Intermediate Filament Protein Vimentin Research Articles

Hydrodynamic and Electrical Characterization of T-vimentin Dimers and Tetramers by Transient Electric Birefringence Measurements

The structure and charge distribution of T-vimentin, which differs from the intact intermediate filament protein vimentin through the absence of the first 70 amino acids, has been studied by transient electric birefringence measurements. It is found that in 0.7 mM phosphate, pH 7.5 buffer, exclusively single dimers (with a hydrodynamic length of 40 to 43 nm) are present, which are considerably bent and/or flexible and which have a relatively large permanent dipole moment. This indicates a parallel alignment of two protein chains. In 0.2 mM phosphate, 0.5 mM MgCl2, pH 7.5, predominantly tetrameric T-vimentin is found with a rigid structure, no permanent dipole moment, and a length of 63 to 68 nm. Tetramer formation is likely to be induced by binding of Mg2+ to the protein. The observed length is in agreement with that of intact vimentin tetramers in which the 1B regions of the rod domains of the dimers overlap (A11 configuration). A minor part of the tetramers may be in a flexible or bent A22 form. The loss of the permanent dipole moment when tetramers are formed is, apart from charge compensation, presumably due to the antiparallel alignment of the constituting dimers in which their dipoles cancel.

Colocalization of Nestin and Vimentin/Desmin in Skeletal Muscle Cells Demonstrated by Three-Dimensional Fluorescence Digital Imaging Microscopy

During skeletal muscle development three intermediate filament proteins are expressed: nestin, vimentin, and desmin. Vimentin and desmin belong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more distantly related, class VI, intermediate filament. It was previously observed by conventional immunocytochemistry that the intracellular patterns of nestin, desmin, and vimentin appeared indistinguishable, despite nestin's more distant evolutionary relationship. We here extend this analysis by applying three-dimensional fluorescence digital imaging microscopy to compare the intracellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells can produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo, i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree of overlap between nestin and desmin/vimentin was very extensive in myoblasts and in multinucleate myotubes in all regions of the cells. In contrast, nestin did not colocalize with tubulin or actin in G6 myoblasts. In particular, nestin immunoreactivity was not detected at the microtubule-organizing center, and it was only sparsely observed at the cell periphery where actin stress fibers were seen. Our data lend further support to the notion that nestin interacts very closely with the two more distantly related class III intermediate filament proteins desmin and vimentin in the entire muscle cell, before and after myotube formation. A comparison of conserved amino acid residues in the different IFs suggests that charged amino acid residues in the α-helical rod domain may play a role in the interaction.

Rapid Changes in the Expression of Glial Cell Proteins Caused by Experimental Retinal Detachment

We examined the expression of several proteins normally present in Müller's glia after the production of experimental retinal detachment in adult cats. Retinas were detached for one-half to seven days, after which the tissue was processed for correlative immunocytochemistry and biochemistry. Previous studies demonstrated that the intermediate filament proteins glial fibrillary acidic protein and vimentin, increase after long-term retinal detachment (30 to 60 days), whereas glutamine synthetase, carbonic anhydrase C, and cellular retinaldehyde-binding protein all decrease to barely detectable levels. Alterations in Müller cell protein expression are rapid and specific events that can be detected as early as two days after retinal detachment. By seven days, levels of protein expression are similar to those in the long-term retinal detachments. Within the first week after injury the Müller cell processes hypertrophy and begin forming glial scars, which indicates that early intervention may be required to halt or reverse the effects of detachment.

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

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

High-affinity binding and localization of the cyclic GMP-dependent protein kinase with the intermediate filament protein vimentin.

