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PubMed1991

Isolation and sequence analysis of two intermediate filament cDNA clones from fish optic nerve.

The high post-traumatic regenerative ability of fish central nervous system has been partially attributed to the hospitable nature of the surrounding non-neuronal cells and their appropriate response to injury. Uncovering the correlation between fish non-neuronal cell structure and behavior might yield a better understanding of what makes them supportive to axonal growth. Towards this goal, structural proteins expressed by fish non-neuronal cells need to be characterized. In the present study we isolated cDNA clones encoding fish intermediate filaments which are prominent structural proteins in astrocytes. Among the isolated clones, one was identified as fish vimentin and another was found identical to the cloned fish keratin 8. Results are discussed with respect to the use of these cDNAs for further understanding of fish non-neuronal cell plasticity.

Amino Acid Sequence↗
PubMed1990

Differentiation and retrodifferentiation of U937 cells: reversible induction and suppression of intermediate filament protein synthesis.

Significant morphological and functional changes were observed when human monoblastoid U937 tumor cells growing in suspension were induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h to differentiate along the monocyte/macrophage pathway. These include adherence of the cells to each other and to the substratum, alterations in cell-surface antigen expression and cessation of autonomous proliferation. In this study, we show by both, hybridization analysis of RNA and immunoblotting that an enhanced expression of the intermediate filament (IF) subunit proteins vimentin, lamin A and lamin C accompanied the TPA-induced differentiation process. After long-term culture of differentiated U937 cells in the absence of TPA (more than 28 days), however, the adherent cells retracted their pseudopodia, detached and started again to proliferate. This "retrodifferentiation" process, not previously described was paralleled by a rapid down-regulation of both, IF mRNA and protein synthesis back to the level of undifferentiated U937 control cells. These data suggest a functional relationship between the expression of vimentin and lamins A and C and the differentiation process taking place in these cells.

Cell Differentiation↗
PubMed1996

The relative roles of specific N- and C-terminal phosphorylation sites in the disassembly of intermediate filament in mitotic BHK-21 cells.

Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disassembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis. Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) effectively prevents mitotic cells from disassembling their IF. In contrast, apparently normal disassembly takes place in cells transfected with cDNA containing mutated p37 kinase phosphorylation sites (thr457:ala/ser458:ala). Transfection of cells with vimentin cDNAs lacking both the N- and C-terminal phosphorylation sites yields a phenotype indistinguishable from that obtained with the single N-terminal mutant. Taken together, our results demonstrate that the site-specific phosphorylation of the N-terminal domain, but not the C-terminal domain of vimentin plays an important role in determining the state of IF polymerization and supramolecular organization in mitotic cells.

Animals↗
PubMed1997

Heterodimeric associations between neuronal intermediate filament proteins.

Formation of protein dimers involving alpha-internexin, peripherin, and the neurofilament (NF) proteins NFH, NFM, and NFL was investigated by partial renaturation of various combinations of individually purified subunits in buffered 2 M urea. Oligomers that were formed were resolved by "blue" native electrophoresis (Schägger, H., Cramer, W. A., and von Jagow, G. (1994) Anal. Biochem. 217, 220-230) modified to include urea in the polyacrylamide gels. Combining this method with Western blot analysis, disulfide cross-linking, and SDS-polyacrylamide gel electrophoresis in the second dimension showed that NFL readily forms significant amounts of heterodimer with NFH, NFM, alpha-internexin, or peripherin in the presence of 2 M urea. alpha-Internexin and peripherin also formed heterodimers with NFH or NFM under these conditions. The modified version of blue native gel electrophoresis described here may be useful in monitoring the impact of post-translational modifications and mutations on the dimerization of intermediate filament proteins.

Animals↗
PubMed1984

Sequence of a cDNA clone encoding mouse glial fibrillary acidic protein: structural conservation of intermediate filaments.

A clone encoding mouse glial fibrillary acidic protein (GFAP) was isolated from a cDNA library constructed so as to express the cloned sequences. The library was screened using a GFAP-specific polyclonal antiserum; a single bacterial colony expressing GFAP was identified. The complete sequence of the cDNA insert in this clone is presented, encompassing 2.5 kilobases and specifying greater than 97% of the GFAP amino acid sequence. The clone includes a long (1.4-kilobase) 3' untranslated region. Within the coding region, the data show extensive homology with other intermediate filament proteins, particularly in those regions predicted to be alpha-helical. RNA blot transfer experiments using the cloned GFAP cDNA probe revealed a single GFAP mRNA species of 2.7 kilobases in mouse brain. Southern blot analysis indicates the existence of at most two genes encoding GFAP in the mouse genome. The mouse GFAP probe cross-hybridizes weakly at high stringency with genomic DNA from diverse eukaryotic species.

