Vimentin and desmin of a cartilaginous fish, the shark Scyliorhinus stellaris: sequence, expression patterns and in vitro assembly

In the shark Scyliorhinus stellaris we have biochemically identified and cDNA-cloned orthologs of human vimentin and desmin, SstV and SstD, as deduced from immunoblotting and sequence alignment with teleost, frog and human vimentin and desmin, respectively. This allowed us to further clarify the relationship of previously identified lower vertebrate intermediate filament proteins to mammalian vimentin and desmin. Immunofluorescence microscopy with antibodies H5 and VIM13.2 showed vimentin expression in shark eye and brain and absence in epithelia, which resembles the situation in higher vertebrates. In addition, SstV is expressed in many mesenchymal cell types which corresponds to the case in terrestrial vertebrates but strongly differs from teleosts. Surprisingly, shark interstitial cells, including fibroblasts, express neither SstV nor keratins but other as yet unidentified intermediate filament proteins as deduced from their reactivity with antibody IFA. In vitro assembly studies of recombinant SstV revealed a temperature optimum for uncompromised filament assembly of 15 degrees C. At 18 degrees C, but more pronounced at 21 degrees C and 24 degrees C, which is notably above the animal's inherent preferred environmental temperature, both, SstV and SstD assemble into thick and inflexible fibers. Thus, environmental temperature apparently is, as a general principle, a driving force for the fine tuning of protein primary structure and eventually 3D structure.     

Vimentin Dephosphorylation by Protein Phosphatase 2A Is Modulated by the Targeting Subunit B55

The intermediate filament protein vimentin is a major phosphoprotein in mammalian fibroblasts, and reversible phosphorylation plays a key role in its dynamic rearrangement. Selective inhibition of type 2A but not type 1 protein phosphatases led to hyperphosphorylation and concomitant disassembly of vimentin, characterized by a collapse into bundles around the nucleus. We have analyzed the potential role of one of the major protein phosphatase 2A (PP2A) regulatory subunits, B55, in vimentin dephosphorylation. In mammalian fibroblasts, B55 protein was distributed ubiquitously throughout the cytoplasm with a fraction associated to vimentin. Specific depletion of B55 in living cells by antisense B55 RNA was accompanied by disassembly and increased phosphorylation of vimentin, as when type 2A phosphatases were inhibited using okadaic acid. The presence of B55 was a prerequisite for PP2A to efficiently dephosphorylate vimentin in vitro or to induce filament reassembly in situ. Both biochemical fractionation and immunofluorescence analysis of detergent-extracted cells revealed that fractions of PP2Ac, PR65, and B55 were tightly associated with vimentin. Furthermore, vimentin-associated PP2A catalytic subunit was displaced in B55-depleted cells. Taken together these data show that, in mammalian fibroblasts, the intermediate filament protein vimentin is dephosphorylated by PP2A, an event targeted by B55.     

The chicken vimentin gene. Nucleotide sequence, regulatory elements, and comparison to the hamster gene

Here we report the nucleotide sequence of the chicken vimentin gene and its deduced primary amino acid sequence. A comparison of this gene to other intermediate filament protein genes demonstrates that both exon size and position are strongly conserved features of this multigene family. In addition, the hamster and chicken vimentin genes exhibit strong identity at the level of nucleotide (74%) and amino acid (80%) sequence. Interestingly, 40% of total sequence diversity is localized to the N terminus or "head" region of these genes whereas other protein domains (rod and C terminus) are remarkably identical in both nucleotide (81%) and amino acid (89%) sequence. Even stronger amino acid identity (100%) is exhibited in certain subdomains which may define regions crucial for filament formation and function. Not surprisingly, vimentin is more homologous across animal species than it is to other intermediate filament protein members (e.g. desmin) within the same species. A comparison of 5'-flanking sequences of the hamster and chicken genes as well as other characterized promoter elements (SV40, HSV-TK) reveals homologous sequence elements which may define common and/or unique sites involved in the modulation of gene expression. The implications of these sequence elements for both tissue-specific and developmental expression of the vimentin gene are discussed.     

