days; umbilical cord occlusion was performed in the experimental group by complete inflation of an occluder cuff for 90 seconds every 30 minutes for 3 to 5 hours each day. Animals were then killed, and the fetal brain was perfusion fixed and processed for immunohistologic examination of the gray and white matter. Immunoreactivity was quantified with an image analysis system and expressed as the fractional area positive stain for each protein. ResultsIn both preterm and near-term animal groups, umbilical cord occlusion caused a large decline in arterial Po2 (to approximately 7 mm Hg), a modest decline in pH (to approximately 7.30), and a modest rise in Pco2 (to approximately 61 mm Hg; all P < .01), with a return to control values after the occluder release and no cumulative acidosis over each day of study. Vimentin and glial fibrillary acidic protein immunoreactivity showed reciprocal changes, with vimentin decreased and glial fibrillary acidic protein increased in both the gray and white matter of the control group from 0.75 to 0.90 of gestation, which can be attributed to the transition of radial glia into mature astrocytes. Myelin basic protein immunoreactivity increased approximately 3-fold in the white matter of the control group with advancing gestation, which likely reflected active oligodendrocyte differentiation and increased myelination at this time of development. Intermittent umbilical cord occlusion over 4 days resulted in an approximately 60% decrease in nestin, vimentin, and glial fibrillary acidic protein immunoreactivity, which was qualitatively similar for both the gray and white matter and likely indicative of altered protein synthesis and/or degradation, but only in the preterm group and with no change in myelin basic protein immunoreactivity. ConclusionThere is considerable change in the immunoreactivity of structural proteins within the ovine fetal brain over the latter part of gestation and consistent with a high rate of protein turnover, as previously reported. Intermittent umbilical cord occlusion as studied with minimal evidence for necrotic cell injury appears capable of altering selected protein synthesis/degradation, more so in younger animals when protein turnover is higher, which might then impact on the brain's development.

Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development.

The efficient functioning of striated muscle is dependent upon the structure of several cytoskeletal networks including myofibrils, microtubules, and intermediate filaments. However, little is known about how these networks function together during muscle differentiation and maintenance. In vitro studies suggest that members of the muscle-specific RING finger protein family (MURF-1, 2, and 3) act as cytoskeletal adaptors and signaling molecules by associating with myofibril components (including the giant protein, titin), microtubules and/or nuclear factors. We investigated the role of MURF-2, the least-characterized family member, in primary cultures of embryonic chick skeletal and cardiac myocytes. MURF-2 is detected as two species (approximately 55 kDa and approximately 60 kDa) in embryonic muscle, which are down-regulated in adult muscle. Although predominantly located diffusely in the cytoplasm, MURF-2 also colocalizes with a sub-group of microtubules and the M-line region of titin. Reducing MURF-2 levels in cardiac myocytes using antisense oligonucleotides perturbed the structure of stable microtubule populations, the intermediate filament proteins desmin and vimentin, and the sarcomeric M-line region. In contrast, other sarcomeric regions and dynamic microtubules remained unaffected. MURF-2 knock-down studies in skeletal myoblasts also delayed myoblast fusion and myofibrillogenesis. Furthermore, contractile activity was also affected. We speculate that some of the roles of MURF-2 are modulated via titin-based mechanisms.

Absence of glial fibrillary acidic protein and vimentin prevents hypertrophy of astrocytic processes and improves post-traumatic regeneration.

The regenerative capacity of the CNS is extremely limited. The reason for this is unclear, but glial cell involvement has been suspected, and oligodendrocytes have been implicated as inhibitors of neuroregeneration (Chen et al., 2000, GrandPre et al., 2000; Fournier et al., 2001). The role of astrocytes in this process was proposed but remains incompletely understood (Silver and Miller, 2004). Astrocyte activation (reactive gliosis) accompanies neurotrauma, stroke, neurodegenerative diseases, or tumors. Two prominent hallmarks of reactive gliosis are hypertrophy of astrocytic processes and upregulation of intermediate filaments. Using the entorhinal cortex lesion model in mice, we found that reactive astrocytes devoid of the intermediate filament proteins glial fibrillary acidic protein and vimentin (GFAP-/-Vim-/-), and consequently lacking intermediate filaments (Colucci-Guyon et al., 1994; Pekny et al., 1995; Eliasson et al., 1999), showed only a limited hypertrophy of cell processes. Instead, many processes were shorter and not straight, albeit the volume of neuropil reached by a single astrocyte was the same as in wild-type mice. This was accompanied by remarkable synaptic regeneration in the hippocampus. On a molecular level, GFAP-/-Vim-/- reactive astrocytes could not upregulate endothelin B receptors, suggesting that the upregulation is intermediate filament dependent. These findings show a novel role for intermediate filaments in astrocytes and implicate reactive astrocytes as potent inhibitors of neuroregeneration.

