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.     

Intermediate-sized filaments of the prekeratin type in myoepithelial cells

Myoepithelial cells from mammary glands, the modified sweat glands of bovine muzzle, and salivary glands have been studied by electron microscopy and by immunofluorescence microscopy in frozen sections in an attempt to further characterize the type of intermediate-sized filaments present in these cells. Electron microscopy has shown that all myoepithelial cells contain extensive meshworks of intermediate-sized (7--11-nm) filaments, many of which are anchored at typical desmosomes or hemidesmosomes. The intermediate-sized filaments are also intimately associated with masses of contractile elements, identified as bundles of typical 5--6-nm microfilaments and with characteristically spaced dense bodies. This organization resembles that described for various smooth muscle cells. In immunofluorescence microscopy, using antibodies specific for the various classes of intermediate-sized filaments, the myoepithelial cells are strongly decorated by antibodies to prekeratin. They are not specifically stained by antibodies to vimentin, which stain mesenchymal cells, nor by antibodies to chick gizzard desmin, which decorate fibrils in smooth muscle Z bands and intercalated disks in skeletal and cardiac muscle of mammals. Myoepithelial cells are also strongly stained by antibodies to actin. The observations show (a) that the epithelial character, as indicated by the presence of intermediate-sized filaments of the prekeratin type, is maintained in the differentiated contractile myoepithelial cell, and (b) that desmin and desmin-containing filaments are not generally associated with musclelike cell specialization for contraction but are specific to myogenic differentiation. The data also suggest that in myoepithelial cells prekeratin filaments are arranged--and might function--in a manner similar to the desmin filaments in smooth muscle cells.     

Simultaneous expression of two different types of intermediate sized filaments in mouse keratinocytes proliferating in vitro.

The intermediate-sized filaments present in epidermal keratinocytes derived from mouse skin and in an established cell line (HEL) derived from spontaneous transformation of murine keratinocytes grown in vitro, have been examined by immunofluorescence microscopy, using antibodies directed against subunit proteins of different classes of intermediate-sized filaments, as well as by electron microscopy and gel electrophoresis of cytoskeletal preparations highly enriched in intermediate-sized filaments. The keratinocytes derived from neonatal skin, which are capable of only limited replication in vitro, show only a single type of intermediate-sized filaments, i.e., the tonofibril-like arrays of filaments containing prekeratin. HEL cells, which proliferate indefinitely in vitro, retain the tonofilament-like structures typical of differentiated epidermal cells but in addition display intermediate-sized filaments of the vimentin type, i.e., the filament system typically found in mesenchymal and mesenchyme-derived cells. We discuss the possibility that (i) the advent of vimentin-type filaments in epidermal cells in culture is related either to the transformed state or the in vitro growth conditions as such and (ii) other differentiated epithelial cells proliferating in vitro may have more than one system of intermediate-sized filaments.     

Simultaneous Expression of Two Different Types of Intermediate Sized Filaments in Mouse Keratinocytes Proliferating in vitro

The intermediate-sized filaments present in epidermal keratinocytes derived from mouse skin and in an established cell line (HEL) derived from spontaneous transformation of murine keratinocytes grown in vitro, have been examined by immunofluorescence microscopy, using antibodies directed against subunit proteins of different classes of intermediate-sized filaments, as well as by electron microscopy and gel electrophoresis of cytoskeletal preparations highly enriched in intermediate-sized filaments. The keratinocytes derived from neonatal skin, which are capable of only limited replication in vitro, show only a single type of intermediate-sized filaments, i.e., the tonofibril-like arrays of filaments containing prekeratin. HEL cells, which proliferate indefinitely in vitro, retain the tonofilament-like structures typical of differentiated epidermal cells but in addition display intermediate-sized filaments of the vimentin type, i.e., the filament system typically found in mesenchymal and mesenchyme-derived cells. We discuss the possibility that (i) the advent of vimentin-type filaments in epidermal cells in culture is related either to the transformed state or the in vitro growth conditions as such and (ii) other differentiated epithelial cells proliferating in vitro may have more than one system of intermediate-sized filaments.     

