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PubMed1986

Peritubular myoid cells of human and rat testis are smooth muscle cells that contain desmin-type intermediate filaments.

We studied the cytoskeletal composition of human and rat testicular myoid cells by using immunofluorescence microscopy with polyclonal and monoclonal antibodies. In adult human and rat testis, the peritubular myoid cell layer was brightly positive for desmin, the muscle type of intermediate filament protein, and a faint reaction was also seen with antibodies to vimentin, the intermediate filament protein of fibroblasts and diverse other mesenchymal cells. The desmin-positive myoid cell layer could already be identified in newborn rat testis but was more compact in appearance 23 days after birth. Both squash preparations and cultured cells from adult rat seminiferous tubules revealed distinct cell populations positive for desmin. The adult myoid cells of both species also showed a strong reaction with antibodies to myosin and p230, a nonerythroid avian alpha-spectrin analogue. The immunostaining results could be confirmed by the western blotting technique: Experiments with isolated seminiferous tubules showed a specific reaction with a 55,000-dalton and a 58,000-dalton polypeptide when desmin and vimentin antibodies were used, respectively. The present results show that the peritubular myoid cells are genuine smooth muscle cells with desmin-type intermediate filament cytoskeleton and suggest that these cells can be identified by this feature before their ultrastructural maturation.

Animals↗
PubMed1993

Organization and expression of intermediate filaments in epithelial cells expressing the HTLV-I Tax protein.

Intermediate filaments (IF) represent major components of the cytoskeletal network. These proteins which are differentially expressed according to the cell type, constitute a dynamic structure which not only contributes to the cell architecture but also defines its state of differentiation. Furthermore, numerous observations have shown that the IF network is altered in cells transformed by tumorigenic viruses. We have previously demonstrated that HTLV-I (human T-cell leukemia virus type I) transformed T cells were characterized by a high level of vimentin transcripts and that the HTLV-I Tax regulatory protein was able to transactivate the vimentin promoter transfected into Jurkat and HeLa cells. To enlarge the scope of this study, we investigated the effects of the Tax protein on the expression and organization of IF of epithelial cells in which the IF network is composed of vimentin and cytokeratin. To this aim, we have developed a model of epithelial cells (HeLa) stably expressing the tax sequences which were introduced by using retrovirus-mediated gene transfer. Half of the Tax expressing HeLa clones were loosely adherent to the culture surface and were displaying remarkable morphological alterations, as ascertained by the presence of round-shaped or spindle-shaped cells. In these cells, expression of this viral protein correlated to a pronounced disruption in the distribution of both the vimentin and the cytokeratin networks, as shown by immunofluorescence and ultrastructural analysis. Indeed, vimentin filaments appeared to be concentrated in discrete spots throughout the cytoplasm, while the cytokeratin filaments appeared to form a dense ring around the nucleus. More importantly, mRNA and protein analysis indicate an enhanced expression of the cytokeratin 7 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelium↗
PubMed1986

Intermediate filaments in the human retina and retinoblastoma. An immunohistochemical study of vimentin, glial fibrillary acidic protein, and neurofilaments.

Fifty-five retinoblastoma specimens were studied by a sensitive immunoperoxidase method to determine the intermediate filament types present in human retina and retinoblastoma. Polyclonal antiserum against vimentin and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and to the 200 kD neurofilament triplet protein were used. In the human retina, Müller's cells coexpressed vimentin and GFAP in most instances, probably as a reactive phenomenon. Surprisingly, the horizontal cells did not stain with any of the antibodies used, and may thus lack intermediate filaments. Also, the meshwork of neural fibers in the inner plexiform layer was unusually sparse. Retinoblastoma cells were found to be devoid of all intermediate filament types studied. The tumors contained, however, vimentin and GFAP in the stromal cells. All neurofilament-positive cells in retinoblastoma apparently derived from infiltrated retina. One retinoblastoma eye was also studied by indirect immunofluorescence microscopy of frozen sections with identical results.