The major receptor protein for cyclic GMP (cGMP) in smooth muscle is the cGMP-dependent protein kinase (cGMP kinase). The more abundant I alpha isoform (subunit M(r) congruent to 78,000) of this enzyme mediates the effects of cGMP to relax contracted vascular smooth muscle preparations. In this study, we have addressed the hypothesis that the cGMP kinase is anchored to intracellular proteins which might serve to target cGMP kinase to protein substrates. Using a gel overlay technique, immunoprecipitation, and a fluorescence binding assay for cGMP kinase, we have identified vimentin as a high-affinity and specific binding protein for cGMP kinase. Binding of cGMP kinase to vimentin is reversible and stoichiometric (one cGMP kinase dimer/vimentin dimer) with a KD of approximately 49 nM. The site of high-affinity binding between cGMP kinase and vimentin did not appear to be localized to the catalytic domain of the kinase since vimentin phosphorylated by cGMP kinase and peptide substrates for cGMP kinase did not compete for high-affinity binding. Neither the proteolytically-derived 69-kDa catalytic fragment nor the 8-kDa N-terminal fragment bound vimentin with high affinity, suggesting that the cGMP kinase dimer was necessary for the interaction. Vimentin was readily phosphorylated in vitro with the dimer, but not the monomeric 69-kDa catalytic fragment even though the monomeric 69-kDa fragment was catalytically active toward other substrates such as histone F2b and peptides. This suggests that the high-affinity interaction between cGMP kinase and vimentin occurs at the N-terminal region, thus allowing the interaction between the phosphorylation site of vimentin and the catalytic site of cGMP kinase to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Charge interactions in the rod domain drive formation of tetramers during intermediate filament assembly

The purpose of this study was to test a long standing hypothesis regarding the forces that drive the assembly of intermediate filaments (IFs). The initial step of IF assembly is the formation of dimeric, alpha-helical coiled coils. On the outside of the coiled coils, charged amino acids are distributed periodically such that positively and negatively charged residues are arranged in alternating zones, 9.5/2 residues wide (Parry et al., 1977; McLachlan and Stewart, 1982). This structural feature has given rise to the hypothesis that, if neighboring coiled coils were staggered axially by an odd multiple of a charged zone, electrostatic interactions between them could provide the driving force for the assembly of higher order oligomers or filaments (Fraser et al., 1986; Parry and Steinert, 1992). Using the IF protein vimentin as a model system, we carried out deletion mutagenesis experiments to test this hypothesis. We generated mutant vimentin proteins lacking 14, 21, and 28 residues in Helix 1B of the rod domain, and analyzed their assembly properties by DNA transfection into IF null cells, in vitro assembly, and chemical cross-linking. Results from these experiments are consistent with, and support, the hypothesis that charge complementation plays a key role in the assembly and stabilization of intermediate filaments.

Structural Elements the Amino-Terminal Head Domain of Vimentin Essential for Intermediate Filament Formation in Vivo and in Vitro

The biological functions of the non-α-helical, N- and C-terminal head and tail domains of intermediate filament (IF) proteins are still ill-defined. Previously, it has been shown that the basic, N-terminal head piece of the type III IF protein vimentin is essential for regular IF assembly and that arginine residues within the N-terminus may be involved. In order to identify particular regions within this domain essential for filament formation and stabilization, N-terminally truncated and arginine substitution forms of vimentin were constructed via site-directed in vitro mutagenesis of murine vimentin cDNA. The de novo filament assembly properties of these modified forms were compared with those of wild-type vimentin after transient expression in vimentin-free, cultured cells. In order to investigate their filament assembly competence in vitro, they were also produced in an E. coli expression system. It could be demonstrated that deletion of the first 10, 13, 17, and 32 amino acid residues, respectively, from the N-terminus of vimentin has an increasingly deleterious effect on filament assembly in vitro and network formation in vivo and that, thus, the highly conserved sequence motif, SSYRRXFGG, located in the N-terminus of various IF proteins and partially or totally removed by the above deletions plays a particularly important role in both activities. These results were confirmed and extended by arginine point mutations in the N-terminal head region, which showed that only one of the two adjacent arginine residues located within the conserved sequence motif is essential for filament assembly and stability in vitro as well as network formation in vivo. The neighboring arginine residues could be replaced by lysine residues without severe effects on the assembly properties of the respective mutant proteins. Distinction between the assembly-promoting potentials of the two arginine residues of the N-terminal doublet was considerably facilitated by a Val380 → Asp substitution toward the carboxy-end of the 2B segment of the vimentin rod domain. The synergistic effect of point mutations in this and the N-terminal region of the vimentin molecule implies the interaction of both protein domains in the process of filament assembly. Mutant vimentin proteins that were characterized by distinct incompetence to assemble into IFs caused a massive collapse of the endogenous vimentin filament system when expressed in mouse skin fibroblasts.