Direct demonstration of the presence of two immunologically distinct intermediate-sized filament systems in the same cell by double immunofluorescence microscopy: Vimentin and cytokeratin fibers in cultured epithelial cells

The epithelial derived cell lines PtK2 and HeLa were characterized by double immunofluorescence microscopy using purified antibodies against vimentin and prekeratin. The results show that both cell types express simultaneously two immunologically distinct intermediate-sized filaments. Use of colcemid-treated cells confirms that the vimentin fibers and not the keratin-related fibers are rearranged into coils around the nucleus. In some cells staining of fibrous fragments is observed, which are perhaps involved in the synthesis or breakdown of this class of filaments. The concept that growing cells derived from differentiated cell types express not only the intermediate-sized filament system typical of the differentiated cell type but in addition contain intermediate-sized filaments of the vimentin type is discussed.     

Antibodies as probes of cellular differentiation and cytoskeletal organization in the mouse blastocyst

Indirect immunofluorescence microscopy has been used to detect cytoskeletal proteins, which allow a distinction between the two cell types present in the mouse blastocyst: i.e. the cells of the inner cell mass (ICM) and the outer trophoblastic cells. Antibodies against three classes of intermediate-sized filaments (cytokeratins, desmin and vimentin), as well as antibodies against actin and tubulin were studied. Antibodies against prekeratin stain the outer trophoblastic cells but not the ICM in agreement with the findings on adult tissues that cytokeratins are a marker for various epithelial cells. Interestingly, vimentin filaments typical of mesenchymal cells as well as of cells growing in culture seem to be absent in both cell types of the blastocyst. Thus, the cytokeratins of the trophoblastic cells seem to be the first intermediate-sized filaments expressed in embryogenesis. Antibodies to tubulin and actin show that microtubules and microfilaments are ubiquitous structures, although microfilaments have a noticeably different organization in the two cell types. In addition, since early embryogenic multipotential cells show close similarities to teratocarcinomic cells, a comparison is made between the cells of the blastocyst, embryonal carcinoma cells (EC cells) and an epithelial endodermal cell line (PYS2 cells) derived from EC cells. EC cells display vimentin filaments whereas PYS2 cells show both vimentin and cytokeratin filaments. The results emphasize the usefulness of antibodies specific for different classes of intermediate filaments in further embryological studies, and suggest that cells of the blastocyst and EC cells differ with respect to vimentin filaments.     

An unusual type of cytokeratin filament in cells of a human cloacogenic carcinoma derived from the anorectal transition zone

Epithelia-derived tumors (carcinomas) can be distinguished from mesenchymally derived tumors by the presence of intermediate-sized filaments of the cytokeratin type, which usually coincides with the absence of other types of intermediate-sized filaments such as vimentin filaments. In the course of diagnostic examinations of human tumors, using immunofluorescence microscopy, we have come across a case of an unusual carcinoma (Primary tumor and lymph node metastasis) positively stained not only with cytokeratin antibodies but also with immunoglobulins present in vimentin antisera. Therefore, this tumor, a cloacogenic carcinoma apparently derived from the rectal-anal transitional region, has been examined in greater detail using both immunofluorescence microscopy and immuno-electron microscopy as well as gel electrophoretic analysis of cytoskeletal polypeptides from total tumor tissue and from microdissected nodules enriched in carcinoma cells. The unusual reaction of the carcinoma cells with immunoglobulins present in seven different (rabbit or guinea pig) antisera raised against vimentin, has been found to be diminished after absorption on purified cytokeratin or total epidermal cytoskeletal material, but not after absorption on purified vimentin. Gel electrophoretic analysis of tumor cytoskeletons showed an unusual complex pattern of cytokeratin polypeptides containing relatively large (Mr 68,000 and Mr 58,000) neutral-to-slightly basic cytokeratins, as are typically found in epidermis and other stratified squamous epithelia, as well as several smaller acidic cytokeratins, including a Mr 40,000 polypeptide found in certain nonstratified epithelial such as colon and small intestine. Total tumor also showed the inclusion of some vimentin which, however, was significantly decreased in analysis of excised carcinoma nodules. Examining antibody binding to polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper, we have found that antisera raised against vimentin contained not only vimentin antibodies but also immunoglobulins which specifically bound to the largest cytokeratin component. We conclude that the unusual reaction of immunoglobulins present in vimentin antisera with cytokeratin filament bundles does not represent specific binding to vimentin in these carcinoma cells, but is due to a component obviously widespread in vimentin antisera which binds specifically to a cytokeratin present in this type of tumor but not in most other carcinomas. It is proposed that use is made in diagnostic examinations of vimentin antisera or affinity-purified vimentin antibodies that have been pre-absorbed on cytokeratin protein, in order to eliminate such disturbing reactions.     