Animals↗
PubMed2000

Regulation of intermediate filament organization during cytokinesis: possible roles of Rho-associated kinase.

Intermediate filaments (IFs), which form the structural framework of cytoskeleton, have been found to be dramatically reorganized during mitosis. Some protein kinases activated in mitosis are thought to control spatial and temporal IF reorganization through phosphorylation of IF proteins. Rho-associated kinase (Rho-kinase), one of the putative targets of the small GTPase Rho, does phosphorylate IF proteins, specifically at the cleavage furrow during cytokinesis. This cleavage furrow-specific phosphorylation plays an important role in the local IF breakdown and efficient separation of IF networks. Recent studies on Rho signaling pathways have introduced new models about the molecular mechanism of rearrangements of cytoskeletons including IFs during cytokinesis.

Animals↗
PubMed2003

Intermediate filaments: novel assembly models and exciting new functions for nuclear lamins.

Intermediate filament (IF) proteins constitute a highly diverse family of fibrous proteins in metazoans, which assemble into 10-nm-thick filaments in the cytoplasm and the nucleus. Novel recent insights into the in vitro assembly mechanism have revealed principal differences in the formation of cytoplasmic and nuclear filaments. Moreover, the past years have seen dramatic developments for the nuclear specific IF proteins, the lamins. While in the past lamins have been assumed to form only a structural scaffold at the nuclear periphery, their discovery in the nuclear interior, the identification of novel lamin-binding proteins and the functional disruption of lamin structures have brought to light essential functions for lamins in fundamental cellular events such as chromatin organization, DNA replication and RNA transcription. Furthermore, mutations in lamins and lamin-binding proteins have been demonstrated to cause various different human diseases, affecting muscle, heart, neuronal, adipose and bone tissue or leading to premature ageing. However, the molecular basis of these diseases is just beginning to emerge.

Humans↗
PubMed1996

Expression of intermediate filaments and N-cadherin adhesion molecule in the thymus of domesticated birds.

Stromal cells of the thymus from the domestical birds (chicken and guinea fowl) were studied by antibodies against their intermediate filaments (keratin, vimentin and desmin). The medulla revealed large irregular shaped keratin free areas, surface of which is dentate but sharply outlined. The keratin free areas are connected with the substance of the interlobular septae which indicates that the medullary microenvironment is rather similar to that of the peripheral lymphoid tissue. The vimentin intermediate filament is expressed by the entire medullary region. The immunologically competent medullary T cells are vimentin positive unlike the immature cortical T cells. The anti-desmin antibody revealed a three dimensional network in the medulla which includes desmin negative "channels". The myoid cells, locating close to the "channels", are possibly the well-differentiated forms of the desmin positive network. N-cadherin is expressed by the cortico-medullary epithelium and it is suggested that this adhesion molecule plays some role in the thymocyte selection. Both keratin and desmin intermediate filaments indicate that the medulla of the thymus has a complex stromal structure which further compartmentalizes the thymic medulla.

Animals↗
PubMed1983

Proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins.

The degradation of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins proceeds in two stages in the form of a limited proteolysis. At first, the reaction is very rapid, with the stepwise and complete removal of a peptide (ca. 9,000 daltons) from the N-terminal of vimentin and desmin. This results in the production of a characteristic "staircase" of degradation products, as seen in two-dimensional polyacrylamide gel electrophoresis. The second stage of proteolysis is characterized by the accumulation of peptides which are resistant to further proteolysis; this is due not to product inhibition but to the fact that these peptides are not substrates for the proteinase and therefore do not protect the latter from inactivation (autodigestion). In vitro phosphorylation of the substrates does not affect proteinase activity, probably because the phosphorylation site is located towards the C-terminal of the molecules. The specific and limited proteolysis of vimentin and desmin results in the deletion of the nucleic acid binding and filament assembly site of these proteins, indicating that the Ca2+-activated proteinase plays a role in regulating the function(s) of these intermediate filament proteins, rather than their simple turnover during the cell cycle.