Molecular and Biophysical Characterization of Assembly-Starter Units of Human Vimentin

We have developed an assembly protocol for the intermediate filament (IF) protein vimentin based on a phosphate buffer system, which enables the dynamic formation of authentic IFs. The advantage of this physiological buffer is that analysis of the subunit interactions by chemical cross-linking of internal lysine residues becomes feasible. By this system, we have analyzed the potential interactions of the coiled-coil rod domains with one another, which are assumed to make a crucial contribution to IF formation and stability. We show that headless vimentin, which dimerizes under low salt conditions, associates into tetramers of the A 22-type configuration under assembly conditions, indicating that one of the effects of increasing the ionic strength is to favor coil 2–coil 2 interactions. Furthermore, in order to obtain insight into the molecular interactions that occur during the first phase of assembly of full-length vimentin, we employed a temperature-sensitive variant of human vimentin, which is arrested at the “unit-length filament” (ULF) state at room temperature, but starts to elongate upon raising the temperature to 37 °C. Most importantly, we demonstrate by cross-linking analysis that ULF formation predominantly involves A 11-type dimer–dimer interactions. The presence of A 22 and A 12 cross-linking products in mature IFs, however, indicates that major rearrangements do occur during the longitudinal annealing and radial compaction steps of IF assembly.

Formin homology domain protein (FHOD1) is a cyclic GMP-dependent protein kinase I-binding protein and substrate in vascular smooth muscle cells.

Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration.

Keratin 9 gene mutations in five Korean families with epidermolytic palmoplantar keratoderma

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant disease characterized clinically by localized palmoplantar thickening and histopathologically by granular degeneration of the epidermis. Recent molecular biological studies have revealed that EPPK is caused by mutations of the keratin 9 gene in sequences mainly encoding the highly conserved 1 A rod domain. Here we demonstrate a novel mutation of N160H (position 8 of the 1 A domain) and two other previously reported mutations, R162W and N160S, in five unrelated Korean families with EPPK. The three-dimensional structure of the 1 A domain of the related vimentin intermediate filament protein chain is now known. Based on its likely similarity to the keratin 9 chain, we predict that inappropriate amino acid substitutions in position 10 of 1 A will likely interfere with coiled-coil dimer stability, and those in position 8 will interfere with tetramer stability. Accordingly, these mutations compromise the structural integrity of the keratin intermediate filaments leading to the pathology of EPPK.

Caspase Proteolysis of Desmin Produces a Dominant-negative Inhibitor of Intermediate Filaments and Promotes Apoptosis

Caspase cleavage of key cytoskeletal proteins, including several intermediate filament proteins, triggers the dramatic disassembly of the cytoskeleton that characterizes apoptosis. Here we describe the muscle-specific intermediate filament protein desmin as a novel caspase substrate. Desmin is cleaved selectively at a conserved Asp residue in its L1-L2 linker domain (VEMD downward arrow M(264)) by caspase-6 in vitro and in myogenic cells undergoing apoptosis. We demonstrate that caspase cleavage of desmin at Asp(263) has important functional consequences, including the production of an amino-terminal cleavage product, N-desmin, which is unable to assemble into intermediate filaments, instead forming large intracellular aggregates. Moreover, N-desmin functions as a dominant-negative inhibitor of filament assembly, both for desmin and the structurally related intermediate filament protein vimentin. We also show that stable expression of a caspase cleavage-resistant desmin D263E mutant partially protects cells from tumor necrosis factor-alpha-induced apoptosis. Taken together, these results indicate that caspase proteolysis of desmin at Asp(263) produces a dominant-negative inhibitor of intermediate filaments and actively participates in the execution of apoptosis. In addition, these findings provide further evidence that the intermediate filament cytoskeleton has been targeted systematically for degradation during apoptosis.

Integrins are involved in synaptogenesis, cell spreading, and adhesion in the postnatal brain

Integrins are a major family of heterodimeric surface glycoproteins that act as adhesion molecules, have a spectrum of extracellular matrix (ECM) molecules as their ligands, and regulate a variety of cellular functions. Integrins are known to be critical to embryonic brain development, and recent studies have indicated their essential role in adult brain function, although their role in postnatal brain development and function has not been examined. Here, we used the organotypic slice culture system to investigate the role of integrins in postnatal hippocampal development by exposing the tissue to either an integrin competitive antagonist, the peptide GRGDSP containing Arg–Gly–Asp (RGD) attachment site, or to function-blocking β 1-integrin antibodies to disrupt integrin interactions. These experiments revealed that β 1-integrin antibodies interfered with spreading of the culture, resulting in a rapid and marked diminution of slice area. β 1-integrin antibodies and RGD peptide disrupted cell adhesion, causing cell detachment and migration of glial cells from the explant. The majority of the detached cells were of macroglial origin and switched to expression of the intermediate filament proteins vimentin and nestin, suggesting a developmental regression. The organotypic organization of slice cultures was not affected, although exposure to either integrin antagonist or antibody resulted in a statistically significant reduction in the number of synapses measured in the apical dendrites of CA1 pyramidal neurons. The results demonstrate that integrins markedly affect postnatal CNS development, in both ultrastructural construction and organizational processes.