Characterization of cells of amniotic fluids by immunological identification of intermediate-sized filaments: Presence of cells of different tissue origin

Summary Antibodies against intermediate-sized filaments, of the prekeratin or vimentin type, were used to investigate the presence of these filaments by indirect immunofluorescence microscopy in cultured and non-cultured amniotic fluid cells, in frozen sections of the placenta and in isolated cells of the amniotic epithelium. Two major classes of cells can be cultured from amniotic fluids, namely cells of epithelial origin containing filaments of the prekeratin type and cells of different origin which contain filaments of the vimentin type but are negative when tested with antibodies to epidermal prekeratin. The presence of prekeratin type filaments correlates with the morphology of colonies of amniotic fluid cell cultures in vitro as classified by Hoehn et al. (1974). Cells of E-type colonies are shown to be of epithelial origin. In contrast our data indicate a different origin of almost all cells of F-type colonies and of the large majority of cells of AF-type colonies. Cells of epithelial origin and positively stained with antibodies to epidermal prekeratin are occasionally scattered in F-type colonies and in variable percentages (up to 30%) in AF-type colonies. Surprisingly, cryostat sections of the amniotic epithelium and isolated groups of amniotic cells showed positive reactions with both antibodies to vimentin and prekeratin. The possibility that amniotic cells may be different from other epithelial cells in that they contain both types of filaments simultaneously already in situ is presently under investigation.Part of this work is included in the doctoral thesis of Irmgard Treiss to be submitted to the Faculty of Medicine of the University of Heidelberg     

Constitutive aggregates of intermediate-sized filaments of the vimentin and cytokeratin type in cultured hepatoma cells and their dispersal by butyrate

Cloned hepatoma cells ($\text{72}\text{22}$) derived from the liver of a rat treated with the carcinogen, diethylnitrosamine, exhibit a genetically stable, large, acentric, juxtanuclear, hyaline aggregate of loosely packed intermediate-sized (7–11 nm) filaments, interspersed with variable but minor amounts of microtubules, polyribosomes and membranous structures. Immunofluorescence microscopy shows that the these filaments react specifically with antibodies to bovine prekeratin and to murine vimentin. The aggregates contain aster-like foci common to the arrangement of both tonofilament-like and vimentin-containing intermediate-sized filaments, although both filament systems show different fibrillar patterns in other cytoplasmic regions. While the cytokeratin filament system is not significantly altered during exposure to colcemid, the vimentin in the abnormal aggregate is rearranged during such treatment into extensive and complex perinuclear ‘whorls’ of filaments. Treatment of the cells with butyrate results in a markedly flattened, hepatocyte-like morphology, a reappearance of typical actin-containing ‘cables’, and a progressive disintegration of the filament aggregate concomitant with a normal display of filaments of both the cytokeratin and vimentin type. The results show that (i) some cells contain aggregates consisting of two different types of intermediate-sized filaments oriented onto a common focal center; (ii) such an abnormal filament arrangement is clonally stable; (iii) the vimentin-type filaments contained in such aggregates are still susceptible to the action of antimitotic drugs and can be rearranged into characteristic perinuclear whorls; and (iv) this abnormal aggregate of intermediate filaments can be reverted to normal patterns upon treatment of the cells with butyrate.     