Amino Acid Sequence↗
PubMed1993

Evidence that the chick lens cytoskeletal protein CP 49 belongs to the family of intermediate filament proteins.

A partial cDNA sequence for chick lens beaded-filament protein CP 49 showed the greatest similarity to the sequence of acidic cytokeratins, especially human cytokeratin 18. The predicted amino acid sequence of chick CP 49 corresponded to the entire coil 1a region of the rod domain of human cytokeratin 18, spacer 1, coil 1b, spacer 2 and about half of coil 2. For this sequence of 242 amino acids, there was an overall 38% identity and 76.8% similarity between the chick CP 49 and human cytokeratin 18. This is further evidence that CP 49 belongs to the family of intermediate filament proteins.

Amino Acid Sequence↗
PubMed2003

Subcellular localization of S100A11 (S100C) in LLC-PK1 renal cells: Calcium- and protein kinase c-dependent association of S100A11 with S100B and vimentin intermediate filaments.

The subcellular localization of the Ca(2+)-modulated protein, S100A11, was investigated in the renal cell line LLC-PK1 by immunofluorescence and confocal laser scanning microscopy under varying experimental conditions. In control cells, S100A11 was detected on the plasma membrane, where the protein co-localized with annexin I (ANXA1) at discrete sites, and found diffusely in the cytoplasm. Elevation of the cytosolic Ca(2+) concentration by means of the Ca(2+) ionophore, ionomycin, caused a significant fraction of S100A11 to associate with vimentin intermediate filament (IF)-bound S100B, another member of the S100 protein family. Under these conditions, ANXA1 underwent a quite different kind of relocation. Translocation of S100A11 onto vimentin IF-bound S100B was also observed upon activation of protein kinase C (PKC). Under these conditions, S100A11 appeared to associate directly with vimentin IFs at cell sites displaying low or no abundance of S100B such as cell processes, and, again, S100A11 and ANXA1 underwent a different relocation. Our data suggest the possibility that the intracellular Ca(2+) level might regulate the subcellular localization of S100A11 and its interaction with definite target proteins, and that S100A11 might serve the function of modulating S100B activities. Interestingly, in spite of the known ability of S100A11 to form heterotetramers with ANXA1, the two proteins underwent a different relocation on elevation of the cytosolic Ca(2+) concentration or activation of PKC, pointing to different regulatory activities of individual proteins in renal cells.

Animals↗
PubMed1997

Patterns of intermediate filaments, VLA integrins and HLA antigens in a new human biliary epithelial cell line sensitive to interferon-gamma.

BACKGROUND/AIMS: Intra-hepatic bile ducts are the primary site of damage in several immunologically mediated liver diseases. However, immunological processes underlying biliary epithelial cell recognition by T lymphocytes are poorly understood. Therefore, a convenient in vitro model that could mimic these immunologic disorders would be of great interest. METHODS: A human cell line (HuGB) was established from a metastasis of gallbladder adenocarcinoma in the liver. Intermediate filament expression was analysed by immunostaining, and gamma-glutamyl transpeptidase and albumin secretion were measured. VLA integrin expression pattern, expression of HLA class I and II antigens and ICAM-1 protein were analysed by flow cytometry and their modulation by interferon-gamma was quantitated using a QIFIKIT commercial kit. RESULTS: Histological analysis showed high similarity between the initial gallbladder adenocarcinoma and the established cell line. Cytokeratins 8 and 19 and vimentin showed strong positive staining in the established cell line. Gamma-glutamyl transpeptidase was secreted by these cells while albumin expression was negative. HuGB cells also expressed VLA-alpha2, VLA-alpha3, VLA-alpha6, VLA-beta1, but not VLA-alpha1, VLA-alpha4 and NCAM, a pattern of adhesion molecule expression compatible with the biliary epithelium. Also, similar to the biliary epithelium found in normal liver, HuGB cells expressed abundant HLA class I but few HLA class II antigens. We found that the expression of HLA antigens and ICAM-1 protein were increased during interferon-gamma treatment of HuGB cell line. CONCLUSIONS: Both phenotypic and morphological characteristics of HuGB cells suggested their biliary origin. Sensitivity of HuGB cells to interferon-gamma suggests that this new cell line could represent a suitable model to investigate the up-regulation of membrane antigens occurring in immune diseases involving biliary epithelial cells.

Adenocarcinoma↗
PubMed1992

Immunohistochemistry for intermediate filaments in the enteric nervous system of the porcine small intestine.

