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PubMed2001

Intermediate filaments regulate astrocyte motility.

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell-type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.

Animals↗
PubMed1990

Interactions of intermediate filaments with cell structures.

Intermediate filaments (IF) are unique components of the cytoskeleton of most eukaryotic cells. Also the nuclear lamins are now recognized to be IF-like proteins, providing the nucleus with a putative skeleton for chromatin attachment. Immunofluorescence and whole-mount electron microscopic studies reveal that IF form a cytoplasmic network that surrounds the nucleus and extends to cell surface, as 'mechanical integrators of cellular space'. It seems however unlikely that IF in the cell accomplish a merely structural role, considering the diversity of IF proteins and the complex regulation of their gene expression. In this work we primarily present electron microscopic data that points to the presence of interactions between IF and several cellular components, namely the nucleus, plasma membrane, other cytoskeletal elements, cytoplasmic organelles and ribonucleoproteins. Although the functional significance of such interactions remains to be demonstrated, assumptions like involvement of IF in information transfer or cytoskeleton-dependent control of gene expression represent attractive hypothesis for future research.

Animals↗
PubMed2005

Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.

The intermediate filament (IF)-binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell-cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 microm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell-cell contact and regulated by actin and DP-IF interactions.

Actin Cytoskeleton↗
PubMed1981

Distribution of intermediate filaments in amphibian oxyntic cells. Biochemical and immunological characterization.

Intermediate filaments of toad oxyntic cells were isolated and analysed by SDS-PAGE. The major proteins of the residue were identified as actin and a 51,000 dalton polypeptide. Immunological crossreactivity between toad oxyntic cell intermediate filament components and anti-prekeratin, was shown by double immunodiffusion tests and indirect immunofluorescence. The immunofluorescent decoration of oxyntic cells and the electron microscope images are coincident in locating the intermediate filaments mainly at the cortical and perinuclear basal zones. Furthermore, the cortical zone appears especially rich in prekeratin-like material at its adluminal third. This results in a cup-like structure that encloses the cell portion occupied by the tubulovesicular system, which does not contain intermediate filaments. The translocation of membranes occurring during the secretory cycle of the oxyntic cell, has been attributed to a system of contractile proteins. The disposition of the prekeratin-like material suggests a role for intermediate filaments in the generation of movement, produced by actin and myosin interaction, by providing a fixed plane for the anchoring of actin microfilaments.

Actins↗
PubMed1999

alphaB-crystallin interacts with cytoplasmic intermediate filament bundles during mitosis.

The small heat-shock protein alphaB-crystallin interacts with intermediate filament proteins. Using cosedimentation assay, we showed previously that in vitro binding of alphaB-crystallin to peripherin and vimentin was temperature-dependent. Furthermore, when NIH 3T3 cells were submitted to different stress conditions a dynamic reorganization of the intermediate filament network was observed concomitantly with the recruitment of alphaB-crystallins on the intermediate filament proteins. Thus, the intracellular state of alphaB-crystallin correlated directly with the remodeling of the intermediate filament network in response to stress. Here, we show data suggesting that alphaB-crystallin is implicated in remodeling of intermediate filaments during cell division. We investigated the intracellular distribution of alphaB-crystallin in naturally occurring mitotic NIH 3T3 cells and in neuroblastoma N2a and N1E115 cells. In NIH 3T3 cells, alphaB-crystallin remained diffused throughout the cell cycle. Subcellular fractionation of alphaB-crystallin showed that alphaB-crystallin remained in the cytosolic compartment during mitosis. Furthermore, alphaB-crystallin accumulated in mitotically arrested NIH 3T3 cells. This increased level of alphaB-crystallin protein was due to an increased level of alphaB-crystallin mRNA in mitotic NIH 3T3 cells. In the neuroblastoma cells, the intermediate filaments were rearranged into thick cable-like structures and alphaB-crystallin was recruited onto them. In neuroblastoma N2a cells the level of expression did not change during the cell cycle. However, a small fraction of alphaB-crystallin switched onto the insoluble fraction in mitotically arrested N2a cells. Our results suggested that depending on the state of rearrangement of the intermediate filament network during mitosis alphaB-crystallin was either recruited onto the intermediate filaments or upregulated in the cytosolic compartment.

3T3 Cells↗
PubMed2000

Association of protein phosphatase 2A with its substrate vimentin intermediate filaments in 9L rat brain tumor cells.

