Intermediate Filament Protein Vimentin Research Articles

Dominant cataract formation in association with a vimentin assembly disrupting mutation

Cataracts are characterized by an opacification of the eye lens, often caused by protein misfolding and aggregation. The intermediate filament protein vimentin, which is highly expressed in lens fiber cells and in mesenchymal tissues, is a main structural determinant in these cells forming a membrane-connected cytoskeleton. Additional functions of vimentin remain to be identified. Here, we demonstrate that a mutation in VIM causes a dominant, pulverulent cataract. We sequenced the complete human VIM gene in 90 individuals suffering from congenital cataract and found a G596A change in exon 1 in a single individual, causing the missense mutation E151K in coil 1B of vimentin. The mutant vimentin formed an aberrant vimentin cytoskeleton and increased the proteasome activity in transfected cells. Furthermore, this mutation causes a severe kinetic defect in vimentin assembly both in vitro and in vivo. Hence, in conjunction with available mouse and cell culture models, our results reveal for the first time an important functional role for vimentin in the maintenance of lens integrity. Finally, this invites novel therapy approaches for cataracts.

Dynamic Expression of Vimentin in Bovine Blastocysts in ExtendedIn VitroCulture

In mammalian blastocysts, the inner cell mass (ICM) differentiates into the epiblast and the hypoblast. In vitro culture of bovine pre-implantation embryos is generally carried out for a limited duration at most up to the blastocyst formation, therefore, it is unclear whether and when the differentiation of ICM occurs in vitro. In bovine species, epiblast formation is characterized by the expression of the intermediate filament protein vimentin. In the present study, the spatial and temporal expression of vimentin in bovine blastocysts maintained under extended in vitro culture (EIVC) was investigated. Bovine blastocysts produced by in vitro maturation, fertilization, and culture were further cultured in vitro in modified synthetic oviduct fluid up to day 12 (fertilization = day 0). Early, expanded, or hatched blastocysts on day 8 and hatched blastocysts on days 9 and 12 were individually subjected to indirect immunofluorescence analysis with an anti-vimentin monoclonal antibody and to semi-quantitative reverse transcription-polymerase chain reaction analysis to examine the vimentin expression. Most of the blastocysts (25/26) did not exhibit any significant immunostaining for the vimentin protein on day 8. On the other hand, the vimentin protein was observed in 59.1% (13/22) and 86.4% (19/22) of the hatched blastocysts on days 9 and 12, respectively. Vimentin was detected as filaments localized within a portion of the ICM, and its expression levels varied among the embryos. Vimentin transcript could not be detected in 50% (3/6) of the blastocysts on day 8, whereas it was detected in all the blastocysts on days 9 and 12. Examination of the day 8 blastocysts revealed that compared with the slow developers, the fast developers (blastocysts which had already expanded by 168 h post-insemination) had significantly higher levels of vimentin transcripts. These results indicate that the ICM differentiates dynamically in bovine blastocysts maintained under EIVC, as reflected by the expression of vimentin and that the higher vimentin expression may reflect the higher developmental competence of embryos.

New Advances in Breast Cancer Metastasis

New Advances in Breast Cancer Metastasis

Protein Quality Control: On IPODs and Other JUNQ

The accumulation of misfolded cytosolic or aggregation-prone proteins leads to cellular stress. To protect the cell, damaged or aggregated proteins are actively sequestered in two newly discovered quality control compartments, JUNQ and IPOD, which are highly conserved in evolution.

Antisense vimentin cDNA combined with chondroitinase ABC reduces glial scar and cystic cavity formation following spinal cord injury in rats

The formation of glial scar and cystic cavities restricts axon regeneration after spinal cord injury. Chondroitin sulphate proteoglycans (CSPGs) are regarded as the prominent inhibitory molecules in the glial scar, and their inhibitory effects may be abolished in part by chondroitinase ABC (ChABC), which can digest CSPGs. CSPGs are secreted mostly by reactive astrocytes, which form dense scar tissues. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. Previously we have shown that retroviruses carrying full-length antisense vimentin cDNA reduce reactive gliosis. Here we administered both antisense vimentin cDNA and ChABC to hemisected rat spinal cords. Using RT-PCR, Western blotting and immunohistochemistry, we found that the combined treatment reduced the formation of glial scar and cystic cavities through degrading CSPGs molecules and inhibiting intermediate filament proteins. The modified intra- and extra-cellular architecture may alter the physical and biochemical characteristics of the scar, and the combined therapy might be used to inhibit glial scar formation.

