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PubMed1983

Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken cardiac muscle.

We studied the localization of desmin (skeletin), the major protein subunit of muscle-type intermediate filaments, in adult chicken cardiac muscle by high resolution immunoelectron microscopic labeling of ultrathin frozen sections of the intact fixed tissues. We carried out single labeling for desmin and double labeling for both desmin and either vinculin or alpha-actinin. In areas removed from the intercalated disk membranes, we observed desmin labeling between adjacent Z-bands in every interfibrillar space. Where these spaces were wide and contained mitochondria, convoluted strands of desmin labeling bridged between the periphery of neighboring Z-bands and the mitochondria. The intermediate filaments appeared to be organized in a more three-dimensional manner within the interfibrillar spaces of cardiac as compared to skeletal muscle. Near the intercalated disks, desmin labeling was intense within the interfibrillar spaces, but was completely segregated from the microfilament attachment sites (fascia adherens) where vinculin and alpha-actinin were localized. Desmin therefore appears to play no role in the attachment of microfilaments to the intercalated disk membrane. We discuss the role of intermediate filaments in the organization of cardiac and skeletal striated muscle in the light of these and other results.

Actinin↗
PubMed1984

Differential expression of intermediate-filament proteins in murine sarcoma 180 ascites or solid tumor.

The intermediate-filament proteins in Sarcoma 180 ascites cells and solid tumors generated by s.c. injection of ascites cells in NMRI or nude mice were analyzed by one- and two-dimensional gel electrophoresis and identified by immunological methods. The ascites form of Sarcoma 180 coexpresses keratin and vimentin, whereas the solid tumor ceases to synthesize keratins but continues to express vimentin. These reversible changes in the expression of intermediate-filament proteins may be due to a change in the differentiation program induced by environmental conditions like growth with or without cell contact.

Animals↗
PubMed1996

Colocalization of three types of intermediate filament proteins in perisinusoidal stellate cells: glial fibrillary acidic protein as a new cellular marker.

The presence and the colocalization of the three intermediate filament proteins, glial fibrillary acidic protein (GFAP) and the marker of mesenchymal liver cells, vimentin, were studied by an immunofluorescence double-labeling technique in cultures of isolated rat perisinusoidal stellate cells (PSC) and hepatocytes, in cocultures of isolated PSC and hepatocytes as well as in cryostat sections of rat liver. GFAP and vimentin immunoreactivities were localized in cultured PSC which were identified by the presence of the cellular marker desmin, another intermediate filament protein, or the stellate morphology to be seen after staining for one of three intermediate filament proteins. Both GFAP and vimentin were strongly expressed in the perinuclear region and the cell processes of cultured PSC. Staining for GFAP highly coincided with that for vimentin or desmin in cultured PSC and with that for vimentin in the liver sections. Desmin-positive cells were always also GFAP-positive. However, of the GFAP-positive cells only an estimated 50% were found desmin-positive. The coexpression of desmin and GFAP in the same cells appear to be unique, since apparently it has not been previously reported for any other cell type. Almost all of the vimentin-positive cells in hepatocyte culture were also expressing GFAP. Since desmin was not found in all of the cultured cells with PSC morphology, GFAP is suggested as a more reliable marker for PSC than desmin.

Animals↗
PubMed1982

Structure of fibroblastic intermediate filaments: analysis of scanning transmission electron microscopy.

