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PubMed1992

Expression of intermediate filaments in chordomas. An immunocytochemical study of five cases.

An immunocytochemical investigation has been carried out on five cases of chordoma (2 of the sacrum, 2 in the spheno-occipital bone and 1 in the parapharyngeal area) to study the expression of the 5 classes of intermediate filaments (IF): cytokeratin (CK), desmin (DES), GFAP, neurofilaments (NF) and vimentin (VIM). Our results show that constant coexpression of CK, NF and VIM does occur in chordomas, whereas DES and GFAP are not demonstrable in tumor cells. The three detected IF are invariably present in all cell types but not in intracellular vacuoles or in the extracellular mucoid substance. The pattern of immunoreactivity of chordomas appears very unique as very few other neoplasms demonstrate the simultaneous occurrence of 3 distinct IF. Only choroid plexus tumors have been shown to manifest CK-NF-VIM immunoreactivity. The complex immunophenotype of chordomas may be related to their supposed origin from the notochord which normally undergoes conspicuous changes in location and morphology during embryonal development. Such changes might require the contemporary presence of multiple IF; IF expression, in fact, is known to be related to cell function and morphology. Notochordal cells and their neoplastic counterpart may consequently express an IF pattern which reflects unique architectural and morphological variations occurring during embryonal and tumor growth. Together with the speculative value of the detection of CK, NF and VIM in chordomas, the unusual immunocytochemical pattern of these tumors might provide useful diagnostic tool in differential diagnosis.

Adult↗
PubMed1995

Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts.

Separate populations of microtubules (MTs) distinguishable by their level of posttranslationally modified tubulin subunits and by their stability in vivo have been described. In polarized 3T3 cells at the edge of an in vitro wound, we have found a striking preferential coalignment of vimentin intermediate filaments (IFs) with detyrosinated MTs (Glu MTs) rather than with the bulk of the MTs, which were tyrosinated MTs (Tyr MTs). Vimentin IFs were not stabilizing the Glu MTs since collapse of the IF network to a perinuclear location, induced by microinjection of monoclonal anti-IF antibody, had no noticeable effect on the array of Glu MTs. To test whether Glu MTs may affect the organization of IFs we regrew MTs in cells that had been treated with nocodazole to depolymerize all the MTs and to collapse IFs; the reextension of IFs into the lamella lagged behind the rapid regrowth of Tyr MTs, but was correlated with the slower reformation of Glu MTs. Similar realignment of IFs with newly formed Glu MTs was observed in serum-starved cells treated with either serum or taxol to induce the formation of Glu MTs. Next, we microinjected affinity purified antibodies specific for Glu tubulin (polyclonal SG and monoclonal 4B8) and specific for Tyr tubulin (polyclonal W2 and monoclonal YL1/2) into 3T3 cells. Both injected SG and 4B8 antibodies labeled the subset of endogenous Glu MTs; W2 and YL1/2 antibodies labeled virtually all of the cytoplasmic MTs. Injection of SG or 4B8 resulted in the collapse of IFs to a perinuclear region. This collapse was comparable to that observed after complete MT depolymerization by nocodazole. Injection of W2, YL1/2, or nonspecific control IgGs did not result in collapse of the IFs. Taken together, these results show that Glu MTs localize IFs in migrating 3T3 fibroblasts and suggest that detyrosination of tubulin acts as a signal for the recruitment of vimentin IFs to MTs.

3T3 Cells↗
PubMed1999

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism.

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.

3T3 Cells↗
PubMedDate not supplied

Clathrin light chains contain brain-specific insertion sequences and a region of homology with intermediate filaments.

The primary structures of four bovine clathrin light chains have been determined. Light chains LCa and LCb are homologous proteins encoded by different genes. In the brain the messenger RNA from these genes undergoes differential splicing to yield proteins having centrally inserted brain-specific sequences. A potentially alpha-helical region of the clathrin light chains shows homology with intermediate filament proteins.

Amino Acid Sequence↗
PubMed1989

[Expression of genes coding for the proteins of intermediate filaments in man].

Cellular morphogenesis is a fundamental phenomenon in our understanding of eucaryotic cells. When, where and how these supra molecular structures generate during differentiation to cellular shape? It seems that the cytoskeleton alone is responsible for the formation and maintenance of the cellular shape. The supra molecular organisation of the intermediate filaments is specific for the cell type, the developmental stage and the type of differentiation. The human genes coding for the different subunits of these polymers have now all been isolated, cloned and characterised. The regulation of their expression is now being analysed.

