Intermediate-sized filaments of human endothelial cells.

Human endothelial cells prepared from unbilical cords are characterized in parallel by electron microscopy and indirect immunofluorescence microscopy using specific antibodies against different classes of intermediate-sized filaments. The strongly developed, loose bundles of intermediate-sized filaments typically found in these cells are not decorated by antibodies against prekeratin or antibodies against smooth muscle desmin. They are, however, strongly decorated by antibodies directed against murine "vimentin," i.e., the 57,000 mol wt polypeptide which is the major protein of the intermediate-sized filaments predominant in various cells of mesenchymal origin. Cytoskeletal preparations greatly enriched in intermediate-sized filaments show the enrichment of a polypeptide band comigrating with murine vimentin. This shows that the intermediate-sized filaments that are abundant in human endothelial cells are predominantly of the vimentin type and can be demonstrated by their cross-reaction with the vimentin of rodents. These data also strengthen the evidence for several subclasses of intermediate-sized filaments, which can be distinguished by immunological procedures.     

Early expression of vimenti in human mammary cultures

Summary The intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the tissue of origin     

Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates.

Specific antibodies against vimentin, the major constitutive protein of intermediate-sized filaments present in cytoskeletons of mesenchymal cells of vertebrates, have been raised in guinea pigs. Antibodies to murine and human vimentin are of three types. The first two types produced against murine vimentin show an exclusive or preferential reaction with vimentin filaments of rodents. The third type raised against murine or human vimentin reacts with intermediate-sized filaments in species as diverse as mammals, birds and amphibia. This latter type is used here to show, both by immunoreplica techniques and by immunofluorescence microscopy, that almost all vertebrate cells growing in culture contain filaments of the vimentin type which are usually present in extended arrays. These immunological findings also suggest that the vimentin molecule contains both sequences conserved during evolution and regions different in different vertebrate species. The cells studied include not only cells of mesenchymal origin, but also cells derived from epithelia, in which it is now possible to demonstrate extensive arrays of vimentin filaments in interphase cells as well as intermediate-sized filaments of the prekeratin type. The data are consistent with the idea that most cells grown in culture contain intermediate-sized filaments of the vimentin type, irrespective of the state of differentiation of the cells from which they are derived.     

Vimentin-typing in diagnostic surgical pathology: a comparative study using four antibodies after different fixations

Vimentin-typing was carried out on various normal and neoplastic tissues using four anti-vimentin antibodies in order to evaluate the effect of different fixation treatments on tissue reactivity in comparison to the results obtained on frozen sections. All antisera were reactive on frozen material; on paraffin embedded material staining of tissues depended on the type of fixation method applied (formalin, methacarn or absolute alcohol) and each antibody behaved differently in relation to the fixative used. Only mesenchymal normal structures were revealed on frozen material whilst on paraffin embedded material three of the four antibodies reacted also with non-mesenchymal normal structures (epithelia, central and peripheral nervous system cells). All four antibodies decorated, regardless of treatment, neoplastic cells of mesenchymal and non-mesenchymal derivation, but not germ cells or germ cell tumors. The reactivity of vimentin to its specific antibodies depends on the fixative used: therefore, in routine pathology more than one antiserum should be available for testing. Furthermore, given the variety of non-mesenchymal structures stained by the anti-vimentin antibodies, the differential diagnosis of undifferentiated tumors must not be based on vimentin positivity alone. The expression of vimentin by non-mesenchymal neoplastic cells seems to parallel that of normal tissues during embryogenesis; therefore, this intermediate filament appears to be not only a marker of mesenchymal cells but also of many immature elements.     

