Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

Attachment of vimentin filaments to desmosomal plaques in human meningiomal cells and arachnoidal tissue

Desmosomal proteins are co-expressed with intermediate-sized filaments (IF) of the cytokeratin type in epithelial cells, and these IF are firmly attached to the desmosomal plaque. In meningiomal and certain arachnoidal cells, however, vimentin IF are attached to desmosomal plaques. Meningiomas obtained after surgery, arachnoid "membranes", and arachnoid granulations at autopsy, as well as meningiomal cells grown in short-term culture have been examined by single and double immunofluorescence and immunoelectron microscopy using antibodies to desmoplakins, vimentin, cytokeratins, glial filament protein, neurofilament protein, and procollagen. In addition, two-dimensional gel electrophoresis of the cytoskeletal proteins has been performed. Using all of these techniques, vimentin was the only IF protein that was detected in significant amounts. The junctions morphologically resembling desmosomes of epithelial cells have been identified as true desmosomes by antibodies specific for desmoplakins and they provided the membrane attachment sites for the vimentin IF. These findings show that anchorage of IF to the cell surface at desmosomal plaques is not restricted to cytokeratin IF as in epithelial cells and desmin IF as in cardiac myocytes, suggesting that binding to desmosomes and hemidesmosomes is a more common feature of IF organization. The co- expression of desmosomal proteins and IF of the vimentin type only defines a new class of cell ("desmofibrocyte") and may also provide an important histodiagnostic criterion.     

An epithelium-type cytoskeleton in a glial cell: astrocytes of amphibian optic nerves contain cytokeratin filaments and are connected by desmosomes

In higher vertebrates the cytoskeleton of glial cells, notably astrocytes, is characterized (a) by masses of intermediate filaments (IFs) that contain the hallmark protein of glial differentiation, the glial filament protein (GFP); and (b) by the absence of cytokeratin IFs and IF-anchoring membrane domains of the desmosome type. Here we report that in certain amphibian species (Xenopus laevis, Rana ridibunda, and Pleurodeles waltlii) the astrocytes of the optic nerve contain a completely different type of cytoskeleton. In immunofluorescence microscopy using antibodies specific for different IF and desmosomal proteins, the astrocytes of this nerve are positive for cytokeratins and desmoplakins; by electron microscopy these reactions could be correlated to IF bundles and desmosomes. By gel electrophoresis of cytoskeletal proteins, combined with immunoblotting, we demonstrate the cytokeratinous nature of the major IF proteins of these astroglial cells, comprising at least three major cytokeratins. In this tissue we have not detected a major IF protein that could correspond to GFP. In contrast, cytokeratin IFs and desmosomes have not been detected in the glial cells of brain and spinal cord or in certain peripheral nerves, such as the sciatic nerve. These results provide an example of the formation of a cytokeratin cytoskeleton in the context of a nonepithelial differentiation program. They further show that glial differentiation and functions, commonly correlated with the formation of GFP filaments, are not necessarily dependent on GFP but can also be achieved with structures typical of epithelial differentiation; i.e., cytokeratin IFs and desmosomes. We discuss the cytoskeletal differences of glial cells in different kinds of nerves in the same animal, with special emphasis on the optic nerve of lower vertebrates as a widely studied model system of glial development and nerve regeneration.     

Transient coexpression of desmin and cytokeratins 8 and 18 in developing myocardial cells of some vertebrate species

