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Astroglial cells are the most abundant cells in the mammalian central nervous system (CNS), yet our knowledge about their function in health and disease has been limited. This review focuses on the recent work addressing the function of intermediate filaments in astroglial cells under severe mechanical or osmotic stress, in hypoxia, and in brain and spinal cord injury. Recent data show that when astrocyte intermediate filaments are genetically ablated in mice, reactive gliosis is attenuated and the course of several CNS pathologies is altered, while the signs of CNS regeneration become more prominent. GFAP is the principal astrocyte intermediate filament protein and dominant mutations in the GFAP gene have been shown to lead to Alexander disease, a fatal neurodegenerative condition in humans.

We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the “endotube”); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called “fence” function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.

Desmin ( DES) mutations have been recognized as a cause of desmin-related myopathy (OMIM 601419), or desminopathy, a disease characterized by progressive limb muscle weakness and accumulation of desmin-reactive granular aggregates in the myofibers. We have studied three families with skeletal or cardioskeletal myopathy caused by small in-frame deletions in the desmin gene. The newly identified in-frame deletions E359_S361del and N366del alter the heptad periodicity within a critical 2B coiled-coil segment. Structural analysis reveals that the E359_S361 deletion introduces a second stutter immediately downstream of the naturally occurring stutter, thus doubling the extent of the local coiled-coil unwinding. The N366del mutation converts the wild-type stutter into a different type of discontinuity, a stammer. A stammer, as opposed to a stutter, is expected to cause an extra overwinding of the coiled-coil. These mutations alter the coiled-coil geometry in specific ways leading to fatal damage to desmin filament assembly. Expression studies in two cell lines confirm the inability of desmin molecules with this changed architecture to polymerize into a functional filamentous network. This study provides insights into molecular pathogenetic mechanisms of desmin mutation-associated skeletal and cardioskeletal myopathy.

Synemin is an intermediate filament protein shown previously to interact with alpha-dystrobrevin and desmin. Immunoblot analysis detects a beta-synemin protein of 170 kDa in human skeletal muscle and an alpha-synemin protein of 225 kDa in monkey brain. Low-resolution immunohistochemical analysis localizes beta-synemin within muscle along the sarcolemma, whereas confocal microscopic analysis further refines localization to the costamere and muscle Z-lines. In addition to these locations, beta-synemin is also enriched at the neuromuscular and myotendinous junctions, other regions that undergo high stress during myofiber contraction. Based on its localization and its expression pattern, it is proposed that beta-synemin functions as a structural protein involved in maintaining muscle integrity through its interactions with alpha-dystrobrevin, desmin, and other structural proteins.

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.

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

In this report, the phenotype associated with the first targeted knockout of the lens specific intermediate filament gene CP49 is described. Several surprising observations have been made. The first was that no cataract was observed despite the fact that the beaded filaments of the lens fibre cells had been disrupted. Light scatter and the lens optical properties had, however, deteriorated in the CP49 knockout lenses compared to litter mate controls. These changes were accompanied by dramatic changes in plasma membrane organisation of the fibre cells as revealed by detailed morphological examinations and providing the second surprising result. The CP49 knockout mouse is therefore an important model to study the functional link between lens transparency, the cytoskeleton and plasma membrane organisation.

In response to stressful stimuli, cells respond by inducing a set of heat shock (stress) proteins (hsps) that play important roles in repair and protective mechanisms. The present study investigates the expression patterns of Hsp27 and Hsp32 in the adult rat hippocampus following whole body hyperthermia. A pronounced induction of these low-molecular-weight stress proteins was apparent in populations of glial cells such as astrocytes and microglia that were identified using cell-specific markers (GFAP for astrocytes and the lectin GSA I-B4 for microglia). Hyperthermia also resulted in a robust induction of the intermediate filament protein, vimentin, in glial cells in the adult rat hippocampus. Interestingly, a rapid induction of both Hsp27 and vimentin was observed in the microvasculature, suggesting that hyperthermic stress may compromise the blood-brain barrier.

Expression of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) helps to establish the origin of biliary and metastatic carcinomas. We investigated the expression of CK7 and CK20 in inflammatory, metaplastic and neoplastic conditions of the bile ducts, and evaluated possible relationships between the CK expression pattern and extrahepatic bile duct/gallbladder carcinomas (EBDCs) or intrahepatic bile duct carcinomas (IBDCs). We used immunohistochemistry for the investigation of 48 formalin-fixed, paraffin-embedded specimens grouped as: A) lithiasic or inflamed surgically resected extrahepatic bile ducts/gallbladders: all were CK7+/CK20+; B) percutaneous liver biopsies from patients with chronic hepatitis C primary biliary cirrhosis and primary sclerosing cholangitis: all were CK7+/CK20-; C) EBDCs: all were CK7+/CK20+, except for two cases which were CK7-/CK20-; D) IBDCs: all were CK7+/CK20-, except for one case showing CK20 positivity. Metaplastic changes were seen only among specimens in groups A and C: in these cases, CK20 was either focally or diffusely expressed. Our study suggests that the expression of cytokeratins under specific stimuli can be different from normal tissues, and that sometimes CK20 expression can be related to and precede the occurrence of metaplastic alterations.

