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PubMed1986

Neurons of the olfactory epithelium in adult rats contain vimentin.

In the developing nervous system, the intermediate filament protein vimentin is found in the proliferating neuroepithelium and neural crest. As development proceeds, postmitotic neurons cease vimentin expression and neurofilament proteins begin to accumulate. We have shown that olfactory receptor neurons deviate from the general pattern of neuronal intermediate filament expression, in that they continue to express vimentin or a highly vimentin-like protein rather than neurofilament proteins in the adult rat. With light-microscopic immunohistochemistry, three independently derived antibodies to vimentin label all portions of the primary olfactory projection, including the sensory neuron cell bodies in the olfactory epithelium, the fascicles of the olfactory nerve, and their axonal arbors in the glomeruli of the olfactory bulb. In contrast, anti-neurofilament antisera stain only rare scattered receptor cells and a small number of axons in the olfactory nerve. Electron-microscopic immunohistochemistry shows dense staining of olfactory axons with anti-vimentin. The vimentin-like immunoreactive material in the olfactory nerve layer was characterized by SDS-PAGE and by immunoblotting. On immunoblots of homogenates of the olfactory nerve, the anti-vimentin monoclonal antibody SBV-21 (Blose et al., 1984) stains only a single protein of Mr = 55 kDa. This band comigrates with vimentin in crude cytoskeletal material from the neonatal rat brain prepared according to the method of Dahl et al. (1981). SBV-21 does not stain neurofilament triplet proteins or glial fibrillary acidic protein, which are also present in these blots. These results demonstrate that the vast majority of olfactory receptor neurons and their axons contain vimentin or a protein of similar immunological character and electrophoretic mobility, while identifiable expression of neurofilament proteins is confined to a very small subpopulation. Hence, the switch in intermediate filament proteins that normally accompanies neuronal maturation is arrested in most olfactory neurons, and a "juvenile" biochemical marker is retained. This population of neurons is also unique among mammalian neurons in several other respects, including that olfactory neurons die during normal adult life or following injury and then are replaced from a proliferating pool of stem cells.

Age Factors↗
PubMed1995

[Vimentin and desmin expression in the type 2 fibers in muscle biopsies from myotonic dystrophy patients].

Immunoreactivity for the intermediate filament proteins vimentin and desmin was assessed in the type 2 fibers of biceps brachii muscle biopsies from 20 adults with myotonic dystrophy (MD). Some type 2C fibers showed strong reactivity with both anti-vimentin and anti-desmin, suggesting that they were regenerating fibers. Some type 2A and 2B fibers of various diameter were weakly reactive with anti-vimentin, but none of the type 1 fibers were reactive with anti-vimentin. Hardly any reactivity with anti-vimentin was demonstrated in patients in the younger age group (10-29 years) with mild illness, but vimentin-positive type 2 fibers increased in the age groups 30 years of age and older. The mean diameters of type 2A and type 2B fibers in vimentin-positive patients were larger than in vimentin-negative patients. In the patients in the 30 years of age and older group, the vimentin-negative patients had more severe muscle weakness than the vimentin-positive patients. These findings suggest a possible relationship between the process of regeneration in type 2 fibers in adult MD patients and the increase in their mean diameter.

Adolescent↗
PubMed1994

Induction of aggregation and augmentation of protein kinase-mediated phosphorylation of purified vimentin intermediate filaments by withangulatin A.

Purified assembly-competent vimentin, an intermediate filament protein, was obtained from bovine lens in this study. The effects of withangulatin A on vimentin assembly with or without phosphorylation were examined by negative-stain electron microscopy. Soluble tetrameric vimentin was assembled into irregular fibrils with lateral associations or short filaments after pretreatment with 50 or 100 microM withangulatin A, respectively. Incubation of assembled vimentin filaments with withangulatin A at 50 or 100 microM resulted in the formation of aggregates, and the degree of aggregation was concentration dependent. The appearance of vimentin filaments was slightly altered after treatment with cAMP-dependent protein kinase or protein kinase C; however, phosphorylation of filamentous vimentin by the protein kinases in the presence of withangulatin A resulted in higher degrees of aggregation of the filaments, compared with those treated only with the drug. Moreover, the level of phosphorylation of filamentous vimentin by the protein kinases was augmented in the presence of withangulatin A. Experimental results indicated that withangulatin A directly and specifically affects the conformation of the vimentin molecules, thereby resulting in alterations in assembly behavior and reactivity toward cAMP-dependent protein kinase and protein kinase C. The data observed further imply that withangulatin A, which directly causes aggregation of vimentin filaments, is a vimentin intermediate filament-targeting drug.