Expression of intermediate filaments in conjunctival melanocytic lesions.

To our knowledge there have been no studies on intermediate filament (IF) expression in conjunctival melanocytic tumors to date. Melanocytic lesions occurring at various body sites are known to express, in addition to the predominant IF protein vimentin, the epithelial-specific cytokeratins (CKs) and the neuronal IF peripherin. The present study was therefore carried out to assess the expression of IF proteins in conjunctival melanocytic lesions. Paraffin-embedded material from 34 tumors - 16 conjunctival nevi, nine specimens of primary acquired melanosis (PAM; eight with and one without atypia), and nine conjunctival melanomas - was assessed after the application of a panel of antibodies directed against diverse IF proteins, including vimentin, CKs and peripherin. The most significant finding of this study was that all the tumors investigated expressed vimentin exclusively. While simple-epithelium CKs were found in epithelial cysts of nevi and in pseudoglandular portions adjacent to the melanoma, they were not identified in the tumor cells themselves. Similarly, peripherin and neurofilaments were not detected within any of the tumor cells. The IF expression pattern of conjunctival melanocytic lesions differs from that seen in melanocytic tumors of other body sites (including uveal melanomas), in that it includes neither CKs nor peripherin.

Visualization of the ER-to-cytosol dislocation reaction of a type I membrane protein.

The human cytomegalovirus gene products US2 and US11 induce proteasomal degradation of MHC class I heavy chains. We have generated an enhanced green fluorescent protein-class I heavy chain (EGFP-HC) chimeric molecule to study its dislocation and degradation in US2- and US11-expressing cells. The EGFP-HC fusion is stable in control cells, but is degraded rapidly in US2- or US11-expressing cells. Proteasome inhibitors induce in a time-dependent manner the accumulation of EGFP-HC molecules in US2- and US11-expressing cells, as assessed biochemically and by cytofluorimetry of intact cells. Pulse-chase analysis and subcellular fractionation show that EGFP-HC proteins are dislocated from the endoplasmic reticulum and can be recovered as deglycosylated fluorescent intermediates in the cytosol. These results raise the possibility that dislocation of glycoproteins from the ER may not require their full unfolding.

Lens membrane fraction associated intermediate filaments of different aged rats

Purpose. To describe the intermediate filament proteins vimentin, filensin and phakinin associated with different fractions isolated from neonatal, 10 day old and 20 day old rat lenses. Methods. Fractions were isolated by differential and density gradient centrifugation of lens homogenates from neonatal, 10 day old and 20 day old rats. Aliquots of the 8M urea soluble proteins of each fraction were separated by SDS PAGE, transferred to PVDF membranes, the membranes were probed with antibodies to vimentin, filensin or phakinin, and analyzed by computer. Results. Over the 20 day growth period, the water soluble fraction increased and the most abundant membrane fraction was characterized by a significant increase in its urea insoluble protein and a significant decrease in its urea soluble protein. There were no significant quantitative changes in any of the other fractions. The concentration of each intermediate filament protein was greatest in the cytoskeletal fraction and over the 20 day period, the amount of vimentin associated with this fraction dramatically decreased, and the amounts of filensin and phakinin dramatically increased. Among the membrane fractions, the greatest concentration of each intermediate filament protein was found in the non sedimenting membrane fraction (NSMF) which was the least abundant fraction recovered. Filensin and phakinin associated with the other three major membrane fractions increased over the 20 day growth period, but the level of vimentin did not significantly change. Conclusions. The NSMF may represent a domain of the lens plasma membrane particularly important in interaction between plasma membrane and cytoskeleton and as the membrane–cytoskeleton protein architecture of rat lens changes over the first 20 days of life, the changes are readily detected in the different membrane fractions.

Role of tyrosine kinases and the tyrosine phosphatase SptP in the interaction of Salmonella with host cells.