The mechanical effects of the intermediate filament protein desmin was examined in desmin deficient mice (Des-/-) and their wild type control (Des+/+). Active force generation was determined in intact soleus muscles and in skinned single fibres from soleus and psoas. A decreased force generation of skinned muscle fibres from Des-/- mice and a tendency towards decreased active force in intact soleus muscle were detected. Concentrations of the contractile protein actin and myosin were not altered in Des-/- muscles. Ca(2+)-sensitivity of skinned single fibres in Des-/- muscles was unchanged compared to Des+/+. Using a protocol with repeated short tetani an increased fatigue resistance was found in the intact soleus muscles from Des-/- mice. In conclusion, desmin intermediate filaments are required for optimal generation or transmission of active force in skeletal muscle. Although other studies have shown that the desmin intermediate filaments appear to influence Ca(2+)-handling, the Ca(2+)-sensitivity of the contractile filaments is not altered in skeletal muscle of Des-/- mice. Previous studies have reported a switch towards slower myosin isoforms in slow skeletal muscle of Des-/- mice. The increased fatigue resistance show that this change is reflected in the physiological function of the muscle.

The intermediate filament protein IFA-2 is essential for the structural integrity of the Caenorhabditis elegans epidermis. It is one of the major components of the fibrous organelle, an epidermal structure comprised of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments that serve to transmit force from the muscle to the cuticle. Mutations of IFA-2 result in epidermal fragility and separation of the apical and basal epidermal surfaces during postembryonic growth. An IFA-2 lacking the head domain fully rescues the IFA-2 null mutant, whereas an IFA-2 lacking the tail domain cannot. Conversely, an isolated IFA-2 head was able to localize to fibrous organelles whereas the tail was not. Taken together these results suggest that the head domain contains redundant signals for IF localization, whereas non-redundant essential functions map to the IFA-2, tail, although the tail is unlikely to be directly involved in fibrous organelle localization.

Desmin is the major intermediate filament protein in muscle tissue. In skeletal and cardiac muscle, desmin filaments distribute in the intermyofibrillar space to link adjacent myofibrils to each other at the z disc level, to the sarcolemma via costameres and sarcoglycan-dystroglycan complexes, and insert into to the nuclear envelope. The desmin filament network contributes to the positioning organelles such as mitochondria. In cardiac muscle, desmin is enriched in the intercalated disk by association with desmosomes. Hence, desmin plays an important role in the structural integrity and mechanical tension transmission across the myocyte and perhaps in the communication between cytoskeleton and extracellular matrix. Germ-line ablation of the desmin gene (DES) in mice does not affect muscle formation in embryos but causes postnatal cardiac and skeletal myopathies that deteriorate with age. A large number of mutations in DES or its partner proteins (e.g., CRYAB) cause desmin-related myopathy (DRM), a heterogeneous group of muscle disease featured by the presence of DES-positive aberrant protein aggregates in myocytes. Both disruption of the DES filament network as result of the mutation and gained toxicity of the mutant proteins may contribute to DRM pathogenesis but the precise pathogenesis remains poorly understood. Studies on DRM patients and DRM cell and animal models have also provided new insight into protein quality control in muscle tissue. Chemical chaperones, priming the proteasome, and enhancing autophagy have been shown to delay the progression of experimental DRM.

The cardiomyopathic (CM) Syrian golden hamster (strain UM-X7.1) exhibits a hereditary cardiomyopathy, which causes premature death resulting from congestive heart failure. The CM animals show extensive cardiac myofibril disarray and myocardial calcium overload. The present study has been undertaken to examine the role of desmin phosphorylation in myofibril disarray observed in CM hearts. The data from skinned myofibril protein phosphorylation assays have shown that desmin can be phosphorylated by protein kinase C (PKC). There is no significant difference in the content of desmin between CM and control hamster hearts. However, the desmin from CM hearts has a higher phosphorylation level than that of the normal hearts. Furthermore, we have examined the distribution of desmin and myofibril organization with immunofluorescent microscopy and immunogold electron microscopy in cultured cardiac myocytes after treatment with the PKC-activating phorbol ester, 12-O-tetradecanylphorbol-13-acetate (TPA). When the cultured normal hamster cardiac cells are treated with TPA, desmin filaments are disassembled and the myofibrils become disarrayed. The myofibril disarray closely mimics that observed in untreated CM cultures. These results suggest that disassembly of desmin filaments, which could be caused by PKC-mediated phosphorylation, may be a factor in myofibril disarray in cardiomyopathic cells and that the intermediate filament protein, desmin, plays an Important role in maintaining myofibril alignment in cardiac cells.

The goldfish visual pathway displays a remarkable capacity for continuous neurogenesis, plasticity, and regeneration. The intermediate filament protein composition of this system differs from that of higher vertebrates, which lack the capacity for continued nerve growth and development. In an effort to determine how intermediate filament proteins are regulated during nerve growth, we isolated and characterized cDNA and genomic clones representing the goldfish neurofilament medium (NF-M) protein. The tissue-specific expression of goldfish NF-M mRNA was analyzed by RNase protection assays and by in situ hybridization. The expression of goldfish NF-M is qualitatively the same as in other species. Although the intermediate filament protein composition of the goldfish visual pathway is unusual when compared with higher vertebrates, the goldfish NF-M protein is similar to higher vertebrate NF-M proteins. In addition, the organization of the goldfish NF-M gene is identical to the NF-M genes in all other vertebrate species. In contrast, the promoter region of the goldfish NF-M gene has several potential regulatory sequences that are not found in the promoter regions of higher vertebrate NF-M genes.

High sulphur proteins (HSPs) form part of the matrix surrounding the intermediate filaments in the cortical cells of the wool fibre. There are three known families of HSPs, comprising in excess of 40 components and their molecular weights range from 10-30 kDa. Here we report the use of the increased resolving power of isoelectric focusing in the first dimension of two-dimensional electrophoresis and modern gel comparison software to investigate the nature of within- and between-breed variations amongst the proteins of three breeds of sheep: Merino, Romney and Corriedale. In agreement with past studies we observed very little variation in the intermediate filament protein content in wool, both between and within these three sheep breeds. Instead, most of the observed variation occurred among the HSPs, along with some minor variation among the high glycine-tyrosine proteins. Breed-specific differences were observed in the HSP patterns in the wool proteome maps. Merino sheep were found to exhibit the simplest HSP expression patterns, with eight major spots linked to form four pairs. In contrast, the Romney and Corriedale HSP patterns exhibited more spots at lower isoelectric point values (around 4.8), while some of the lower molecular weight HSPs were less prevalent in Romney sheep and absent from the Corriedales.