Animals↗
PubMed1994

Visualization and function of vimentin phosphorylation by cdc2 kinase during mitosis.

To investigate the role of intermediate filament (IF) protein phosphorylation by cdc2 kinase during mitosis, we developed a monoclonal antibody 4A4 recognizing Ser55-phosphorylated vimentin. Western blotting indicated that this antibody reacted with vimentin phosphorylated by cdc2 kinase but not with non-phosphorylated vimentin or with vimentin phosphorylated by other kinases such as cAMP-dependent protein kinase, protein kinase C, or Ca(2+)-calmodulin-dependent protein kinase II. Immunofluorescence and immunoelectron microscopy showed that vimentin Ser55 residues distributed in the entire cytoplasmic vimentin filament system are phosphorylated when the cells enter mitosis and dephosphorylated in cytokinesis. All cell lines examined showed a similar appearance of immunoreactivity with antibody 4A4. Fractionation of mitotic cell extracts on Mono-Q Sepharose revealed a single peak of vimentin Ser55 kinase activity, and the anti-p34cdc2 antibody reacted with the 34 kDa band in the kinase containing fractions. Vimentin Ser55 kinase activities were nil in the interphase cell extract. Immunofluorescent evidence using antibody 4A4 and biochemical analysis using vimentin Ser55 peptide showed that the degree of disassembly of vimentin filament of various cell types at early mitotic phase correlated well with the amount of mitotically activated cdc2 kinase.

Amino Acid Sequence↗
PubMed1992

Anti-vimentin monoclonal antibody recognizes a cell with dendritic appearance in the chicken's bursa of Fabricius.

The bursa of Fabricius was studied by immunohistochemical method using anti-vimentin monoclonal antibody (clone 3B4). This monoclonal antibody identified a vimentin positive cell in the medulla of the bursal follicle. During the first 2 weeks of life the vimentin positive cells located along the corticomedullary border and later became prominent in the medulla with the exception of a narrow zone adjacent to the corticomedullary border. After hatching the accumulation of vimentin-type intermediate filaments on one side of the nucleus endowed the vimentin positive cells with a polarized appearance. This "cap-like" vimentin positive area of the cytoplasm determined the position of the major cell process. Within the medulla the Ia positive secretory dendritic cells contained secretory granules in one of the cell processes. The distribution, shape, and polarized appearance of the vimentin positive cells were identical with that of the secretory dendritic cells. Therefore, the anti-vimentin monoclonal antibody proved to be useful for identification of the bursal secretory dendritic cells. During rapid bursal growth the number of secretory dendritic cells increased, possibly, by proliferation of vimentin negative secretory dendritic cell precursors located along the corticomedullary border.

Animals↗
PubMed1995

Dendritic cells in the bursal follicles and germinal centers of the chicken's caecal tonsil express vimentin but not desmin.

BACKGROUND: Immunohistochemical studies with anti-vimentin and anti-desmin monoclonal antibodies were designed to determine the origin of bursal secretory dendritic cells (SDC) and follicular dendritic cells. METHODS: The binding sites of anti-vimentin, anti-desmin, and anti-chicken-IgG specific monoclonal antibodies were visualized with a biotinylated anti-mouse-IgG, ABC Elite kit, and 4-chloronaphthol. Cells were double stained (anti-vimentin and rabbit anti-chicken-IgG Fc) to determine if the vimentin positive cells possessed surface IgG. RESULTS: Vimentin positive cells were observed in the cortex and medulla of the bursa and germinal center and lymphoepithelial compartment of the caecal tonsil. The mesenchymal reticular cell, the basic supporting cell of the germinal center, was stained prominently by anti-vimentin and anti-desmin. Both antibodies stained the bursal cortex but only anti-vimentin bound the bursal secretory dendritic cell of the medulla. In addition to being vimentin positive and desmin negative, the bursal secretory dendritic cell possessed and the follicular dendritic cell appeared to possess IgG on their surfaces. In all the observations, B-cells were vimentin negative. CONCLUSION: These studies suggest that follicular dendritic cells and mesenchymal reticular cells in the caecal tonsil's germinal centers may be functionally different cell populations while the bursal secretory dendritic cell and follicular dendritic cell of the caecal tonsil may have a common origin.

Animals↗
PubMed2002

Regulation of the association of alpha 6 beta 4 with vimentin intermediate filaments in endothelial cells.