Salmonella has evolved an intimate functional interface with its host. Central to this interface is a battery of bacterial proteins delivered into host cells via a specialized organelle termed the type III secretion system. A subset of these bacterial proteins stimulates cellular responses by activating the Rho family GTPases Cdc42 and Rac. Stimulation of these responses leads to actin cytoskeleton reorganization and the activation of cellular transcription factors that result in bacterial uptake and proinflammatory cytokine production. Remarkably, the cellular responses stimulated by Salmonella are quickly reversed by another bacterial protein, SptP, which exerts its function as a GTPase-activating protein (GAP) for Cdc42 and Rac. In addition to its GAP activity located within its amino-terminus, the carboxy-terminal domain of SptP possesses potent tyrosine phosphatase activity. We show here that the tyrosine phosphatase activity of SptP is involved in reversing the MAP kinase activation that results from Salmonella infection. We also demonstrate an important role for tyrosine kinases, including ACK, in the cellular responses induced by Salmonella. We also found that a potential target for the tyrosine phosphatase activity of SptP is the intermediate filament protein vimentin, which is recruited to the membrane ruffles stimulated by Salmonella.

Polycystin-1 Interacts with Intermediate Filaments

Polycystin-1, the protein defective in a majority of patients with autosomal dominant polycystic kidney disease, is a ubiquitously expressed multi-span transmembrane protein of unknown function. Subcellular localization studies found this protein to be a component of various cell junctional complexes and to be associated with the cytoskeleton, but the specificity and nature of such associations are not known. To identify proteins that interact with the polycystin-1 C-tail (P1CT), this segment was used as bait in a yeast two-hybrid screening of a kidney epithelial cell library. The intermediate filament (IF) protein vimentin was identified as a strong polycystin-1-interacting partner. Cytokeratins K8 and K18 and desmin were also found to interact with P1CT. These interactions were mediated by coiled-coil motifs in polycystin-1 and IF proteins. Vimentin, cytokeratins K8 and K18, and desmin also bound directly to P1CT in GST pull-down and in in vitro filament assembly assays. Two observations confirmed these interactions in vivo: (i) a cell membrane-anchored form of recombinant P1CT decorated the IF network and was found to associate with the cytoskeleton in detergent-solubilized cells and (ii) endogenous polycystin-1 distributed with IF at desmosomal junctions. Polycystin-1 may utilize this association for structural, storage, or signaling functions.

Immunocytochemical analysis of vimentin expression patterns in porcine embryos suggests mesodermal differentiation from day 9 after conception.

The expression of the intermediate filament proteins vimentin and keratin in porcine embryos was studied by whole-mount immunocytochemistry between day 7 and day 11 after conception. Expression of vimentin was first detected in the inner cell mass of about 50% of the 9-day-old embryos. In elongated 11-day-old embryos, cells expressing vimentin were observed in the epiblast (after disappearance of Rauber's membrane) and in cells migrating from the epiblast between the trophoblast and the underlying hypoblast layer. A keratin-positive response was observed in trophectoderm cells at all stages. These findings suggest that inner cell mass cells in the pig start differentiating into mesodermal cells not later than day 9 after conception. While the delamination of the mesodermal germ layer is known to correlate with the loss of pluripotency of the inner cell mass cells, the early onset of mesodermal differentiation in the porcine embryo, characterized by vimentin expression and in contrast to the mouse, could in part be responsible for the lack of success in establishing pluripotent embryonic stem cell lines in this species. Our results suggest that further attempts to isolate inner cell mass-derived pluripotent cells should be attempted well before day 9 after conception.

A new marker for cup cells in the rabbit small intestine: expression of vimentin intermediate filament protein.

Cup cells in the intestinal lining in mammals are largely limited to the ileum, suggesting a specific but undetermined function. The investigation of these cells has been hindered by the lack of a suitable marker for immunohistochemistry. The small intestine of rabbits was examined for a potential molecular marker. Segments of New Zealand white rabbit small intestine, with and without Peyer's patches, were removed, rinsed, fixed with periodate-lysine-2% paraformaldehyde (PLP), and frozen for immunohistochemistry, or fixed in 2.5% glutaraldehyde solution for electron microscopy. The sections were stained with monoclonal antibodies (V9, 3B4) against vimentin, which is known to be absent in enterocytes other than antigen-sampling M cells, and observed by immunohistochemistry and immunoelectron microscopy. Epithelial expression of vimentin intermediate filament proteins was limited to a subpopulation of villus-associated cells and to cells over Peyer's patch domes. Immunoreactivity with vimentin antibodies was selectively found in cells with a cup-like indentation in the brush border in villous epithelium and in M cells in Peyer's patch follicle epithelium. Immunoelectron microscopy revealed that the labeled cells in villous epithelium had shorter microvilli than the adjacent absorptive cells, and small, sparse, mitochondria, which are ultrastructural characteristics of cup cells. Vimentin filaments were recognized in the area surrounding the nucleus and extending beneath the plasma membrane of cup cells. In rabbit small intestine, vimentin is useful for the detection of cup cells, as well as M cells, and may facilitate the investigation of these little understood cells.

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.