Vimentin’s tail interacts with actin-containing structures in vivo

The tail domain of the intermediate filament (IF) protein vimentin is unnecessary for IF assembly in vitro. To study the role of vimentin's tail in vivo, we constructed a plasmid that directs the synthesis of a 'myc-tagged' version of the Xenopus vimentin-1 tail domain in bacteria. This polypeptide, mycVimTail, was purified to near homogeneity and injected into cultured Xenopus A6 cells. In these cells the tail polypeptide co-localized with actin even in the presence of cytochalasin. Two myc-tagged control polypeptides argue for the specificity of this interaction. First, a similarly myc-tagged lamin tail domain localizes to the nucleus, indicating that the presence of the myc tag did not itself confer the ability to co-localize with actin (Hennekes and Nigg (1994) J. Cell Sci. 107, 1019-1029). Second, a myc-tagged polypeptide with a molecular mass and net charge at physiological pH (i.e. -4) similar to that of the mycVimTail polypeptide, failed to show any tendency to associate with actin-containing structures, indicating that the interaction between mycVimTail and actin-containing structures was not due to a simple ionic association. Franke (1987; Cell Biol. Int. Rep. 11, 831) noted a similarity in the primary sequence between the tail of the type I keratin DG81A and vimentin. To test whether the DG81A tail interacted with actin-containing structures, we constructed and purified myc-tagged DG81A tail polypeptides. Unexpectedly, these keratin tail polypeptides were largely insoluble under physiological conditions and formed aggregates at the site of injection. While this insolubility made it difficult to determine if they associated with actin-containing structures, it does provide direct evidence that the tails of vimentin and DG81A differ dramatically in their physical properties. Our data suggest that vimentin's tail domain has a highly extended structure, binds to actin-containing structures and may mediate the interaction between vimentin filaments and microfilaments involved in the control of vimentin filament organization (Hollenbeck et al. (1989) J. Cell Sci. 92, 621; Tint et al. (1991) J. Cell Sci. 98, 375).

The presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human SW-13 cells.

Human SW-13 cells express the intermediate filament protein vimentin in a mosaic pattern (Hedberg, K. K. and Chen, L. B. (1986). Exp. Cell Res. 163, 509-517). We have isolated SW-13 clones that do (vim+) or do not (vim-) synthesize vimentin as analyzed using anti-intermediate filament immunofluorescence, electron microscopy and two-dimensional gel analysis of detergent-extracted preparations. Vimentin is the only cytoplasmic intermediate filament protein present in the vim+ cells, and the vim- cells do not contain any detectable cytoplasmic intermediate filament system. The presence or absence of intermediate filaments did not observably affect the distribution of mitochondria, endoplasmic reticulum, microtubules or actin stress fibers when these structures were visualized by fluorescence microscopy. However, electron microscopy and anti-lamin A/C immunofluorescence studies showed that nuclear morphology in vim- cells was frequently characterized by large folds or invaginations, while vim+ cells had a more regular or smooth nuclear shape. When vim- cells were transfected with a mouse vimentin expression plasmid, the synthesis of a mouse vimentin filament network restored the smooth nuclear morphology characteristic of vim+ cells. Conversely, when vim+ cells were transfected with a carboxy-terminally truncated mutant vimentin, expression of the mutant protein disrupted the organization of the endogenous vimentin filaments and resulted in nuclei with a prominently invaginated morphology. These results indicated that in SW-13 cells the vimentin filament system affects the shape of the nucleus.

Immunohistochemical identification and characterization of a special type of desmin-producing stromal cells in human placenta and other fetal tissues

Abstract An unusual type of stromal cells has been found to be abundantly present in chorionic villi of human placenta of gestational weeks 6, 17, 35–42 and in tissues of early stages of fetal development (gestational weeks 16–21). These mesenchymal cells are loosely arranged throughout the villous interior and contain the intermediate filament (IF) proteins vimentin and desmin; however the smooth muscle (sm) markers sm-α-actin and sm-myosin are absent. Typical myoid stromal cells that are positive for both desmin and sm-α-actin also occur in this tissue but are restricted to certain dispersed cell clusters associated with blood vessels. Similar disperse desmin-positive, sm-α-actin-negative stromal cells have also been identified, although more sparsely, in the chorionic plate of the placenta and in other diverse fetal tissues such as the interstitium of the kidney, of testis and epididymis, and in cells surrounding Hassall bodies of thymus. The biological nature of these desmin-containing but sm-α-actin-negative stromal cells is discussed in relation to myoid cell differentiation. It is emphasized that despite their synthesis of considerable amounts of desmin they cannot be considered myogenic as the occurrence of desmin in the cells may represent an isolated expression of an individual IF protein gene, independent of the synthesis of other muscle proteins.