Amino Acid Sequence↗
PubMed2006

Spatial patterns of the pathological changes in neuronal intermediate filament inclusion disease (NIFID): an alpha-internexin immunohistochemical study.

Neuronal intermediate filament inclusion disease (NIFID) is characterized by alpha-internexin positive neuronal cytoplasmic inclusions (NCI), swollen achromatic neurons (SN), neuronal loss, and gliosis. This study tested: 1) whether the spatial patterns of the lesions was topographically organized in areas of the frontal and temporal lobe and 2) whether a spatial relationship exists between the NCI and SN. The NCI were distributed in regular clusters and in a quarter of these areas, the clusters were 400-800 microm in diameter approximating to the size of the cells of origin of the cortico-cortical pathways. Variations in the density of the NCI were positively correlated with the SN. Hence, cortical degeneration in NIFID appears to be topographically organized and may affect the cortico-cortical projections, the clusters of NCI and SN developing within the same vertical columns of cells.

Adult↗
PubMed2000

Immunofluorescent localization of intermediate filaments (IFs) in helminths using anti-mammalian IFs monoclonal antibody.

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

Animals↗
PubMed1993

Demonstration of cytokeratin intermediate filaments in oocytes of the developing and adult human ovary.

The intermediate filaments (IF) present in the various cells of human ovaries were studied by immunolocalization using antibodies to cytokeratins (CKs), vimentin, desmin and alpha-smooth muscle (alpha-SM) actin. Oocytes revealed a single paranuclear aggregate, which reacted with antibodies to CKs 8, 18 and 19 both in adult and fetal ovaries. The existence of this aggregate was also documented by electron microscopy. Ovarian surface epithelium and granulosa cells consistently coexpressed CKs 8, 18, 19 and vimentin. During follicle maturation vimentin remained unchanged in the granulosa layer while CKs content decreased, showing variation in the amount and distribution of the different CK-types. Thecal cells of secondary and mature follicles showed alpha-SM actin positivity. These contractile fibres increased in mature follicles. Ordinary fibrous stromal cells showed isolated cells which were desmin and alpha-SM actin positive. A similar pattern of IF expression and distribution existed in all stages of development in fetal and embryonic ovaries. These results indicate that CKs are present in human oocytes and that the coexpression of vimentin and CKs can be regarded as a peculiar feature of all ovarian cell types except oocytes and ordinary stromal cells. Contractile properties have been documented associated with a modification in expression of IF proteins. This is likely to represent an integral part of folliculogenesis along with the functional hormone-dependent changes.

Adult↗
PubMed1997

The intermediate filament peripherin is expressed in cutaneous melanocytic lesions.

Peripherin is an intermediate filament involved in growth and development of the peripheral nervous system and is localized to neurons, some other cells derived from neural tube and neural crest, and some neuroendocrine cells (e.g. beta cells of islets of Langerhans). Peripherin also has been demonstrated in neuroblastomas and cutaneous neuroendocrine (Merkel cell) carcinomas. The expression of peripherin by other cells derived from the neural crest is unknown. We evaluated by immunohistochemistry 74 cutaneous melanocytic lesions including primary invasive malignant melanoma (IMM), melanoma in situ (MIS), atypical nevus (nevus with architectural disorder and cytologic atypia of melanocytes) (AN), spindle and epithelioid cell nevus (Spitz nevus) (SN), blue nevus (BN), and common intradermal benign melanocytic nevus (BMN) for expression of peripherin. Peripherin was detected in a cytoplasmic distribution within tumor cells in 14/14 IMM and 8/10 MIS. For IMM, peripherin localized to both the intraepidermal and invasive dermal components. Peripherin was detected in 10/10 AN and 9/9 SN, being localized to the intraepidermal component and, focally, to the superficial dermal component of the lesions. The dendritic nevus cells in 15/15 BN also expressed peripherin. For most of the BMN, expression of peripherin was absent or limited to rare, scattered cells in the superficial portion of the lesions. Melanocytes in adjacent normal skin were not labeled in any of the lesions studied. These results indicate that expression of peripherin is common in both benign and malignant melanocytic lesions, but not in normal resting adult melanocytes. Among benign lesions, expression of peripherin in the dermal component is rare except in the dendritic cells of BN. These findings provide evidence that the expression of peripherin, a marker of neuronal differentiation, is maintained by IMM, MIS, and BN, but is lost in the normal maturational sequence of the dermal component of other melanocytic lesions.