Transgenic expression of the muscle-specific intermediate filament protein desmin in nonmuscle cells

The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.     

Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly

We have assessed the mechanism of intermediate filament assembly by assaying the sites of incorporation of chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. The localization of newly synthesized vimentin was determined by immunofluorescence and immunoelectron microscopy at short time periods of induced synthesis, using antibodies specific for chicken vimentin. Under conditions where neither the soluble subunit pools nor the steady-state distribution of endogenous filaments are affected, newly synthesized vimentin incorporates into the vimentin filament network at numerous and discrete sites throughout the cell. Over time, the pattern of newly assembled vimentin converts to a continuous array coincident with preexisting vimentin filaments. These results are consistent with a novel mechanism of intermediate filament assembly, whereby growth of intermediate filaments occurs by topographically restricted and localized subunit addition, necessitating a transient disruption of filament integrity.     

Vimentin gene: expression in human lymphocytes and in Burkitt''s lymphoma cells.

We have isolated a human genomic clone for the intermediate filament subunit vimentin with a DNA probe encoding chicken vimentin. We show that the gene for this protein exists as a single copy in the haploid human genome and is transcribed into one mature RNA species of 2 kb. In vitro translation of poly(A)+ mRNA in a rabbit reticulocyte cell-free system showed that vimentin is a major product of RNA from normal lymphocytes but not of RNA extracted from Burkitt cells. 2-kb vimentin mRNA can be detected with a DNA probe in normal lymphocytes and in fibroblasts, but not in cell lines derived from Burkitt's lymphoma (JI, JBL2, BJAB, DAUDI). The abundance of vimentin mRNA is correlated with the quantity of vimentin present in the cells, suggesting that the level of expression is regulated by the abundance of mRNA. The half-lives of vimentin mRNA were found identical in both fibroblasts and lymphocytes and belong to the class of stable mRNA.     

Vimentin cDNA clones covering the complete intermediate-filament protein are found in an EHS tumor cDNA library

Intermediate filaments are part of the cytoskeleton of most cells. To analyze changes in intermediate filament synthesis, we have isolated two cDNA clones (pV-C25, pV-C877) that cover the complete coding sequence of the murine intermediate filament protein vimentin. The cDNA clones were isolated from a murine Engelbreth-Holm-Swan (EHS) tumor cDNA library by screening under (i) non-stringent conditions with a synthetic oligodeoxynucleotide (oligo), LW-36, which is specific for type-IV collagen, and (ii) stringent conditions with oligo LW75, which was derived from the vimentin clone pV-C25. The cDNA clones contain 38 nucleotides (nt) of the 5'-untranslated region, 1398 nt of the coding region and 7 nt of the 3'-untranslated region. Comparing the mouse sequence with the published sequence for vimentin from hamster, human and chicken, we find shared identities of 99, 97 and 87%, respectively. Since the cDNA clones have been isolated from a basement membrane tumor (EHS) cDNA library, we measured the vimentin mRNA production in EHS tumor cells in culture, and found that this mRNA is half as abundant as mRNA for type-IV mRNA.     

Plectin deficiency affects precursor formation and dynamics of vimentin networks

Plectin is a typical cytolinker protein that connects intermediate filaments to the other cytoskeletal filament systems and anchors them at membrane-associated junctional sites. One of the most important binding partners of plectin in fibroblasts is the intermediate filament subunit protein vimentin. Previous studies have demonstrated that vimentin networks are highly dynamic structures whose assembly and disassembly is accomplished stepwise via several intermediates. The precursor forms as well as polymerized (filamentous) vimentin are found in the cells in a dynamic equilibrium characterized by the turnover of the subunits within the polymer and the movement of the smaller precursors. To examine whether plectin plays a role in intermediate filament dynamics, we studied vimentin filament formation in plectin-deficient compared to wild-type fibroblasts using GFP-tagged vimentin. Monitoring vimentin and plectin in spreading and dividing cells, we demonstrate that plectin is associated with vimentin from the early stages of assembly and is required for vimentin motility as well as for the stepwise formation of stable filaments. Furthermore, plectin prevents vimentin networks from complete disassembly during mitosis, facilitating the rebuilding of the intermediate filament network in daughter cells.     