Antibodies against the cytoskeletal neurofilament protein 200 and gliafilament acidic protein were used for an immunohistochemical staining of nerve and glia cells in porcine small intestine. In sections as well as in whole mount preparations, the morphological and topographical features of the enteric nerve plexus could be demonstrated. The enteric glia cells are characterized by an abundance of immunoreactive GFAP, which allows a subsequent staining of the plexus. NFP 200 is immunohistochemically recognized only in a part of the neurons. This immunoreactive neuronal population can be identified morphologically as Typ II-neurons, which are defined as adendritic and pseudouniaxonal to multiaxonal. The immunostaining of intermediate filaments is an easy and reproducible means for studying the enteric nervous system and invaluable for the histopathological diagnosis of its morphological abnormalities.

Animals↗
PubMed2001

Polycystin-1 interacts with intermediate filaments.

Polycystin-1, the protein defective in a majority of patients with autosomal dominant polycystic kidney disease, is a ubiquitously expressed multi-span transmembrane protein of unknown function. Subcellular localization studies found this protein to be a component of various cell junctional complexes and to be associated with the cytoskeleton, but the specificity and nature of such associations are not known. To identify proteins that interact with the polycystin-1 C-tail (P1CT), this segment was used as bait in a yeast two-hybrid screening of a kidney epithelial cell library. The intermediate filament (IF) protein vimentin was identified as a strong polycystin-1-interacting partner. Cytokeratins K8 and K18 and desmin were also found to interact with P1CT. These interactions were mediated by coiled-coil motifs in polycystin-1 and IF proteins. Vimentin, cytokeratins K8 and K18, and desmin also bound directly to P1CT in GST pull-down and in in vitro filament assembly assays. Two observations confirmed these interactions in vivo: (i) a cell membrane-anchored form of recombinant P1CT decorated the IF network and was found to associate with the cytoskeleton in detergent-solubilized cells and (ii) endogenous polycystin-1 distributed with IF at desmosomal junctions. Polycystin-1 may utilize this association for structural, storage, or signaling functions.

Amino Acid Sequence↗
PubMed1990

Changes in intermediate filament immunolabeling occur in response to retinal detachment and reattachment in primates.

The immunolabeling patterns for vimentin and glial fibrillary acidic protein (GFAP) were studied in five rhesus monkeys that had undergone retinal detachment or detachment and reattachment. Anti-vimentin and anti-GFAP labeling intensity increased in Müller cells after 2 days of detachment. Weak anti-vimentin labeling of the basal RPE cytoplasm, which was absent in control tissue, was detected 2 days after detachment. After detachment for 7 days and reattachment for 7 or 14 days, the pattern and extent of intermediate filament (IF) labeling changed. In Müller cells, the labeling, which in controls was restricted to processes near the vitreal border of the retina, was present in Müller processes spanning the entire retina. In retinal pigment epithelium (RPE) cells, prominent anti-vimentin labeling was identified in the basal and basolateral cytoplasm. The extent of RPE and Müller cell IF labeling in two animals whose retinas had been detached and then reattached for 150 days was different from that found at either the 7- or 14-day reattachment time points. This suggests that the abnormal IF distribution triggered by detachment may be attenuated after a lengthy period of reattachment.

Animals↗
PubMed1987

Intermediate filaments and desmosomal plaque proteins in testicular seminomas and non-seminomatous germ cell tumours as revealed by immunohistochemistry.

Seminomas and non-seminomatous testicular germ cell tumours were studied for the presence of cytokeratin and vimentin filaments and desmosomes using immunohistochemical methods. In the majority of the classical seminomas and in seminomatous area of mixed tumours most tumour cells appeared to lack cytokeratin filaments. Some seminomas contained a focally variable proportion of cells exhibiting cytokeratin-positive structures while other cases contained only few seminoma cells with a well developed fibrillar cytokeratin network. Gel electrophoresis of cytoskeletal proteins from microdissected regions revealed cytokeratin polypeptides nos. 8 and 18 typical of simple epithelia. In one seminoma, however, all, or almost all, tumour cells contained cytokeratin filaments. This finding is in line with the assumption of transitional forms between seminoma and embryonal carcinoma. Despite the lack - or variable expression - of cytokeratin filaments most seminoma cells contained desmosomes, although often few in number and irregularly distributed at the circumference of the cells. Loosely arranged and often very sparse vimentin fibrils were found in many, but not all seminoma cells. Double label immunofluorescence microscopy suggested that the majority of desmosomes was associated with intermediate filaments of the vimentin type. In contrast, in carcinoma cells of malignant teratomas, in well differentiated epithelial cells of intermediate-type malignant teratomas and in trophoblastic cells present in trophoblastic-type malignant teratomas cytokeratin filament bundles as well as desmosomes were decorated. The arrangement and density of the cytokeratin filament skeleton and of desmosomes varied with degree of maturation of the tissue. The most regular distribution and intensive staining of cytokeratin filaments and desmoplakin was found in "mature" tissues. Vimentin was demonstrated in mesenchymal areas and stroma cells. The results show that seminomas are distinguished from most other germ cell and non-germ cell tumours by the presence of true desmosomes together with scanty vimentin filaments in most tumour cells. In addition, they indicate that seminoma cells can be heterogeneous in their cytoskeletal complement and may include cells with cytokeratin expression, indicative of a multipotential character of the initially transformed cell(s).