Cell Differentiation↗
PubMed2001

Sites of nucleic acid binding in type I-IV intermediate filament subunit proteins.

A combination of enzymatic and chemical ladder sequencing of photo-cross-linked protein-single-stranded oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry was employed to identify the amino acid residues responsible for the stable binding of nucleic acids in several intermediate filament (IF) subunit proteins. The IF proteins studied included the type I and type II cytokeratins K8, K18, and K19; the type III proteins desmin, glial fibrillary acidic protein (GFAP), peripherin, and vimentin; and the type IV neurofilament triplet protein L (NF-L). The site of nucleic acid binding was localized to the non-alpha-helical, amino-terminal head domain of all of the IF proteins tested. GFAP, which has the shortest head domain of the proteins tested, cross-linked via only two amino acid residues. One of these residues was located within a conserved nonapeptide domain that has been shown to be required for filament formation. One or more cross-linked residues were found in a similar location in the other proteins studied. The major binding site for nucleic acids for most of the proteins appears to be localized within the middle of the head domain. The two exceptions to this generalization are GFAP, which lacks these residues, and NF-L, in which a large number of cross-linked residues were found scattered throughout the first half of the head domain. Control experiments were also done with two bacteriophage ssDNA-binding proteins, as well as actin and tubulin. The single sites of cross-linkage observed with the bacteriophage proteins, Phe(183) for the T4 gene 32 protein and Phe(73) for the M13 gene 5 protein, were in good agreement with literature data. Actin and tubulin could not be cross-linked to the oligonucleotide. Aside from the insight into the biological activity of IF proteins that these data provide, they also demonstrate that this analytical method can be employed to study a variety of protein-nucleic acid interactions.

Amino Acid Sequence↗
PubMed1984

Distribution of intermediate filaments and actin microfilaments in parotid autoimmune sialoadenitis of Sjögren syndrome.

In order to study the importance of myoepithelial cells in the formation of parotid epi-myoepithelial islands of the autoimmune sialoadenitis in Sjögren's syndrome, the distribution of cytoskeletal elements has been investigated by electron and immunofluorescence microscopy. Antibodies to intermediate filament proteins--prekeratins, desmin and vimentin--were used as well as a 'DNase I/anti-DNase I' immunohistochemical procedure to detect actin microfilaments for characterization of myoepithelial cells compared to the other types of parotid epithelial cells. Two types of prekeratin antibodies were raised. One of these reacted preferentially with myoepithelial cells. The same type of antiserum also showed a bright fluorescence in cells located peripherally in the epi-myoepithelial islands. These island cells, however, exhibited only a very low actin microfilament content and were thus not ordinary myoepithelial cells. The other type of prekeratin antibodies reacted with all types of parotid epithelia, except acinar secretory epithelium. They also stained all island epithelial cells, but the reactivity was less in cell regions with pronounced lymphocyte infiltration. The heterogeneity between the island epithelial cells, immunohistochemically as well as ultrastructurally, indicates different stages of island formation. Interepithelial lymphocytes showed less vimentin and actin filament content in comparison with lymphoid cells outside the islands. Conclusively, typical epi-myoepithelial islands are composed of metaplastic epithelial cells of intercalated duct origin and invaded lymphoid cells, and ordinary myoepithelial cells are lacking.

Actins↗
PubMed1986

Efficient degradation in vitro of all intermediate filament subunit proteins by the Ca2+-activated neutral thiol proteinase from Ehrlich ascites tumor cells and porcine kidney.

Vimentin, desmin, glial fibrillary acidic protein, neurofilament triplet proteins, and a mixture of cytokeratins were digested with Ca2+-activated neutral thiol proteinase isolated from Ehrlich ascites tumor (EAT) cells and porcine kidney. All intermediate filament proteins were degraded by the proteinase, although with different rates and Ca2+ optima. These results are in part at variance with our previous statement that the Ca2+-activated proteinase from EAT cells is specific for vimentin and desmin.

Animals↗
PubMed1989

Obplacental giant cells of the domestic rabbit: development, morphology, and intermediate filament composition.