Association of glycosphingolipids with intermediate filaments of mesenchymal, epithelial, glial, and muscle cells.

We reported recently that two glycosphingolipids (GSLs), globoside (Gb4) and ganglioside GM3, colocalized with vimentin intermediate filaments of human umbilical vein endothelial cells. To determine whether this association is unique to endothelial cells or to vimentin, we analyzed a variety of cell types. Double-label immunofluorescent staining of fixed, permeabilized cells, with and without colcemid treatment, was performed with antibodies against glycolipids and intermediate filaments. Globoside colocalized with vimentin in human and mouse fibroblasts, with desmin in smooth muscle cells, with keratin in keratinocytes and hepatoma cells, and with glial fibrillary acidic protein (GFAP) in glial cells. Globoside colocalization was detected only with vimentin in MDCK and HeLa cells, which contain separate vimentin and keratin networks. GM3 ganglioside also colocalized with vimentin in human fibroblasts. Association of other GSLs with intermediate filaments was not detected by immunofluorescence, but all cell GSLs were detected in cytoskeletal fractions of metabolically labelled endothelial cells. These observations indicate that globoside colocalizes with vimentin, desmin, kertain and GFAP, with a preference for vimentin in cells that contain both vimentin and keratin networks. The nature of the association is not yet known. Globoside and GM3 may be present in vesicles associated with intermediate filaments (IF), or bound directly to IF or IF associated proteins. The prevalence of this association suggests that colocalization of globoside with the intermediate filament network has functional significance. We are investigating the possibility that intermediate filaments participate in the intracellular transport and sorting of glycosphingolipids.     

Vimentin and 70K Neurofilament Protein Co-exist in Embryonic Neurones from Spinal Ganglia

Abstract: The mesenchymal intermediate filament protein vimentin and the 70K component of neurofilament were detected by two-dimensional gel electrophoresis in cultures of pure sensory and sympathetic neurones derived from chick embryos. The identities of these neuronal intermediate filament proteins were confirmed by comparison of their molecular weights, isoelectric points, and peptide patterns from limited papain digestions with those of the corresponding proteins from fibroblasts and brain, respectively. A specific antibody to vimentin stained filamenteous structures and colcemid-induced coils in both neurones and associated satellite cells. In contrast, a specific antibody to the 70K neurofilament protein stained these structures solely in neurones. This neurone-specific staining, as well as its molecular weight and isoelectric point, distinguishes the 70K neurofilament protein from the 68K neurofilament as sociated protein described by others, which has been claimed to resemble the tubulin assembly protein.     

A novel fibrillar structure in cultured cells detected by a monoclonal antibody

Using a monoclonal antibody, we have detected an antigen present in a unique fibrillar structure in the cytoplasm of cultured cells by immunofluorescence. These structures have been identified by transmission electron microscopy and ultrastructural immunocytochemistry as large single paracrystalline arrays of individual filaments morphologically similar to intermediate filaments. The antibody detects these structures in fibroblastic and epithelioid cultured cell lines of mouse, rat, bovine, and human origin but not of avian origin. Only a small percentage of the cells in a culture contains these structures; each cell usually contains only one, although two or more have been observed in a single cell. The structures are elongated vermiform arrays of filaments in the cytoplasm (approximately 0.5 X 3 microns) which have a thread-like or toroidal appearance. Because of this shape, we have named the putative antigen recognized by this antibody "nematin." Double-label experiments showed that these structures had no relationship to tubulin or vimentin. Immunocytochemical localization in human tissues revealed a high concentration of a reactive antigen in the stratum granulosum of skin and in what probably are neuroglial cells in the central nervous system. This monoclonal antibody may detect a novel intermediate filament protein and/or a shared determinant of different intermediate filament proteins.     