The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

BackgroundColocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed.ResultsThe results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-α-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association.ConclusionWe hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.

Vimentin-immunopositive cells in the rat telencephalon after experimental ischemic stroke

The aim of the present work was to perform immunocytochemical studies of cells synthesizing the intermediate filament protein vimentin in the telencephalon of intact rats and rats subjected to unilateral permanent occlusion of the middle cerebral artery, which models ischemic stroke. In the intact rat brain, vimentin-containing cells were seen in the brain barriers. At 14 days from occlusion of the middle cerebral artery, there were numerous vimentin-immunopositive cells in the perifocal damage zone, and these accounted for a significant proportion of the cells in the regenerating nervous tissue at the boundary with undamaged tissue. The subependymal proliferative zone contained a significant number of vimentin-negative small cells, located between the long processes of vimentin-immunopositive cells running towards the lesioned zone. These data provide evidence of the predominant location of vimentin-immunopositive brain cells (in both intact and lesioned animals) in the zones forming barrier structures.

Targets of Caspase-6 Activity in Human Neurons and Alzheimer Disease

Caspase-6 activation occurs early in Alzheimer disease and sometimes precedes the clinical manifestation of the disease in aged individuals. The active Caspase-6 is localized in neuritic plaques, in neuropil threads, and in neurofibrillary tangles containing neurons that are not morphologically apoptotic in nature. To investigate the potential consequences of the activation of Caspase-6 in neurons, we conducted a proteomics analysis of Caspase-6-mediated cleavage of human neuronal proteins. Proteins from the cytosolic and membrane subcellular compartments were treated with recombinant active Caspase-6 and compared with undigested proteins by two-dimensional gel electrophoresis. LC/MS/MS analyses of the proteins that were cleaved identified 24 different potential protein substrates. Of these, 40% were cytoskeleton or cytoskeleton-associated proteins. We focused on the cytoskeleton proteins because these are critical for neuronal structure and function. Caspase-6 cleavage of alpha-Tubulin, alpha-Actinin-4, Spinophilin, and Drebrin was confirmed. At least one Caspase-6 cleavage site was identified for Drebrin, Spinophilin, and alpha-Tubulin. A neoepitope antiserum to alpha-Tubulin cleaved by Caspase-6 immunostained neurons, neurofibrillary tangles, neuropil threads, and neuritic plaques in Alzheimer disease and co-localized with active Caspase-6. These results imply that the early and neuritic activation of Caspase-6 in Alzheimer disease could disrupt the cytoskeleton network of neurons, resulting in impaired neuronal structure and function in the absence of cell death. This study provides novel insights into the pathophysiology of Alzheimer disease.

Innate Immune-Mediated Neuronal Injury Consequent to Loss of Astrocytes

Neuronal injury and loss are recognized features of neuroinflammatory disorders, including acute and chronic encephalitides and multiple sclerosis; destruction of astrocytes has been demonstrated in cases of Rasmussen encephalitis. Here, we show that innate immune cells (i.e. natural killer [NK] and gammadelta T cells) cause loss of neurons from primary human neuron-enriched cultures by destroying the supporting astrocytes. Interleukin 2-activated NK cells caused loss of astrocytes within 1 hour, whereas neurons were lost at 4 hours. Time-lapse imaging indicated that delayed neuron loss was due to early destruction of supporting astrocytes. Selective blocking of astrocyte death with anti-NKG2D antibodies reduced neuron loss, as did blocking of CD54 on astrocytes. gammadelta T cells also induced astrocyte cytotoxicity, leading to subsequent neuronal displacement. In astrocytes, NK cells caused caspase-dependent fragmentation of the intermediate filament proteins glial fibrillary acidic protein and vimentin, whereas anti-CD3-activated T cells produced fragmentation to a lesser extent and without measurable cytotoxicity. Glial fibrillary acidic protein fragmentation was also demonstrated in lysates from chronic multiple sclerosis plaques but not from normal control white matter. These data suggest that non-major histocompatibility complex-restricted immune effector cells may contribute to neuron loss in neuroinflammatory disorders indirectly through injury of glia.

