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PubMed1982

O-phosphoserine content of intermediate filament subunits.

Purified subunits of intermediate filaments obtained from a variety of tissues and cell types contain O-phosphoserine and, in some cases, smaller amounts of O-phosphothreonine. The O-phosphoserine content was estimated by reaction of performic acid oxidized subunits with methylamine in NaOH. Decamin of BHK-21 and CHO fibroblasts contained about 1 mol/mol. Avian and mammalian desmin consists of two subunits, an acidic (alpha) subunit which contained 2 mol/mol and a more basic (beta) nonphosphorylated subunit. The principal (Mr approximately 60 000) subunit of squid brain neurofilaments contained 5 mol/mol. Most mouse and bovine keratin subunits contained 3--6 mol/mol, although certain bovine subunits of higher molecular weight contained none. The O-phosphoserine contents of keratin subunits purified from the viable and stratum corneum layers were the same. The O-phosphoserine was located in non-alpha-helical regions of the subunits which presumably project out from the alpha-helical wall of the intermediate filaments. Most subunits could be partially dephosphorylated in vitro with alkaline phosphatase. It was found that the capacity of such partially dephosphorylated subunits for assembly into native-type filaments in vitro was independent of their phosphate content.

Animals↗
PubMed1999

Intermediate filament proteins in developing human arteries.

The distribution of intermediate filament proteins in adult human blood vessels and in human fetal elastic arteries is relatively well-known. However, the distribution of these proteins in the course from neonate to adult has not been established. In this investigation, human postnatal arteries were studied with immunohistochemistry, using antibodies targeted on the intermediate filament proteins desmin, vimentin and cytokeratins 8, 18 and 19. Vimentin was present in most smooth muscle cells in all vessels and at all ages. The proportions of desmin-expressing cells increased in the elastic arteries during the first year of life and was higher in the pulmonary trunk than in the aorta. In the muscular arteries, the proportion of desmin-labelled cells increased in the coronary and the deep femoral arteries, but remained constant in the renal and the cerebral arteries. Cytokeratins were detected in the pulmonary trunk earlier than in the aorta. Cytokeratins were present throughout the wall of the ductus arteriosus, but desmin was present only in some cells. Thus, there are postnatal changes in the distribution of intermediate filament proteins in the elastic arteries and in some muscular arteries, whereas the intermediate filament pattern remains unchanged in other muscular arteries.

Adolescent↗
PubMed1995

Semiquantitative postembedding characterization of intermediate filaments in central nervous system lesions using immunoelectron microscopy.

Standardized postembedding immunoelectron microscopy was performed to demonstrate glial fibrillary acidic protein (GFAP) and vimentin in individual intermediate filaments to determine the diagnostic value of demonstrating ultrastructural and immunophenotypic characteristics of intermediate filaments in routine brain biopsy specimens. Dual expression of GFAP and vimentin was observed in the astroblastoma and astrocytes of Alexander's disease. The antigen availability for vimentin, however, was too low to allow reliable assessment of the GFAP:vimentin ratio in individual intermediate filaments and/or filament bundles. In meningioma, only vimentin positive intermediate filaments were found. GFAP positive intermediate filaments were present in all other specimens except the oligodendroglial components of the mixed glioma, which were devoid of intermediate filaments. GFAP positivity in the filamentous periphery and electron-dense core of Rosenthal fibers was demonstrated. Technical and tissue processing factors had a significant effect on particle density values obtained for individual specimens. Although the number, distribution, and density of glial intermediate filaments varies in different astroglial entities, correlation of particle density values determined by immunoelectron microscopy with relative GFAP concentrations in different lesions requires utmost caution. Nevertheless, application of the postembedding approach to routinely fixed biopsy specimens indicated an association of different entities with the exclusive presence of GFAP and/or vimentin in individual intermediate filaments, thus emphasizing the diagnostic value of intermediate filament typing for pathological characterization.

Adolescent↗
PubMed1994

Rearrangement of intermediate filament network of BHK-21 cells infected with vaccinia virus.