The adhesion of microvascular endothelial cells to their underlying basement membrane is important for the maintenance of vascular integrity. Most integrins function in endothelial cell adhesion by forming a transmembrane link between their basement membrane ligand and the actin microfilament cytoskeleton. The alpha 6 beta 4 laminin-binding integrin, however, associates with vimentin intermediate filaments (IFs) in microvascular endothelial cells and therefore is likely to uniquely contribute to the barrier function of the endothelium. In this study, we examined the regulation of alpha 6 beta 4-vimentin IF association. We first tested the requirement for alpha 6 beta 4-laminin interactions and actin microfilament assembly. We found that alpha 6 beta 4 associated with vimentin IFs when cells were adherent to either laminin 5 or fibronectin, indicating that this association can occur independent of alpha 6 beta 4-ligand interactions. Additionally, we found that alpha 6 beta 4 was associated with vimentin IFs prior to cell spreading, indicating that changes in the microfilament cytoskeleton associated with changes in cell shape are also not required. Thus, although the association of alpha 6 beta 4 with vimentin IFs may strengthen cell adhesion by providing endothelial cells with an additional transmembrane linkage between the basement membrane and the cytoskeleton, this association is not itself regulated by alpha 6 beta 4-mediated adhesion. Finally, we tested the role of plectin in the association of alpha 6 beta 4 with vimentin IFs. Plectin is known to bind in vitro to both IFs and the beta 4 cytoplasmic domain (beta 4 tail), suggesting that it may be important for this linkage. Therefore, we generated deletion mutants of the beta 4 tail and compared the ability of alpha 6 beta 4 containing these deletions to associate with vimentin IFs. We targeted the two regions of the beta 4 tail known to bind to plectin IN VITRO: the N-terminal and C-terminal plectin binding sites. We found that deletion of the N-terminal binding site inhibited the association of alpha 6 beta 4 with vimentin IFs. Thus, plectin-beta 4 tail interactions may play an important role in connecting alpha 6 beta 4 with vimentin IFs and may prove to be important targets in the regulation of this association in endothelial cells.

Animals↗
PubMed1988

Keratin and vimentin expression in early organogenesis of the rabbit embryo.

The expression of vimentin and keratins is analysed in the early postimplantation embryo of the rabbit at 11 days post conceptionem (d.p.c.) using a panel of monoclonal antibodies specific for single intermediate filament polypeptides (keratins 7, 8, 18, 19 and vimentin) and a "pan-epithelial" monoclonal keratin antibody. Electrophoretic separation of cytoskeletal preparations obtained from embryonic tissues, in combination with immunoblotting of the resulting polypeptide bands, demonstrates the presence of the rabbit equivalents of human keratins 8, 18, and vimentin in 11-day-old rabbit embryonic tissues. Immunohistochemical staining shows that several embryonic epithelia such as notochord, surface ectoderm, primitive intestinal tube, and mesonephric duct, express keratins, while others (neural tube, dermomyotome) express vimentin, and a third group (coelomic epithelia) can express both. Similarly, of the mesenchymal tissues sclerotomal mesenchyme expresses vimentin, while somatopleuric mesenchyme (abdominal wall) expresses keratins, and splanchnopleuric mesenchyme (dorsal mesentery) expresses both keratins and vimentin. While these results are in accordance with most results of keratin and vimentin expression in embryos of other species, they stand against the common concept of keratin and vimentin specificity in adult vertebrate tissues. Furthermore, keratin and vimentin are not expressed in accordance with germ layer origin of tissues in the mammalian embryo; rather the expression of these proteins seems to be related to cellular function during embryonic development.

Animals↗
PubMed1983

TPA induces simultaneous alterations in the synthesis and organization of vimentin.

We have found an increased rate of vimentin synthesis in TPA-treated chicken embryonic fibroblasts, as shown by two-dimensional electrophoretic separation of newly synthesized polypeptides. The capacity of TPA to elicit this effect correlated with its activity as a tumor promoter. Treatment with TPA also altered the organization of the vimentin-containing intermediate filaments, as revealed by immunofluorescence. Treatments which inhibited the TPA-induced rearrangements of vimentin filaments did not prevent the increase in the rate of vimentin synthesis, indicating that gross alterations in cytoskeletal organization were not the immediate cause of the altered vimentin synthesis. On the other hand, alterations in the rate of vimentin synthesis may be a necessary, but not sufficient condition for alterations in vimentin filament organization. TPA as a positive modulator of vimentin synthesis may serve as a useful probe for an eventual understanding of the dynamics of the mechanisms that control the assembly and organization of vimentin filaments.

