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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Proteins IEF (isoelectric focusing) 31 and IEF 46 are keratin-type components of the intermediate-sized filaments: keratins of various human cultured epithelial cells

Mouse polyclonal antibodies have been raised against two human proteins (IEF [isoelectric focusing] 31, Mr = 50,000; IEF 46, Mr = 43,500) that have previously been shown to be present in HeLa cytoskeletons enriched in intermediate-sized filaments. Immunoprecipitation studies show that both proteins share common antigenic determinants with each other and with the putative human keratins IEF 36 and 44, also present in HeLa cytoskeletons. Indirect immunofluorescence studies showed that both antibodies revealed similar filamentous networks in various cultured epithelial cells of human origin. These included AMA (transformed amnion), HeLa (cervical carcinoma), normal amnion cells, Fl-amnion (transformed amnion), WISH-amnion (transformed amnion), Chang liver (liver), and Detroid-98 (sternal marrow). Human cells that did not react with both antibodies included skin fibroblasts, lung fibroblasts (WI-38), SV40-transformed lung fibroblasts, Molt 4 (leukemia), lymphocytes, and monocytes. These results were in complete agreement with the presence or absence of both proteins in two-dimensional gels of the different cell types. Exposure of AMA cells to demecolcine (24 h; 10 micrograms/ml) caused the total collapse of vimentin filaments but, as seen by indirect immunofluorescence, caused only a partial redistribution of the IEF 31 and 46 filaments. These results are taken to suggest that both proteins are components of the intermediate-sized filaments of the "keratin" type. The antibodies could be clearly differentiated by staining human bladder carcinoma EJ 19 cells, as only the IEF 46 antibody stained a filamentous network in these cells The occurrence of keratins IEF 31, 36, 44, and 46 in different cultured human epithelial cells has been studied using two-dimensional gel electrophoresis.     

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     

An in vitro study of the effects of androgens on the cytoskeleton of ovarian granulosa cells with special reference to actin

Ovarian granulosa cells from small antral follicles from immature rats were cultured in a serum-free medium for 1-6 days with or without the presence of 10(-5) M dehydroepiandrosterone (DHEA) or 10(-5) M-androstenedione (delta 4-A). Control cultures reveal that the cells are flattened and contain many filamentous bundles organized as stress fibers, numerous scattered cytoplasmic actin filaments, microtubules and vimentin. Alpha actinin and myosin were shown by immunocytochemistry to have a punctate pattern along the stress fibers. For the most part, cells exposed to androgens did not flatten; however, they assumed a varied shape and contained fewer stress fibers and actin filaments. Many of these cells did not develop stress fibers and those that did develop were fewer in number and displayed--actinin and myosin in a punctate pattern. Microtubules and vimentin filaments remained unaltered when compared to controls. It is believed that the deficiency of actin filaments, coupled with certain other degenerative changes which express themselves in other cellular compartments, leads to an early atresia of the granulosa cell cultured in high concentrations of androgens.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

In vitro differentiation of mouse teratocarcinoma cells monitored by intermediate filament expression

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.     

In vitro Differentiation of Mouse Teratocarcinoma Cells Monitored by Intermediate Filament Expression

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.     

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).     

Expression of intermediate filament-associated proteins paranemin and synemin in chicken development

The expression of two intermediate filament-associated proteins, paranemin (280,000 mol wt) and synemin (230,000 mol wt), was investigated with respect to the expression of two core intermediate filament proteins, desmin and vimentin, in various embryonic and adult chicken muscle and nonmuscle cells. All developing muscle cells, regardless of their type, simultaneously express desmin, vimentin, paranemin, and synemin. However, a difference is observed in the expression of paranemin in adult muscle. This protein is removed during differentiation of both fast and slow skeletal muscle, visceral smooth muscle, and the smooth muscle of muscular arteries, but remains in mature myocardial cells, cardiac conducting fibers, and the smooth muscle cells of elastic arteries. Some of these cells express vimentin, others desmin, and still others a mixture of the two. On the other hand, synemin is expressed in all the above types of adult muscle cells except myocardial cells. Adult myocardial cells also lack vimentin, and its presence is gradually reduced after hatching. Since in adult striated muscle all expressed intermediate filament proteins are found predominantly in association with the peripheries of myofibrillar Z discs, these results suggest that a change in the composition of skeletal and cardiac muscle Z discs occurs during chicken development and maturation. Erythrocytes that express synemin and vimentin do not express paranemin, while both embryonic and adult Schwann cells co- express paranemin and vimentin, but not synemin. Endothelial cells of muscular vessels express paranemin, while those of elastic vessels do not, and neither contains synemin. Paranemin and synemin are not expressed in neurons, epithelial, and most glial cells, suggesting that these two polypeptides are expressed only in conjunction with desmin or vimentin. These results suggest that the composition of intermediate filaments changes during chicken development, not only with respect to their core subunit proteins but also with respect to two associated polypeptides, particularly in muscle cells.