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell lines in vitro and in vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-rasH, v-rasKi, v-mos, SV40T and c-myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determined in vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection with ras, despite similar ras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-rasH + SV40T and v-rasH + v-mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentin-positive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.

Immunohistochemical localisation of cytokeratin and vimentin intermediate filament proteins in canine mammary tumours

Monoclonal antibodies to human stratifying keratin (StK), simple keratin ( sk), broad spectrum keratin ( bsk) and vimentin were applied to 47 canine mammary tumours (three benign hyperplasia, 26 benign mammary tumours, one malignant mixed tumour, two malignant complex tumours, seven lobular carcinomas, three papillary carcinomas and five squamous cell carcinomas). In benign hyperplasia the SK antibody reacted with acinar and duct epithelial cells; the StK and bsk antibodies reacted strongly with duct epithelial cells and myoepithelium. Neoplastic epithelial cells in the canine mammary tumours expressed mainly keratins of stratified epithelia rather than those of simple epithelia. Myoepithelial cells in complex and mixed tumours can express StK and vimentin. The finding might be helpful in the assessment of complex tumours where stromal reactions may be confused with myoepithelial neoplastic proliferation. Stromal mesenchymal tissues, including precartilage in complex tumours, and clearly differentiated myoepithelial cells always stained positively for vimentin. Occasional carcinoma cells stained positively for vimentin.

Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions.

The reaction of microglial and a stroglial cells to anterograde axonal degeneration was studied in the fascia dentata of adult rats at various timepoints after removal of the entorhinal perforant path projection. Microglial cells were identified by histochemical staining for nucleoside diphosphatase (NDPase) at light and electron microscopical levels. Astroglial cells were stained immunocytochemically for glial fibrillary acidic protein (GFAP). Activated astroglial cells and some microglial cells also stained immunocytochemically for the intermediate filament protein vimentin. Phagocytotic activity was detected by histochemical staining for acid phosphatase. The postlesional connective reorganization of the cholinergic septohippocampal projection was monitored by histochemical staining for acetylcholinesterase. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path zones of the fascia dentata and the adjacent neuropil reacted by shortening and coarsening of processes and an increase in NDPase reactivity. These changes occurred prior to a noticeable increase in GFAP immunoreactivity and hypertrophy of astroglial cells (first evident on postlesional day 2) or sprouting of cholinergic septohippocampal fibres (first evident on day 3). There was evidence of an early, local proliferation of microglial cells in the denervated perforant path zones and migration into these zones of microglial cells from adjacent intact areas. The specific accumulation of strongly stained microglial cells within the denervated parts of the dentate molecular layer persisted for at least 4 weeks, while the astroglial reaction subsided at 3 weeks. The results demonstrate an early activation of microglial cells by axonal degeneration, and indicate that these cells may play a pivotal, inductive role in the subsequent glial and neural events.

Retinoylation of vimentin in the human myeloid leukemia cell line HL60.

Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in many eukaryotic cell lines. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. The regulatory subunits of cyclic AMP-dependent protein kinase are retinoylated in the human myeloid leukemia cell line HL60 (Takahashi, N., Liapi, C., Anderson, W. B., and Breitman, T. R. (1991) Arch. Biochem. Biophys. 290, 293-302), and cytokeratins are retinoylated in normal human keratinocytes (Takahashi, N., Jetten, A. M., and Breitman, T. R. (1991) Biochem. Biophys. Res. Commun. 180, 393-400). We show, in this study, that the intermediate filament protein vimentin is retinoylated in HL60 cells during a 24-h exposure to 100 nM [3H]RA. We found that a retinoylated protein of M(r) 55,000 coeluted on anion exchange chromatography and comigrated on either one- or two-dimensional polyacrylamide gel electrophoresis with a protein that also was stained on immunoblots by an anti-vimentin antibody. About 50% of the [3H]RA was released from this M(r) 55,000 retinoylated protein after hydrolysis with either NH2OH (1 M, pH 10) or CH3OH, 0.1 M KOH. These results indicated that a large fraction of the RA was bound to vimentin by an ester bond. Both the M(r) 55,000 retinoylated protein and immunoreactive vimentin were associated with cell nuclei isolated by two procedures. They were detached during exposure to a nonionic detergent buffer, suggesting that they are bound to the nuclear envelope. These results indicate that retinoylation is a new modification of vimentin that may be an early event in RA-induced differentiation of HL60 cells.