Association between vaccinia virus (VV) structures and intermediate filaments in specific areas of the cytoplasm of infected cells (virus "factories") suggests that VV infection interferes with the cellular architecture by modifying the intermediate filament network. To analyse this question, we examined the array of intermediate filaments of BHK-21 cells infected with VV by laser scanning confocal microscopy using an anti-vimentin mouse monoclonal antibody. We observed a marked reorganization of intermediate filaments around the nucleus of infected cells. Bidimensional analysis of 32PO4-labeled intermediate filament proteins revealed that the acidic isoform of vimentin and two isoforms of desmin have increased phosphorylation levels in infected cells. Our results suggest that the reorganization of intermediate filaments observed during VV infection could be promoted by an increase in the phosphorylation level of the intermediate filament proteins, vimentin and desmin.

Animals↗
PubMed1990

On the relation between distinct components of the cytoskeleton: an epitope shared by intermediate filaments, microfilaments and cytoplasmic foci.

Monoclonal antibodies were generated against detergent-insoluble cytoskeletal proteins isolated from low-density membrane fractions of rat liver. By immunofluorescence, one of the antibodies stains three distinct structures in cultured rat fibroblast and hepatocyte lines as well as the PtK2 rat-kangaroo kidney epithelial line. These structures are: i) many tangled filaments similar to intermediate filaments (IFs), ii) fewer and variable numbers of straight filaments, and iii) punctate cytoplasmic foci, often most intense around the nucleus. All three of these structures are resistant to extraction by non-ionic detergent. Close examination reveals that the tangled and straight filaments are not stained uniformly, but as a series of bright patches. In cells treated with nocodazole, the antibody reacts strongly with a perinuclear filamentous cage. Very few tangled filaments are detected in these cells, however, the straight filaments and punctate cytoplasmic staining are resistant to nocodazole treatment. Double-label immunofluorescence shows that, even though tangled filament distribution and nocodazole sensitivity are similar to the behavior of vimentin IFs, there is only partial coincidence of staining with either vimentin or cytokeratin IFs. The straight filaments coincide with some actin stress fibers, but the punctate cytoplasmic staining is not related to IFs, actin, or tubulin. Thus, this monoclonal antibody stains a novel group of three seemingly unrelated cytoskeletal structures, including a previously undescribed insoluble nonfilamentous pool. Taken as a whole, two hypotheses are consistent with these data. i) The antigen recognized may be a protein which has a large insoluble cytoplasmic pool and binds both IFs and actin, but only binds to a subset of each class of filaments.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗
PubMed1987

[Intermediate filaments: structure, regulation of expression and possible function].

New data are reviewed on intermediate filaments, i.e. on one of the cytoskeleton components. Structural proteins of intermediate filaments, their enzymatic modification, filament-associated proteins and the peculiarities of filament assembly are dealt with. The regularities of expression of intermediate filament proteins in normal tissues are analysed, as well as during differentiation and cultured cell growth. In the final part of the paper possible functions of intermediate filaments are discussed.

Animals↗
PubMed1986

Homology and diversity between intermediate filament proteins of neuronal and nonneuronal origin in goldfish optic nerve.

The predominant intermediate filament proteins of the goldfish optic nerve have molecular weights of 58K. They can be separated into a series of four major isoelectric variants of neuronal (ON1 and ON2) and nonneuronal (ON3 and ON4) origin. The extent of homology between the goldfish 58K intermediate filament proteins themselves and to rat optic nerve vimentin and glial fibrillary acidic protein (GFAP) was investigated. Unlabeled and [32P]orthophosphate-labeled proteins were subjected to partial hydrolysis by V8 protease, chymotrypsin, and CNBr. The results show that the goldfish intermediate filament proteins share with vimentin and GFAP a 40K chymotrypsin-resistant core fragment. Phosphorylated moieties appear to be located outside the core region since they are preferentially cleaved off by chymotrypsin and not found associated with the 40K core. In addition, the goldfish ON proteins contain the antigenic site within the core that is common to most intermediate filaments. V8 or CNBr digestion indicates that many fragments that are common to ON1 and ON2 are clearly distinct from fragments that are common to ON3 and ON4. In addition, structural variability is observed between the goldfish intermediate filament proteins and vimentin and GFAP. The results are discussed in terms of intermediate filament structure and their possible role in nerve growth.