Epithelial-Mesenchymal Transition and the Stem Cell Phenotype

Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire the motile, migratory properties of mesenchymal cells. In a recent issue of Cell, Mani et al. (2008) show that induction of EMT stimulates cultured breast cells to adopt characteristics of stem cells.

Modification of Vimentin

In a recent study, we were able to show that the intermediate filament protein vimentin aggregates in human dermal fibroblasts because of modification by the advanced glycation endproduct carboxymethyllysine (CML). In this work, we investigated the formation of intracellular CML in relation to the concentration of glucose in the culture medium. The natural degradation product of glucose, methylglyoxal, was able to induce the aggregation of vimentin. This dicarbonyl leads to the formation of the modifications MG-H1 and carboxyethyllysine (CEL) as a result of the reaction with arginine and lysine residues of proteins. Furthermore, we found that the protein vimentin was modified, not only by CML and CEL, but also by pentosidine and pyrraline. These findings underline the special position of vimentin as a preferential target of the Maillard reaction in human skin.

Intermediate filaments in smooth muscle

The intermediate filament (IF) network is one of the three cytoskeletal systems in smooth muscle. The type III IF proteins vimentin and desmin are major constituents of the network in smooth muscle cells and tissues. Lack of vimentin or desmin impairs contractile ability of various smooth muscle preparations, implying their important role for smooth muscle force development. The IF framework has long been viewed as a fixed cytostructure that solely provides mechanical integrity for the cell. However, recent studies suggest that the IF cytoskeleton is dynamic in mammalian cells in response to various external stimulation. In this review, the structure and biological properties of IF proteins in smooth muscle are summarized. The role of IF proteins in the modulation of smooth muscle force development and redistribution/translocation of signaling partners (such as p130 Crk-associated substrate, CAS) is depicted. This review also summarizes our latest understanding on how the IF network may be regulated in smooth muscle.

Expression and Distribution of Intermediate‐filament Proteins and Laminin during the Development of the Bovine Müllerian Duct

The expression pattern of several intermediate-filament proteins (vimentin, cytokeratin 8, 18 and 19) and the basal lamina component laminin was investigated in the Wolffian and the Müllerian ducts of bovine embryos and fetuses. The material studied comprised sexually undifferentiated stages [crown-rump length (CRL) 0.9 cm/1.0 cm/1.2 cm/1.9 cm/2.5 cm] and female stages (CRL 3.0 cm/4.2 cm/5.1 cm). Laminin could be demonstrated in the basal lamina of the developing Wolffian and Müllerian duct as well as in the stroma surrounding the Müllerian duct. The intermediate-filament protein vimentin was expressed in the mesothelium of the funnel field and in the epithelium of the Müllerian duct in all studied specimens, whereas the epithelial cells of the Wolffian duct only showed vimentin expression from a CRL of 2.2 cm onwards. In the cranial part of the Müllerian ducts only a few cells stained with pan-cytokeratin antibodies, whereas mesothelium and epithelium of the Wolffian duct showed as distinct immunostaining in all investigated stages. Both genital ducts showed no immunostaining with the antibody against cytokeratin 19 at any time of development. We conclude from our immunohistochemical results that the epithelial cells of the Wollfian duct do not contribute cells to the developing Müllerian duct.