Morphological integrity of single adult cardiac myocytes isolated by collagenase treatment: immunolocalization of tubulin, microtubule-associated proteins 1 and 2, plectin, vimentin, and vinculin

Single cardiac myocytes were isolated from hearts of 9 to 12-week-old rats by means of collagenase (100 U/ml). After assessment of their functional integrity they were processed for immunofluorescence microscopy of the cytoskeletal proteins tubulin, microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2), plectin, vimentin, and vinculin. Antibodies to tubulin decorated a delicate filamentous network that apparently was unrelated to any sarcomeric organization. The distribution of MAP-1 and MAP-2 was strikingly different from that of tubulin, as both antigens were confined to Z-line structures. These structures were also prominently stained by affinity-purified antibodies to plectin and a monoclonal antibody to vimentin. Co-distribution of plectin and vimentin was also observed at the former intercalated disk region of the heart cell. Anti-vinculin antibodies decorated an intricate meshwork consisting of delicate filaments with predominantly irregular orientation and occasional assembly into whorls. These immunolocalization data indicate that the cell shape and cytoskeletal architecture characteristic of cardiac myocytes in tissues is maintained in single isolated cells. Furthermore, intermediate filaments rather than microtubules seem to be instrumental in the preservation of cell morphology.     

LN-6: a monoclonal antibody to vimentin expressed in non-hematopoietic mesenchymal cells and derived tumors and reactive in B5-fixed, paraffin-embedded tissues

We generated a monoclonal antibody (MAb), designated LN-6, directed against human vimentin, which retains its immunoreactivity in B5-fixed, paraffin-embedded tissues. Like other anti-vimentin MAb, LN-6 was found to be reactive with a wide spectrum of human sarcomas and normal cells of mesenchymal derivation. However, unlike other similar reagents, LN-6 was unreactive with normal and malignant human lymphoid cells and therefore displays a more restricted immunoreactivity. Because of its ability to stain routinely processed pathological tissues and its marked reactivity with human sarcomas, LN-6 is a unique reagent for the immunohistochemical diagnosis of human cancer.     

Epinemin: a new protein associated with vimentin filaments in non-neural cells

In this report I describe a new protein, defined by a monoclonal antibody, which is associated with vimentin filaments in a variety of cultured cells and in skeletal muscle. By immunofluorescence it is absent in smooth muscle, in cells without vimentin, and in neural vimentin containing cells. This protein has a molecular weight of 44,500, a pl of 5, a two-dimensional tryptic peptide fingerprint pattern different from vimentin, is unrelated to actin by Cleveland peptide analysis and by light and electron microscopy, and is not recognized by either a polyclonal antivimentin antibody (Frank, E.D., and L. Warren, 1981, Proc. Natl. Acad. Sci. USA, 78:3020-3024) or a monoclonal antibody against all classes of intermediate filaments (Pruss, R.M., R. Mirsky, M.C. Raff, R. Thorpe, A.J. Dowding, and B.H. Anderton, 1981, Cell, 27:419-428). The protein is resistant to nonionic detergent extraction, is soluble in high salt and can thus be removed from vimentin filaments, but fragments with vimentin in either low salt or anionic detergent and collapses with vimentin in colchicine-treated cells. By light microscopy, the distribution of the protein is indistinguishable from vimentin filaments and appears uniform along them. In contrast, immunoferritin electron microscopy reveals that the molecule is distributed in an intermittent pattern on vimentin filaments. Adopting the terminology of Granger and Lazarides (1980, Cell, 30:263-275), the molecule is called epinemin, meaning "upon filaments."     

Microinjection of monoclonal antibodies to vimentin, desmin, and GFA in cells which contain more than one IF type

Microinjection of antibodies to vimentin into fibroblast cell lines causes intermediate filaments (IFs) to build perinuclear caps. We have extended these findings by microinjection of monoclonal antibodies specific for different IF types to non-epithelial cell lines of human origin, which co-express two different IF proteins. Thus GFA and vimentin IgGs have been microinjected in separate experiments into a glioma cell line, desmin and vimentin IgGs into RD cells, and vimentin IgGs into a cell line which co-expresses neurofilaments and vimentin. In all instances, microinjection of a single antibody causes the formation of perinuclear caps in which the two different IF proteins co-localize, suggesting that vimentin and the second IF type present in each cell line localize to the same 10-nm filaments. Immunoelectron microscopy using desmin and vimentin antibodies made in different species and appropriate second antibodies labelled with 5 and 20 nm gold particles confirm this result for RD cells. When Fab' fragments of the vimentin IgGs are microinjected into different cell types, formation of perinuclear caps is observed in immunofluorescence microscopy. In RD cells immunoelectron microscopy shows that the Fab' fragments induce caps which appear less dense than the caps seen after microinjection of IgGs.     