Animals↗
PubMed1998

Chemotactic peptide-induced changes of intermediate filament organization in neutrophils during granule secretion: role of cyclic guanosine monophosphate.

In neutrophils activated to secrete with formyl-methionyl-leucyl-phenylalanine, intermediate filaments are phosphorylated transiently by cyclic guanosine monophosphate (cGMP)-dependent protein kinase (G-kinase). cGMP regulation of vimentin organization was investigated. During granule secretion, cGMP levels were elevated and intermediate filaments were transiently assembled at the pericortex to areas devoid of granules and microfilaments. Microtubule and microfilament inhibitors affected intermediate filament organization, granule secretion, and cGMP levels. Cytochalasin D and nocodazole caused intermediate filaments to assemble at the nucleus, rather than at the pericortex. cGMP levels were elevated in neutrophils by both inhibitors; however, with cytochalasin D, cGMP was elevated earlier and granule secretion was excessive. Nocodazole did not affect normal cGMP elevations, but specific granule secretion was delayed. LY83583, a guanylyl cyclase antagonist, inhibited granule secretion and intermediate filament organization, but not microtubule or microfilament organization. Intermediate filament assembly at the pericortex and secretion were partially restored by 8-bromo-cGMP in LY83583-treated neutrophils, suggesting that cGMP regulates these functions. G-kinase directly induced intermediate filament assembly in situ, and protein phosphatase 1 disassembled filaments. However, in intact cells stimulated with formyl-methionyl-leucyl-phenylalanine, intermediate filament assembly is focal and transient, suggesting that vimentin phosphorylation is compartmentalized. We propose that, in addition to changes in microfilament and microtubule organization, granule secretion is also accompanied by changes in intermediate filament organization, and that cGMP regulates vimentin filament organization via activation of G-kinase.

Actin Cytoskeleton↗
PubMed1997

Association of hnRNP S1 proteins C2 and D2 with vimentin intermediate filaments.

S1 proteins A-D are hnRNP proteins which were originally isolated from nuclei of various tissues, by selective extraction of pH 4.9 from the supernatants of nuclei mildly treated with DNase I or RNase A. In the present study, a hybridoma was isolated which produced a monoclonal antibody that reacted specifically with S1 proteins C2 and D2. When the antibody was used in indirect immunofluorescence staining of cultured cells, it stained, in addition to the nuclei, the cytoskeleton-like fibrous structures in the cytoplasm. We demonstrate that the cytoskeletal filaments are vimentin intermediate filaments. This is the first report on the hnRNP protein-association with cytoskeleton, and will help to clarify cytoplasmic mRNA localization as well as cytoplasmic distribution of hnRNP proteins.

Animals↗
PubMed1989

Intermediate filament collapse is an ATP-dependent and actin-dependent process.

In this study, we have investigated the properties of intermediate filament rearrangements using experimentally induced collapse of vimentin intermediate filaments in mouse fibroblasts. In these cells, depolymerizing microtubules by colchicine or vinblastine treatment at 37 degrees C results in a two-stage collapse of intermediate filaments. First, the vimentin filaments aggregate into large cables; then, the cables coil into a dense mass surrounding the nucleus. By using inhibitors of oxidative phosphorylation along with glucose deprivation to lower intracellular ATP levels by 95%, we have found that both stages of intermediate filament collapse require ATP. However, once collapse has occurred, only the second stage can be reversed in the absence of microtubules by lowering ATP levels. An additional difference between the two stages of collapse was revealed by treating cells with cytochalasin D: the formation of intermediate filament cables still occurs after disruption of the actin filament system by cytochalasin, but the subsequent coiling of cables to form a perinuclear mass is strongly inhibited by these conditions, and can be reversed by applying cytochalasin to cells in which intermediate filaments have already undergone complete collapse. We propose that the formation of vimentin cables involves a phosphorylation event, while the coiling of cables into a perinuclear mass relies on interaction of intermediate filaments with a component of the actin cortex.

Actins↗
PubMed1985

Survey of intermediate filament proteins in optic nerve and spinal cord: evidence for differential expression.

