Vimentin filaments and centrosomes: Are they associated?

HeLa cells were examined by immunofluorescence using anti-vimentin and anti-centrosphere anti-bodies, and by transmission electron microscopy (TEM), after vimentin redistribution induced by the action of nocodazole or taxol. A redistribution of vimentin bundles in the centriolar area was observed after nocodazole treatment, although no direct interaction between centrioles and vimentin filaments could be detected. After taxol treatment, the juxtanuclear accumulation of vimentin filaments and the centrioles were rarely observed in the same area. Our results do not support the concept of a direct association between centrioles and vimentin filaments.     

Altered Intermediate Filament Expression in Human Neuroblastoma Cells Transformed by a Growth-Promoting Agent Derived from Schizophrenic CSF

1. Transformed (TR) cell lines showed faster doubling times and higher cell densities at confluence, as well as altered morphology, changing from flat epitheloid to smaller round or bipolar shapes. Since such morphological changes are suggestive of alterations in intermediate filaments, we have analyzed the expression of both vimentin and neurofilament.2. Immunohistochemical analysis of vimentin showed a redistribution from a cytoplasmic network to a perinuclear accumulation in TR cell lines.3. Western blot analysis demonstrated that the vimentin content was decreased 60–90%. The content of the 70-kD neurofilament protein was also decreased in TR cells, but its intracellular distribution was indistinguishable from that in the control cell lines.     

Cell configuration-related control of vimentin biosynthesis and phosphorylation in cultured mammalian cells

The cell configuration-related control of a cytoskeletal protein (vimentin) expression was examined by varying cell shape between flat and spherical. Cultivation of cells in monolayer or in a spherical configuration on poly-2-hydroxyethylmethacrylate-coated plates revealed a preferential down regulation of vimentin synthesis during suspension culture. The mechanism(s) regulating the decrease in the expression of vimentin in spherical cells appears to be at the level of translation, because mRNAs extracted from monolayer and suspension-cultured cells were equally active in directing vimentin synthesis in the rabbit reticulocyte cell-free system. When after prolonged suspension culture, the cells were allowed to reattach and spread, vimentin synthesis recovered rapidly to the control monolayer rate. The phosphorylation of vimentin was also reduced dramatically during suspension culture. However, unlike the rapid recovery of vimentin biosynthesis upon reattachment (less than 6 h), the recovery in the rate of vimentin phosphorylation was much slower (greater than 20 h) and paralleled the recovery to the monolayer growth rate. Although the control of vimentin biosynthesis in suspension culture is a cell configuration-related process, the decrease in the rate of vimentin phosphorylation in suspension culture appears to be the result of the slower growth rate and may reflect the reported correlation between the rate of vimentin phosphorylation and the accumulation of cells in mitosis.     

ROCK is Involved in Vimentin Phosphorylation and Rearrangement Induced by Dengue Virus

Our previous study showed that dengue virus 2 (DENV2) infection induces rearrangement of vimentin into dense structures at the perinuclear area. However, the underlying mechanism of this phenomenon is poorly characterized. In the present work, we found that vimentin and Ser71 phosphorylated vimentin display similar distributions in DENV2-infected cells. DENV2 infection also induced ROCK activation and phosphorylation of vimentin at Ser71 as the DENV2 infection progressed. Furthermore, Ser71 phosphorylation and vimentin rearrangement induced by DENV2 infection were blocked by the ROCK inhibitor Y-27632. In addition, DENV2 led to endoplasmic reticulum (ER) redistribution in the perinuclear region of the host cells, which was partially blocked by pretreatment with Y-27632. Together, these data support indicate that ROCK may have a role in governing regulating vimentin and ER rearrangement during DENV2 infection. We hypothesize that DENV2 infection, via ROCK activation, induces both vimentin rearrangement and ER redistribution around the perinuclear region, which may play a structural role in anchoring DENV2 to replication sites.     