Obplacental giant cells are large (less than or equal to 210 microns) polyploid cells that appear in the stroma of the pregnant uterus of the rabbit following ovoimplantation. Histological examination of a complete developmental series indicates that obplacental giant cells arise from trophoblastic knobs that have traversed the uterine epithelium during early implantation. During maturation, the cells undergo a massive (approximately 6,000%) increase in volume and penetrate deeply into the uterine stroma and myometrium, where they often become associated with blood vessels and smooth muscle cells. Giant cells at mid-gestation contain one or two large nuclei with prominent nucleoli and appear to be amitotic. They are rich in Golgi complexes, RER, SER, and cortically distributed cytoplasmic filaments, and contain intracellular canaliculi lined by microvilli. Giant cells vary with respect to the occurrence of lipid droplets, phagocytotic inclusions, lysosomal structures, and electron-dense granules. Immunocytochemistry demonstrates that the giant cells exhibit intermediate filaments related to cytokeratin and vimentin, but are negative for desmin and for an endothelial cell marker, Factor VIII-related antigen. The cells are positive for cytokeratin from their inception, but only become vimentin-positive between Days 12 and 15 of pregnancy, a change seemingly related to their detachment from epithelial tissue to take on an independent existence. Our findings indicate that the giant cells originate from obplacental trophoblast and, at maturity, exhibit cytoskeletal characteristics of isolated epithelial cells, as well as a complement of organelles suggestive of synthetic activity.

Animals↗
PubMed2004

Assessing the flexibility of intermediate filaments by atomic force microscopy.

Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.

Actin Cytoskeleton↗
PubMed1998

Regulation of a hair follicle keratin intermediate filament gene promoter.

During hair growth, cortical cells emerging from the proliferative follicle bulb rapidly undergo a differentiation program and synthesise large amounts of hair keratin proteins. To identify some of the controls that specify expression of hair genes we have defined the minimal promoter of the wool keratin intermediate filament gene K2.10. The region of this gene spanning nucleotides -350 to +53 was sufficient to direct expression of the lacZ gene to the follicle cortex of transgenic mice but deletion of nucleotides -350 to -150 led to a complete loss of promoter activity. When a four base substitution mutation was introduced into the minimal functional promoter at the binding site for lymphoid enhancer factor 1 (LEF-1), promoter activity in transgenic mice was decreased but specificity was not affected. To investigate the interaction of trans-acting factors within the minimal K2.10 promoter we performed DNase I footprinting analyses and electrophoretic mobility shift assays. In addition to LEF-1, Sp1, AP2-like and NF1-like proteins bound to the promoter. The Sp1 and AP2-like proteins bound sequences flanking the LEF-1 binding site whereas the NF1-like proteins bound closer to the transcription start site. We conclude that the LEF-1 binding site is an enhancer element of the K2.10 promoter in the hair follicle cortex and that factors other than LEF-1 regulate promoter tissue- and differentiation-specificity.

Animals↗
PubMed2002

DEDD regulates degradation of intermediate filaments during apoptosis.

Apoptosis depends critically on regulated cytoskeletal reorganization events in a cell. We demonstrate that death effector domain containing DNA binding protein (DEDD), a highly conserved and ubiquitous death effector domain containing protein, exists predominantly as mono- or diubiquitinated, and that diubiquitinated DEDD interacts with both the K8/18 intermediate filament network and pro-caspase-3. Early in apoptosis, both cytosolic DEDD and its close homologue DEDD2 formed filaments that colocalized with and depended on K8/18 and active caspase-3. Subsequently, these filamentous structures collapsed into intracellular inclusions that migrated into cytoplasmic blebs and contained DEDD, DEDD2, active caspase-3, and caspase-3-cleaved K18 late in apoptosis. Biochemical studies further confirmed that DEDD coimmunoprecipitated with both K18 and pro-caspase-3, and kinetic analyses placed apoptotic DEDD staining prior to caspase-3 activation and K18 cleavage. In addition, both caspase-3 activation and K18 cleavage was inhibited by expression of DEDDDeltaNLS1-3, a cytosolic form of DEDD that cannot be ubiquitinated. Finally, siRNA mediated DEDD knockdown cells exhibited inhibition of staurosporine-induced DNA degradation. Our data suggest that DEDD represents a novel scaffold protein that directs the effector caspase-3 to certain substrates facilitating their ordered degradation during apoptosis.

Animals↗
PubMed1990

Intermediate filaments in the inner ear of normal and experimentally damaged guinea pigs.