The distribution of intermediate filament proteins in optic nerve and spinal cord from rat, hamster, goldfish, frog, and newt were analyzed by two-dimensional gel electrophoresis. General as well as specific monoclonal and polyclonal antibodies were reacted against putative intermediate filament proteins. In vitro incubations of excised optic nerve in the presence of [35S]methionine distinguished between neuronal and nonneuronal intermediate filament proteins. The proteins of the intermediate filament complex in the two tissues for rat and hamster were similar. The typical neurofilament triplet and glial fibrillary acidic protein (GFAP) were observed. Vimentin was more concentrated in the optic nerve than in the spinal cord. The goldfish, newt, and frog contained neurofilament proteins in the 145-150K range and in the 70-85K range. In addition, predominant neurofilament proteins in the 58-62K molecular-weight range were found in all three species. In contrast to mammalian species, the goldfish, newt, and frog displayed extensive heterogeneity between optic nerve and spinal cord in the expression of both neuronal and nonneuronal intermediate filament proteins. The distinctive presence of low-molecular-weight intermediate filament proteins and their high concentration in the optic nerve and spinal cord of these nonmammalian vertebrates is discussed in terms of neuronal development and regeneration.

Amphibians↗
PubMed1995

Re-expression of the intermediate filament nestin in reactive astrocytes.

The intermediate filament nestin is highly expressed in multipotential stem cells of the developing central nervous system (CNS). During neuro- and gliogenesis, nestin is replaced by cell type-specific intermediate filaments, e.g. neurofilaments and glial fibrillary acidic protein (GFAP). In this study, we demonstrate that nestin expression is re-induced in reactive astrocytes in the lesioned adult brain. Following ischaemic and mechanical lesioning, a strong and sustained expression of nestin was noted in GFAP-positive cells surrounding the lesion site. Lesion experiments in transgenic mice carrying the lacZ gene under control of regulatory sequences from the nestin gene suggested that the upregulation of nestin in reactive astrocytes is mediated via the same sequences that control nestin expression during CNS development. These observations and recent data on the co-expression of glial and neuronal marker antigens in reactive astrocytes point to a close relationship between proliferating astrocytes and neuroepithelial precursor cells.

Animals↗
PubMed1997

The importance of intermediate filaments in the adaptation of tissues to mechanical stress: evidence from gene knockout studies.

Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.

Adaptation, Physiological↗
PubMedDate not supplied

Interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules--a quadruple fluorescence labeling study.

To study the interrelationships of endoplasmic reticulum, mitochondria, intermediate filaments, and microtubules, we have developed a quadruple fluorescence labeling procedure to visualize all four structures in the same cell. We applied this approach to study cellular organization in control cells and in cells treated with the microtubule drugs vinblastine or taxol. Endoplasmic reticulum was visualized by staining glutaraldehyde-fixed cells with the dye 3,3'-dihexyloxacarbocyanine iodide. After detergent permeabilization, triple immunofluorescence was carried out to specifically visualize mitochondria, vimentin intermediate filaments, and microtubules. Mitochondria in human fibroblasts were found to be highly elongated tubular structures (lengths up to greater than 50 microns), which in many cases were apparently fused to each other. Mitochondria were always observed to be associated with endoplasmic reticulum, although endoplasmic reticulum also existed independently. Intermediate filament distribution could not completely account for endoplasmic reticulum or mitochondrial distributions. Microtubules, however, always codistributed with these organelles. Microtubule depolymerization in vinblastine treated cells resulted in coaggregation of endoplasmic reticulum and mitochondria, and in the collapse of intermediate filaments. The spatial distributions of organelles compared with intermediate filaments were not identical, indicating that attachment of organelles to intermediate filaments was not responsible for organelle aggregation. Mitochondrial associations with endoplasmic reticulum, on the other hand, were retained, indicating this association was stable regardless of endoplasmic reticulum form or microtubules. In taxol-treated cells, endoplasmic reticulum, mitochondria, and intermediate filaments were all associated with taxol-stabilized microtubule bundles.

Animals↗
PubMed1998

GFAP-deficient astrocytes are capable of stellation in vitro when cocultured with neurons and exhibit a reduced amount of intermediate filaments and an increased cell saturation density.