Differences in the expression of prekeratin and vimentin in organ and monolayer cultures of rat hepatocytes

Results obtained by the indirect immunofluorescence method employing specific monoclonal antibodies show that during the first 24 hours of cultivation in a monolayer there appears another protein of intermediate filaments--vimentin, which is a characteristic of most mesenchymal cells. At the same time, in the organ liver culture maintained in the same culture medium, no expression of vimentin was observed up to 5-7 days of cultivation. Vimentin was revealed only in cells that migrated from a tissuepiece to collagen. Besides the vimentin expression in these migrating cells and monolayer cultures of hepatocytes, a redistribution of prekeratin filaments took place: the cytoplasmic network appeared instead of thick fibers underlying membranes. The results of the present work suggest that the vimentin expression and the prekeratin filament redistribution in epithelial liver cells in vitro do not depend on the changes of natural humoral factors for the components of culture medium but are due to damages of the intact liver tissue structure.     

Immunofluorescent localization of vimentin, prekeratin and actin during odontoblast and ameloblast differentiation

The localization of constitutive proteins of different types of cytoskeletal components (prekeratin, vimentin, and actin) was examined in embryonic mouse molars using specific antibodies and immunofluorescence microscopy on frozen sections. Prekeratin and actin were found in the enamel organ. Preameloblasts demonstrated uniform staining, whereas ameloblasts demonstrated an apical accumulation of both prekeratin and actin. Vimentin and actin were observed in the dental papilla. A redistribution of vimentin accompanied the polarization of odontoblasts. A possible transmembranous control of cytoskeletal activities by the extracellular matrix is discussed.     

Immunofluorescent Localization of Vimentin, Prekeratin and Actin during Odontoblast and Ameloblast Differentiation

The localization of constitutive proteins of different types of cytoskeletal components (prekeratin, vimentin, and actin) was examined in embryonic mouse molars using specific antibodies and immunofluorescence microscopy on frozen sections. Prekeratin and actin were found in the enamel organ. Preameloblasts demonstrated uniform staining, whereas ameloblasts demonstrated an apical accumulation of both prekeratin and actin. Vimentin and actin were observed in the dental papilla. A redistribution of vimentin accompanied the polarization of odontoblasts. A possible transmembranous control of cytoskeletal activities by the extracellular matrix is discussed.     

Role of caveolin-1 and cytoskeletal proteins, actin and vimentin, in adipogenesis of bovine intramuscular preadipocyte cells

We investigated the involvement of caveolin-1 and the cytoskeletal proteins, actin and vimentin, in the adipogenesis of bovine intramuscular preadipocyte (BIP) cells. Immunoblot analysis demonstrated that levels of caveolin-1 and actin gradually increased during adipose conversion in BIP cells, whereas a slight decrease was observed for vimentin. We found that part of the vimentin was clearly distributed to caveolin-1-enriched membrane fractions in BIP cells, but actin was not. During adipogenesis of BIP cells, treatment with the tubulin depolymerizer, nocodazole, significantly increased intracellular triglyceride accumulation compared to non-treated cells. Immunocytochemical analysis showed that actin microfilaments were significantly disrupted in nocodazole-treated cells. Also, a decrease in the localization of vimentin in caveolin-1-enriched fractions and a failure of vimentin to co-immunoisolate with caveolin-1 were observed in nocodazole-treated cells. These results suggest that a rearrangement of cytoskeletal proteins has a role in the intracellular accumulation of lipid droplets during adipogenesis of BIP cells.     