Differential location of different types of intermediate-sized filaments in various tissues of the chicken embryo.

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11--20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to--and specific for--epithelial cells; vimentin filaments are seen--at this stage of embryogenesis--only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structurees provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Epithelial cell components immunoreact with antiserum thymic factor (FTS) antibodies: Possible association with intermediate-sized filaments

By indirect immunofluorescence microscopy, an antiserum raised in rabbit against serum thymic factor (FTS) was found to decorate the epithelial cells not only in the thymus, but also in the kidney, uterus, urinary bladder, prostatic glands, stomach, ileum, colon, submaxillary glands, trachea, epidermis and epidermal appendages of mouse. The staining ability was completely absorbed with an FTS-binding immunoabsorbent, and affinity-purified anti-FTS IgG showed the same staining patterns as the original antiserum. The staining profiles resembled those described for tissues stained with antiprekeratin and antikeratin antibodies in both distribution in tissue and localization in the epithelial cells. In primary-cultured cells from mouse kidney medullae, the anti-FTS antibodies decorated the cytoplasmic fiber network. The fibers were wavy, bundled together and branched. They were dense in the perinuclear cytoplasm and spread in the cytoplasm toward the cell periphery. This decoration was resistant to colchicine and cytochalasin B, but sensitive to pretreatment with formaldehyde. The organization and shape of the fiber network were similar to those of the networks of intermediate-sized filaments containing cytokeratins, keratins and vimentin. However, the antiserum did not give a precipitin band in immunodiffusion test with prekeratin from bovine muzzle, keratin from human epidermis or 3T3 vimentin. Neither tubulin nor actin formed precipitin bands with the antiserum. These results show that the epithelial cells of various mouse tissues contain FTS or substances close to FTS in chemical structure and suggest that they are associated with the intermediate-sized filaments.     

Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.     

Differential Location of Different Types of Intermediate-Sized Filaments in Various Tissues of the Chicken Embryo

The location of constitutive proteins of different types of intermediate-sized (about 10 mm) filaments (cytokeratin, vimentin, desmin, brain filament protein) was examined in various tissues of 11–20 day chick embryos, using specific antibodies against the isolated proteins and immunofluorescence microscopy on frozen sections and on isolated serous membrane. The tissues studied which contained epithelia were small intestine, gizzard, esophagus, crop, liver, kidney, thymus, mesenteries, and epidermis. The results show that the different intermediate filament proteins, as seen in the same organ, are characteristic of specific lines of differentiation: Cytokeratin filaments are restricted to – and specific for – epithelial cells; vimentin filaments are seen – at this stage of embryogenesis – only in mesenchymal cells, including connective tissue, endothelial and blood cells, and chondrocytes; filaments containing protein(s) related to the subunit protein prepared from gizzard 10 nm filaments (i.e., desmin) are significant only in muscle cells; and intermediate filament protein of brain, most probably neurofilament protein, is present only in nerve cells. We conclude that for most tissues the expression of filaments of cytokeratin, vimentin, desmin, and neurofilament protein is mutually exclusive, and that these protein structures provide useful markers for histochemical and cytochemical differentiation of cells of epithelial, mesenchymal, myogenic, and neurogenic differentiation.     