Animals↗
PubMed1989

Astrocytes and guidance of outgrowing corticospinal tract axons in the rat. An immunocytochemical study using anti-vimentin and anti-glial fibrillary acidic protein.

In the present investigation the role of astrocytes and their precursors in guidance of outgrowing corticospinal tract axons in the rat is studied. Antibodies against glial fibrillary acidic protein and vimentin are used to analyse immunogen expression of glial cells, whereas the postnatal outgrowth of corticospinal tract axons through the spinal cord was studied using anterogradely transported horseradish peroxidase. The first, leading corticospinal tract axons, being the objective of the present study, are characterized by dilatations at their distal ends, the growth cones. Growth cones of pioneer corticospinal tract axons are randomly distributed in the presumptive corticospinal tract area of the ventral most part of the dorsal funiculus. A dramatic change in glial cell labelling is found from the majority being vimentin immunoreactive and glial fibrillary acidic protein-negative at birth to almost all being the reverse at the end of the fourth postnatal week. From double labelling experiments it can be concluded that the vimentin-glial fibrillary acidic protein transition occurs within astrocyte precursor cells. The absence of glial fibrillary acidic protein-immunoreactive glial cells during the outgrowth period of pioneer corticospinal tract axons indicates that they cannot play a role in the guidance of outgrowing corticospinal tract pioneer axons. Vimentin-immunoreactive glial cells are present throughout the presumptive corticospinal tract area at the time of arrival of the leading corticospinal tract fibres. The vimentin-immunoreactive glial cells, which themselves are orientated perpendicular to the outgrowing corticospinal tract axons, are mainly arranged in longitudinal tiers parallel to the rostrocaudal axis. Electron microscopically, growth cones of pioneer corticospinal tract axons frequently exhibit protrusions into vimentin-immunoreactive glial cell processes, suggesting an adhesive type of contact. Therefore, in addition to a positional role, vimentin-immunoreactive glial cells probably play a chemical role in guidance of pioneer corticospinal tract axons. A prominent vimentin-immunoreactive glial septum was noted during corticospinal tract outgrowth in the midline raphe of the medulla oblongata and spinal cord whereas it is absent in the decussation area of corticospinal tract fibres. After the first postnatal week the major vimentin-immunoreactive glial barrier either completely disappears (medullary levels) or gradually reduces to a minor glial fibrillary acidic protein-immunoreactive one (spinal cord levels).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Co-expression of GFAP and vimentin in astrocytes proliferating in response to injury in the mouse cerebral hemisphere. A combined autoradiographic and double immunocytochemical study.

Changes in the distribution of proliferating astrocytes expressing glial fibrillary acidic protein (GFAP) and/or vimentin were examined in the injured cerebral hemisphere in adult mice. The injury was followed by [3H]thymidine injections at different time intervals. The brain sections were doubly immunostained for GFAP and vimentin and subjected to autoradiography. In that way three cell types were distinguished immunocytochemically: (1) astrocytes co-expressing glial fibrillary acidic protein (GFAP) and vimentin, (2) astrocytes expressing only GFAP and (3) astrocyte-like cells expressing vimentin. Thereafter, numbers of immunopositive and autoradiographically labelled cells and their locations within the region of injury were recorded at each stage of the experiment. Two hours as well as 1 day after the injury proliferation of GFAP-positive astrocytes and of those co-expressing GFAP and vimentin could only be seen as statistically insignificant phenomena. On day 2 the reactive proliferation of each immunocytochemically defined cell type was already maximal, then gradually decreased and its last signs were recorded on day 8. On day 2, among all the proliferating GFAP-positive astrocytes, 67.2% were also vimentin-positive. Later, the proportion declined to 50.7% and 38.5% on days 4 and 8, respectively. The labelled astrocyte-like vimentin-positive cells were located closest to the lesion margins. In comparison, the astrocytes co-expressing GFAP and vimentin and those expressing exclusively GFAP, occupied regions progressively farther from the lesion site. At the initial stages of the response to injury, vimentin expression in cells starting their reactive proliferation did not precede the expression of GFAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Histogenetic consideration of ovarian sex cord-stromal tumors analyzed by expression pattern of cytokeratins, vimentin, and laminin. Correlation studies with human gonads.

