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

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

Intermediate filament proteins in nonfilamentous structures: Transient disintegration and inclusion of subunit proteins in granular aggregates

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2–4 nm diameter, and intermediate filament protein is included in numerous, variously sized (2–15 μm) spheroidal aggregates containing densely stained granular particles of 5–16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.     

Human immunodeficiency virus type 1 Vif is efficiently packaged into virions during productive but not chronic infection

Packaging of the human immunodeficiency virus type 1 Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. Here, we compared the efficiency of packaging of Vif into virions derived from acutely and chronically infected H9 cells. We found that Vif was efficiently packaged into virions from acutely infected cells (60 to 100 copies per virion), while packaging into virions from chronically infected H9 cells was near the limit of detection (four to six copies of Vif per virion). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild-type virus was readily packaged (30 to 40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were not due to gross differences in the steady-state distribution of Vif in chronically or acutely infected cells but are likely due to differences in the relative rates of de novo synthesis of Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild-type infectivity. The results from our study provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging.     

Cytoplasmic network arrays demonstrated by immunolocalization using antibodies to a high molecular weight protein present in cytoskeletal preparations from cultured cells

Antisera were raised in rabbits against the two major components of intermediate filament preparations from glia-derived C6 cells, polypeptides of M_r around 300 000 and 58 000 (vimentin). These, and a third antiserum raised against microtubule proteins from hog brain, were shown to be specific for their respective immunogens. The assay employed involved the separation of components of crude cell extracts or filament preparations by SDS-polyacrylamide gel electrophoresis, and their subsequent transfer to and immobilization on nitrocellulose sheets. Cross-reacting counterparts of the immunogens were found in various cell lines, including C6, BALB/c 3T3, SV101, CHO, HeLa and PtK2 cells. In indirect immunofluorescence studies, antibodies to the high-M_r polypeptide component stained dense cytoplasmic network arrays of seemingly short, irregularly oriented fibres and lines of dots, in fibroblasts and in HeLa cells, but not in PtK2 cells. In well spread cells these networks were clearly distinguishable in morphology from the fibres decorated by antibodies to either microtubule protein or vimentin. The network arrays were resistant towards treatments with Triton X-100 and colcemid. By double immunofluorescence microscopy of single cells, using an additional antibody preparation to vimentin raised in guinea pigs, it was shown that after prolonged colcemid treatment of BALB/c 3T3 cells both; vimentin filaments and the structures stained by antibodies to the high-M_r component, accumulated in corresponding areas of the cytoplasm. The possibilities are discussed that this novel network-like structure is of the intermediate filament type and that it might function as a cross-linker of cytoplasmic—in particular cytoskeletal—elements. To signify its fluorescent localization and its possible linking role it is proposed to call the high-M_r component of intermediate filament preparations from cultured cells ‘plectin’.     

Interaction of Theiler’s Virus with Intermediate Filaments of Infected Cells

Theiler’s murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler’s virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.     

Subcellular localization of the Vif protein of human immunodeficiency virus type 1.

The Vif (viral infectivity factor) protein of human immunodeficiency virus type 1 (HIV-1) has been shown to dramatically enhance the infectivity of HIV-1 virus particles during virus production. The subcellular localization of Vif was examined to elucidate cellular pathways which may be important for Vif function. Indirect immunofluorescence staining of Vif demonstrated a diffuse cytoplasmic distribution and showed that most Vif was not associated with the Golgi complex, a proposed site of localization (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). Subcellular fractionation of transfected COS cells and HIV-1-infected Jurkat and CEM cells demonstrated that Vif is a cytoplasmic protein which exists in both a soluble cytosolic form and membrane-associated form. The membrane-associated form of Vif is a peripheral membrane protein which is tightly associated with the cytoplasmic side of cellular membranes. The C terminus of Vif was required for the stable association of Vif with membranes. The C terminus was also essential for Vif function, suggesting that the association of Vif with membranes is likely to be important for its biological activity. The highly conserved regions at residues 103 to 115 and 142 to 150 were important for Vif function but did not affect membrane association, indicating that these regions are likely to be important for other, as-yet-unknown functions.     

The Vif protein of human and simian immunodeficiency viruses is packaged into virions and associates with viral core structures.