Lymphocyte mechanical response triggered by cross-linking surface receptors

Using a recently developed method (Petersen, N. O., W. B. McConnaughey, and E. L. Elson, 1982, Proc. Natl. Acad. Sci. USA., 79:5327-5331), we have measured changes in the deformability of lymphocytes triggered by cross-linking cell surface proteins. Our study was motivated by two previously demonstrated phenomena: the redistribution ("capping") of cross-linked surface immunoglobulin (sIg) on B lymphocytes and the inhibition of capping and lateral diffusion ("anchorage modulation") of sIg by the tetravalent lectin Concanavalin A (Con A). Both capping and anchorage modulation are initiated by cross-linking cell surface proteins and both require participation of the cytoskeleton. We have shown that the resistance of lymphocytes to deformation strongly increased when sIg or Con A acceptors were cross-linked. We have measured changes in deformability in terms of an empirical "stiffness" parameter, defined as the rate at which the force of cellular compression increases with the extent of compression. For untreated cells the stiffness was approximately 0.15 mdyn/micron; for cells treated with antibodies against sIg or with Con A the stiffness increased to approximately 0.6 or 0.4 mdyn/micron, respectively. The stiffness decreased after completion of the capping of sIg. The increases in stiffness could be reversed to various extents by cytochalasin D and by colchicine. The need for cross-linking was demonstrated by the failure both of monovalent Fab' fragments of the antibodies against sIg and of succinylated Con A (a poor cross-linker) to cause an increase in stiffness. We conclude that capping and anchorage modulation involve changes in the lymphocyte cytoskeleton and possibly other cytoplasmic properties, which increase the cellular viscoelastic resistance to deformation. Similar increases in cell stiffness could be produced by exposing cells to hypertonic medium, azide ions, and to a calcium ionophore in the presence of calcium ions. These results shed new light on the capabilities of the lymphocyte cytoskeleton and its role in capping and anchorage modulation. They also demonstrate that measurements of cellular deformability can characterize changes in cytoskeletal functions initiated by signals originating at the cell surface.     

Ontogeny of B-lymphocyte function. XIV. Maturation of the capacity of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the ability of the B-cell population to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit anti-mouse immunoglobulin (RAMIg) was studied in LAF1, C57BL/6, and C57L mice. As demonstrated by previous workers, the B-cell population from immature mice failed to re-express sIg after treatment with RAMIg. We have shown that the age at which the B-cell population acquires the capacity to re-express sIg is different in different strains and that the order in which the B-cell population of the different strains acquires the capacity to re-express sIg is different from the order in which their B-cell populations acquire the capacity to produce high-affinity antibodies. This suggests that these represent distinct differentiation events in the development of the B-cell population. In all of the strains studied the maturation of the capacity to re-express sIg occurred in two steps. After the first maturation step the B-cell population was able to re-express sIg after treatment with RAMIg for 1 hr but did not re-express sIg after treatment with RAMIg for 24 hr. After the second maturation step the B-cell population could re-express sIg even after 24 hr treatment with RAMIg. It has been suggested by previous workers that the inability of the immature B-cell population to re-express sIg could represent one of the mechanisms responsible for the development of B-cell self-tolerance. It is suggested here that the existence of a period during which cells become tolerant only upon prolonged exposure to antigen could protect the developing B cells from becoming unresponsive to transiently experienced foreign antigens but still permit them to become tolerant to self antigens which are continuously present.     

Vimentin dynamics during the mitogenic stimulation of mouse splenic lymphocytes

We have used double immunofluorescence and electron microscopy to examine the distribution of tubulin and vimentin during the stimulation of mouse splenic lymphocytes by the mitogen concanavalin A. In unstimulated cells, vimentin forms a filamentous network partially coincident with the radial pattern of microtubules. In stimulated cells, the numbers of microtubules assembled from the centrosome have increased and vimentin is organized as an aggregate located near the centrosome. When these cells enter mitosis, vimentin is arranged into a filamentous cage enclosing the mitotic apparatus. During cytokinesis, the polar centrosomes are observed at a position adjacent to the midbody and vimentin is detected as an aggregate, similar to that seen prior to mitosis, close to the centrosome in each daughter cell. Using several agents, such as colchicine, colcemid, nocodazole, and taxol, which affect microtubule assembly, we have observed that the vimentin system, although closely related spatially to the microtubule complex in lymphocytes, can still reorganize independently as these cells progress through the cell cycle. Throughout mitogenic stimulation in the continued presence of taxol, microtubules are reorganized into a few thick bundles while the vimentin system undergoes a sequence of rearrangements similar to those observed during normal stimulation. These data suggest that vimentin dynamics may be important in the progression of lymphocytes through the cell cycle in response to mitogen.     