Microscopic characterization of rat retinal progenitor cells

A progenitor cell line was developed from a postnatal day 2 (P2) rat retina to study the effects of secreted proteins of the retinal pigment epithelium (RPE) on isolated retinal progenitor cells and markers for immature and differentiated retinal cells. Progenitor cells were cloned from a P2 explant grown in secreted proteins of cultured RPE cells. A cell line was cloned from a single progenitor cell. During the period of RPE-secreted protein stimulation the cells were transformed with the psi AE1A virus. Progenitor cells formed extensive processes when grown in serum and proliferated from the explant when grown in secreted proteins of RPE cells as demonstrated by bromodeoxyuridine (BrdU). All progenitor cells at early and late passages including a cloned cell line (D4) expressed Pax6, a transcription factor essential for eye development, which was verified by Western blotting. All cells expressed nestin, an early neuroepithelial cell marker. These two traits showed the immature character of these rat retinal progenitor cells. All cells expressed the intermediate filament protein vimentin, an intermediate filament protein. Interestingly, most progenitor cells grown in serum expressed the mature cell markers opsin, but few cells expressed glial fibrillary acidic protein (GFAP). The progenitor cells responded to proteins secreted by cultured RPE cells by forming large clusters, while cells grown in retinoic acid formed long thin processes that extended from a round cell body. These progenitor cells, following treatment with secreted proteins of the RPE, will be tested for their therapeutic effect in diseased rat retinas.

Evidence for the epithelial to mesenchymal transition in biliary atresia fibrosis

The epithelial to mesenchymal transition has recently been implicated as a source of fibrogenic myofibroblasts in organ fibrosis, particularly in the kidney. There is as yet minimal evidence for the epithelial to mesenchymal transition in the liver. We hypothesized that this process in biliary epithelial cells plays an important role in biliary fibrosis and might be found in patients with especially rapid forms, such as is seen in biliary atresia. We therefore obtained liver tissue from patients with biliary atresia as well as a variety of other pediatric and adult liver diseases. Tissues were immunostained with antibodies against the biliary epithelial cell marker CK19 as well as with antibodies against proteins characteristically expressed by cells undergoing the epithelial to mesenchymal transition, including fibroblast-specific protein 1, the collagen chaperone heat shock protein 47, the intermediate filament protein vimentin, and the transcription factor Snail. The degree of colocalization was quantified using a multispectral imaging system. We observed significant colocalization between CK19 and other markers of the epithelial to mesenchymal transition in biliary atresia as well as other liver diseases associated with significant bile ductular proliferation, including primary biliary cirrhosis. There was minimal colocalization seen in healthy adult and pediatric livers, or in livers not also demonstrating bile ductular proliferation. Multispectral imaging confirmed significant colocalization of the different markers in biliary atresia. In conclusion, we present significant histologic evidence suggesting that the epithelial to mesenchymal transition occurs in human liver fibrosis, particularly in diseases such as biliary atresia and primary biliary cirrhosis with prominent bile ductular proliferation.

Proteomic analysis of embryonic stem cell–derived neural cells exposed to the antidepressant paroxetine

Antidepressant drugs can have significant effects on the mood of a patient suffering from major depression or other disorders. The pharmacological actions of these drugs generally affect the uptake or metabolism of the neurotransmitters serotonin, noradrenalin, and, to a lesser extent, dopamine. However, many aspects of antidepressant action are not understood. We conducted a proteomic analysis in a neuronal cell culture model in an attempt to identify molecules important to the operation of pathways functionally relevant to antidepressant action. The model involved generating cultures containing mixed neural and glial cells by controlled differentiation of mouse embryonic stem cells, followed by exposure to 1 microM paroxetine for 14 days. After antidepressant exposure, we observed increased expression or modification of sepiapterin reductase (SPR), heat shock protein 9A, RAS and EF-hand domain containing, and protein disulfide isomerase associated 3 and decreased expression or modification of creatine kinase, actin, prohibitin, a T-cell receptor alpha chain, defensin-related cryptdin 5, and the intermediate filament proteins glial fibrillary acidic protein and vimentin. SPR, the most strongly up-regulated protein observed, controls production of tetrahydrobiopterin, an essential cofactor for the synthesis of many neurotransmitters including serotonin, making it a plausible and intriguing candidate protein for involvement in mood control and antidepressant drug action. SPR and the other proteins identified may represent links to molecular processes of importance to mood dysregulation and control, and their respective genes may be novel candidates for the study of antidepressant pharmacogenetics.