The vif gene of human and simian immunodeficiency viruses (HIV and SIV) encodes a late gene product that is essential for viral infectivity in natural target cells. Virions produced in the absence of Vif are abnormal in their ultrastructural morphology and are severely impaired in the ability to complete proviral DNA synthesis upon entry into new target cells. Because previous studies failed to detect Vif protein in virus particles, Vif is believed to influence virus infectivity indirectly, by affecting virion assembly, release, and/or maturation. In this report, we reexamined the possibility that Vif is a virion-associated protein. Utilizing high-titer Vif-specific antibodies, a sensitive immunoblot technique, and highly concentrated virus preparations, we detected a 23-kDa Vif-reactive protein in wild-type HIV type 1 (HIV-1) and a 27-kDa Vif-reactive protein in wild-type SIVSM virions. Neither protein was present in virions derived from vif-deficient HIV-1 and SIVSM proviral constructs. Vif protein content was similar among different strains of HIV-1 and was independent of the cell type (permissive or nonpermissive) used to produce the virus. To determine the subvirion localization of Vif, HIV-1 virions were treated with proteinase K or Triton X-100 to remove virion surface proteins and the viral membrane, respectively, purified through sucrose, and analyzed by immunoblot analysis. Vif protein content was not affected by the removal of external surface proteins or by the removal of the viral membrane and submembrane p17Gag matrix protein. Instead, Vif colocalized with viral core structures which sedimented at a density of 1.25 g/ml on linear sucrose gradients (enveloped HIV-1 particles sediment at a density of 1.17 g/ml). Finally, the amount of Vif protein packaged into virions was estimated to be on the order of 1 molecule of Vif for every 20 to 30 molecules of p24Gag, or between 60 and 100 molecules of Vif per particle. These results indicate that Vif represents an integral component of HIV and SIV particles and raise the possibility that it plays a direct role in early replication events.     

上皮细胞分裂过程中中等纤维的变化

Immunofluorescence microscopy was used to follow the rearrangement of keratin filaments and vimentin filaments during mitosis in Vero and HeLa cell lines. The experiment results showed that the three dimensional organization and structure of intermediate filaments changed drastically during mitosis. The behavior of intermediate filaments was different in these two epithelial cell lines. In mitotic Vero cells the keratin filaments and vimentin filaments maintained their filamentous structure and formed a cage around the mitotic apparatus. In mitotic HeLa cells the keratin filaments and vimentin filaments reorganized extensively and formed granular cytoplasmic bodies. The ratio of granular cytoplasmic body formation changed in different mitotic phase. The interphase intermediate filament network was reconstructed after mitosis. It is proposed that the state of intermediate filament network in these cells is cell cycle-dependent and intermediate filaments may have some skeletal role in mitosis.     

上皮细胞分裂过程中中等纤维的变化

应用制备的血清抗体,采用免疫细胞化学方法观察了两株培养上皮细胞的分裂过程中IF的动态变化过程。实验结果显示,在上皮细胞分裂过程中,IF形态结构及空间分布发生了显著变化,不同细胞之间存在差异,分裂的Vero细胞中角蛋白纤维和波形纤维都维持纤维形态,围绕分裂器形成纤维网罩或纤维束环,随着细胞分裂的进行,IF网的空间组织结构和外观发生动态变化;分裂的HeLa细胞中,角蛋白纤维和波形纤维广泛重组形成颗粒状胞质小体,分裂结束后重建IF网。实验结果表明,IF变化具有细胞周期依赖性和一定的细胞特异性。本文对IF在细胞分裂过程中的功能意义作了讨论。     

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.     

Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography

Vimentin polymerizes via complex lateral interactions of coiled-coil dimers into long, flexible filaments referred to as intermediate filaments (IFs). Intermediate in diameter between microtubules and microfilaments, IFs constitute the third cytoskeletal filament system of metazoan cells. Here we investigated the molecular basis of the 3-D architecture of vimentin IFs by cryo-electron microscopy (cryo-EM) as well as cryo-electron tomography (Cryo-ET) 3-D reconstruction. We demonstrate that vimentin filaments in cross-section exhibit predominantly a four-stranded protofibrilar organization with a right-handed supertwist with a helical pitch of about 96 nm. Compact filaments imaged by cryo-EM appear surprisingly straight and hence appear very stiff. In addition, IFs exhibited an increased flexibility at sites of partial unraveling. This is in strong contrast to chemically fixed, negatively stained preparations of vimentin filaments that generally exhibit smooth bending without untwisting. At some point along the filament unraveling may be triggered and propagates in a cooperative manner so that long stretches of filaments appear to have unraveled rapidly in a coordinated fashion.     

Plectin deficiency affects precursor formation and dynamics of vimentin networks

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

Assembly of amino-terminally deleted desmin in vimentin-free cells

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin- free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.     

人体肝癌细胞和HeLa细胞中等纤维的比较研究

本文用兔抗角蛋白抗体、豚鼠抗波形纤维蛋白抗体和抗角蛋白单抗AE1的间接免疫荧光抗体法比较了两个人体肝癌细胞系(BEL-7402和BEL-7404)和HeLa细胞中等纤维的分布式样,同时用SDS-PAGE法分析了上述细胞的中等纤维抽提物的多肽组成。结果表明:三种上皮细胞均含有两套不同类型的中等纤维系统:角蛋白纤维和波形纤维。但是,人体肝癌细胞和HeLa细胞的中等纤维分布式样和角蛋白多肽组成均有明显的差别。其中最明显的差别是HeLa细胞具有丰富的桥粒-张力纤维复合物和分子量为40 kd的角蛋白多肽,而在两个人体肝癌细胞系中看不到。     