Glial fibrillary acidic protein (GFAP) is an intermediate filament protein predominantly expressed in cells of astroglial origin. To allow for the study of the biological functions of GFAP we have previously generated GFAP-negative mice by gene targeting [Pekny et al. (1995) EMBO J. 14, 1590-1598]. Astrocytes in culture, similar to reactive astrocytes in vivo, express three intermediate filament proteins: GFAP, vimentin, and nestin. Using primary astrocyte-enriched cultures from GFAP-negative mice, we now report on the effect of GFAP absence on (i) the synthesis of other intermediate filament proteins in astrocytes, (ii) intermediate filament formation, (iii) astrocyte process formation (stellation) in response to neurons in mixed cerebellar astrocyte/neuron cultures, and (iv) saturation cell density in vitro. GFAP-/- astrocytes were found to produce both nestin and vimentin. At the ultrastructural level, the amount of intermediate filaments as revealed by transmission electron microscopy was reduced in GFAP-/- astrocytes compared to that in GFAP+/+ astrocytes. GFAP-/- astrocytes retained the ability to form processes in response to neurons in mixed astrocyte/neuron cultures from the cerebellum. GFAP-/- astrocyte-enriched primary cultures exhibited an increased final cell saturation density. The latter leads us to speculate that the loss of GFAP expression observed focally in a proportion of human malignant gliomas may reflect tumor progression toward a more rapidly growing and malignant phenotype.

Animals↗
PubMed2004

Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds.

Intermediate filaments are cytoskeletal polymers encoded by a large family of differentially expressed genes that provide crucial structural support in the cytoplasm and nucleus of higher eukaryotes. Perturbation of their function accounts for several genetically determined diseases in which fragile cells cannot sustain mechanical and non-mechanical stresses. Recent studies shed light on how this structural support is modulated to meet the changing needs of cells, and reveal a novel role whereby intermediate filaments influence cell growth and death through dynamic interactions with non-structural proteins.

Animals↗
PubMed1983

Malignant melanomas contain only the vimentin type of intermediate filaments.

Six malignant melanomas have been examined for the type of intermediate filament they contain. All six cases showed positive staining of intermediate filaments with antibodies to vimentin, with cells containing large numbers of melanosomes being stained less strongly in general. The tumor cells did not react with antibodies to keratin, desmin, neurofilaments or glial fibrillary acidic protein. Thus typing of intermediate filaments can distinguish melanoma from undifferentiated carcinoma, but not from lymphoma or sarcoma. Since melanocytes are known to be vimentin positive, and since most of the samples we studied were from metastases, these results are a further indication that the intermediate filament type typical of the parental cell is retained in the metastases, as well as in the primaries of solid tumours. The implications of vimentin positivity for the histiogenesis of the melanocyte are also discussed.

Cytoskeleton↗
PubMed1998

The pathway of assembly of intermediate filaments from recombinant alpha-internexin.

The pathway of filament assembly from the neuronal intermediate filament alpha-intermexin was investigated. Optimal assembly occurred in solutions of pH 6.5 to 7 and moderate ionic strength at 37 degrees C. Short filaments formed upon dialysis at 24 degrees C, which elongated further when incubated at 37 degrees C. Soluble forms of alpha-internexin were characterized by analytical ultracentrifugation and electron microscopy. In 10 mM Tris, pH 8, conditions that favor formation of tetramers and other small oligomers for other intermediate filament proteins, alpha-internexin formed 10.5 S particles, apparently unit-length half-filaments in the form of rods 10.6 nm in diameter and 68 nm long. Dialysis vs the same buffer with added 10 mM NaCl yielded 16 S rods, probably unit-length filaments, of the same length but 13.0 nm in diameter. At 50 mM NaCl, rods about 13 nm in diameter and heterogeneous in length were observed in electron micrographs, apparently formed from longitudinal annealing of unit-length rods. The results favor a model of assembly in which coiled coil dimers aggregate laterally to form first "unit-length half-filaments" (Herrmann, H., and Aebi, U. (1998) Curr. Opin. Struct. Biol. 8, 177-185) and then "unit-length filaments," which subsequently elongate by annealing.

Amino Acid Sequence↗