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PubMed1998

A novel taxol-induced vimentin phosphorylation and stabilization revealed by studies on stable microtubules and vimentin intermediate filaments.

To understand how protein phosphorylation modulates cytoskeletal organization, we used immunofluorescence microscopy to examine the effects of okadaic acid, a serine/threonine protein phosphatase inhibitor, and taxol, a microtubule-stabilizing agent, on stable (acetylated and detyrosinated) microtubules, vimentin intermediate filaments and other cytoskeletal elements in CV-1 cells. Okadaic acid caused major changes in both stable microtubules and vimentin intermediate filaments, but through independent mechanisms. At 300 nM, okadaic acid caused apparent fragmentation and loss of stable microtubules which was not prevented by prior exposure to K252a. In contrast, major reorganization of vimentin intermediate filaments elicited at 750 nM okadaic acid was prevented by prior exposure to K252a. Taxol pretreatment blocked the effects of okadaic acid on stable microtubules and vimentin intermediate filaments. Recent reports have revealed that taxol can activate cellular signal transduction pathways in addition to its known ability to promote microtubule stabilization, so the possibility that taxol-induced resistance of vimentin intermediate filaments to okadaic acid was through a microtubule-independent mechanism involving direct phosphorylation of intermediate filament proteins was explored. Vimentin immunoprecipitation from cytoskeletal extracts from 32P-labeled cells revealed that taxol (4 microM, 1 or 2 hours) caused about a 2-fold increase in vimentin phosphorylation. This phosphorylation was recovered exclusively in cytoskeletal vimentin, in contrast to the increased phosphorylation of soluble and cytoskeletal vimentin caused by exposure to 750 nM okadaic acid. Phosphorylation of soluble and cytoskeletal vimentin from cells exposed to taxol (4 microM, 1 hour) then okadaic acid (750 nM, 1 hour) was comparable to taxol-treatment alone. These findings demonstrate a novel new activity of taxol, induction of vimentin phosphorylation, that may impact on vimentin organization and stability.

Acetylation↗
PubMed1994

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.

Amino Acid Sequence↗
PubMed2000

Organization of desmin-containing intermediate filaments during differentiation of mouse decidual cells.

Decidualization of the mouse endometrium consists of a redifferentiation of the endometrial stromal fibroblasts. During decidualization these fibroblasts undergo growth, change of shape, multinucleation, and establishment of intercellular junctions. One feature of rodent decidual cells is the accumulation of intermediate filaments. In spite of the fact that fibroblasts normally have vimentin intermediate filaments, they acquire a large amount of desmin intermediate filaments while they undergo decidualization. The light and electron microscope immunocytochemical results of the present work show that during the initial stages of decidual transformation the desmin intermediate filaments accumulate around the nuclei, often forming caps around the nuclear envelope. As the decidual cells grow, the filaments form bundles and nets that radiate from the nuclei toward the cell surface. During the final stages of differentiation, on day 8 of pregnancy, staining of differentiated decidual cells decreases and most filaments accumulate under the cell surface. A role for intermediate filaments is suggested for decidualization of mouse endometrial cells.

Animals↗
PubMed1984

Intermediate filaments in the cytoskeletons of fish chromatophores.

When fish pigment cells (melanophores, erythrophores) are lysed by a modified Kleinschmidt method on a buffer-air interface and examined by electron microscopy, large numbers of intermediate filaments are observed. The intermediate filament networks are distinct from actin and tubulin, and entrap the pigment as determined by stereo viewing of freeze-dried rotary-shadowed specimens. During lysis, under conditions that do not preserve actin filaments or microtubules, the area covered by dispersed pigment granules reaches a maximum size and remains stable for many minutes, suggesting that intermediate filaments are responsible for holding the pigment in position and preventing further cytoplasmic dispersion. These observations demonstrate that fish pigment cells contain large numbers of intermediate filaments and suggest that they may be important for coordinating pigment granule movement.