During myogenesis the intermediate-sized filament (IF) cytoskeleton is characterized by increasing proportions of desmin. While skeletal and smooth muscle formation occurs in free mesenchymal cells containing vimentin-type IFs, myocardial development starts from a polar epithelium containing cytokeratin IFs and desmosomes. Therefore, we have studied the formation of the epicardium and the myocardium in different vertebrate species, combining light and electron microscopic immunolocalization techniques with gel-electrophoretic analyses of cytoskeletal proteins of microdissected myocardial tissue at differing developmental stages. In this report, we describe results obtained from advanced stages of myocardial differentiation. In all species studied the myocardial cell possess IFs abundant in desmin, often together with smaller amounts of vimentin, and the mesothelial layer of the epicardium contains cytokeratin IFs. However, we have observed remarkable interspecies differences with respect to the occurrence of cytokeratins in embryonic myocardial cells. In fetal human myocardium, from week 10 of pregnancy on, but not in juvenile and adult myocardium, and in chicken myocardium of all stages examined (until several days after hatching) specific immunostaining was seen with certain broad-range cytokeratin antibodies as well as with antibodies specific for cytokeratins 18 (in both species) and 8 (showing significant reaction only in human). This cytokeratin immunoreaction, however, did not appear in IFs extending throughout the cytoplasm or at Z-lines, but was localized in punctate arrays representing aggregates of dense material. The aggregates were often enriched at, but not restricted to, the desmosomal plaques of the intercalated discs. These observations were supported by gel-electrophoretic demonstration of small but significant amounts of cytokeratins 18 (in both species) and 8 (detected only in human) in microdissected myocardial tissue. We also observed cytokeratins in smooth muscle cells of some cardiac blood vessels. In contrast, bovine myocardium of advanced fetal age as well as rat and mouse myocardium (from fetal day 12 on) were negative for cytokeratins with all methods, although epicardial cytokeratin IFs were demonstrable. These observations are discussed in relation to myocardial histogenesis and to general problems of cytokeratin gene expression control in epithelial and nonepithelial 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).     

Low level expression of cytokeratins 8, 18 and 19 in vascular smooth muscle cells of human umbilical cord and in cultured cells derived therefrom, with an analysis of the chromosomal locus containing the cytokeratin 19 gene

Of the various intermediate filament (IF) proteins certain cytokeratins, usually a hallmark of epithelial differentiation, can also be detected in some non-epithelial cells in low amounts. We have studied a representative case of this atypical expression, the smooth muscle cells of the blood vessel walls of the human umbilical cord, at the protein and nucleic acid level, by light and electron microscopic immunolocalization, gel electrophoresis and immunoblotting of cytoskeletal proteins, and mRNA identification by Northern blotting. For the latter we have used sensitive probes for various cytokeratins, including new probes for cytokeratin 19. We also describe the chromosome 17 locus comprising the genes for cytokeratins 15 and 19, and we emphasize the occurrence of several unusual and evolutionarily stable sequence elements in the introns of the cytokeratin 19 gene. Most, perhaps all smooth muscle cells of these blood vessels, positively identified by the presence of desmin and smooth muscle type alpha-actin, are immunostained by antibodies specific for cytokeratins 8 and 18, and a subpopulation also contains cytokeratin 19. Immunoelectron microscopy indicates that these cytokeratins are arranged in IFs that are distributed differently from the majority of the IFs formed by desmin and vimentin. Gel electrophoresis of cytoskeletal proteins from microdissected vascular wall tissue shows that the amounts of cytokeratins 8 and 18 present in these tissues are very low, representing less than 1% of the total IF protein, and that cytokeratin 19 is present only in trace amounts. Correspondingly, the contents of mRNAs for cytokeratins 8, 18 and 19 in these tissues are much lower than those present in epithelial cells examined in parallel. We have also established cell cultures derived from umbilical cord vascular smooth muscles that have maintained the expression of cytokeratins 8, 18 and 19, together with vimentin and the smooth muscle type alpha-actin, but do not synthesize desmin. In these cell cultures the cytokeratins are present in much higher amounts than in the original tissue and form IFs that, surprisingly, show a similar distribution as the vimentin IFs and, upon treatment of the cells with colcemid, collapse into juxtanuclear aggregates, often even more effectively than the vimentin IFs do. We conclude that in a certain subtype of smooth muscle cells, the genes encoding cytokeratins of the "simple epithelial type", i.e., cytokeratins 8, 18 and 19, are expressed and that the low level expression of these genes is compatible with myogenic differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytokeratins in certain endothelial and smooth muscle cells of two taxonomically distant vertebrate species, Xenopus laevis and man