The structure of fibroblastic intermediate filaments from Chinese hamster ovary cells has been investigated by scanning transmission electron microscopy. Freshly extracted (native) filaments were compared with filaments reassembled in vitro from purified decamin. From digital micrographs of unstained specimens, direct measurements of linear mass density were performed on many individual filaments. Native filaments beyond a certain minimal length constitute a homogeneous population, averaging 38 +/- 4 kilodaltons (kDal)/nm. A minor but distinct polymorphic variant (23 +/- 4 kDal/nm) was also present as very short filaments or end-segments; these may represent breakdown products or assembly intermediates. Analysis of reassembled filaments demonstrates that the in vitro assembly reaction is--in the main--faithful, although the distribution of their mass measurements is appreciably broader than that of the native data. In addition to the predominant type at 37 +/- 4 kDal/nm and a minor component at 26 +/- 4 kDal/nm, small amounts of a third, more massive, polymorphic variant at 52 +/- 5 kDal/nm were also present. Micrographs of negatively stained specimens clearly demonstrate that the filaments are composed of bundles of protofilaments--each 2-3 nm in diameter--and also reveal an axial periodicity of about 46 nm. The implications of these findings are discussed for three classes of model previously proposed for the structure of intermediate filaments.

Animals↗
PubMed1997

Ultrastructure of intermediate filaments of nestin- and vimentin-immunoreactive astrocytes in organotypic slice cultures of hippocampus.

Glial cells in rat hippocampal slices cultured for 4 weeks were examined with immunocytochemical and cryoelectron microscopical methods. Astrocytes possessing long processes were similarly stained with antibodies against nestin, vimentin, and glial fibrillary acidic protein as seen by confocal microscopy. The three antibodies also labeled intermediate filaments in these astrocytes. In order to examine the fine structure of these intermediate filaments, slices were rapid-frozen for freeze-substitution and freeze-etching. By freeze-substitution the processes of the astrocytes were packed with large hundles of intermediate filaments. In rapid-freeze deep-etched slices, these filaments were often interconnected with filamentous cross-bridges. These cross-bridges were rather uniform in size and shape (mean 2.9 nm thick and 14.8 nm long). These results suggest that the filament network with these cross-linkers is important for shaping the long processes of nestin- and vimentin-immunoreactive astrocytes in slice cultures.

Animals↗
PubMed2000

Bundle formation of smooth muscle desmin intermediate filaments by calponin and its binding site on the desmin molecule.

Smooth muscle basic calponin, a major actin-, tropomyosin-, and calmodulin-binding protein, has been examined for its ability to interact with desmin intermediate filaments from smooth muscle cells using sedimentation analysis, turbidity changes, chemical cross-linking, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/MS), and electron microscopic observations. Calponin interacted with desmin intermediate filaments in a concentration-dependent manner in vitro. The binding of calponin to desmin produced dense aggregates at 30 degrees C. The dense aggregates were observed by electron microscopy to be composed of large anisotropic bundles of desmin filaments, indicating that calponin forms bundles of desmin filaments. The addition of calmodulin or S100 to the mixture of calponin and desmin caused the removal of calponin from the desmin filaments and inhibited bundle formation in the presence of Ca(2+), but not in the presence of EGTA. Calponin-related proteins including G-actin, tropomyosin, and SM22, had little effect on the binding of calponin to desmin filaments, whereas tubulin weakly inhibited the binding. Desmin had little influence on the calponin-actin and calponin-tubulin interactions using the zero-length cross-linker, EDC. Domain mapping with chymotryptic digestion showed that the binding site of calponin resides within the central a-helical rod domain of the desmin molecule. The chemical cross-linked products of calponin and synthetic peptides (TQ27, TNEKVELQELNDRFANYIEKVRFLEQQ; EE24, EEELRELRRQVDALTGQRARVEVE) derived from the rod domain were detected by MALDI TOF/MS. Furthermore, the calponin-desmin interaction was significantly inhibited by the addition of EE24, but only slightly by TQ27. These results suggest that calponin may act as a cross-linking protein between desmin filaments as well as among intermediate filaments, microfilaments and microtubules in smooth muscle cells.

Actins↗
PubMed1985

Intermediate filaments in cultured human pleomorphic adenomas. An immunohistochemical study.