Adolescent↗
PubMed1999

Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments.

The small GTPase Rho and one of its targets, Rho-associated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can specifically recognize distinct phosphorylated sites on glial fibrillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate filament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of anti-Rho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/moesin (ERM) proteins and Ser19-phosphorylated myosin light chain. These results raise the possibility that Rho-kinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

Cell Division↗
PubMed1988

The association of prosomes with some of the intermediate filament networks of the animal cell.

The small RNP complexes of defined morphology and biochemical composition termed prosomes, first isolated from the cytoplasm associated with repressed mRNA (Martins de Sa, C., M.-F. Grossi de Sa, O. Akhayat, F. Broders, and K. Scherrer. J. Mol. Biol. 1986. 187:47-493), were found also in the nucleus (Grossi de Sa, M.-F., C. Martins de Sa, F. Harper, O. Coux, O. Akhayat, P. Gounon, J. K. Pal, Y. Florentin, and K. Scherrer. 1988. J. Cell Sci. 89:151-165). Immunofluorescence, immunoelectron microscopy, and immunochemical studies using mAbs directed against some of the prosomal proteins of duck erythroblasts indicate that in the cytoplasm of HeLa and PtK cells, prosome antigens are associated with the intermediate filament network of the cytokeratin type.

Actin Cytoskeleton↗
PubMed1983

Intermediate filaments of the cytoskeleton in glandular cells of the rat fundic mucosa: immunofluorescence and electron microscopy study.

The organization of intermediate filaments (IF) in cells of the rat fundic mucosa was studied by electron microscopy and immunofluorescence microscopy using specific antiprekeratin antibodies on frozen sections and isolated cells. Our results suggest that mucous cells lining the gastric surface and the gastric pits, which appeared strongly decorated, are the most rich in IF. These cells displayed coarse bundles of IF oriented in all directions as well as desmosome-attached tonofibrils. Mucous neck cells contained fewer bundles of IF located preferentially toward the apical region. Zymogen cells showed a strong staining along the contour of the luminal border, together with a faint decoration of a fine meshwork extending throughout the cytoplasm. A poorly defined fibrillar cortex present underneath the secretory plasma membrane and sparse bundles of IF among the elements of the rough endoplasmic reticulum were seen in thin sections. In contrast, parietal cells appeared brightly stained and the prekeratinlike material formed a cortical polygonal meshwork especially visible in isolated cells. A developed system of IF formed by conspicuous bundles located underneath the secretory canaliculi, among the mitochondria and in the vicinity of the basal plasma membrane, was observed in the electron microscope.

Animals↗
PubMed1982

Giant axonal neuropathy: an inborn error of organization of intermediate filaments.

Light microscopic examination of cultured skin fibroblasts from three patients with giant axonal neuropathy (GAN) revealed large aggregates of intermediate filaments (IF). However, electrophoretic analysis of these cells showed normal amounts of vimentin, the fibroblast IF subunit protein. The isoelectric point and peptide mapping of vimentin were identical to controls. Moreover, vimentin was synthesized and phosphorylated at a normal rate. These findings demonstrate that GAN is an inborn error of the organization of IF and suggest that the basic genetic defect in GAN does not involve primarily the IF subunit proteins.

Axons↗
PubMed1991

Intermediate filament expression in carcinoid tumours.

Twenty-eight carcinoid tumours were stained with monoclonal antibodies to epithelial and neural related intermediate filaments. All were found to express epithelial markers, and none expressed neural markers. These results support the theory that carcinoids are of epithelial, rather than neural crest origin.

Antibodies, Monoclonal↗
PubMed1985

Glial fibrillary acidic protein (GFAP): the major protein of glial intermediate filaments in differentiated astrocytes.

The glial fibrillary acidic protein (GFA protein or GFAP) is the major protein constituent of glial intermediate filaments in differentiated fibrous and protoplasmic astrocytes of the central nervous system. Proteins having similar molecular weights, isoelectric points, and immunoreactivity with GFAP have been found in cells of neural crest and ectodermal origin. A putative function ascribed to glial filaments is its role as a component of the cytoskeleton in defining and maintaining the shape of the astrocyte. Since 1980, over 350 reports have utilized antisera to GFAP for immunochemical and immunocytochemical studies.

Animals↗