Cytoplasmic accumulation of wild-type p53 in tumor cells indicates that the tumor suppressor is inactive with regard to growth suppressive functions. Whether this occurs randomly during tumor development or characterizes a certain tumor cell subset is not known. Here we assayed primary glioblastomas for expression and subcellular localization of p53 and determined a correlation with expression of intermediate filament proteins characterizing glial cell development. Sixty-nine percent of the tumors were p53 positive in immunohistochemistry. A significant number of tumors (23%) accumulated wild-type p53 in the cytoplasm, which correlated with the presence of vimentin and glial fibrillary acidic protein, except for 1 case. Tumors with exclusive nuclear p53 contained none or only one of these intermediate filament proteins. In an alternative approach, tumors positive for glial fibrillary acidic protein were screened for expression of p53 and vimentin. Thirty-eight percent of these tumors showed cytoplasmic p53, and all of those also expressed vimentin. Tumors with only nuclear p53 were vimentin negative, except for 1 case. No mutation was detected in p53 exons 5 to 8 in tumors with cytoplasmic p53, suggesting that they express wild-type p53. The data indicate that a cytoplasmic accumulation of wild-type p53 in human primary glioblastomas correlates with a certain intermediate filament protein expression, suggesting that it identifies a certain subset of tumors.

In preparation for blastocyst implantation, uterine luminal epithelial cells express new cell adhesion molecules on their apical plasma membrane. Since one mechanism epithelial cells employ to regulate membrane polarity is the establishment of specific membrane-cytoskeletal interactions, this study was undertaken to determine if new cytokeratin (CK) intermediate filament assemblies are expressed in endometrial epithelial cells during developmental stages related to blastocyst implantation. Type-specific CK antibodies were used for immunocytochemical and immunoblot analyses of 1) intermediate filament networks of the endometrial epithelium during embryo implantation in rabbits and 2) proliferative and secretory phases of the human menstrual cycle. CK18, a type I CK found in most simple epithelia, was expressed in all luminal and glandular epithelial cells of both the human and rabbit endometrium at all developmental stages analyzed; it was also strongly expressed in trophectoderm of the implanting rabbit blastocyst. In contrast, CK13, another type I cytokeratin, exhibited a regulated expression pattern in luminal, but not glandular, epithelial cells of secretory phase human and peri-implantation stage rabbit endometrium. Furthermore, in the rabbit implantation chambers, CK13 was predominately localized at the cell apex of luminal epithelial cells, where it assembled into a dense filamentous network. These data suggest that the stage-specific expression of CK13 and a reorganization of the apical intermediate filament cytoskeleton of uterine luminal epithelial cells may play important functions in preparation for the implantation process.

We recently identified a novel protein called syncoilin, a putative intermediate filament protein that interacts with α-dystrobrevin, a member of the dystrophin-associated protein complex. Syncoilin is found at the neuromuscular junction, sarcolemma, and Z-lines and is thought to be important for muscle fiber integrity. Based on the similar protein structure and cellular localization of syncoilin and desmin, we proposed that these proteins interact in vivo. The data presented confirm an interaction between syncoilin and desmin and demonstrate their co-localization in skeletal muscle. Intriguingly, whereas these proteins interact, COS-7 cell expression studies show that desmin and syncoilin do not assemble into heterofilaments. Furthermore, fractionation assay and immunofluorescence study of H2K myoblasts and myotubes suggest that, unlike typical intermediate filament proteins, syncoilin does not participate in filament formation with any protein. However, it is possible that syncoilin is involved in the anchoring of the desmin intermediate filament network at the sarcolemma and the neuromuscular junction. This interaction is likely to be important for maintaining muscle fiber integrity and may also link the dystrophin-associated protein complex to the cytoskeleton. The dysfunction or absence of syncoilin may result in the disruption of the intermediate filament network leading to muscle necrosis. Syncoilin is therefore an ideal candidate gene for muscular dystrophies and desmin-related myopathies.

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.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 8 M 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.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.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.

Epithelial- to-mesenchymal transition (EMT) is a critical event in the induction of cell motility and increased survival both under physiological situations like wound healing and during development as well as in malignant cells undergoing invasion and metastasis. Vimentin is an intermediate filament protein which is characteristically upregulated in cells undergoing EMT. For decades vimentin has been considered as a marker for EMT but its functional contribution to the process has remained unclear. Our data demonstrate that vimentin contributes to EMT via upregulating the gene expression of several EMT-linked genes. Especially, we find that vimentin regulates EMT associated induced migration via upregulation of the expression of receptor tyrosine kinase Axl. In addition to our data, several other exciting recent studies support the notion that vimentin in fact functions as a positive regulator of EMT and upregulation of vimentin appears to be a prerequisite for EMT induction.

Background Nestin is a type VI intermediate filament protein known as a marker for progenitor cells that can be mostly found in tissues during the embryonic and fetal periods. In our study, we aimed to determine the expression of nestin in meninges covering the brain tissue at different developmental stages and in the new born. Methods In this study 10 human fetuses in different development stages between developmental weeks 9-34 and a newborn brain tissue were used. Fetuses in paraffin section were stained with H+E and nestin immunohistochemical staining protocol was performed. Results In this study, in the human meninges intense nestin expression was detected as early as in the 9th week of development. Intensity of this expression gradually decreased in later stages of development and nestin expression still persisted in a small population of newborn meningeal cells. Conclusion In the present study, nestin positive cells gradually diminished in the developing and maturing meninges during the fetal period. This probably depends on initiation of a decrease in nestin expression and replacement with other tissue-specific intermediate filaments while the differentiation process continues. These differences can make significant contributions to the investigation and diagnosis of various pathological disorders (Tab. 1, Fig. 3, Ref. 36).