Using immunolocalization techniques, electron microscopy, and gel electrophoresis combined with immunoblotting, we have noted remarkable interspecies differences in the expression of cytokeratins in certain nonepithelial cells. In the present study we describe, in two taxonomically distant vertebrate species, the African clawed toad Xenopus laevis and man, endothelial and smooth muscle cells which express cytokeratin intermediate filaments (IFs), in addition to vimentin and/or desmin IFs. In Xenopus, all endothelia seem to produce both vimentin and cytokeratin IFs. As well, certain smooth muscle bundles located in the periphery of the walls of the esophagus and the urinary bladder produce small amounts of cytokeratin IFs in addition to IFs containing vimentin or desmin or both. The amphibian equivalents of human cytokeratins 8 and 18 have been identified in these nonepithelial tissues. In human endothelial cells, immunocytochemical reactions with certain cytokeratin antibodies are restricted to a rare subset of blood vessels. Vessels of this type were first noted in synovial and submucosal tissues, but also occur in some other locations. Cytokeratins have also been detected in certain groups of smooth muscles, such as those present in the walls of some blood vessels in synovial tissue and umbilical cord. Here, the synthesis of low levels of cytokeratins 8 and 18, sometimes with traces of cytokeratin 19, has been demonstrated in smooth muscle cells by colocalization with myogenic marker proteins, such as desmin and/or the smooth-muscle-specific isoform of alpha-actin. Possible reasons for the differences in cytokeratin expression between adjacent endothelia in man, and smooth-muscle structures in both species, as well as biologic and histodiagnostic implications of these findings, are discussed.     

Patterns of expression of cytoskeletal proteins in human thyroid gland and thyroid carcinomas

By two-dimensional gel electrophoresis of proteins insoluble in detergents and high-salt buffer and immunofluorescence microscopy with a panel of polypeptide-specific antibodies to proteins of intermediate filaments (IF) and desmosomes, we have characterized the cytoskeletons of normal human thyroid gland, several kinds of benign lesion (goiter, Hashimoto's and Graves' diseases, adenomas), and the major thyroid carcinomas (follicular, papillary, medullary, and anaplastic). In all these tissues, desmoplakins and cytokeratins 7, 8, 18, and 19 were identified. While cytokeratins 8 and 18 occurred in all epithelial cells and cytokeratin 7 was also rather widespread, cytokeratin 19 occurred in amounts variable between the different types of tissues and in normal thyroid gland was restricted to certain clusters of follicular epithelial cells. Of all samples studied, in none did we detect cytokeratins commonly associated with stratified epithelia such as cytokeratins 4-6, 10, and 13-17, indicating that these are infrequent, if at all present, in such tissues. Coexpression of cytokeratins with vimentin appears to occur constitutively in follicular epithelial cells of normal thyroid gland and is also frequent in the diverse carcinomas, though to various degrees. Medullary carcinomas are exceptional, not only because they express neuroendocrine markers, but also because they coexpress combinations of cytokeratin IFs with neurofilaments and/or vimentin IFs in some cases, but not all. The results are discussed in relation to states of cell differentiation in normal and diseased thyroid gland and with respect to their value in tumor diagnosis.     

Arachnoid mater of the bullfrog,Rana catesbeiana

Summary In the bullfrog, the meninges surrounding the central nervous system include an arachnoid mater that contains layers of cells with abundant intermediate filaments (IFs) having unique organizational characteristics. This membrane contains an inner lamina of cells that resemble fibroblasts and an outer lamina of flattened cells that are almost filled with IFs. The IFs of the outer arachnoid are arranged in compact, arching bundles that lie parallel to the outer surface of the central nervous system. Thus, sections cut tangentially to the membrane reveal bending of filament bundles, whereas transverse sections do not. In some cells bordering the subdural space, bundles of filaments are organized into highly-ordered spiral arrays. Attachments to the numerous desmosomes and, apparently, to the nuclear envelope suggest anchoring of cytoplasmic structures by the IF system. Microtubules occur primarily near the plasma membrane and the nucleus. Numerous caveolae also are associated with the plasma membrane.The unusual abundance, organization, and cytoplasmic relations of IFs in the bullfrog arachnoid suggest that this membrane may serve as an important model for study of fundamental cytoskeletal relations and function.     