Actins↗
PubMed1984

Distinction of nephroblastomas from other childhood tumors using antibodies to intermediate filaments.

Ten nephroblastomas were investigated by antibodies to intermediate filaments. In seven cases, which in light microscopy were characterized by the presence of blastema and tubules, immunofluorescence microscopy with IF-specific antibodies reveals expression of cytokeratin and vimentin in blastema cells, while tubules were only labelled by the cytokeratin antibodies. This result was independent of whether the conventional cytokeratin antibody or monoclonal antibodies specific for cytokeratin 18 were used. Stroma cells were vimentin-positive. In two cases nephroblastomas were undifferentiated and also lacked tubuli formation. In both these tumors blastema cells were vimentin-positive and cytokeratin-negative. Finally one case of clear cell sarcoma of the kidney could only be labelled by the vimentin antibody. Thus antibodies to intermediate filaments seem to be useful tools to distinguish nephroblastomas from neuroblastomas or rhabdomyosarcomas, especially in cases of metastasis.

Child↗
PubMed1999

Intermediate filament proteins during carcinogenesis and apoptosis (Review).

The intermediate filament network spreading from the cell periphery to the nucleus forms dynamic linkages between nuclear matrix, actin microfilaments, and the extracellular matrix. The six different types (types I-VI) of IF proteins consisting of nearly 50 different proteins form at least nine different kinds of filaments depending on the tissue types: keratins, lamins, vimentin, desmin, neurofilaments, peripherin, alpha-internexin, glial fibrillary acidic protein and nestin. Their tissue specific expression in normal cells and differential expression/assembly in neoplasia has been of immense value in tumor diagnosis. At the same time, recent in vitro studies point out that keratins, lamins and vimentin are subject to caspase-mediated proteolysis in an apoptosis-related manner. We reviewed the experimentally demonstrated P4-P1 motif specificities of caspases in the selection of substrates in the IF protein family. In addition, we provided clues to possible cleavage of additional IF proteins during programmed cell death, based on acceptable cut site motifs indicated by searches using the PIR protein sequence database. The present review concludes with presentation of evidence on the emerging roles of IFs in association with intermediate filament associated proteins in the dynamic remodeling of the cell during development of neoplastic phenotype and execution of apoptosis.

Amino Acid Sequence↗
PubMed1981

Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody.

Monoclonal antibodies (JLB1 and JLB7) that recognize minor components of the intermediate filament system of cultured cells were introduced into living fibroblasts by microinjection. Several minutes after injection of the JLB7 antibody virtually all of the intermediate filaments of the cells were found to be aggregated into tight bundles near or around the nucleus. In contrast, injection of the JLB1 antibody caused little or no aggregation of the intermediate filaments. Electron microscopy showed that the perinuclear bundles that formed after injection of the JLB7 antibody each consisted of ten or more filaments apparently crosslinked together. Double-label immunofluorescence microscopy showed that virtually all of the vimentin-containing intermediate filaments in the JLB7 antibody-injected cells were redistributed to the perinuclear region and remained there for at least 24 hr. The distributions of actin microfilaments and microtubules were seemingly undisturbed following microinjection. No obvious changes in cell morphology or behavior were apparent in the cells injected with JLB7 antibody; the cells displayed a flat appearance, showed a polarity, were able to ruffle and bleb and even appeared to show the normal saltatory movements of intracellular vesicles, granules and mitochondria, suggesting that intermediate filaments are not involved in these activities. The microinjection of highly specific monoclonal antibodies that recognize and alter components of the cell provides an additional approach to determine the in vivo functions of intracellular elements.

Animals↗
PubMed1984

Monoclonal antibodies to human intermediate filament proteins. II. Distribution of filament proteins in normal human tissues.