14-3-3 Expression in Denervated Hippocampus after Entorhinal Cortex Lesion Assessed by Culture-Derived Isotope Tags in Quantitative Proteomics

Activation of astrocytes accompanies many brain pathologies. Reactive astrocytes have a beneficial role in acute neurotrauma but later on might inhibit regeneration. 2D-gel electrophoresis and mass spectrometry were applied to study the proteome difference in denervated hippocampus in wildtype mice and mice lacking intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin (GFAP-/-Vim-/-) that show attenuated reactive gliosis and enhanced posttraumatic regeneration. Proteomic data and immunohistochemical analyses showed upregulation of the adapter protein 14-3-3 four days postlesion and suggested that 14-3-3 upregulation after injury is triggered by reactive gliosis. Culture-derived isotope tags (CDIT) and mass spectrometry demonstrated that 14-3-3 epsilon was the major isoform upregulated in denervated hippocampus and that its upregulation was attenuated in GFAP-/-Vim-/- mice and thus most likely connected to reactive gliosis.

Increased Neurogenesis and Astrogenesis from Neural Progenitor Cells Grafted in the Hippocampus of GFAP−/−Vim−/− Mice

After neurotrauma, ischemia, or neurodegenerative disease, astrocytes upregulate their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin (Vim), and nestin. This response, reactive gliosis, is attenuated in GFAP(-/-)Vim(-/-) mice, resulting in the promotion of synaptic regeneration after neurotrauma and improved integration of retinal grafts. Here we assessed whether GFAP(-/-)Vim(-/-) astrocytes affect the differentiation of neural progenitor cells. In coculture with GFAP(-/-)Vim(-/-) astrocytes, neural progenitor cells increased neurogenesis by 65% and astrogenesis by 124%. At 35 days after transplantation of neural progenitor cells into the hippocampus, adult GFAP(-/-)Vim(-/-) mice had more transplant-derived neurons and astrocytes than wild-type controls, as well as increased branching of neurite-like processes on transplanted cells. Wnt3 immunoreactivity was readily detected in hippocampal astrocytes in wild-type but not in GFAP(-/-)Vim(-/-) mice. These findings suggest that GFAP(-/-)Vim(-/-) astrocytes allow more neural progenitor cell-derived neurons and astrocytes to survive weeks after transplantation. Thus, reactive gliosis may adversely affect the integration of transplanted neural progenitor cells in the brain. Disclosure of potential conflicts of interest is found at the end of this article.

Time course of expression of intermediate filament protein vimentin, nestin and desmin in rat renal glomerular injury

Time course of expression of intermediate filament protein vimentin, nestin and desmin in rat renal glomerular injury

Novel GKAPs for cGMP-dependent protein kinase type II: identification and functional significance

Many protein kinases are maintained in specific subcellular microenvironments by association to anchoring proteins. Proper functioning of the cGMP-dependent protein kinase II (cGK II) involves membrane targeting through myristoylation. To determine whether additional binding to anchoring proteins is also important for its targeting, in analogy with G-kinase anchoring proteins (GKAPs) for cGK I, and to identify potential new components of the cGMP/cGK signalling pathway we performed a yeast twohybrid screen of novel cGK II binding partners. By screening a brain cDNA library using cGK II as a bait the intermediate filament protein GFAP (glial fibrillary acidic protein) was obtained as a positive clone. The interaction was confirmed by overlayand pull-down assays and by immunoprecipitation. Interestingly, the highly homologous intermediate filament protein vimentin has previously been identified as a high affinity binding partner for cGK Iα. We determined which domains are required for the interaction and found, surprisingly, that the least conserved N-terminal domain of the cGK's (aa 1–108 of cGKII and 1–94 of cGK Iα) interacts with the most strongly conserved core domain of the intermediate filaments (aa 237–386 of GFAP). The physiological function of the interaction is under current investigation.