Heterogeneity of intermediate filament expression in human testicular seminomas

Testicular seminoma has in the past been considered to represent a germ cell tumor incapable of further differentiation. In recent years this view has been challenged on the basis of morphologic and chromosomal studies. Moreover, studies of intermediate filaments (IF) of seminoma cells have provided evidence of the capability of seminoma cells to differentiate in different directions. In the present study of the IF protein profile of 26 human testicular seminomas, using frozen as well as formalin-fixed, paraffin-embedded tissues, we report evidence of a heterogeneous differentiation potential inherent in these neoplasms. Thus, in 4 of the seminomas neither cytokeratins nor vimentin were detected; 3 showed vimentin positive cells but no cytokeratins; in 4 seminomas only cytokeratins were detected. In the remaining 15 cases both cytokeratins and vimentin were present, with occasional cells demonstrating coexpression of cytokeratin and vimentin. While the cytokeratins present were mostly of the "simple epithelial type", in 2 instances seminoma cells also contained cytokeratins 4 and 17, normally found in stratified and/or complex glandular epithelia. Furthermore, in 3 cases scattered tumor cells stained for desmin and in 2 other seminomas neurofilaments were identified. All of the cases showed variable positive staining for desmoplakins and desmoglein, indicative of the presence of desmosomes. It can therefore be concluded that, while some seminomas seem to be devoid of IFs, most of them show varied differentiation patterns usually with epithelial features but occasionally also with components commonly regarded as characteristic of myogenic or neurogenic differentiation. These observations may help to elucidate the relationship of seminomas to other germ cell tumors, and also contribute to our understanding of the histogenesis of these neoplasms.     

An epithelial cell line with elongated myoid morphology derived from bovine mammary gland : Expression of cytokeratins and desmosomal plaque proteins in unusual arrays

Cells of a clonal line (BMGE + HM) selected from bovine mammary gland epithelial cell cultures are described which, after reaching confluence, do not assume typical epithelioid morphology, but form elongated cells with long slender processes extending over the surfaces of other cells. However, cells of this line which display non-epithelioid morphology and are exceptionally rich in actin microfilaments are identified as epithelial cells by their synthesis of cytokeratins and desmosomal plaque proteins, as demonstrated by immunofluorescence and immunoelectron microscopy and by gel electrophoresis of cytoskeletal proteins. The cells do not produce vimentin and desmin filaments. The specific cytokeratin polypeptides of these myoid cells are identical to those present in normal epithelioid BMGE + H cells but are arranged in unusual arrays of meshworks of finely dispersed, non-fasciated filaments and granular structures. Desmosomal plaque proteins, notably desmoplakins, are abundant, but the electron microscopic appearance of the desmosomes is abnormal in that most of them are associated with a second accessory plaque formed at a distance of 0.1-0.15 micron from the normal desmosomal plaque. Both cytokeratin filaments and desmosomal structures are found throughout the whole cytoplasm, including the extended cell processes. The existence of an epithelial cell line with such an unusual morphology demonstrates the importance of non-morphological criteria in identifying epithelium-derived cells. Our findings also indicate that dramatic differences of cell shape and organization of epithelial cells need not necessarily be associated with changes in the expression of specific cytoskeletal proteins. The possible origin of this cell line from myoepithelial cells is discussed.     

Cytokeratin filament modulation in pulmonary microvessel endothelial cells by vasoactive agents and culture confluency.