Monoclonal antibodies generated against different human intermediate filament (IF) proteins were assayed on fixed, embedded tissue by the biotin-avidin-immunoperoxidase method for evaluation of the tissue specificity of these antibodies. An antibody (43 beta E8) made to fibroblast IF protein stains mesenchymal tissue such as endothelium, histiocytes, stromal fibroblasts, and Schwann cells but does not stain epithelium, skeletal muscle, lymphocytes, or neurons. Three different anti-cytokeratin antibodies decorate epithelium in three unique patterns. One (35 beta H11) stains all nonsquamous epithelium but fails to recognize squamous epithelium. Antibody 34 beta E12 stains the full thickness of squamous epithelium and ductular epithelium but does not react with hepatocytes, pancreatic acinar cells, proximal renal tubules, or endometrial glands. Antibody 34 beta B4 stains only the suprabasal portion of squamous epithelium. None of these three anti-cytokeratin antibodies reacts with nerve or mesenchymal tissue. Two anti-neurofilament antibodies recognize only neurons, failing to react with epithelial or mesenchymal tissue. We conclude that these anti-intermediate filament antibodies can be used as tissue-specific markers. Neoplasms retain the same intermediate filament patterns as the normal parental tissue; therefore, these antibodies can be used as diagnostic aids in surgical pathology.

Antibodies, Monoclonal↗
PubMed1999

The cardiomyopathy and lens cataract mutation in alphaB-crystallin alters its protein structure, chaperone activity, and interaction with intermediate filaments in vitro.

Desmin-related myopathy and cataract are both caused by the R120G mutation in alphaB-crystallin. Desmin-related myopathy is one of several diseases characterized by the coaggregation of intermediate filaments with alphaB-crystallin, and it identifies intermediate filaments as important physiological substrates for alphaB-crystallin. Using recombinant human alphaB-crystallin, the effects of the disease-causing mutation R120G upon the structure and the chaperone activities of alphaB-crystallin are reported. The secondary, tertiary, and quaternary structural features of alphaB-crystallin are all altered by the mutation as deduced by near- and far-UV circular dichroism spectroscopy, size exclusion chromatography, and chymotryptic digestion assays. The R120G alphaB-crystallin is also less stable than wild type alphaB-crystallin to heat-induced denaturation. These structural changes coincide with a significant reduction in the in vitro chaperone activity of the mutant alphaB-crystallin protein, as assessed by temperature-induced protein aggregation assays. The mutation also significantly altered the interaction of alphaB-crystallin with intermediate filaments. It abolished the ability of alphaB-crystallin to prevent those filament-filament interactions required to induce gel formation while increasing alphaB-crystallin binding to assembled intermediate filaments. These activities are closely correlated to the observed disease pathologies characterized by filament aggregation accompanied by alphaB-crystallin binding. These studies provide important insight into the mechanism of alphaB-crystallin-induced aggregation of intermediate filaments that causes disease.

Cardiomyopathies↗
PubMed2006

"Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights.

Intermediate filaments (IFs) are major components of the mammalian cytoskeleton. They are among the most abundant cellular phosphoproteins; their phosphorylation typically involves multiple sites at repeat or unique motifs, preferentially within the "head" or "tail" domains. Phosphorylation and dephosphorylation are essential for the regulation of IF dynamics by modulating the intrinsic properties of IFs: solubility, conformation and filament organization, and, in addition, for the regulation of other IF post-translational modifications. These phosphorylation-regulated properties dictate generalized and context-dependent IF functions that reflect their tissue-specific expression. Most important among IF phosphorylation-mediated functions are the regulation of IF cellular or subcellular compartmentalization, levels and turnover, binding with associated proteins, susceptibility to cell stresses (including apoptosis), tissue-specific functions and IF-associated disease pathogenesis (where IF hyperphosphorylation also serves as a tissue-injury marker).

Animals↗
PubMed2003

Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis.

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase-promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.

Animals↗
PubMed2004

Clinical and neuropathologic variation in neuronal intermediate filament inclusion disease.