Adult↗
PubMed1982

Diagnosis of human childhood rhabdomyosarcoma of antibodies to desmin, the structural protein of muscle specific intermediate filaments.

Four embryonal rhabdomyosarcomas, one tumor diagnosed as an undifferentiated sarcoma, probably a rhabdomyosarcoma, and six different non-muscular sarcomas were investigated with antibodies specific for different intermediate filament types. The tumor cells in the rhabdomyosarcomas and the undifferentiated tumor were stained clearly by antibodies to desmin, the intermediate filament type characteristic of muscle. The staining of tumor cell by antibodies to vimentin, the intermediate filament type characteristic of certain cell types of mesenchymal origin including myoblasts, was different in these 5 cases. In one case of embryonal rhabdomyosarcoma nearly all tumor cells were stained, but in the remaining cases few or no tumor cells were positive with the vimentin antibody. In these rhabdomyosarcomas not only the large rhabdomyoblasts, but also the small undifferentiated cells were labeled by antibodies to desmin. In the latter cell type the desmin filaments were arranged typically in coils. In contrast, tumor cells in the non-muscular mesenchymal sarcomas were stained only by antibodies to vimentin but not by antibodies to desmin or prekeratin. The retention of the desmin marker characteristic of normal muscle in cases of rhabdomyosarcoma not only allowed the undifferentiated desmin-positive sarcoma to be classified as rhabdomyosarcoma but also suggests that the use of antibodies to desmin could be very helpful in the future for the diagnosis of undifferentiated rhabdomyosarcomas.

Adult↗
PubMed1986

Association of spectrin with desmin intermediate filaments.

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament-erythrocyte inside-out vesicle preparations. Desmin filaments bound to erythrocyte inside-out vesicles in a spectrin-dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration-dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.

Animals↗
PubMed1986

Epithelial and mesenchymal cell differentiation in the fetal rat genital ducts: changes in the expression of cytokeratin and vimentin type of intermediate filaments and desmosomal plaque proteins.

Mesonephric and paramesonephric ducts develop in different ways in male and female fetuses. We have analyzed the changes in the expression of cytokeratin and vimentin type of intermediate filaments and desmosomal plaque proteins in progressing and regressing genital ducts of rat fetuses. The concomitant changes in the basement membranes were detected by laminin antibody. Epithelial cells of the indifferent (Day 15) male and female mesonephric and paramesonephric ducts contained faint vimentin positivity which, however, later disappeared. Indifferent mesonephric duct epithelium stained strongly for cytokeratin, whereas in the corresponding paramesonephric duct only a weak and spotty positivity was seen. Immunocytochemical localization of cytokeratin filaments and desmosomal plaque proteins correlated with the ultrastructural differences in the apical junctional complexes of the mesonephric and paramesonephric ducts. Regardless of the ongoing regression of the male paramesonephric duct, cytokeratin positivity increased in the disorganizing epithelium; the most weak and a granular immunoreaction was seen in the cells found in the intensively vimentin-positive periductal mesenchyme. In the regressing female mesonephric duct cytokeratin positivity was lost before the final dissolution of the basement membrane. Immunoblotting analysis of cytokeratin and vimentin polypeptides of the individual genital ducts were in agreement with the immunocytochemical results obtained in 15- and 16-day-old fetuses. The results suggest that the expression of vimentin type intermediate filaments is an indication of the mesothelial origin of the genital ducts. The increase in cytokeratin positivity of the regressing paramesonephric duct epithelium suggests that the degenerative changes are initiated by the mesenchyme. Cytokeratin-positive cells found in the periductal mesenchyme of the male paramesonephric duct may be epithelial cells transforming into mesenchyme. The results emphasize a close relationship between the changes of the intermediate filament system and extracellular matrix upon differentiation of the fetal genital ducts.