Identification and Characterization of Epithelial Cells in Mammalian Tissues by Immunofluorescence Microscopy Using Antibodies to Prekeratin

The occurrence of intermediate-sized filaments containing prekeratin-like proteins ('cytokeratins') has been examined in various organs of rat and cow by electron microscopy and by immunofluorescence microscopy on frozen sections using antibodies to defined constitutive proteins of various types of intermediate-sized filaments (prekeratin, vimentin, desmin). Positive cytokeratin reaction and tonofilament-like structures have been observed in the following epithelia: epidermis; ductal, secretory, and myoepithelial cells of sweat glands; mammary gland duct; myoepithelial cells of lactating mammary gland; milk secreting cells of cow; ductal, secretory, and myoepithelial cells of various salivary glands; tongue mucosa; bile duct; excretory duct of pancreas; intestinal mucosa; urothelium; trachea; bronchi; thymus reticulum, including Hassall corpuscles; mesothelium; uterus; and ciliated cells of oviduct. None of the epithelial cells mentioned has shown significant reaction with antibodies to vimentin, the major component of the type of intermediate-sized filaments predominant in mesenchymal cells. The widespread, if not general occurrence of cytokeratin filaments in epithelial cells is emphasized, and it is proposed to use this specific structure as a criterion for true epithelial character or origin.     

The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments.

When cultured cells of the rat kangaroo cell line PtK2 grown on plastic or glass surfaces are lysed and extracted with combinations of low and high salt buffers and the non-ionic detergent Triton X-100 cytoskeletal preparations are obtained that show an enrichment of 6 to 11 nm thick filaments. The arrays of these filaments have been examined by various light and electron microscopic techniques, including ultrathin sectioning, whole mount transmission electron microscopy, negative staining, and indirect immunofluorescence microscopy. In addition, 6 to 11 nm filaments isolated from these cells with similar extraction procedures and with centrifugation techniques have been examined by electron microscopy. The arrays of these isolated intermediate-sized filaments, their ultrastructure and their specific decoration by certain antibodies present in normal rabbit sera as well as by guinea pig antibodies against purified bovine prekeratin is demonstrated. When preparations enriched in these intermediate-sized filaments are examined by SDS-polyacrylamide gel electrophoresis a corresponding enrichment of three polypeptide bands with apparent molecular weights of about 45 000, 52 000 and 58 000 (the latter component sometimes appears split into two bands) is observed, besides some residual actin and a few high molecular weight bands. The morphology of the isolated filaments, their immunological reaction with antibodies decorating prekeratin-containing structures, and the sizes of their constitutive polypeptides suggest that these filaments are closely related to prekeratin-containing filaments observed in a variety of epithelial cells.     

Identification of the cytoskeletal proteins in lens-forming cells, a special epithelioid cell type

Proteins of contractile and cytoskeletal elements have been studied in bovine lens-forming cells growing in culture as well as in bovine and murine lenses grown in situ by immunofluorescence microscopy using antibodies to the following proteins: actin, myosin, tropomyosin, α-actinin, tubulin, prekeratin, vimentin, and desmin. Lens-forming cells contain actin, myosin, tropomyosin, and α-actinin which in cells grown in culture are enriched in typical cable-like structures, i.e. microfilament bundles. Antibodies to tubulin stain normal, predominantly radial arrays of microtubules. In the epithelioid lens-forming cells of both monolayer cultures grown in vitro and lens tissue grown in situ intermediate-sized filaments of the vimentin type are abundant, whereas filaments containing prekeratin-like proteins (‘cytokeratins’) and desmin filaments have not been found. The absence of cytokeratin proteins observed by immunological methods is supported by gel electrophoretic analyses of cytoskeletal proteins, which show the prominence of vimentin and the absence of detectable amounts of cytokeratins and desmin. This also correlates with electron microscopic observations that typical desmosomes and tonofilament bundles are absent in lens-forming cells, as opposed to a high density of vimentin filaments. Our observations show that the epithelioid lens-forming cells have normal arrays of (i) microfilament bundles containing proteins of contractile structures; (ii) microtubules; and (iii) vimentin filaments, but differ from most true epithelial cells by the absence of cytokeratins, tonofilaments and typical desmosomes. The question of their relationship to other epithelial tissues is discussed in relation to lens differentiation during embryogenesis. We conclude that the lens-forming cells either represent an example of cell differentiation of non-epithelial cells to epithelioid morphology, or represent a special pathway of epithelial differentiation characterized by the absence of cytokeratin filaments and desmosomes. Thus two classes of tissue with epithelia-like morphology can be distinguished: those epithelia which contain desmosomes and cytokeratin filaments and those epithelioid tissues which do not contain these structures but are rich in vimentin filaments (lens cells, germ epithelium of testis, endothelium).     