Renal Cells Express Different Forms of Vimentin: The Independent Expression Alteration of these Forms is Important in Cell Resistance to Osmotic Stress and Apoptosis

Osmotic stress has been shown to regulate cytoskeletal protein expression. It is generally known that vimentin is rapidly degraded during apoptosis by multiple caspases, resulting in diverse vimentin fragments. Despite the existence of the known apoptotic vimentin fragments, we demonstrated in our study the existence of different forms of vimentin VIM I, II, III, and IV with different molecular weights in various renal cell lines. Using a proteomics approach followed by western blot analyses and immunofluorescence staining, we proved the apoptosis-independent existence and differential regulation of different vimentin forms under varying conditions of osmolarity in renal cells. Similar impacts of osmotic stress were also observed on the expression of other cytoskeleton intermediate filament proteins; e.g., cytokeratin. Interestingly, 2D western blot analysis revealed that the forms of vimentin are regulated independently of each other under glucose and NaCl osmotic stress. Renal cells, adapted to high NaCl osmotic stress, express a high level of VIM IV (the form with the highest molecular weight), besides the three other forms, and exhibit higher resistance to apoptotic induction with TNF-α or staurosporin compared to the control. In contrast, renal cells that are adapted to high glucose concentration and express only the lower-molecular-weight forms VIM I and II, were more susceptible to apoptosis. Our data proved the existence of different vimentin forms, which play an important role in cell resistance to osmotic stress and are involved in cell protection against apoptosis.     

Immunocytochemical demonstration of a new vimentin-associated protein in 3T3 fibroblasts

Summary Using a xanthophore cytoskeletal preparation as immunogen, we have produced a monoclonal antibody, A2, which recognized a 160 kDa protein in 3T3 fibroblasts. This protein makes up a cytoplasmic filamentous system, which colocalizes with vimentin filaments. When microtubules and actin filaments are dissolved by high salt extraction, staining with antibody A2 is unaffected. Immunoblot analysis confirms that the 160 kDa protein is co-isolated with vimentin duringin vivo high salt extraction. Following vinblastine treatment, both the 160 kDa protein and vimentin become localized to perinuclear caps, as do other intermediate filaments and their associated proteins; after vinblastine removal, the immunostaining produced by A2 becomes filamentous. Immunoelectron microscopy demonstrates that antibody A2 stains a filament system with a diameter of about 10 nm. Our observations suggest that the 160 kDa protein may be a new vimentin-associated protein which differs from the intermediate filament-associated proteins previously reported, and is widely distributed in several cell types.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network

Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.     

Posttranslational regulation of keratins: degradation of mouse and human keratins 18 and 8.

Human keratin 18 (K18) and keratin 8 (K8) and their mouse homologs, Endo B and Endo A, respectively, are expressed in adult mice primarily in a variety of simple epithelial cell types in which they are normally found in equal amounts within the intermediate filament cytoskeleton. Expression of K18 alone in mouse L cells or NIH 3T3 fibroblasts from either the gene or a cDNA expression vector results in K18 protein which is degraded relatively rapidly without the formation of filaments. A K8 cDNA containing all coding sequences was isolated and expressed in mouse fibroblasts either singly or in combination with K18. Immunoprecipitation of stably transfected L cells revealed that when K8 was expressed alone, it was degraded in a fashion similar to that seen previously for K18. However, expression of K8 in fibroblasts that also expressed K18 resulted in stabilization of both K18 and K8. Immunofluorescent staining revealed typical keratin filament organization in such cells. Thus, expression of a type I and a type II keratin was found to be both necessary and sufficient for formation of keratin filaments within fibroblasts. To determine whether a similar proteolytic system responsible for the degradation of K18 in fibroblasts also exists in simple epithelial cells which normally express a type I and a type II keratin, a mutant, truncated K18 protein missing the carboxy-terminal tail domain and a conserved region of the central, alpha-helical rod domain was expressed in mouse parietal endodermal cells. This resulted in destabilization of endogenous Endo A and Endo B and inhibition of the formation of typical keratin filament structures. Therefore, cells that normally express keratins contain a proteolytic system similar to that found in experimentally manipulated fibroblasts which degrades keratin proteins not found in their normal polymerized state.