Animals↗
PubMed1989

Phosphorylation of peripherin, an intermediate filament protein, in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons.

Peripherin, an intermediate filament protein, described recently, is expressed in well defined neuronal populations. We studied the phosphorylation, in vivo, of this protein in mouse neuroblastoma NIE 115 cell line and in sympathetic neurons labelled with [32P]-orthophosphate. The autoradiograms of proteins separated on two-dimensional polyacrylamide gels were compared with the Coomassie-blue stainings. The results show that peripherin occurs as a mixture of phosphorylated and non-phosphorylated isoforms, and that these forms coexist in both differentiated and non-differentiated cells. We demonstrate by cleavage at the unique tryptophan residue, a characteristic shared by most other intermediate filament proteins (IFP), that the phosphorylation sites are located on the amino-terminal half of peripherin as it is for vimentin and desmin. These results are discussed in relation to the organization of the filamentous network constituted by peripherin.

Animals↗
PubMed1995

Intermediate filaments in the testis of the teleost mosquito fish Gambusia affinis holbrooki: a light and electron microscope immunocytochemical study and western blotting analysis.

A light and electron microscope immunocytochemical study and Western blotting analysis has been performed on intermediate filaments (vimentin, desmin and cytokeratins) in the testis of the teleost fish Gambusia affinis holbrooki. An immunoreaction to vimentin was observed in the epithelium of the efferent ducts, testicular canal and their surrounding peritubular cells. Positive vimentin immunostaining was also observed in the cells located around seminiferous tubules (boundary cells), Leydig cells, interstitial fibroblasts, chromatophores, and blood vessel endothelial cells. In contrast to mammals, no vimentin immunoreactivity was found in the Sertoli cells. Immunoreactivity to desmin was weak in the epithelial cells of the efferent ducts and testicular canal and intense in the peritubular cells that surrounded these ducts. Desmin immunoreactivity was also observed in the seminiferous tubule boundary cells. The immunoreactivity was weak in the boundary cells that surrounded germ cell cysts containing spermatogonia or spermatocytes and intense in the boundary cells around cysts with elongated or mature spermatids. Immunoreactivity towards cytokeratins was observed only in testicular blood vessels. Cytokeratin immunolabelling was intense in the endothelium and weak in the vascular smooth muscle cells. No cytokeratin immunoreactivity was found in the Sertoli cells, germ cells, interstitial cells or in the efferent duct epithelium. The absence of intermediate filaments in the Sertoli cells, the absence of cytokeratins in the epithelium of the sperm excretory ducts, and the presence of desmin filaments in these epithelial cells are the most important differences with regards to the intermediate filament phenotype in mammalian testes.

Animals↗
PubMed1988

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins.

A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture. Unlike what is commonly observed on glass or plastic, where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state.

Basement Membrane↗
PubMed1986

cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins.

The amino acid sequences deduced from cDNA clones of human lamin A and lamin C show identity between these two lamins except for an extra 9.0-kDa carboxyl-terminal tail that is present only in lamin A. Both lamins A and C contain an alpha-helical domain of approximately 360 residues that shows striking homology to a corresponding alpha-helical rod domain that is the structural hallmark of all intermediate filament proteins. However, the lamin alpha-helical domain is 14% larger than that of the intermediate filament proteins. In addition to the extensive homology to intermediate filament proteins as reported [McKeon, F., Kirschner, M. & Caput, D. (1986) Nature (London) 319, 463-468], a different 82-amino acid residue stretch at the carboxyl terminus of lamin A has been deduced and verified by amino acid sequencing. This region contains sequence homology to amino- and carboxylterminal domains of type I and type II epidermal keratins. Implications of the presence of these and other domains in lamins A and C for the assembly of the nuclear lamina are discussed.

Amino Acid Sequence↗