The importance of protein phosphatases in maintaining the integrity of intermediate filaments is supported by the fact that intermediate filaments would undergo a massive reorganization in cells treated with inhibitors of protein phosphatases 1 and 2A. Herein we used okadaic acid to investigate the differential roles of protein phosphatases 1 and 2A in the maintenance of intermediate filament integrity in 9L rat brain tumor cells. Protein phosphatase 2A activity was substantially inhibited after treatment with 400 nM okadaic acid for 2 h, whereas the activity of protein phosphatase 1 was only slightly affected. Furthermore, protein phosphatase 2A shows selective specificity toward phosphovimentin, which was immunologically precipitated from isotopically labeled and okadaic acid-treated cells. Further biochemical fractionation and microscopic studies revealed that vimentin intermediate filaments were colocalized with protein phosphatase 2A, but not protein phosphatase 1, in control cells. On okadaic acid treatment, vimentin filament disassembled and protein phosphatase 2A redistributed throughout the cytoplasm, suggesting that these two proteins separate from each other, whereas protein phosphatase 2A was inhibited. This working hypothesis was further supported by treatment with a low concentration (40 nM) of okadaic acid, which causes the same phenomenon. Taken together, our results showed that protein phosphatase 2A could be assigned to the intermediate filaments to serve the physiological role in maintaining the proper phosphorylation level of intermediate filaments in normal cells. This finding should pave the way for the elucidation of the regulatory mechanism of intermediate filament organization governed by protein phosphorylation.

Animals↗
PubMed1992

Intermediate filament structure.

In the past year, several new developments concerning the structure of intermediate filament proteins and their assembly into intact intermediate filaments have been made: the coiled-coil structure of a rod domain has been elucidated; the basis of the chain interaction and its role in intermediate filament assembly has been specified; the organization of nearest-neighbour molecules in keratin intermediate filaments has been determined; and the glycine loop structures of the terminal domains of epidermal keratin chains have been defined. In addition, mutations in intermediate filament chains that promote pathology have been reported for the first time.

Animals↗
PubMed1988

Intermediate filaments in mouse taste bud cells.

The intermediate filaments in mouse taste bud cells were studied by immunocytochemistry using antikeratin antibodies, and by conventional electron microscopy. Taste bud cells (types I, II, and III) possessed less densely aggregated bundles of intermediate filaments than the surrounding epithelial cells. Type III cells, however, contained more densely aggregated bundles than type I or II cells. Basal cells in the taste buds showed aggregations of filaments as dense as those seen in the epithelial cells, although their bundles were more slender than those of the epithelial cells. The antibodies to keratins from the bovine muzzle and human stratum corneum stained all types of the taste bud cells as well as the surrounding epithelial cells. PKK2 antibody reacted with the surrounding epithelial cells, but did not react with the taste bud cells. These results show that keratins are present in both taste bud and surrounding epithelial cells, although the keratin subtype differs between those cells. This finding has led us to the supposition that all cell types comprising the taste buds--including type III (receptor) cells--originate from the epithelial cells surrounding the taste buds. It is also suggested that both keratin subtypes and aggregation patterns of intermediate filament bundles change during differentiation from surrounding epithelial cells to taste bud cells, and from basal cells in the taste buds to types I, II, or III cells.

Animals↗
PubMed1987

Neurons and glia in purified retinal cultures identified by monoclonal antibodies to intermediate filaments.

Two monoclonal antibodies known to bind intermediate filaments were used to identify neurons and glia from embryonic chick neural retina. Neurofilament specific antibody RT-97-F1, bound neuroepithelial cells, migrating neurons, as well as the photoreceptor layer, plexiform layers and optic fiber layer. The other, 3A7, bound intermediate filaments of Müller cells. In purified neuronal cultures, RT-97-F1 bound 75, 83 and 98% of the cells present at 24, 48 and 72 h respectively, while 3A7 bound 26, 15 and 0.3% for the same times in vitro. In purified glial cultures, RT-97-F1 showed a weak perinuclear binding, while 3A7 strongly bound intermediate filaments of nearly 100% of the cells. These antibodies confirmed and quantitated the high purity of our cultures.

Animals↗
PubMed1994

Relationship of nuclear invaginations to perinuclear rings composed of intermediate filaments in MIA PaCa-2 and some other cells.

There have been few investigations of the cause of nuclear invagination and lobule formation. The human pancreatic cancer cells MIA PaCa-2 often show nuclear lobulation and well-developed juxtanuclear aggregates of intermediate filaments with thick bundles of intermediate filaments developed from them. Therefore MIA PaCa-2 cells were used as model cells to examine whether or not there is any relationship between the shape of the nucleus and intermediate filaments. Immunoblotting showed that the intermediate filament proteins in MIA PaCa-2 cells are vimentin, and keratins 8, 18, and 19. Fluorescence microscopy and confocal laser scanning fluorescence microscopy revealed perinuclear rings composed of intermediate filaments, that is, thick bundles of both vimentin and keratin filaments run along deep invaginations in the nucleus and, together with juxtanuclear intermediate filament aggregates, they form closed rings around nuclear invaginated and constricted sites. In other cells such as human bladder carcinoma T24 cells, human melanoma G-361 cells, and human cervix carcinoma HeLa/S3 cells, there were also thick bundles of intermediate filaments cutting into the nucleus at the nuclear invagination site. Thus, it seems that the formation of perinuclear intermediate filament rings may be involved in nuclear invagination and lobule formation in some mammalian cells.