Polymerization of additional actin is not required for capping of surface antigens in B-lymphocytes

CH12 is a murine B-cell lymphoma whose surface immunoglobulin (sIg) and concanavalin A (Con A) receptors patch and cap readily. Actin may be involved in CH12 patching and capping, since fodrin and F-actin collect under the cap, and cytochalasin D inhibits sIg capping. We have examined the state of the actin cytoskeleton during patching and capping. A wide range of concentrations of rabbit anti-mouse antibody (RAM) and Con A were used to patch or cap CH12 cells. G-actin was quantitated by DNase I inhibition, F-actin was quantitated by fluorescence-activated cell sorter analysis of fluorescent phalloidin staining, and actin nucleation sites were measured by pyrene actin polymerization. None of these methods detected any significant changes in actin when compared to control cells or untreated cells, leading us to conclude that increased actin polymerization is not necessary for capping to occur. The significance of these data to the membrane flow and cytoskeletal models of capping is discussed.     

Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner.     

Ontogeny of B lymphocyte function. XV. A role for thymus cells in the maturation of the capacity of a subpopulation of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the C57BL/6 B cell population to be able to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit antimouse Ig (RAMIg) was studied in a cell transfer system. It was found that thymus cells were required for the maturation of a subset of the B cell population to be able to re-express sIg. The B cell population of irradiated, thymectomized mice reconstituted with spleen cells from donors under 2 wk of age remained deficient in their ability to re-express sIg even after 4 wk residence in the cell transfer recipient. In contrast, if adult thymus cells were transferred together with the immature B cells, the B cell population matured to be able to re-express sIg after treatment with RAMIg. Approximately one-third of the B cell population appears to require thymus cells for this maturation. The maturation of the thymus cell population to be capable of mediating this maturation of the B cell population occurs in two steps: between 2 and 3 and between 3 and 4 wk of age. This timing corresponds to the age at which the B cell population of C57BL/6 mice normally acquires the capacity to re-express sIg, which we have previously shown to also occur in two steps. Thymus cells from 3-wk-old donors can mediate the first step in B cell maturation to be able to re-express sIg, but cannot mediate the second step in this maturation of the B cell population. Thymus cells from 4-wk-old donors can mediate both steps in the maturation of the B cell population. The results suggest that thymus cells are involved in regulating some aspects of B cell differentiation.     

Induction of vimentin modification and vimentin-HSP72 association by withangulatin A in 9L rat brain tumor cells

Withangulatin A induced cell rounding up and the morphological alteration resulted from the reorganization of all of the major cytoskeletal components, i.e., vimentin, tubulin, and actin, as revealed by immunofluorescence techniques. When the withangulatin A-treated cells changed to a round-up morphology, vimentin intermediate filaments were found to be collapsed and clustered around the nucleus. The alteration was accompanied by characteristic changes of vimentin molecules, including augmentation of phosphorylation, retardation of electrophoretic mobility, and decrease in detergent extractability. The levels of vimentin phosphorylation were augmented by 2.5- and 1.8-fold in cells incubated with 50 μM withangulatin A for 1 and 3 h, respectively. The electrophoretic mobility of vimentin was partially retarded in cells treated with withangulatin A for 1 h at 10 μM and a completely upshift mobility was observed after 5 h treatment at 50 μM. In addition, vimentin molecules became less extractable by nonident P-40 after the cells were treated with withangulatin A and this effect was dose dependent. The decrease in solubility of vimentin was accompanied by the redistribution of HSP72 into the detergent nonextractable fraction and these two events were well correlated. Our results suggest that withangulatin A induced the modification of vimentin, which resulted in the alteration of cell morphology and redistribution of intracellular HSP72, an event that may play an important role in the induction of heat-shock response.     

Src SH3/2 domain-mediated peripheral accumulation of Src and phospho-myosin is linked to deregulation of E-cadherin and the epithelial-mesenchymal transition