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.     

Intermediate filaments of the vimentin-type and the cytokeratin-type are distributed differently during mitosis

Immunofluorescence microscopy has been used to follow the rearrangement of intermediate-sized filaments during mitosis in rat kangaroo PtK2 cells. These epithelial cells express two different intermediate filament systems: the keratin-related tonofilament-like arrays typical of epithelial cells, and the vimentin-type filaments characteristic of mesenchymal cells in vivo, and of many established cell lines. The two filament systems do not appear to depolymerize extensively during mitosis, but show differences in their organization and display which may indicate different functions. The most striking rearrangements have been seen with the vimentin filaments, and in particular in prometaphase a transient cage-like structure of vimentin fibers surrounding the developing spindle is formed. In metaphase, this cage disappears, and vimentin fibers are found in an elliptical band surrounding the chromosomes and the interzone. In telophase, these bands separate, usually breaking first on the side closest to where the cleavage furrow has started to form. Double label experiments with tubulin and vimentin antibodies have indicated that the microtubules and the chromosomes are contained within the thick crescents of vimentin filaments and suggest that the vimentin intermediate filaments may be involved in the orientation of the spindle and/or the chromosomes during mitosis. In contrast, extensive arrays of cytokeratin filaments are present throughout mitosis on the substrate-attached side of the cell and also in other cellular areas, although they are usually not present in the spindle region. Thus the cytokeratin filaments probably continue to play a cytoskeletal role during mitosis and may be responsible for the flat shape that certain epithelial cells such as PtK2 cells continue to maintain during mitosis.     

De novo synthesis and specific assembly of keratin filaments in nonepithelial cells after microinjection of mRNA for epidermal keratin

Poly(A)+ RNA isolated from bovine muzzle epidermis was microinjected into nonepithelial cells containing only intermediate-sized filaments of the vimentin type. In recipient cells keratin polypeptides are synthesized and assemble into intermediate-sized filaments at multiple dispersed sites. We describe the time course and the pattern of de novo assembly of keratin filaments within living cells. These filaments were indistinguishable, by immunofluorescence and immunoelectron microscopic criteria, from keratin filament arrays present in true epithelial cells. The presence of extended keratin fibril meshworks in these injected cells is compatible with cell growth and mitosis. Double immunolabeling revealed that newly assembled keratin was not codistributed with microfilament bundles, microtubules or vimentin filaments. We suggest that assembly mechanisms exist which in vivo sort out newly synthesized cytokeratin polypeptides from vimentin.     

Visualization of the 10-nm filament vimentin rings in vascular endothelial cells in situ : Close resemblance to vimentin cytoskeletons found in monolayers in vitro

Surface staining of the intact vascular endothelial cell layer lining the lumen of guinea pig thoracic aorta with antibodies to vimentin revealed that at least 70% of the cells contained intact perinuclear rings of 10-nm filaments. This correlated with the observations made on these cells in culture: 60–80% of the endothelial cells at confluence have complete perinuclear rings. By one- and two-dimensional gel electrophoresis and immunoprecipitation we confirmed that vimentin [17, 18] is the major constituent polypeptide of the 10-nm filaments in guinea pig endothelial cells. These results indicate that the vimentin [17] 10-nm filament cytoskeleton found in guinea pig endothelial cells in vitro is similar to the cytoskeleton found in situ.