Antibodies, Monoclonal↗
PubMed1988

Cyclic AMP-dependent protein kinase-induced vimentin filament disassembly involves modification of the N-terminal domain of intermediate filament subunits.

The intermediate filament protein vimentin was phosphorylated with cAMP-dependent protein kinase under conditions that induce filament disassembly. Digestion of phosphorylated vimentin with lysine-specific endoprotease and subsequent tryptic peptide mapping indicated that a 12 kDa N-terminal fragment contained all the phosphorylation sites found in the intact molecule. Analysis of cyanogen bromide digests indicated that two phosphorylated peptides were produced, with the major 32P-labeled species representing amino acid position 14-72, and a minor 32P-labeled peptide representing amino acid positions 1-13. These results demonstrate that phosphorylation of sites within the N-terminal head domain of vimentin are associated with phosphorylation induced filament disassembly.

Adenosine Triphosphate↗
PubMed1993

Skelemin, a cytoskeletal M-disc periphery protein, contains motifs of adhesion/recognition and intermediate filament proteins.

In striated muscle, myofibrils are anchored to an interconnecting cytoskeleton of desmin intermediate filaments. Skelemin (195 kDa) may be a link between myofibrils and the intermediate filament cytoskeleton. Skelemin partitions with desmin to the insoluble cytoskeleton, and increases the thickness of reconstituted intermediate filaments. Concentrated at the M-disc periphery, skelemin may also contact myosin filaments. We used immunoscreening to isolate a mouse muscle cDNA which encodes a protein with a calculated molecular mass of 185 kDa. Anti-skelemin antibodies bound to the protein products of each of three nonoverlapping regions of the open reading frame. Antibodies directed against the protein products of each one-third of the cDNA react with a 195-kDa muscle protein and stain the M-disc indistinguishably from the original anti-skelemin antibodies, suggesting that the cDNA encodes skelemin. A single skelemin mRNA is detected in muscle but not non-muscle tissues, consistent with immunostaining results. Skelemin is a member of a family of myosin-associated proteins containing fibronectin type III and immunoglobulin superfamily C2 motifs. Skelemin is unique in this family in having intermediate filament core-like motifs, one near each terminus. We hypothesize that skelemin could interact with myosin or myosin-associated proteins through its fibronectin and/or immunoglobulin motifs, and with intermediate filaments through intermediate filament-like motifs.

Amino Acid Sequence↗
PubMed1985

Clonal variations in keratin. Intermediate filament expression by human somatic cell hybrids.

The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.

Cell Fusion↗
PubMed2002

Caspase proteolysis of desmin produces a dominant-negative inhibitor of intermediate filaments and promotes apoptosis.

Caspase cleavage of key cytoskeletal proteins, including several intermediate filament proteins, triggers the dramatic disassembly of the cytoskeleton that characterizes apoptosis. Here we describe the muscle-specific intermediate filament protein desmin as a novel caspase substrate. Desmin is cleaved selectively at a conserved Asp residue in its L1-L2 linker domain (VEMD downward arrow M(264)) by caspase-6 in vitro and in myogenic cells undergoing apoptosis. We demonstrate that caspase cleavage of desmin at Asp(263) has important functional consequences, including the production of an amino-terminal cleavage product, N-desmin, which is unable to assemble into intermediate filaments, instead forming large intracellular aggregates. Moreover, N-desmin functions as a dominant-negative inhibitor of filament assembly, both for desmin and the structurally related intermediate filament protein vimentin. We also show that stable expression of a caspase cleavage-resistant desmin D263E mutant partially protects cells from tumor necrosis factor-alpha-induced apoptosis. Taken together, these results indicate that caspase proteolysis of desmin at Asp(263) produces a dominant-negative inhibitor of intermediate filaments and actively participates in the execution of apoptosis. In addition, these findings provide further evidence that the intermediate filament cytoskeleton has been targeted systematically for degradation during apoptosis.

Animals↗
PubMed1990

Human immunodeficiency virus type 1 protease cleaves the intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein.

The intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein are cleaved in vitro by human immunodeficiency virus type 1 protease (HIV-1 PR). Microsequencing showed that HIV-1 PR cleaved both human and murine vimentin between leucine-422 and arginine-423 within the sequence between positions 418 and 427, Ser-Ser-Leu-Asn-Leu/Arg-Glu-Thr-Asn-Leu (SSLNL/RETNL). Minor cleavages at other sites were also observed. Heat-denatured vimentin was cleaved by HIV-1 PR less efficiently than native vimentin. A decapeptide containing the sequence SSLN-LRETNL was also cleaved in vitro by HIV-1 PR as predicted. The presence of a charged residue (arginine) at the primary cleavage site distinguishes this from other known naturally occurring cleavage sites. Microinjection of HIV-1 PR into cultured human fibroblasts resulted in a 9-fold increase in the percentage of cells with an altered and abnormal distribution of vimentin intermediate filaments. Most commonly, the intermediate filaments collapsed into a clump with a juxtanuclear localization. These results support the possibility that intermediate filament proteins may serve as substrates within HIV-1-infected cells.

Amino Acid Sequence↗
PubMed1985

Immunofluorescent localization of intermediate filament subunits for the differential diagnosis of malignant melanoma.

Intermediate filament subunits in normal cells and in their malignant derivatives can be used as specific markers for their histogenetic origins. We have studied five neoplasms of the skin in which positive identification of vimentin containing intermediate filaments by indirect immunofluorescence microscopy helped to establish the diagnosis of malignant melanoma. All of the neoplasms included in this study posed problems in differential diagnosis by conventional light microscopy and yielded equivocal results by conventional histochemistry. Thus, definitive distinction between poorly differentiated carcinoma and poorly differentiated melanoma could not be made by conventional microscopy. In all of the neoplasms described here, immunolabeling with antibodies against different intermediate filaments demonstrated positive staining for vimentin only. This intermediate filament subunit is present in melanocytes (as well as in many mesenchymal cells) but not in epithelial cells. Our study indicates that this technique may be valuable in differential diagnosis of malignant melanoma, particularly in instances where cells lack melanin or show other atypical morphologic features.

Aged↗
PubMed2001

Molecular polymorphism of the intermediate filament protein transitin.

Transitin is an avian intermediate filament protein whose transient expression in the progenitor cells of the muscle and nerve tissues is similar to that of mammalian nestin. Both proteins contain an alpha-helical core domain flanked by a short N-terminal head and a long C-terminal extremity. However, the tail region of transitin is significantly different from that of nestin in that it harbors a unique motif containing more than 50 leucine zipper-like heptad repeats which is not found in any other intermediate filament protein. Despite the absence of introns in this region of the transitin gene, it was reported that different isoforms of the protein were produced by exclusion or inclusion of a number of repeats generated by an unusual splicing mechanism recognizing consensus 5' and 3' splice sites contained within the coding sequence of the heptad repeat domain [Napier et al. (1999) J Mol Neurosci 12:11-22]. Two monoclonal antibodies (mAbs) reacting with repeated epitopes of this motif were used to monitor transitin expression during in vitro myogenesis of the quail myogenic cell line QM7. Confocal microscopy revealed that the subcellular domains decorated with mAbs A2B11 and VAP-5 were mutually exclusive: the intermediate filament network visualized with mAb VAP-5 appeared to abut on a submembranous domain defined by mAb A2B11. When QM7 cells were induced to differentiate by switching to medium containing low serum components, an early effect was the local loss of A2B11 cortical staining at the points of cell-cell contacts. The A2B11 signal also disappeared before that of VAP-5 in newly formed myotubes. Unexpectedly, the mutually exclusive staining pattern of the mAbs could not be explained by alternative splicing since both epitopes mapped to a repeated element preceding the consensus 5' splice sites of the heptad repeat domain. An alternative explanation would be that the central repeat domain of transitin is a polymorphic structure from which different conformations exist depending on the local context. This hypothesis is strengthened by the observation that in cultured neural crest cells, the A2B11 antigen is preferentially expressed by freely migrating crest cells whose intracellular pH and calcium concentrations are different from those of non-migrating cells.

Amino Acid Sequence↗
PubMed2000

Fingerprinting taste buds: intermediate filaments and their implication for taste bud formation.

Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non-gustatory adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primordia, cytokeratin 20, is not detectable prior to the ingrowth of nerve fibres into the epithelium, which supports the hypothesis that nerve fibres are necessary for initiating taste bud development. Another intermediate filament protein, vimentin, occurs in derivatives of mesoderm, but usually not in epithelium. In humans, vimentin immunoreactivity is expressed mainly in border (marginal) epithelial cells of taste bud primordia, while in chick, vimentin expression occurs in most taste bud cells, whereas non-gustatory epithelium is vimentin immunonegative. Our chick data suggest a relationship between the degree of vimentin expression and taste bud cell proliferation especially during the perihatching period. It is suggested that surrounding epithelial cells (human) and mesenchymal cells (chick) may be contributing sources of developing taste buds. The dense perinuclear network of intermediate filaments especially in dark (i.e. non-sensory) taste disc cells of Xenopus indicates that vimentin filaments also might be associated with cells of non-gustatory function. These results indicate that the mechanisms of taste bud differentiation from source tissues may differ among vertebrates of different taxa.

Animals↗