Activation of the protein kinase a signal transduction pathway by granulocyte‐macrophage colony‐stimulating factor or by genetic manipulation reduces cytoskeletal organization in lewis lung carcinoma variants

Granulocyte-macrophage colony-stimulating factor (GM-CSF) that is produced by metastatic Lewis lung carcinoma (LLC-LN7) cells functions as an autocrine stimulator of tumor-cell motility through protein kinase A (PKA) signal transduction. This GM-CSF-mediated enhancement of LLC-LN7 cell motility coincides with a reduction in the level of polymerized F-actin. In contrast, non-metastatic LLC-C8 tumor cells, which have a diminished level of PKA signaling, do not produce GM-CSF and do not respond to exogenous GM-CSF, since they remain non-motile and retain a high content of filamentous actin. The capacity of PKA to regulate the cytoskeletal organization of tumor cells was further studied with the use of LLC variants that had been stably transfected to over-express the C alpha subunit of PKA (CEV cells) or to express a mutant cAMP-resistant PKA RI alpha subunit resulting in a defective PKA (REV cells). When compared with wild-type metastatic LLC-LN7 cells, in which the F-actin staining was too diffuse to be clearly visualized microscopically, the PKA-defective REV-LN7 transfectants had an increased level of F-actin. In comparison with the wild-type non-metastatic LLC-C8 cells, which had a high content of F-actin, the CEV-C8 transfectants that over-expressed PKA activity had a reduced level of F-actin. The reduced polymerization of actin in these CEV-C8 transfectants was accompanied by reduced levels of the intermediate filament protein vimentin and a shift in the distribution both of F-actin and of vimentin to the periphery of the cells. These results show reduced cytoskeletal organization in metastatic LLC-LN7 cells as compared with that of non-metastatic LLC-C8 cells, and indicate that elevation of PKA activity, either by autologous GM-CSF or by genetic manipulation, diminishes cytoskeletal organization.

Modulation of contractile proteins in embryonic and fetal chick cardiac cells by phorbol ester, gamma-interferon, 5-azacytidine and diacylglycerols

We studied changes in the concentration of tropomyosin, actin, desmin and vimentin in cultured myocardiocytes from Hamburger and Hamilton's stages 29 and 39 chick embryos (HH29 and HH39) (1), treated with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), 5-azacytidine (AZA), gamma interferon (INF) and dyacylglycerols (DAG). In embryonic myocardiocytes at HH29, the first three agents modified the intracellular distribution of the thin filament proteins tropomyosin and actin, increasing their cytoplasmic concentration and decreasing their cytoskeletal concentration. The concentration of the intermediate filament proteins desmin and vimentin increased in both subcellular fractions after treatment with these drugs. In fetal myocardiocytes at HH39, total protein content decreased after treatment with these drugs. Cytoplasmic and cytoskeletal concentrations of actin and tropomyosin decreased to different degrees after treatment with TPA, AZA or DAG in HH39 myocardiocytes. TPA, AZA and DAG decreased desmin in the cytoplasmic and cytoskeletal fractions. These findings suggest that the drugs tested alter the normal protein composition in cultured myocardiocytes, and have different effects depending on the developmental stage in which the embryo is treated.

In vitro assembly properties of vimentin mutagenized at the beta-site tail motif.