The hypothesis that proteins known to occur in glial cells in the central nervous system may be present in inner-ear supporting cells was investigated. Immunocytochemical techniques were used to look for the existence of two classes of intermediate filaments, vimentin and glial fibrillary acidic protein (GFAP), in cellular elements of the inner-ear epithelium in normal and experimentally damaged guinea-pig cochleas. Vimentin is present in two types of supporting cells in the normal organ of Corti: Deiters' cells and inner pillar cells. Differences in intensity and distribution of vimentin immunostaining are observed across the three rows of Deiters' cells. GFAP immunoreactivity was not detected in any supporting-cell type in the organ. Cochlear hair cells were not labeled for either GFAP or vimentin. Following hair-cell destruction by exposure to noise or the administration of aminoglycosides, GFAP and vimentin are not present in phalangeal scars replacing lost hair cells.

Animals↗
PubMed1986

[Immunomorphological research on human breast tumors using monoclonal antibodies to intermediate filament proteins. The proliferative epithelial structures in fibrocystic disease (dysplasia) and benign tumors].

Proliferating epithelial structures were studied immunomorphologically in 16 cases of fibrocystic disease and benign tumours. Monoclonal antibodies were used to prekeratin (PK) C12, normally specific for the lining epithelium, to prekeratin (PK) E3 and to the protein of intermediate filaments in mesenchymal cells--vimentin, normally specific for myoepithelium. The immunofluorescent analysis of the most common proliferating structures has shown that there are elements with a variable combination of PK C12, PK E3 and vimentin among the proliferating cells. While there are cells similar to normal lining epithelium (PK C12) or myoepithelium (PK E3) and/or vimentin), many cells have no analogues either among normal cells, or among cells from ductal, lobular and tubular tumour forms. Since in the most common forms of breast cancer only cells containing PK C12 were found, the immunofluorescent study with the application of monoclonal antibodies to PK C12, PK E3 and vimentin can be recommended in difficult and dubious cases for the recognition of carcinogenic or dysplastic nature of morphologically similar proliferating structures.

Antibodies, Monoclonal↗
PubMedDate not supplied

Intermediate filaments of human Sertoli cells in germinal alterations.

Immunohistochemical techniques were used to examine testis biopsy specimens in subjects of different ages, in whom histological examination of the seminiferous tubules revealed a reduction in number, immaturity or absence of germinal elements. Our aim was to detect changes in the expression of the vimentin and cytokeratin (8-18-19) intermediate filaments in Sertoli cells. The use of anti-vimentin antibodies demonstrated intense Sertoli cell positivity in all the cases studied, confirming that germinal alterations do not interfere with the expression of these filaments. The Sertoli cells of the subjects affected by pathological conditions also reacted positively to anti-cytokeratin antibodies. This finding was constant in all the testes, showing evident signs of germinal immaturity. In contrast, anti-cytokeratin positivity was not observed in the control specimens after the pre-pubertal stage. Our findings show that persisting co-expression of vimentin and cytokeratin filaments in the Sertoli cells of productive subjects may be a marker of germinal cell degeneration.

Adolescent↗
PubMed2004

Alpha-keto-beta-methylvaleric acid increases the in vitro phosphorylation of intermediate filaments in cerebral cortex of young rats through the gabaergic system.

In this study we investigated the effects of alpha-ketoisovaleric (KIV) and alpha-keto-beta-methylvaleric acids (KMV), metabolites accumulating in the inherited neurometabolic disorder maple syrup urine disease (MSUD), on the in vitro incorporation of 32P into intermediate filament (IF) proteins from cerebral cortex of young rats during development (9-21 days of age) We observed that KMV significantly increased the in vitro incorporation of 32P into the IF proteins studied in cortical slices of 12-day-old rats through the PKA and PKCaMII, with no alteration at the other ages. In contrast, KIV was ineffective in altering the phosphorylating system associated with IF proteins at all ages examined. A similar effect on IF phosphorylation was achieved by incubating cortical slices with gamma-aminobutiric acid (GABA). Furthermore, by using specific GABA antagonists, we verified that KMV induced a stimulatory effect on IF phosphorylation of tissue slices from 12-day-old rats mediated by GABA(A) and GABA(B) receptors. In conclusion, our results indicate the involvement of the GABAergic system in the alterations of IF phosphorylation caused by KMV, one of the branched-chain keto acids accumulating in MSUD.

Age Factors↗