Elevated Src kinase in epithelial cancer cells induces adhesion changes that are associated with a mesenchymal-like state. We recently showed that Src induces dynamic integrin adhesions in KM12C colon cancer cells, whereas E-cadherin-dependent cell-cell contacts become disorganized. This promotes a fibroblastic-like morphology and expression of the mesenchymal marker vimentin. Furthermore, Src-induced deregulation of E-cadherin, and the associated mesenchymal transition, is dependent on integrin signaling (Avizienyte et al., Nat. Cell Biol. 2002, 4, 632-638), although the nature of downstream signals that mediate these Src- and integrin-dependent effects are unknown. Here we show that the SH2 and SH3 domains of Src mediate peripheral accumulation of phospho-myosin, leading to integrin adhesion complex assembly, whereas loss of SH2 or SH3 function restores normal regulation of E-cadherin and inhibits vimentin expression. Inhibitors of MEK, ROCK, or MLCK also suppress peripheral accumulation of phospho-myosin and Src-induced formation of integrin-dependent adhesions, whereas at the same time restoring E-cadherin redistribution to regions of cell-cell contact. Our data therefore implicate peripheral phospho-myosin activity as a point of convergence for upstream signals that regulate integrin- and E-cadherin-mediated adhesions. This further implicates spatially regulated contractile force as a determinant of epithelial cell plasticity, particularly in cancer cells that can switch between epithelial and mesenchymal-like states.     

Organization of the vimentin system and its spatial relationship to the microtubule complex during the division of mammalian cells growing attached and in suspension

We have used immunofluorescence staining with antibodies that detect vimentin, tubulin and the centrioles to compare the distributions of these respective antigens during the division of several suspension and attached cultured cells. Our observations demonstrate that 1) from distinct interphase organizations in suspension and attached cells, the vimentin system consistently rearranges with the onset of mitosis into a filamentous cage-like structure enclosing the spindle, 2) during cytokinesis, the polar centrosomes relocalize near the midbody in suspension cells while they remain at the pole opposite to it in attached cells, and 3) the vimentin cage is disintegrated and aggregated on each side of the midbody during cytokinesis in lymphoid cells but may be retained in other suspension cells.     

Function and behavior of surface immunoglobulin receptors in antigen-specific T cell-B cell interaction

The expression and function of surface immunoglobulin (sIg) receptors was analyzed using antigen-specific monoclonal B cells and T cells. When B cells were incubated with antigen (hapten carrier), hapten-specific sIg receptor expression was greatly reduced. The specific antigen-presentation activity of the B cells was also markedly reduced. Following the removal of antigen, expression of the sIg receptors was restored to original levels within 3-4 hr. Specific antigen-presentation activity of these B cells was concomitantly restored. Reduction of sIg receptor expression and specific antigen presentation were inhibited by the addition of competing free hapten, suggesting that crosslinking of sIg receptors is required for the observed changes in sIg receptor expression and that specific antigen presentation is hapten specific. Using oligopeptide-specific Th cells, we could show the requirement for processing of antigen in the interaction between hapten-specific B cells and carrier-specific T cells. These results indicated that the expression of sIg receptors for a specific antigen and the ability to present the antigen are interrelated.     

Membrane Ig-cytoskeletal interactions. III. Receptor cross-linking results in the formation of extensive filamentous arrays of vimentin

The proposed function of intermediate filaments is to provide a cell type-specific structural framework that maintains cell shape and organelle distribution and mediates signal transduction through its connections with the plasma membrane and the nucleus. Vimentin is the intermediate filament protein expressed in B lymphocytes. Immunocytochemical analysis of the high salt-stable cytoskeletons from B cells stimulated with anti-Ig revealed an increased accumulation of vimentin in the cytoskeleton compared to nontreated controls. This increased accumulation of vimentin in the cytoskeleton was manifested by the organization of vimentin into extensive filamentous arrays (EFA) as viewed in the fluorescent microscope. In contrast to the effects of anti-Ig, activation of B cells with LPS did not induce the organization of vimentin into EFA. This suggested that signals unique to anti-Ig directed EFA formation. Immunocytochemical results were verified by biochemical analysis showing that vimentin was more abundant in isolated cytoskeletons from anti-Ig activated B cells, than cytoskeletons isolated from LPS-activated B cells. These observations established a relationship between increased content of vimentin in the cytoskeleton and the formation of EFA. By testing a wide variety of activating agents, we were able to correlate increased vimentin expression in the cytoskeleton to activating agents that cross-link membrane Ig. It appeared that treatment of B cells with LPS prohibited the induction of EFA by anti-Ig because cotreatment with both anti-Ig and LPS resulted in decreased vimentin accumulation in the cytoskeleton to a level less than that in resting cells. The significance of these results with regard to B cell biology is discussed.