Nestin has been considered a neural stem cell marker, and represents an intermediate filament protein likely involved in restructuring the cytoskeleton in different cell types. The present ultrastructural study has immunodetected nestin especially in the wound epidermis, regenerating myotubes and in the growing nerves of the regenerating tail of lizards. In keratinocytes of the stratified wound epidermis nestin is present in the irregular electron-paler meshwork located along the cell perimeter and among keratin bundles converging into desmosomes. In the regenerating muscles nestin-immunoreactivity remains confined to some external regions along the myotubes and in the cytoplasmic ends of the myotubes not occupied by myofibrils. A diffuse nestin immunolabeling is also present among the neurofilaments of growing axons, in Schwann cells and in ependymal cells of the regenerating spinal cord of the tail. The localization of nestin in sites of cytoskeletal remodeling in keratinocytes, myotubes, ependymal cells and axons, suggests that this protein is associated to the reassembling of keratin tonofilaments in moving keratinocytes, assembling of contractile proteins in myotubes, and in the organization of neurofilaments during the growth and myelination of axons within the regenerating lizard tail.

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

Submit Manuscript | http://medcraveonline.com Vimentin, a 57kDa intermediate filament protein, is normally expressed in mesenchymal cells. As a major cytoskeleton component of mesenchymal cells, it maintains cellular integrity, stabilizes cytoskeleton interactions and provides resistance under mechanical stress [1]. Biological characterization of Vimentin null cells suggested that it is important for mechanical stability and motility in vitro [2]. Furthermore Vimentin null mice had impaired wound healing without causing any other major physiological disorders [3]. Based on these studies, it can be inferred that Vimentin might have a supporting role in mechanical stability and integrity of cellular structure. In the recent years, understanding of the biological significance of Vimentin has been increasing steadily after its discovery as an epithelial mesenchymal transition (EMT) marker in aggressive cancer cells of epithelial originsbreast, prostate, colon, skin, lung and brain [4]. Inducible expression of Vimentin in large number of epithelial cancer correlates well with metastatic phenotypes in patients [5,6]. The possible explanation for Vimentin expression in epithelial cancer cells undergoing EMT process is to enhance migration and invasiveness [7].

Lamin A is an intermediate filament protein which is cleaved by the enzyme, FACE 1 at VTRSY↓L. The cleavage is the final step in the production of the mature lamin A protein. The mature lamin A protein localizes in the inner membrane of the nucleus. The mutation in the lamin A gene causes many diseases, including accelerated aging. It is known that the protein is not expressed in neuronal cells of the brain. Many splicing variants of the lamin A gene have been reported. In this study, the amino acid sequence VTRSY (a penta-peptide repeat) was found in three different sites of the C-terminal end of the lamin A protein, the protein expressed in cells of ear cartilage tissues is shorter than the protein expressed in cells of the skin tissues. Using two lamin A antibodies, it was found that the amino acid sequence between penta-peptide 2 and 3 is missing in lamin A protein that was expressed in the cells of mouse ear cartilage tissue, besides the RT-PCR data confirmed that the corresponding coding sequence between the penta repeat 2 and 3 is intact. Cleavage may occur at the penta-peptide (VTRSY) at site 3 in the lamin A tail of mouse ear cartilage.

Peripherin is a type III intermediate filament protein, the expression of which is associated with the acquisition and maintenance of a terminally differentiated neuronal phenotype. Peripherin up-regulation occurs during acute neuronal injury and in degenerating motor neurons of amyotrophic lateral sclerosis. The functional role(s) of peripherin during normal, injurious, and disease conditions remains unknown, but may be related to differential expression of spliced isoforms. To better understand peripherin function, we performed a yeast two-hybrid screen on a mouse brain cDNA library using an assembly incompetent peripherin isoform, Per-61, as bait. We identified new peripherin interactors with roles in vesicular trafficking, signal transduction, DNA/RNA processing, protein folding, and mitochondrial metabolism. We focused on the interaction of Per-61 and the constitutive isoform, Per-58, with SNAP25 interacting protein 30 (SIP30), a neuronal protein involved in SNAP receptor-dependent exocytosis. We found that peripherin and SIP30 interacted through coiled-coil domains and colocalized in cytoplasmic aggregates in SW13vim(−) cells. Interestingly, Per-61 and Per-58 differentially altered the subcellular distribution of SIP30 and SNAP25 in primary motor neurons. Our findings suggest a novel role of peripherin in vesicle trafficking. The functional roles of the intermediate filament peripherin remain enigmatic. We performed a two-hybrid screen and identified that peripherin interacts with SIP30 (a), a vesicle trafficking protein. Per-58 and toxic Per-61 isoforms coaggregated with SIP30 in SW13vim(−) cells and altered SIP30 and SNAP25 subcellular localization in primary motor neurons (b). Our results support a role for peripherin in vesicle trafficking.

Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (< 10 μM), or large inclusions (≥ 10 μM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48 h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS.

Intermediate filament (IF) related diseases are linked to defects in proteins that are associated with the contractile apparatus of skeletal and cardiac muscle, and with costameres. Synemin is a la...

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 exam-ined 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.