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

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

Distribution of fast myosin heavy chain isoforms in thick filaments of developing chicken pectoral muscle

Colloidal gold-conjugated monoclonal antibodies were prepared to stage-specific fast myosin heavy chain (MHC) isoforms of developing chicken pectoralis major (PM). Native thick filaments from different stages of development were reacted with these antibodies and examined in the electron microscope to determine their myosin isoform composition. Filaments prepared from 12-d embryo, 10-d chick, and 1-yr chicken muscle specifically reacted with the embryonic (EB165), neonatal (2E9), and adult (AB8) antimyosin gold-conjugated monoclonal antibodies, respectively. The myosin isoform composition was more complex in thick filaments from stages of pectoral muscle where more than one isoform was simultaneously expressed. In 19-d embryo muscle where both embryonic and neonatal isoforms were present, three classes of filaments were found. One class of filaments reacted only with the embryonic antibody, a second class reacted only with the neonatal-specific antibody, and a third class of filaments were decorated by both antibodies. Similar results were obtained with filaments prepared from 44-d chicken PM where the neonatal and adult fast MHCs were expressed. These observations demonstrate that two myosin isoforms can exist in an individual thick filament in vivo. Immunoelectron microscopy was also used to determine the specific distribution of different fast MHC isoforms within individual filaments from different stages of development. The anti-embryonic and anti-adult antibodies uniformly decorated both homogeneous and heterogeneous thick filaments. The neonatal specific antibody uniformly decorated homogeneous filaments; however, it preferentially decorated the center of heterogeneous filaments. These observations suggest that neonatal MHC may play a specific role in fibrillogenesis.     

Transient change of organization of vimentin filaments during mitosis as demonstrated by a monoclonal antibody

A monoclonal antibody specific for vimentin is described which, by immunofluorescence and immunoelectron microscopy, decorates fibrillar and/or granular structures in mitotic and early postmitotic cells but does not react with vimentin filaments of interphase stages of various cultured cells (rat vascular smooth muscle-derived cell line RVF-SM; SV40-transformed human fibroblasts; bovine kidney epithelial cells of line MDBK). These observations indicate that the organization of vimentin filaments varies during the cell cycle, undergoing a perimitotic change of filament organization. These changes of vimentin filaments are described in relation to those reported for cytokeratin filaments of various epithelial and carcinoma cells. The possible functional implications of filament protein rearrangements both during the cell cycle and in cell differentiation processes are discussed.     

Vimentin and keratin intermediate filament systems in cultured PtK2 epithelial cells are interrelated.

Certain cultured epithelial cells contain separate vimentin and keratin-type intermediate filament networks. The intracellular injection of monoclonal antibodies directed against either vimentin or keratin filaments into PtK2 cultured epithelial cells specifically disrupted the organization of both filament types. Neither antibody had any effect when injected into cells which, while containing vimentin or keratin filaments, lacked the specific filament type which that antibody recognized. These experiments suggest that keratin and vimentin filament networks are associated in some way with one another.     