A total of 30 sex cord-stromal tumors including 9 adult type and 5 juvenile type granulosa cell tumors (GCTs), 4 Sertoli-Leydig cell tumors (SLTs), 1 gynandroblastoma, 5 thecomas, 2 fibromas and 3 sclerosing stromal tumors were immunohistochemically evaluated by means of cytokeratins of different molecular weight, vimentin and laminin with regard to the histogenesis of these tumors and to the embryogenesis of the sex cord and stroma of developing gonads. For comparison, 7 embryonic gonads, 9 fetal and 9 adult ovaries, 14 fetal and 5 postnatal testes, and 1 gonadoblastoma were also examined. The coelomic epithelium of all gonads were positive for both cytokeratins (CAM 5.2 and AE1) and vimentin. In fetal ovaries, the granulosa cells of primordial follicles express low molecular weight cytokeratins only and those cells of more maturing follicles did not express any cytokeratin or vimentin. In adult ovaries, the granulosa cells of primordial follicles coexpressed low molecular weight cytokeratins and vimentin, but those cells of more maturing follicles expressed vimentin only. In fetal testes before 20 weeks gestational age, the Sertoli and Leydig cells did not express any cytokeratins and vimentin. After that time, both cells expressed vimentin only throughout life. The rete ovarii and rete testis from fetal to adult life coexpressed both low molecular weight cytokeratins and vimentin. The rete ovarii in all ages and rete testis in prenatal and childhood ages were surrounded by the laminin-positive basement membrane, however, the rete testis in adult were not. In neoplasia, the GCTs, thecomas, fibromas, and sclerosing stromal tumors expressed vimentin only.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2002

Sarin causes early differential alteration and persistent overexpression in mRNAs coding for glial fibrillary acidic protein (GFAP) and vimentin genes in the central nervous system of rats.

Neurotoxic effects of single dose of 0.5 x LD50 sarin (O-isopropylmethylphosphonoflouridate) on central nervous system (CNS) of male Sprague-Dawley rats were studied. We investigated the mRNA expression of the astroglial marker genes glial fibrillary acidic protein (GFAP) and vimentin to evaluate the fate of astroglial and neuronal cells, because reactive gliosis is very often used to assess the extent of CNS damage. Rats were treated with 50 microg/kg/ml of sarin and terminated at the time-points 1 and 2 hours and 1, 3, and 7 days post-treatment. Control rats were treated with normal saline. Total RNA was extracted and Northern blots were hybridized with cDNA probes for GFAP and vimentin, as well as 28S RNA (control). The data obtained indicate that a single dose of sarin (0.5 x LD50) showed induction in the transcript levels of GFAP and vimentin in the cortex, cerebellum, brainstem and midbrain, and spinal cord. The induction showed distinct spatial-temporal differences for each tissue studied. Both GFAP and vimentin were induced at 1 hour in all the tissues studied except brainstem, where moderate and high levels of GFAP induction were noted at 1 and 3 days. Overexpressed transcript levels of GFAP and vimentin remained high in more responsive tissues such as the brainstem and midbrain. Other tissues, such as the cortex, spinal cord, and cerebellum showed a more downward trend for either GFAP or vimentin, or both, transcript levels at 7 days. It is noteworthy that both cortex (318 +/- 12%) and spinal cord (368 +/- 12%) showed relatively higher induction of GFAP, whereas cortex alone showed the highest level of overexpressed vimentin transcript levels (284 +/- 11%). Overall it is also clear that both GFAP and vimentin are needed for the effective recovery involving co-ordinated alternating up- and downregulation of these two key astrocyte genes, depending on tissue specificity. The changes seen in the transcript levels of GFAP and vimentin may be the result of astrocyte dysfunction and loss, accompanied by compensatory proliferation and dedifferentiation of the astroglia. These changes could affect the neuronal cell types, thus altering the neuron-glia homeostasis.

Animals↗
PubMed2003

Vimentin is secreted by activated macrophages.

Vimentin is a widely expressed intermediate filament protein thought to be involved mainly in structural processes, such as wound healing. We now demonstrate that activated human macrophages secrete vimentin into the extracellular space. The maturation of blood-derived monocytes into macrophages involves several signalling pathways. We show that secretion of vimentin, which is phosphorylated at serine and threonine residues, is enhanced by the phosphatase inhibitor okadaic acid and blocked by the specific protein kinase C inhibitor GO6983. These findings are consistent with previous observations that phosphorylation of vimentin affects its intracellular localization and that vimentin is a substrate for protein kinase C (PKC). We also show that the anti-inflammatory cytokine interleukin-10 (IL-10), which inhibits PKC activity, blocks secretion of vimentin. In contrast, the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can trigger secretion of vimentin. Finally, we found that extracellular vimentin is involved in bacterial killing and the generation of oxidative metabolites, two important functions of activated macrophages. These data establish that vimentin is secreted by macrophages in response to pro-inflammatory signalling pathways and is probably involved in immune function.