Transitin is an avian intermediate filament protein whose transient expression in the progenitor cells of the muscle and nerve tissues is similar to that of mammalian nestin. Both proteins contain an alpha-helical core domain flanked by a short N-terminal head and a long C-terminal extremity. However, the tail region of transitin is significantly different from that of nestin in that it harbors a unique motif containing more than 50 leucine zipper-like heptad repeats which is not found in any other intermediate filament protein. Despite the absence of introns in this region of the transitin gene, it was reported that different isoforms of the protein were produced by exclusion or inclusion of a number of repeats generated by an unusual splicing mechanism recognizing consensus 5' and 3' splice sites contained within the coding sequence of the heptad repeat domain [Napier et al. (1999) J Mol Neurosci 12:11-22]. Two monoclonal antibodies (mAbs) reacting with repeated epitopes of this motif were used to monitor transitin expression during in vitro myogenesis of the quail myogenic cell line QM7. Confocal microscopy revealed that the subcellular domains decorated with mAbs A2B11 and VAP-5 were mutually exclusive: the intermediate filament network visualized with mAb VAP-5 appeared to abut on a submembranous domain defined by mAb A2B11. When QM7 cells were induced to differentiate by switching to medium containing low serum components, an early effect was the local loss of A2B11 cortical staining at the points of cell-cell contacts. The A2B11 signal also disappeared before that of VAP-5 in newly formed myotubes. Unexpectedly, the mutually exclusive staining pattern of the mAbs could not be explained by alternative splicing since both epitopes mapped to a repeated element preceding the consensus 5' splice sites of the heptad repeat domain. An alternative explanation would be that the central repeat domain of transitin is a polymorphic structure from which different conformations exist depending on the local context. This hypothesis is strengthened by the observation that in cultured neural crest cells, the A2B11 antigen is preferentially expressed by freely migrating crest cells whose intracellular pH and calcium concentrations are different from those of non-migrating cells.

In a total of 104 individuals who had sustained traumatic brain injury (TBI), the course of glial immunoreactivity was investigated at the injured cortical area during the first 30 weeks after the trauma, in order to provide data for a forensic wound age estimation. Glial cells were stained with antibodies against the intermediate filament protein vimentin, the extracellular matrix protein tenascin and the serine protease inhibitor alpha1-antichymotrypsin (alpha1-ACT). Injury-induced glial staining reactions could be observed, at the earliest, after a post-infliction interval of 3.1 h for alpha1-ACT, 22 h for vimentin and 7 days for tenascin.

Desmin, a muscle-specific intermediate filament protein, is expressed in all muscle tissues. Its absence leads to a multisystemic disorder involving cardiac, skeletal, and smooth muscles. In skeletal muscle, structural abnormalities include lack of alignment of myofibrils, Z disk streaming, and focal muscle degeneration. In this study, we have examined the consequences of an absence of desmin on the mechanisms of regeneration and the integrity of the neuromuscular junction. The muscles of desmin knock-out and wild-type mice were made to regenerate by injecting cardiotoxin and were examined 7 to 42 days following the injection. The absence of desmin resulted in a delayed and modified regeneration and an accumulation of adipocytes. This was associated with a persistence of small diameter muscle fibers containing both N-CAM and developmental myosin isoforms. The amount of the slow myosin was increased, whereas there was a decrease in the fast isoform in the regenerated muscles of desmin knock-out mice. Both regeneration and aging led to the appearance of elongated neuromuscular junctions with diffuse acetylcholinesterase staining and a decrease in the overall acetylcholinesterase activity in the muscles of these mice. The neuromuscular junctions were markedly disorganised and in some cases postjunctional folds were absent. We conclude that desmin is essential for terminal muscle regeneration, maturation of muscle fibers, and maintaining the complex folded structure of the postsynaptic apparatus of the neuromuscular junctions. Cell Motil. Cytoskeleton 49:51–66, 2001. © 2001 Wiley-Liss, Inc.

Intermediate filament (IF) proteins show specific spatial and temporal expression during development of skeletal muscle. Nestin, the least known muscle IF, has an important role in neuronal regeneration. Therefore, we analyzed the expression pattern of nestin as related to that of vimentin and desmin during skeletal muscle regeneration. Nestin and vimentin appear at 6 h post-injury in myoblasts, with maximum expression around day 3-5 post-injury. Thereafter, vimentin expression ceases completely, whereas that of nestin is downregulated to remain only in the sarcoplasm next to neuromuscular and myotendinous junctions. Desmin appears at 6-12 h post-injury and becomes the predominant IF in myofibers simultaneously with the appearance of cross-striations. The expression pattern and colocalization of nestin and vimentin, known to form heteropolymers, suggests that they are essential during the early dynamic phase of the myofiber regeneration when migration, fusion, and structural modeling of myogenic cells occurs, whereas desmin is responsible for keeping myofibrils in register in mature myofibers. In conclusion, the expression of nestin is dynamically orchestrated with that of vimentin and desmin during skeletal muscle regeneration and recapitulates that seen during myogenesis, i.e. these IFs have key functional roles in the construction and restoration of skeletal myofibers.

Caspases are key mediators of apoptosis. Using a novel expression cloning strategy we recently developed to identify cDNAs encoding caspase substrates, we isolated the intermediate filament protein vimentin as a caspase substrate. Vimentin is preferentially cleaved by multiple caspases at distinct sites in vitro, including Asp85 by caspases-3 and -7 and Asp259 by caspase-6, to yield multiple proteolytic fragments. Vimentin is rapidly proteolyzed by multiple caspases into similar sized fragments during apoptosis induced by many stimuli. Caspase cleavage of vimentin disrupts its cytoplasmic network of intermediate filaments and coincides temporally with nuclear fragmentation. Moreover, caspase proteolysis of vimentin at Asp85 generates a pro-apoptotic amino-terminal fragment whose ability to induce apoptosis is dependent on caspases. Taken together, our findings suggest that caspase proteolysis of vimentin promotes apoptosis by dismantling intermediate filaments and by amplifying the cell death signal via a pro-apoptotic cleavage product.

Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathway, is associated with the intermediate filament protein vimentin, in NIH 3T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylase inhibitor 5′-amino benzylacyclouridine linked to an agarose matrix. Vimentin copurification with UPase was confirmed using both Western blot analysis and MALDI-MS methods. Separation of cytosolic proteins using gel filtration chromatography yields a high molecular weight complex containing UPase and vimentin. Purified recombinant UPase and recombinant vimentin were shown to bind in vitro with an affinity of 120 pm and a stoichiometry of 1:2. Immunofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtubule system using nocodazole results in UPase remaining associated with the collapsed intermediate filament, vimentin. Our data demonstrate that UPase is associated with both the soluble and insoluble pools of vimentin. Approximately 60–70% of the total UPase exists in the cytosol as a soluble protein. Sequential extraction of NIH 3T3 or colon 26 cells liberates an additional 30–40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demonstrate for the first time that UPase is associated with vimentin and the existence of an enzymatically active cytoskeleton-associated UPase.

The heterogeneity and differentiation potential of mitotically active cells in the adult brain were studied by labeling adult rats with BrdU, and isolating an enriched population of cycling cells from neocortex and from subcortical white matter. The majority of this population isolated from either brain region labeled with O4, an early oligodendrocyte marker. In tissue culture, these O4+ progenitors acquired galactocerebroside, a glycolipid of mature oligodendrocytes, but not GFAP, an intermediate filament of astrocytes. A minority population expressed the intermediate filament protein, vimentin, but not O4. This population expressed GFAP after several days in culture. A third population of cycling cells, expressing the gangliosides labeled with the A2B5 antibody, represented a minority population in subcortical white matter, but one of the major cycling populations in cortex, with substantial overlap with O4. Small populations of cycling NG2+ cells also were observed. Thus, the cycling cells in the adult brain are heterogeneous, and the majority appear to belong to glial lineages. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 75–86, 2001

The human mesonephros is currently regarded as a simplified version of the foetal metanephros, primarily due to the close morphological resemblance between these two structures. The aim of the present study was to define whether human mesonephric and foetal metanephric nephrons share immunophenotypical traits in their corresponding structures (glomeruli, proximal and distal tubules). For this purpose we first investigated immunohistochemically the overall expression and topographical distribution of cytokeratins 7, 8, 18, 19, and 20, vimentin and α-smooth muscle actin in mature mesonephric nephrons and compared the results with those obtained in maturing-stage foetal metanephric nephrons. No expression of cytokeratins 7 and 20 was found. Cytokeratins 8, 18, and 19 and vimentin showed a restricted and basically coincident expression along the different components of both mesonephric and metanephric nephrons. These findings indicate that the intermediate filament protein profile of human mature mesonephric nephrons closely recapitulates that observed in developing metanephros and thereby strengthens the concept that human mesonephros, a transient ontogenic structure, is largely similar to the foetal metanephros.

The GLUT4 glucose transporter resides mostly in perinuclear membranes in unstimulated 3T3-L1 adipocytes and is acutely translocated to the cell surface in response to insulin. Using a novel method to purify intracellular GLUT4-enriched membranes, we identified by mass spectrometry the intermediate filament protein vimentin and the microtubule protein alpha-tubulin as components of these membranes. Immunoelectron microscopy of the GLUT4-containing membranes also revealed their association with these cytoskeletal proteins. Disruption of intermediate filaments and microtubules in 3T3-L1 adipocytes by microinjection of a vimentin-derived peptide of the helix initiation 1A domain caused marked dispersion of perinuclear GLUT4 to peripheral regions of the cells. Inhibition of the microtubule-based motor dynein by brief cytoplasmic acidification of cultured adipocytes also dispersed perinuclear GLUT4 and inhibited insulin-stimulated GLUT4 translocation to the cell surface. Insulin sensitivity was restored as GLUT4 was again concentrated near the nucleus upon recovery of cells in physiological buffer. These data suggest that GLUT4 trafficking to perinuclear membranes of cultured adipocytes is directed by dynein and is required for optimal GLUT4 regulation by insulin.

Vimentin is a class III intermediate filament protein widely expressed in the developing embryo and in cells of mesenchymal origin in the adult. Vimentin knock-out mice develop and reproduce without any obvious defect. This is an unexpected finding in view of the high degree of conservation of the vimentin gene among vertebrates. However, it does not exclude the possibility of a role for vimentin in pathological conditions, like tumorigenesis. To address this question directly, we have used a teratocarcinoma model involving the injection of ES cells into syngeneic mice. ES cells lacking vimentin were generated from 129/Sv Vim-/- mice with high efficiency. The absence of vimentin did not affect ES cell morphology, viability or growth rate in vitro. Tumours were induced by subcutaneous injection of either Vim-/- or Vim+/+ ES cells into Vim+/+ and Vim-/- mice, in order to analyse the effect of the absence of vimentin in either the tumorigenic cells or the host mice. No significant differences were found in either tumour incidence, size or vascularization of teratocarcinomas obtained with all possible combinations. Vim-/- ES-derived tumours showed the same cellular composition typical of teratocarcinomas induced by wild-type ES cells together with a very similar apoptotic pattern. Taken together, these results demonstrate that in this model vimentin is not essential for efficient tumour growth and differentiation in vivo.

Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non–gustatory, adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primordia, cytokeratin 20, is not detectable prior to the ingrowth of nerve fibres into the epithelium, which supports the hypothesis that nerve fibres are necessary for initiating taste bud development. Another intermediate filament protein, vimentin, occurs in derivatives of mesoderm, but usually not in epithelium. In humans, vimentin immunoreactivity is expressed mainly in border (marginal) epithelial cells of taste bud primordia, while in chick, vimentin expression occurs in most taste bud cells, whereas non–gustatory epithelium is vimentin immunonegative. Our chick data suggest a relationship between the degree of vimentin expression and taste bud cell proliferation especially during the perihatching period. It is suggested that surrounding epithelial cells (human) and mesenchymal cells (chick) may be contributing sources of developing taste buds. The dense perinuclear network of intermediate filaments especially in dark (i.e. non–sensory) taste disc cells ofXenopusindicates that vimentin filaments also might be associated with cells of non–gustatory function. These results indicate that the mechanisms of taste bud differentiation from source tissues may differ among vertebrates of different taxa.

14-3-3 proteins bind their targets through a specific serine/threonine-phosphorylated motif present on the target protein. This binding is a crucial step in the phosphorylation-dependent regulation of various key proteins involved in signal transduction and cell cycle control. We report that treatment of COS-7 cells with the phosphatase inhibitor calyculin A induces association of 14-3-3 with a 55-kDa protein, identified as the intermediate filament protein vimentin. Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires the phosphopeptide-binding domain of 14-3-3. The region necessary for binding to 14-3-3 is confined to the vimentin amino-terminal head domain (amino acids 1-96). Monomeric forms of 14-3-3 do not bind vimentin in vivo or in vitro, indicating that a stable complex requires the binding of a 14-3-3 dimer to two sites on a single vimentin polypeptide. The calyculin A-induced association of vimentin with 14-3-3 in vivo results in the displacement of most other 14-3-3 partners, including the protooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vitro. Concomitant with 14-3-3 displacement, calyculin A treatment blocks Raf activation by EGF; however, this inhibition is completely overcome by 14-3-3 overexpression in vivo or by the addition of prokaryotic recombinant 14-3-3 in vitro. Thus, phosphovimentin, by sequestering 14-3-3 and limiting its availability to other target proteins can affect intracellular signaling processes that require 14-3-3.

Mice lacking the gene encoding for the intermediate filament protein desmin have a surprisingly normal myofibrillar organization in skeletal muscle fibers, although myopathy develops in highly used muscles. In the present study we examined how synemin, paranemin, and plectin, three key cytoskeletal proteins related to desmin, are organized in normal and desmin knock-out (K/O) mice. We show that in wild-type mice, synemin, paranemin, and plectin were colocalized with desmin in Z-disc-associated striations and at the sarcolemma. All three proteins were also present at the myotendinous junctions and in the postsynaptic area of motor endplates. In the desmin K/O mice the distribution of plectin was unaffected, whereas synemin and paranemin were partly affected. The Z-disc-associated striations were in general no longer present in between the myofibrils. In contrast, at the myotendinous and neuromuscular junctions synemin and paranemin were still present. Our study shows that plectin differs from synemin and paranemin in its binding properties to the myofibrillar Z-discs and that the cytoskeleton in junctional areas is particularly complex in its organization.

Nearly all intermediate filament proteins exhibit a highly conserved amino acid motif (YRKLLEGEE) at the C-terminal end of their central α-helical rod domain. We have analyzed its contribution to the various stages of assembly by using truncated forms of Xenopus vimentin and mouse desmin, VimIAT and DesIAT, which terminate exactly before this motif, by comparing them with the wild-type and tailless proteins. It is surprising that in buffers of low ionic strength and high pH where the full-length proteins form tetramers, both VimIAT and DesIAT associated into various high molecular weight complexes. After initiation of assembly, both VimIAT and DesIAT aggregated into unit-length-type filaments, which rapidly longitudinally annealed to yield filaments of around 20 nm in diameter. Mass measurements by scanning transmission electron microscopy revealed that both VimIAT and DesIAT filaments contained considerably more subunits per cross-section than standard intermediate filaments. This indicated that the YRKLLEGEE-motif is crucial for the formation of authentic tetrameric complexes and also for the control of filament width, rather than elongation, during assembly. To determine the structure of the YRKLLEGEE domain, we grew crystals of peptides containing the last 28 amino acid residues of coil 2B, chimerically fused at its amino-terminal end to the 31 amino acid-long leucine zipper domain of the yeast transcription factor GCN4 to facilitate appropriate coiled-coil formation. The atomic structure shows that starting from Tyr400 the two helices gradually separate and that the coiled coil terminates with residue Glu405 while the downstream residues fold away from the coiled-coil axis.

Synemin, a developmentally regulated protein first described in muscle cells, has recently been recognized as an intermediate filament (IF) protein. Because IF proteins are invaluable markers of cell origin within the nervous system, we were interested in determining the expression pattern of synemin in the brain. Our results show that, during development of the rat cortex, synemin is expressed only in a subpopulation of astrocytic cells expressing GFAP as well as vimentin and nestin. Unlike GFAP, however, synemin is not expressed in mature astrocytes and, unlike vimentin and nestin, synemin is not present in astrocytic precursors before GFAP expression. Taken together with morphological evidence, the time course of synemin expression, as determined by Western blotting, suggests that synemin is expressed in radial glial cells undergoing morphological transformation into astrocytes. Studies of synemin expression in vitro demonstrate that, early in primary culture, the majority of polygonal astrocytes are derived from synemin(+) radial glial cells. With time in culture, however, polygonal astrocytes either stop expressing synemin or are overgrown by cells not expressing synemin. The unique pattern of synemin expression, both in vivo and in vitro, suggests that the use of synemin as a marker will add a new dimension to studies of astrocytic differentiation.