Microinjection of monoclonal antibodies specific for one intermediate filament protein in cells containing multiple keratins allow insight into the composition of particular 10 nm filaments

Monoclonal antibodies specific for vimentin (V9), keratin 7 (CK 7) and keratin 18 (CK5) have been microinjected into three human epithelial cell lines: HeLa, MCF-7 and RT-4. The effect of the injection on other keratin polypeptides and vimentin filaments has been observed by double label immunofluorescence and in some instances by immunoelectron microscopy using gold labels of different sizes. Microinjection of V9 into HeLa cells causes the vimentin to collapse into a perinuclear cap leaving the keratin filaments unaffected. Injection of CK5 does not affect the vimentin filaments but disrupts the keratin filaments revealing keratin aggregates similar to those seen in some epithelial cell lines during mitosis. The keratin aggregates obtained after microinjection in HeLa contain the keratins 8 and 18 and probably also other keratins, as no residual keratin filaments are observed with a keratin polyclonal antibody of broad specificity. Aggregates in mitotic HeLa cells contain at least the keratins 7, 8, and 18. In MCF-7 cells keratins 8, 18, and 19 are observed in the aggregates seen 3 h after microinjection which, however, show a different morphology from those seen in HeLa cells. In MCF-7 cells a new keratin filament is built within 6 h after the injection which is composed mainly of keratin 8 and 19. The antibody-complexed keratin 18 remains in spherical aggregates of different size. The results suggest that in HeLa cells vimentin and keratin form independent networks, and that individual 10 nm filaments in epithelial cell lines can contain more than two keratins.     

Keratin and vimentin expression in early organogenesis of the rabbit embryo

Summary 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.Supported by the Deutsche Forschungsgemeinschaft and by the Netherlands Cancer Foundation     

Regulation of protein phosphorylation of the intermediate-sized filament vimentin in the ciliary epithelium of the mammalian eye

The intermediate-sized filaments of vimentin-type (Mr = 57,000) have been identified biochemically and immunochemically as a major cytoskeleton component in the ciliary epithelium of the mammalian eye. When human or rabbit ciliary processes, or cultured ciliary epithelial-derived cells were incubated in serum-free medium containing [32P]orthophosphate and any of the following agents: 1) beta-adrenergic agonists (isoproterenol or epinephrine), 2) direct activators of adenylate cyclase (cholera toxin or forskolin), 3) analogs of cyclic AMP (8-Br-cAMP), or 4) prostaglandin E1, the phosphorylation of vimentin was significantly enhanced. The maximal enhancement ranged, in vivo and in vitro, from about 3-fold in human to 5-fold in rabbit, with either 1 mM 8-Br-cAMP or 0.1 microM forskolin. Phosphorylation of vimentin increased in the presence of beta-adrenergic agonists and could be blocked by the antiglaucoma beta-adrenergic antagonist timolol. The alpha-adrenergic agonist phenylephrine had no effect on phosphorylation of vimentin. Indirect immunofluorescence microscopy using a monoclonal antibody, anti-vimentin, allowed the localization of vimentin filaments in cultured ciliary epithelial cells. Treatment of these cells in culture with the catecholamine hormone, isoproterenol (1 microM), resulted in a profound reorganization of vimentin filaments. This may be correlated with the enhanced levels of phosphorylated vimentin observed upon increasing cellular cyclic AMP.     

Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression

The cell of origin of the nonparenchymal epithelioid cells that emerge in liver cell cultures is unknown. Cultures of rat hepatocytes and several types of nonparenchymal cells obtained by selective tissue dispersion procedures were typed with monoclonal antibodies to rat liver cytokeratin and vimentin, polyvalent antibodies to cow hoof cytokeratins and porcine lens vimentin, and monoclonal antibodies to surface membrane components of ductular oval cells and hepatocytes. Immunoblot analysis revealed that, in cultured rat liver nonparenchymal epithelial cells, the anti-rat hepatocyte cytokeratin antibody recognized a cytokeratin of relative mass (Mr) 55,000 and the anti-cow hoof cytokeratin antibody reacted with a cytokeratin of Mr 52,000, while the anti-vimentin antibodies detected vimentin in both cultured rat fibroblasts and nonparenchymal epithelial cells. Analyses on the specificity of anti-cytokeratin and anti-vimentin antibodies toward the various cellular structures of liver by double immunofluorescence staining of frozen tissue sections revealed unique reactivity patterns. For example, hepatocytes were only stained with anti-Mr 55,000 cytokeratin antibody, while the sinusoidal cells reacted only with the anti-vimentin antibodies. In contrast, epithelial cells of the bile ductular structures and mesothelial cells of the Glisson capsula reacted with all the anti-cytokeratin and anti-vimentin antibodies. It should be stressed, however, that the reaction of the anti-vimentin antibodies on bile ductular cells was weak. The same analysis on tissue sections using the anti-ductular oval cell antibody revealed that it reacted with bile duct structures but not with the Glisson capsula. The anti-hepatocyte antibody reacted only with the parenchymal cells. The differential reactivity of the anti-cytokeratin and anti-vimentin antibodies with the various liver cell compartments was confirmed in primary cultures of hepatocytes, sinusoidal cells, and bile ductular cells, indicating that the present panel of antibodies to intermediate filament constituants allowed a clear-cut distinction between cultured nonparenchymal epithelial cells, hepatocytes, and sinusoidal cells. Indirect immunofluorescence microscopy on nonfixed and paraformaldehyde-fixed cultured hepatocytes and bile ductular cells further confirmed that both anti-hepatocyte and anti-ductular oval cell antibodies recognized surface-exposed components on the respective cell types.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distinction of nephroblastomas from other childhood tumors using antibodies to intermediate filaments

Ten nephroblastomas were investigated by antibodies to intermediate filaments. In seven cases, which in light microscopy were characterized by the presence of blastema and tubules, immunofluorescence microscopy with IF-specific antibodies reveals expression of cytokeratin and vimentin in blastema cells, while tubules were only labelled by the cytokeratin antibodies. This result was independent of whether the conventional cytokeratin antibody or monoclonal antibodies specific for cytokeratin 18 were used. Stroma cells were vimentin-positive. In two cases nephroblastomas were undifferentiated and also lacked tubuli formation. In both these tumors blastema cells were vimentin-positive and cytokeratin-negative. Finally one case of clear cell sarcoma of the kidney could only be labelled by the vimentin antibody. Thus antibodies to intermediate filaments seem to be useful tools to distinguish nephroblastomas from neuroblastomas or rhabdomyosarcomas, especially in cases of metastasis.     

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.     

Ultrastructural immunogold labelling of vimentin filaments on postembedding ultrathin sections of arachnoid villi and meningiomas

An immunoelectron microscopic technique for the labelling of vimentin intermediate filaments on postembedding ultrathin sections is reported. Arachnoid villi obtained at autopsy and meningiomas at surgery were fixed in 1% paraformaldehyde for 30 minutes, embedded without postfixation in Epon-Araldite mixture and polymerized at 37 degrees C for 3 weeks. Ultrathin sections were etched in 2% KOH for 3 minutes and incubated with anti-vimentin monoclonal antibodies which were subsequently labelled with goat anti-mouse IgG coupled to colloidal golds. All of these labelling procedures were consistently performed within 4 hours. In both arachnoidal and meningioma cells, immunogolds preferentially decorated the intermediate filaments in proportion to the concentration. Very few gold particles were seen over the nucleus, Golgi zone, mitochondria and the extracellular connective tissue fibres. The present technique may be applied to the immunogold labelling of intermediate filaments on postembedding ultrathin sections.     

Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo

It is known that the chicken flank somatopleure also has a limb-forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo. Further study revealed that this antibody is specific to vimentin. Time course of vimentin expression in the somatopleural mesoderm during the development was studied. It was revealed to be biphasic. Somatopleural mesoderm expressed vimentin at stage 10, but not at stage 16. Flank somatopleural mesoderm began to express vimentin again at stage 18, whereas limb bud mesenchymal cells did not until stage 27. The earlier re-expression of vimentin at the flank somatopleure suggests that certain physiological changes take place in cells at this region.