Cells, Cultured↗
PubMed2005

PKCepsilon-mediated phosphorylation of vimentin controls integrin recycling and motility.

PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.

Animals↗
PubMed2001

Protein kinases required for segregation of vimentin filaments in mitotic process.

Vimentin, one of type III intermediate filament (IF) proteins, is expressed not only in mesenchymal cells but also in most types of tumor cells. In the present study, we introduced several types of vimentin mutated at putative phosphorylation sites in its amino-terminal head domain into type III IF-negative T24 cells. Site-specific mutation induced the formation of an unusually long bridge-like IF structure between the unseparated daughter cells, although these mutants formed the filament network similar to wild type in interphase cells. Together with sites phosphorylated by Rho-kinase and protein kinase C (PKC), vimentin-Ser72, which can not be phosphorylated by any known vimentin kinase, was one of the mutation sites essential for this phenotype. We further demonstrated that vimentin-Ser72 was phosphorylated specifically at the cleavage furrow during cytokinesis. These observations suggest the existence of a novel protein kinase responsible for vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. We propose that Rho-kinase, PKC, and an unidentified vimentin-Ser72 kinase may play important roles in vimentin filament separation during cytokinesis.

Actin Cytoskeleton↗
PubMed1982

Analysis of desmin and vimentin phosphopeptides in cultured avian myogenic cells and their modulation by 8-bromo-adenosine 3',5'-cyclic monophosphate.

The intermediate filament proteins desmin and vimentin are two of the major 32P phosphate acceptors in chicken myotubes differentiating in tissue culture. Analysis of the desmin and vimentin phosphopeptides by two-dimensional tryptic peptide mapping shows that both proteins are phosphorylated at multiple sites, giving rise to 5 phosphopeptides in desmin and as many as 11 in vimentin. Addition of the cAMP analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) to the culture medium of mature (8-day-old) myotubes results in a 2- to 3-fold increase in PO4 incorporation into desmin and vimentin. Two-dimensional tryptic analysis of desmin and vimentin from 8-BrcAMP-treated myotubes shows increased 32PO4 incorporation into a subset of the phosphopeptides observed in control cells. Comparison of phosphopeptides from the two proteins shows the presence of at least three comigrating peptides. All three comigrating peptides exhibit cAMP-dependent increases in 32PO4 incorporation in vimentin, while only two of the three exhibit 8-BrcAMP-dependent responses in desmin. While these peptides are the only two that are sensitive to 8-BrcAMP in desmin, vimentin contains additional peptides that exhibit increased 32PO4 incorporation in response to 8-BrcAMP. This result suggests the existence of both common and distinct phosphorylation sites between desmin and vimentin that may be differentially regulated by cAMP. Thus, desmin and vimentin, even though structurally related, may be capable of responding differently to physiological stimuli.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells.

Intermediate-sized filaments (IF) are among the most insoluble intracellular protein polymer structures. We have analyzed the small amounts of soluble vimentin, an IF protein, present in cytosol fractions obtained from lysis of cultured cells [rat RVF-SM cells, simian virus 40-transformed human fibroblasts, and human rhabdomyosarcoma (RD line) cells]. The molecular form of this soluble vimentin was determined by sucrose density gradient centrifugation, using vimentin-specific antibodies for subsequent ELISA and immunoblotting analyses. The majority of the soluble vimentin appeared in a distinct form indistinguishable in its sedimentation behavior from reconstituted tetrameric subunits of purified vimentin arrested at low ionic strength. The tetrameric coiled-coil nature of the soluble form of vimentin was indicated by the digestion pattern with chymotrypsin and by chemical crosslinking with copper-1,10-phenanthroline and dimethylsuberimidate. The competence of this soluble vimentin to assemble into IF at higher salt concentrations was demonstrated by electron microscopy. Pulse-chase experiments showed that the soluble form was not an exclusively posttranslational intermediate. We propose that in the living cell a small pool of a distinct soluble tetrameric form of vimentin exists which may exchange with polymeric IF vimentin.

Animals↗