BACKGROUND: Recently described neuronal intermediate filament inclusion disease (NIFID) shows considerable clinical heterogeneity. OBJECTIVE: To assess the spectrum of the clinical and neuropathological features in 10 NIFID cases. METHODS: Retrospective chart and comprehensive neuropathological review of these NIFID cases was conducted. RESULTS: The mean age at onset was 40.8 (range 23 to 56) years, mean disease duration was 4.5 (range 2.7 to 13) years, and mean age at death was 45.3 (range 28 to 61) years. The most common presenting symptoms were behavioral and personality changes in 7 of 10 cases and, less often, memory loss, cognitive impairment, language deficits, and motor weakness. Extrapyramidal features were present in 8 of 10 patients. Language impairment, perseveration, executive dysfunction, hyperreflexia, and primitive reflexes were frequent signs, whereas a minority had buccofacial apraxia, supranuclear ophthalmoplegia, upper motor neuron disease (MND), and limb dystonia. Frontotemporal and caudate atrophy were common. Histologic changes were extensive in many cortical areas, deep gray matter, cerebellum, and spinal cord. The hallmark lesions of NIFID were unique neuronal IF inclusions detected most robustly by antibodies to neurofilament triplet proteins and alpha-internexin. CONCLUSION: NIFID is a neuropathologically distinct, clinically heterogeneous variant of frontotemporal dementia (FTD) that may include parkinsonism or MND. Neuronal IF inclusions are the neuropathological signatures of NIFID that distinguish it from all other FTD variants including FTD with MND and FTD tauopathies.

Adult↗
PubMed1997

Immunocytochemical localization of a novel radial glial intermediate filament protein.

We have examined by immunocytochemistry the subcellular localization of a chick radial glial protein, named transitin, that by molecular cloning has been shown to be a novel member of the intermediate filament protein superfamily. In astrocytes cultured from E10 chick brain, transitin is localized to the intermediate filament network in accordance with its structural properties. Using confocal microscopy we examined the expression of transitin, vimentin and glial fibrillary acidic protein (GFAP) in cultured astrocytes, and show that transitin co-distributes with these other glial intermediate filament proteins. The expression of transitin, vimentin and GFAP was also compared in embryonic chick spinal cord and brain radial glia, with these studies showing that these intermediate filament proteins display distinct expression patterns during CNS development. Of particular note is the absence of vimentin and GFAP in spinal cord midline radial glia that express transitin protein, and a transient expression of transitin in brain midline radial glia that continue to express vimentin. Our studies presented here therefore indicate that transitin, a novel radial glial intermediate filament protein, may have functions that are unrelated to GFAP or vimentin during CNS development, since transitin is localized to the processes of midline radial glia and is transiently expressed during chick CNS development.

Animals↗
PubMedDate not supplied

Immunohistochemical and ultrastructural observations on adenoid cystic carcinoma of salivary glands. With special reference to intermediate filaments and proteoglycan particles.

In adenoid cystic carcinoma (ACC) of salivary gland origin, intermediate filaments of tumour cells were investigated by immunohistochemical techniques and complex carbohydrates within the pseudocysts by ultrastructural methods. The intermediate filament proteins vimentin and cytokeratin were found in most neoplastic cells. The pseudocysts of the tumours were found to contain proteoglycan particles stainable after glutaraldehyde fixation--but not after osmium fixation alone. Ruthenium red (cationic dye) enhanced the staining whereas periodic acid-chromic acid-silver methenamine staining or phosphotungstic acid staining did not visualize the proteoglycan particles mainly containing acid proteoglycans which are normally produced by mesenchymal tissue. The cells of adenoid cystic carcinoma thus have an intermediate filament content and produce complex carbohydrates characteristic of both epithelial and mesenchymal tissue. Hence, an origin in an undifferentiated pluripotential cell seems plausible.

Carcinoma, Adenoid Cystic↗
PubMed1991

Association of glycosphingolipids with intermediate filaments of human umbilical vein endothelial cells.