The intermediate filament (IF) proteins vimentin, desmin and peripherin share a 9-residue sequence motif (beta-site) located near the end of their COOH-terminal tail domain. Peptide inhibition experiments have previously suggested that the beta-site is involved in interactions that limit the lateral growth of IFs and prevent inappropriate filament-filament associations. To investigate this question further, we have constructed and expressed, in Escherichia coli, hamster vimentin bearing different mutations in the beta-site. We show here that a single exchange of glycine 450 with a valine residue, or an internal deletion of amino acids 444-452, strongly interferes with the normal assembly of IFs under in vitro conditions. These mutants polymerize into irregular fibrils that have a strong tendency to anastomose and laterally aggregate under isotonic conditions. In contrast, a non-conservative substitution of arginine 448 for glutamic acid does not significantly interfere with filament structure and yields subunits that polymerize into long, smooth filaments that show a slight aberration in thickness. All mutant proteins are soluble in low salt and form oligomers similar to the ones formed by wild-type vimentin. On the basis of these findings and on related observations, we propose that the tail domain of type III IF proteins contains important structural elements involved in lateral protofilament-protofilament interactions.

Induction of adipose conversion in 3T3-L1 cells is associated with an early phosphorylation of a protein partly homologous with mouse vimentin

Induction of adipose conversion in 3T3-L1 by bezafibrate has been previously shown to be enhanced by dibutyryl cAMP and to be associated with an early phosphorylation of a 60 kDa acidic protein [Biochim. Biophys. Acta 1054 (1990) 219-224; FEBS Lett. 285 (1991) 63-65]. We describe here the isolation and sequencing of two peptides of the protein concerned. Both appear to be homologous with two respective amino acid sequences of the mouse intermediate filament protein vimentin.

Vimentin expression is differentially regulated by IL-2 and IL-4 in murine T cells.

IL-2 and IL-4 are T cell growth factors that are produced by different T cell subsets and have distinct roles in lymphocyte biology. Despite their importance in the immune system, little is known about the genes that these lymphokines may specifically control and the interaction of these lymphokines in regulating the expression of their target genes. In this paper, we use the factor-dependent murine T cell line (CT.4R) to investigate the interaction of IL-2 and IL-4 in regulating gene expression. We report that the intermediate filament protein vimentin is differentially regulated by these lymphokines. Cells grown in IL-2 typically express 10- to 20-fold more vimentin and vimentin RNA than those grown in IL-4, but express similar levels of other cytoskeletal proteins including actin and tubulin. Vimentin was specifically induced by IL-2 and apparently suppressed by IL-4 in normal lymph node T cells, suggesting that its differential regulation by these lymphokines is physiologically relevant. We investigated the synergy between IL-2 and IL-4 in regulating the expression of vimentin RNA and compared it to that of two other lymphokine-responsive genes, pancreatic lipase and the IL-2R alpha subunit. Complex regulatory interactions were revealed: IL-4 suppressed the ability of IL-2 to induce vimentin RNA but not IL-2R alpha RNA, whereas IL-2 inhibited the ability of IL-4 to induce lipase RNA. These results indicate that IL-2 and IL-4 can cross-regulate lymphokine-responsive genes and can simultaneously exert both positive and negative regulation of different genes within the same cell.

Differential Modulation of the Expression of the Intermediate Filament Proteins Vimentin and Nuclear Lamins A and C by Differentiation Inducers in Human Myeloid Leukemia (U-937, HL-60) Cells

Earlier reports seemed to indicate that the cytoplasmic intermediate filament protein vimentin and the nuclear intermediate-type filament proteins A and C lamins are expressed in a coordinate manner in human myeloid cells. We have comparatively studied the expression of the vimentin and the A/C lamin genes at the RNA and protein levels in human U-937 promonocytic and HL-60 promyelocytic cells treated with differentiation inducers. 12-O-Tetradecanoyl phorbol-13-acetate and cytosine arabinoside produced a coordinate and stable stimulation of both vimentin and A/C lamin expression in U-937 cells. A stable increase in vimentin expression was also produced by sodium butyrate and by dibutyryl cyclic AMP in U-937 cells and by dimethyl sulfoxide in HL-60 cells. In contrast, these agents produced only a transient increase in A/C lamin expression (maximum mRNA levels at 6-24 h), which later returned to expression levels similar to or even lower than those in untreated cells. Retinoic acid greatly inhibited vimentin expression in HL-60 cells, but it had little effect on A/C lamin expression. Taken together, the present results suggest that there are important differences in the mechanisms which regulate the expression of the vimentin and nuclear lamin genes, as well as in their implication in the differentiation of human myeloid cells.