We have examined the gene expression of two radial glia intermediate filament proteins, transitin and vimentin, in the developing chick CNS. Despite global similarities in their mRNA distributions, marked regional differences are observed. Most notably, we show that transitin mRNA is localized along radial glial processes and is localized to radial glia endfeet, whereas vimentin mRNA is not localized in radial glia. Localization of transitin mRNA is best shown in the diencephalic radial glia, as well as cerebellar Bergmann glia. In addition, in the early embryonic optic tectum, telencephalon, and retina, transitin mRNA is highly localized to radial glia endfeet, which is suggestive of its transport in these cells. These in vivo demonstrations of transitin mRNA localization are confirmed by in situ hybridization analysis of cultured chick brain radial glia, which demonstrates the presence of granular staining for transitin mRNA in glial processes. Transitin mRNA distribution in developing muscle also shows a highly regulated expression pattern, especially along the Z-lines of myofibrils. As further support for the transport and localization of transitin mRNA in radial glia and muscle, we have identified a consensus RNA transport signal in transitin mRNA that is absent from vimentin. These data suggest that the local regulation of transitin protein synthesis may contribute to its function as an intermediate filament protein in radial glia.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. Here, we report an increase in vimentin gene transcription induced by the cytokine interferon-gamma (IFN-gamma). Northern blot analysis and reporter gene assays reveal that IFN-gamma induces vimentin gene transcription in HeLa cells. However, no increase in vimentin mRNA synthesis was observed de novo in MCF-7 cells, which do not already express vimentin. Band shift analysis shows that the Stat1 alpha protein mediates vimentin induction by IFN-gamma. A human mutant fibroblast cell line (U3A), which lacks Stat1 alpha but expresses vimentin mRNA, yields no increase in vimentin mRNA levels on the addition of IFN-gamma. These results suggest that the induction of vimentin gene expression might be an important part of a complex cellular response to IFN-gamma.

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

To analyze the cell cycle-dependent desmin phosphorylation by Rho kinase, we developed antibodies specifically recognizing the kinase-dependent phosphorylation of desmin at Thr-16, Thr-75, and Thr-76. With these antibodies, phosphorylation of desmin was observed specifically at the cleavage furrow in late mitotic Saos-2 cells. We then found that treatment of the interphase cells with calyculin A revealed phosphorylation at all the three sites of desmin. We also found that an antibody, which specifically recognizes vimentin phosphorylated at Ser-71 by Rho kinase, became immunoreactive after calyculin A treatment. This calyculin A-induced interphase phosphorylation of vimentin at Ser-71 was blocked by Rho kinase inhibitor or by expression of the dominant-negative Rho kinase. Taken together, our results indicate that Rho kinase is activated not only in mitotic cells but also interphase ones, and phosphorylates intermediate filament proteins, although the apparent phosphorylation level is diminished to an undetectable level due to the constitutive action of type 1 protein phosphatase. The balance between intermediate filament protein phosphorylation by Rho kinase and dephosphorylation by type 1 protein phosphatase may affect the continuous exchange of intermediate filament subunits between a soluble pool and polymerized intermediate filaments.

Desmin is the muscle-specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. There have been increasing reports of human cardiomyopathies associated with abnormal accumulation and aggregation of desmin filaments. Recently identified desmin mutations in humans suffering from skeletal muscle myopathy and cardiomyopathy suggest that these diseases might arise as a consequence of impaired function of desmin filaments. Previous generation of desmin null mice in our laboratory demonstrated that the absence of desmin results in myocyte ultrastructural defects and myocyte cell death leading to fibrosis and calcification of the myocardium. However, the effects that these defects have on cardiac function were not addressed. To further our understanding of desmin function in vivo, and in order to address the direct involvement of desmin in cardiomyopathy, we investigated the effect of the absence of desmin on myocardial mass, myocyte size and shape, changes in gene expression and cardiac systolic and diastolic function in mice. Morphometric characterization of isolated cardiomyocytes demonstrated a 24% increase in cell volume in the desmin null mice, solely due to an increase in transverse section area, suggesting for the first time that mice lacking the intermediate filament protein desmin develop concentric cardiomyocyte hypertrophy. This type of hypertrophy was accompanied by induction of embryonic gene expression and later by ventricular dilatation, and compromised systolic function. These results demonstrate that desmin is essential for normal cardiac function, and they suggest that the absence of an intact desmin filament system, rather than accumulation of the protein, may be responsible for the pathology seen in some of the desmin associated cardiomyopathies.

Synemin is a cytoskeletal protein originally identified as an intermediate filament (IF)-associated protein because of its colocalization and copurification with the IF proteins desmin and vimentin in muscle cells. Our sequencing studies have shown that synemin is an unusually large member (1,604 residues, 182,187 Da) of the IF protein superfamily, with the majority of the molecule consisting of a long C-terminal tail domain. Molecular interaction studies demonstrate that purified synemin interacts with desmin, the major IF protein in mature muscle cells, and with α-actinin, an integral myofibrillar Z-line protein. Furthermore, expressed synemin rod and tail domains interact, respectively, with desmin and α-actinin. Analysis of endogenous protein expression in SW13 clonal lines reveals that synemin is coexpressed and colocalized with vimentin IFs in SW13.C1 vim+ cells but is absent in SW13.C2 vim− cells. Transfection studies indicate that synemin requires the presence of another IF protein, such as vimentin, in order to assemble into IFs. Taken in toto, our results suggest synemin functions as a component of heteropolymeric IFs and plays an important cytoskeletal cross-linking role by linking these IFs to other components of the cytoskeleton. Synemin in striated muscle cells may enable these heterofilaments to help link Z-lines of adjacent myofibrils and, thereby, play an important role in cytoskeletal integrity.