Recently, bovine pulmonary microvascular endothelial cells (PMV) were shown to contain cytokeratin 8 and 19 intermediate filaments (Patton et al., 1990). In this study, we examine the effect of culture contiguity and vasoactive agents on the content and assembly of cytokeratins in PMV. Immunofluorescent staining of PMV cultures show a progressive increase in cytokeratin filament assembly. In freshly plated PMV, keratin appears as hazy staining (less than 4 hr) and later organizes into keratin 'plaques' (4 days) associated with cell-cell contacts; post confluent (greater than 7 days) PMV cultures contain fully assembled cytokeratin filaments which extend to the cell periphery and approach filaments in apposed cells. Vimentin filaments are also present in freshly plated PMV cultures but unlike cytokeratins, become less filamentous at confluency. This cell density-dependent modulation of cytokeratins is also demonstrated by densitometric analysis of autoradiographs of 35S-methionine labeled keratins in which PMV keratin content is elevated at high cell densities, while vimentin content remains constant. Desmoplakins I and II, components of desmosomes, could not be demonstrated in PMV by immunoblotting. PMV treated with permeability modulating agents (4 x 10(-3) M EGTA, 1 microM cytochalasin B, 1 microM bradykinin, 1 microM A23187, and 1 microM PMA) exhibit border retraction and altered keratin filament staining. From these studies we conclude: 1) cytokeratin 8 and 19 containing intermediate filaments are present in confluent PMV cultures with vimentin but without desmosomes, 2) the state of assembly of PMV cytokeratin and vimentin filaments appears to be oppositely affected by culture contiguity, and 3) treatment of monolayers with vasoactive agents alters the state of assembly of cytokeratin filaments. We speculate that modulation of cytokeratin assembly in PMV may be involved in regulation of pulmonary microvascular structure and function.     

Changes in the distribution of intermediate-filament types in Japanese quail embryos during morphogenesis

We examined the distribution of intermediate filaments in early quail embryos in order to determine whether these cytoskeletal proteins play a role in the epithelial-mesenchymal transitions that commonly occur during embryogenesis, e.g., the separation of neural-crest cells from the neural epithelium. The distribution of cytokeratins, vimentin, and desmin was examined in frozen sections of quail embryos at stages during which dramatic reorganizations of tissues take place. All embryonic tissues were found to contain either vimentin or cytokeratins, but the distribution of these cytoskeletal proteins was characteristic neither of the cellular organization (e.g., epithelium vs. mesenchyme) nor of the germ-layer derivation of the tissues. Cytokeratin monoclonal antibodies stained most embryonic epithelia (defined here as being sheet-like tissue with an underlying basement membrane), including epidermis and extraembryonic membranes derived in part from the ectoderm, splanchnopleure and kidney tubules derived from mesoderm, and endoderm. Cytokeratin antibodies did not stain some epithelia, including the neural tube, neural plate, and dermatome/myotome. Whereas the cytokeratin antibodies exclusively stained epithelia, the vimentin antibodies labeled both epithelial (the neural tube, dermatome/myotome, and somatic and splanchnic mesoderm) and mesenchymal tissues (the sclerotome and neural-crest cells), regardless of their germ-layer derivation. In early embryos, antibodies against desmin only stained the myotome and, in 4-day embryos, the heart and mesenchyme around the pharynx. As the distribution of intermediate-filament types did not reflect tissue organization or germ-layer derivation, we propose that the distribution of intermediate filaments in early avian embryos reflects the motile capacity of an embryonic cell and/or the presence of specialized cell junctions, i.e., desmosomes.     

Distribution of vimentin, cytokeratins, and desmosomal-plaque proteins in human nephroblastoma as revealed by specific antibodies: co-existence of cell groups of different degrees of epithelial differentiation

The distribution of cytokeratins, desmosomal-plaque proteins (desmoplakins), and vimentin in nephroblastoma tissue was studied by immunofluorescence microscopy using specific antibodies. In undifferentiated blastema cells, desmosomes, as revealed by antibodies to desmoplakins, preceded the advent of significant amounts of cytokeratins, indicating that desmosomes are early and sensitive markers of epithelial differentiation. Cytokeratin-positive tumor cells were seen in the following distribution patterns: groups of loosely arranged and scattered cells containing only scant cytokeratin fibrils surrounded by negative stroma cells; focal accumulation of cytokeratin-positive cells with cytokeratin-specific cytoplasmic fibril meshwork staining; rosettes of cytokeratin-positive cells without formation of distinct lumina, showing concentration of cytokeratin staining in the center; tubules with distinct lumina made up of cytokeratin-positive cells, with cytokeratin staining concentrated in the subapical cell portions. In cytokeratin-positive cells, the numbers of desmoplakin-positive dots were generally increased; in well-formed tubules, enrichment of desmoplakin-positive spots, corresponding to the subapical skeletal disks, was most conspicuous. Vimentin was demonstrated in stromal areas, but also in blastema cells showing coexpression of desmosomes and vimentin filaments. Moreover, in certain blastema cells, an overlap of cytokeratin and vimentin immunostaining was observed. Epithelial cells of nephroblastoma tubules did not react with vimentin antibodies. Our results show that the appearance of desmosomal plaques, as demonstrated by antibodies to desmoplakins, may be a very early feature of epithelial differentiation, and they also emphasize the value of antibodies to desmoplakins in tumor cell typing and diagnosis.     

Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow’s udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.     

Cytoskeletal components of lymphoid organs

Using light and electron microscopic immunolocalization with antibodies to cytoskeletal proteins, we have characterized the nonlymphoid cells of various human lymphoid organs (lymph nodes, tonsils, spleen). In all these tissues, the lymphoid follicles contain a three-dimensional meshwork of "dendritic reticulum cells" which are characterized by the presence of desmosomal junctions, as demonstrated by positive punctate staining with antibodies to the desmosome-specific proteins desmoplakin I and desmoglein, and by intermediate-sized filaments (IFs) of the vimentin type only. In contrast, the extrafollicular regions are characterized by an extended meshwork of other types of reticulum cells, which also contain vimentin IFs but lack desmosomal proteins. In addition, a considerable, although variable proportion of these extrafollicular reticulum cells forms IFs containing cytokeratins 8 and 18 and/or desmin-containing IFs. The occurrence of cytokeratins 8 and 18 in lymph nodes has also been shown by gel electrophoresis and immunoblotting. Results of double-label immunolocalization indicate that some of the extrafollicular reticulum cells coexpress all three kinds of IF protein. A large proportion of these cells also synthesizes another marker of myogenic differentiation, i.e., the isoform of alpha-actin specific for smooth muscle. This proportion includes some cells that are negative for desmin. Comparison of the distribution of cells expressing cytokeratins and/or desmin with that of reticulum cells showing strong alkaline phosphatase activity (as a marker for the so-called "fiber-associated (fibroblastic) reticulum cells") suggests that the former represent a subset of the latter. The biological meaning of these different patterns of expression in reticulum cells and of the resulting cell-type heterogeneity as well as possible implications of these observations for tumor diagnosis, notably of lymph-node metastases and lymphomas, are discussed.     

High frequency of cytokeratin-producing smooth muscle cells in human atherosclerotic plaques

Using immunofluorescence microscopy we show that cells expressing cytokeratins 8 and 18 are frequently enriched in human vascular wall tissue pathologically altered by the appearance of intimal thickenings and atherosclerotic plaques. These cytokeratins occur in cells which also synthesize IFs containing vimentin and/or desmin, and a considerable proportion of the cytokeratin-positive cells has been identified as smooth muscle cells by colocalization of desmin and/or smooth muscle type alpha-actin. The presence of extremely low concentrations of these cytokeratins in such vascular tissues has been confirmed by gel electrophoresis with immunoblotting as well as by Northern blot hybridization using specific cytokeratin cRNA probes. The results are discussed in relation to the recent demonstration that low-level synthesis of cytokeratins 8 and 18 occurs in other muscular tissues and to the specific proliferative activity of these cells.     

The ultrastructural organization of peripheral nerves in two insect species (Periplaneta americana and Schistocerca gregaria)

The ultrastructural organization of various peripheral nerves, including the crural nerve, has been investigated in the locust and cockroach. In some cases the larger nerves are ensheathed by a fat body layer which is not always complete. However, like many nervous connectives, they do possess a continuous acellular neural lamella and a perineurial cell layer which surround the glial-axonal mass. Adjacent perineurial cells are associated with one another by septate desmosomes, gap junctions and tight junctions. These last may represent the morphological basis of the ‘blood-brain barrier’ observed electrophysiologically in these peripheral nerves in another report. Very small nerves of the cockroach, however, although lying embedded in a neural lamella, do not possess a specialized perineurial layer displaying junctional complexes, unless they contain one or more large axons. If they have only one or more small axons, these small nerves may either appear naked, or display a single glial cell process loosely enveloping them; in either case there is no structural basis for a ‘barrier’ system. Various comparisons have been made between locust crural nerve and the cockroach central nervous connectives in an attempt to correlate some aspects of their ultrastructural organization with relevant electrophysiological information.     