Reorganization of the system of intermediate filaments and detection of the organization centers after centrifugation of cells attached to a substrate

Cultured pig kidney epithelial cells were centrifuged at 20,000 gav so that the centrifugation force was oriented parallel to the substrate, fixed and processed for indirect immunofluorescent staining with tubulin and vimentin antibodies. After a 2 hour centrifugation vimentin filaments aggregated in the centripetal parts of the cells (probably, because of their association with floating lipid vesicles). Microtubule-organizing centers were found near the centripetal poles of the nuclei, which migrated in the direction of the centrifugal force. The distribution of the cytoplasmic microtubules did not change during centrifugation. The staining of the cultures one hour after centrifugation revealed vimentin-containing spots with radiating intermediate filaments in most of the cells. These spots were localized near the cell nuclei; double immunofluorescent staining with tubulin and vimentin antibodies showed that their position was identical to that of the microtubule-organizing centers. Similar foci of vimentin filaments were seen in the cells after a 3-4 hour centrifugation. Probably, these structures participate in organizing the intermediate filament cytoskeleton in cells.     

Highly Purified Human Immunodeficiency Virus Type 1 Reveals a Virtual Absence of Vif in Virions

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1-encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.     

The enhanced association of keratin with hepatoma cell nuclei

A monoclonal antibody to rat hepatoma keratin demonstrates a close association of intermediate filaments with the nucleus in hepatoma cells. Immunoblot analysis of nuclear fractions and immunofluorescence of nuclei both prepared by standard procedures, indicate that intermediate filament proteins are consistently present. Sodium citrate extraction of these preparations diminishes the amount of intermediate filament proteins but does not totally remove the antigenic moieties, suggesting a tight association of intermediate filaments with nuclei. The results from both immunoblot analysis and immunofluorescent localization demonstrate the increased amount of keratins associated with hepatoma cell nuclei.     

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

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

Expression of new proteins of the intermediate filament protein family in differentiating F9 embryonal carcinoma cell cytoskeleton

Differentiation of F9 embryonal carcinoma cells by retinoic acid treatment results in extraembryonic endoderm-like cells. The effects of this process on the protein composition of the intermediate filaments were studied by two-dimensional gel electrophoresis and by immunoblotting. By this approach, two new proteins induced in differentiating cells, p57 and p54, were identified in cytoskeletal preparations enriched in intermediate filaments. The 57-kDa protein could be resolved into at least three components (pI 5.6-5.9), and the 54-kDa protein into at least two components (pI approximately 5.6). Both proteins reacted with a monoclonal antibody which recognizes an antigenic determinant common to all intermediate filaments. Based on these results, the two proteins were identified as members of the intermediate filament protein family. Partial digestion with V8 protease showed that p57 was different from vimentin, another intermediate filament protein present in these cells. p57 and p54 were also immunodetected by a polyclonal anti-keratin anti-serum, which suggests that these proteins share some homology with the keratins. These two proteins are different from the endodermal cytoskeletal protein A and B (endo A and endo B) keratins, which are known to be present in extraembryonic endoderm-like cells. They were also more abundant than endo A and endo B in differentiating F9 embryonal carcinoma cells, but almost undetectable in terminally differentiated extraembryonic endoderm-like cells, where endo A and endo B are readily detectable. This suggests that p57 and p54 have a different pattern of expression than endo A and endo B.     

Regulated expression of vimentin cDNA in cells in the presence and absence of a preexisting vimentin filament network

Human cells were transfected with a mouse vimentin cDNA expression vector containing the hormone response element of mouse mammary tumor virus. The distribution of mouse vimentin after induction with dexamethasone was examined by indirect immunofluorescence with antivimentin antibodies specific for either mouse or human vimentin. In stably transfected HeLa cells, which contain vimentin filaments, addition of dexamethasone resulted in the initial appearance of mouse vimentin in discrete areas, usually perinuclear, that always corresponded to areas of the human filament network with the most intense fluorescence. Within 20 h after addition of dexamethasone, the mouse and human vimentin immunofluorescence patterns were identical. However, in stably transfected MCF-7 cells, which lack vimentin filaments, induction of mouse vimentin synthesis resulted in assembly of vimentin filaments throughout the cytoplasm without any obvious local concentrations. Transient expression experiments with SW-13 cell subclones that either lack or contain endogenous vimentin filaments yielded similar results to those obtained with MCF-7 and HeLa transfectants, respectively. Further experiments with HeLa transfectants were conducted to follow the fate of the mouse protein after synthesis had dropped after withdrawal of dexamethasone. The mouse vimentin-specific fluorescence was initially lost from peripheral areas of the cells while the last detectable mouse vimentin always corresponded to the human filament network with the most intense fluorescence. These studies are consistent with a uniform assembly of vimentin filaments throughout the cytoplasm and suggest that previous observations of polarized or vectorial assembly from a perinuclear area to more peripheral areas in cells may be attributable to the nonuniformly distributed appearance of vimentin filaments in immunofluorescence microscopy.