Our previous studies of glycosphingolipids (GSLs) of human umbilical vein endothelial cells (HUVECs) established that globoside and ganglioside GM3 are the most abundant GSLs of HUVECs. Both compounds are located intracellularly, as well as on the cell surface. In this study, we demonstrate that the intracellular globoside and GM3 antigens are associated with the vimentin intermediate filaments of the HUVEC cytoskeleton. Immunofluorescence staining of fixed, permeabilized HUVECs showed colocalization of globoside and GM3 with vimentin but not with tubulin or actin. Both GSLs remained associated with intermediate filaments after perinuclear collapse of the filaments induced by colcemid. Indirect evidence that the globoside epitope is present on a GSL is the loss of staining by anti-globoside after methanol fixation and the absence of anti-globoside reactivity with HUVEC proteins on immunoblots. Colocalization of anti-globoside and anti-vimentin was also demonstrated in cryosections of endothelial cells, which indicates that the observed association was not an artifact induced by exposure of cells to detergent or organic solvent. Association of globoside with intermediate filaments was confirmed by immunoelectron microscopy, which demonstrated the presence of antigen along intermediate filaments, as well as on the cell surface and on lipid vesicles. Interferon-gamma decreased the ratio of surface to filamentous globoside staining, but had the opposite effect on GM3 distribution. Less abundant HUVEC GSLs, including Gb3, nLc4, IV2FucnLc4, and IV3NeuAcnLc4, were not detected along filaments. This is the first report of the association of GSLs with intermediate filaments. We suggest that intermediate filaments may play a role in the transport of GSLs.

Cell Membrane↗
PubMed1996

Experimental coexpression of vimentin and keratin intermediate filaments in human melanoma cells augments motility.

Intermediate filaments have been used as cell-type-specific markers in differentiation and pathology; however, recent reports have demonstrated the coexpression of vimentin (a mesenchymal marker) and keratins (epithelial markers) in numerous neoplasms, including melanoma, which has been linked to metastatic disease. To test the hypothesis that coexpression of vimentin and keratins by melanoma cells contributes to a more migratory and invasive phenotype, we co-transfected a vimentin-positive human melanoma cell line, A375P (of low invasive ability), with cDNAs for keratins 8 and 18. The resultant stable transfectants expressed vimentin- and keratin-positive intermediate filaments showed a two- to threefold increase in their invasion of basement membrane matrix and migration through gelatin in vitro. These findings were further corroborated by video cinematography. During attachment and spreading on fibronectin, the transfectants containing vimentin and keratins 8 and 18 demonstrated an increase in focal adhesions that stained positive for beta 1 integrin and phosphotyrosine, along with enhanced membrane ruffling and actin stress fiber formation. From these data, we postulate that coexpression of vimentin and keratins results in increased cytoskeletal interactions at focal contacts within extracellular matrices involving integrin cell signaling events, which contributes to a more migratory behavior.

Cell Adhesion↗
PubMed1986

Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles.

The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position.

Acrylamide↗
PubMed1997

Association of calponin with desmin intermediate filaments.

Our previous immunoelectron microscopy studies of chicken gizzard smooth muscle cells showed that in certain areas the distribution of anti-calponin exhibits a high degree of overlap with beta-actin, filamin, and in particular, desmin, suggesting that in situ a fraction of calponin may be associated with intermediate filaments of the cytoskeleton. In this work we further explore this idea by studying the interaction between calponin and desmin. We found that at physiological salt concentrations, calponin bound only weakly to synthetic desmin intermediate filaments. On the other hand, calponin bound strongly to nonfilamentous desmin tetramers and was incorporated into intermediate filaments when the two proteins were mixed in a buffer containing 6 M urea and dialyzed into a buffer containing 0.15 M NaCl. Anti-calponin was found to label a portion of intermediate filaments and dense bodies isolated from gizzard tissues. Our findings suggest that in chicken gizzard smooth muscle cells, calponin may be an integral component of desmin intermediate filaments in the vicinity of dense bodies. Since calponin is also known to bind actin, we hypothesize that one of the functions of calponin might be to bridge intermediate filaments with actin in dense bodies.

Animals↗