Simultaneous immunoenzyme staining of vimentin and cytokeratins with monoclonal antibodies as an aid in the differential diagnosis of malignant mesothelioma from pulmonary adenocarcinoma

The occurrence and coexpression of the cytoskeletal proteins vimentin and cytokeratins were studied in malignant mesotheliomas and pulmonary carcinomas. For this purpose a double immunoenzyme staining with monoclonal antibodies was developed which made it possible to visualize vimentin and cytokeratins simultaneously within the same cell. A clear distinction between stromal cells (vimentin only) and tumour cells was also obtained. A total of 12 mesotheliomas (six mixed type and six epithelioid type) and 13 carcinomas (eight adenocarcinomas and five large cell undifferentiated carcinomas) were studied. The results revealed a clear difference between mesotheliomas and adenocarcinomas: 11 of 12 mesotheliomas showed coexpression of vimentin and cytokeratins in at least 50% of the tumour cells, while in seven of the eight adenocarcinomas none or only a few cells could be seen with this coexpression. In the undifferentiated large cell carcinomas three of five expressed both components, but in less than 25% of the cells. It is concluded that a reliable double immunoenzyme staining of vimentin and cytokeratins can be used as an additional means to distinguish malignant mesothelioma from pulmonary adenocarcinoma.     

Differences of expression of cytoskeletal proteins in cultured rat hepatocytes and hepatoma cells

Cytoskeletal elements, enriched in intermediate-sized filaments and insoluble in buffers of high salt concentrations and Triton X-100, were isolated from various cultures of rat hepatocytes and hepatoma cells, and their proteins were studied by one- and two-dimensional gel electrophoresis and immunofluorescence microscopy. The cells examined included several permanent cell lines (MH₁C₁, HTC, hepatoma 72/22, clone 12 from Gunn rat hepatocytes, and cell clones from normal rat hepatocytes), as well as freshly dissociated hepatocytes that were cultured and allowed to attach to substratum for increasing periods of time, beginning at 24 h after removal of the liver from the animal. Filaments containing vimentin, which were not found in hepatocytes grown in liver tissue, were detected in most of the cultured hepatocytes and hepatoma cells, except in MH₁C₁ cells, and were shown to be newly synthesized during the first days of primary culture. Maintenance of expression of filaments containing proteins immunologically related to epidermal prekeratin (‘cytokeratins’) was observed in all cells examined but HTC cells. Detailed comparison of the cytokeratin polypeptides present in various hepatocyte and hepatoma cell cultures showed that, in some of the cultured epithelial liver cells, cytokeratins are expressed which are identical with, or similar to, those of normal hepatocytes grown in the liver. On the other hand, differences in cytokeratin polypeptides were also found among different hepatocyte-derived cell cultures. Changes of expression of cytoskeletal proteins were found to occur even in cloned cell populations, and cells positive for certain cytokeratins could be seen next to other cells that were negative.The results demonstrate that profound changes of cytoskeletal composition, especially concerning intermediate filament protein patterns, can occur during culturing in vitro. Moreover, we show that different intermediate filament proteins can be expressed in different hepatocyte-derived cell cultures and that changes of cytoskeletal composition can occur in a given cell population, without obvious effects on cell growth rate and cell morphology. During culturing of hepatocytes and hepatoma cells, there seems to be a general tendency to induce the production of vimentin filaments as well as to maintain the production of cytokeratins similar to the hepatocyte-specific cytokeratins in liver tissue. However, the demonstrated exceptions speak against a role of these filament proteins as prerequisites for the growth of an epithelial cell in vitro. Rather, the presence of filaments containing certain cytokeratins and of desmosomes in epithelial cells growing in vitro seems to reflect the synthesis of specific differentiation markers which may be lost, independently, in some cells during culturing.