Primary cultures of 9 benign human pleomorphic adenomas were analyzed by immunofluorescence and antibodies against the different intermediate filaments and microfilaments. The cultured cells were also cytogenetically characterized by G- and C-banding techniques. The adenoma cells expressed at least three different classes of intermediate filaments, viz. prekeratin, vimentin and glial fibrillary acidic protein (GFA). No correlation between chromosomal pattern and intermediate filament expression was found. The frequency of cells stained for each of the different antibodies varied considerably among the tumours. Prolonged culturing appeared to induce a reduction of cells positive for prekeratin. This was probably a true loss of antigens and not an effect of overgrowth of stromal cells. It is concluded that the cells studied, on the basis of several criteria, are of neoplastic origin. This in vitro system appears to be well suited for further histogenetic studies on pleomorphic adenomas.

Actin Cytoskeleton↗
PubMed1983

Visualization of longitudinally-oriented intermediate filaments in frozen sections of chicken cardiac muscle by a new staining method.

When ultrathin frozen sections of chicken cardiac muscle were osmicated, dehydrated in ethanol, embedded in ethyl cellulose, and stained with acidic uranyl acetate, filaments of 10-12 nm width were visualized in wide interfibrillar spaces. Immunostaining of the frozen sections for desmin resulted in exclusive labeling of such filaments. These observations indicated that longitudinally oriented networks of intermediate filaments were present in the interfibrillar spaces, in addition to the transversely oriented networks that surround myofibrils at the level of Z band. As in skeletal muscle (Tokuyasu, K. T., A. H. Dutton, and S. J. Singer, 1983, J. Cell Biol. 97:1727-1735), desmin in chicken cardiac muscle is believed to be largely, if not entirely, in the form of intermediate filaments.

Animals↗
PubMed1982

Immunoglobulin M autoantibody to vimentin intermediate filaments.

Serum from a patient with the CREST Syndrome and systemic lupus erythematosus contained an IgM antibody that reacted at dilutions up to 1:800 with a fibrous cytoplasmic network in several epithelioid and fibroblastic cell lines. The antibody was shown by immunofluorescence microscopy to label a specific subset of cytoskeletal polymers, the intermediate filaments. The reactive antigen from this biochemically heterogeneous group of filaments was established as the 58,000-mol wt protein, vimentin: (a) the patient's serum reacts with a range of cell lines that contain intermediate filaments composed of vimentin, but not with cells whose intermediate filaments are composed of different protein subunits; (b) in PTK2 epithelioid cells the serum reacts with the class of filaments that coils around the nucleus after colchicine treatment (vimentin) and not with the filaments that remain dispersed after colchicine (prekeratin); and (c) the component of reactive cells that combines with the serum is shown by immunoelectrophoresis to be a 58,000-mol wt protein antigen. A similar antibody that binds intermediate filaments of PTK2 cells was encountered at lower titer in some sera from other patients with connective tissue diseases and in control sera. Previous routine antinuclear antibody assays using mouse liver or commercially prepared HEp-2 cells have failed to reveal anticytoskeletal antibodies in patient sera, perhaps due to inadequate presentation or preservation of cytoplasmic antigens.

Adult↗
PubMed2001

A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton.

The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.

Actins↗
PubMed1998

Decreased synthesis of glycosphingolipids in cells lacking vimentin intermediate filaments.

We are studying defects in glycosphingolipid synthesis in cells lacking vimentin intermediate filaments (vimentin-). Sugars can be incorporated into glycolipids whose ceramide is synthesized either de novo (pathway 1) or from sphingoid bases salvaged from hydrolysis of sphingolipids (pathway 2) and into glycolipids recycling from the endosomal pathway through the Golgi (pathway 3). Vimentin- embryonic fibroblasts, obtained from vimentin-knockout mice, incorporate less sugar into glycolipids than vimentin+ fibroblasts. Using two inhibitors of ceramide synthesis, beta-chloroalanine and fumonisin B1, we found the major defect in synthesis to be in pathway 2 and not in de novo synthesis. We used two additional approaches to analyze the functions of pathways 2 and 3. First, we used exogenous glucosylthioceramide ([14C]C8-Glc-S-Cer), a synthetic, nonhydrolyzable glycosphingolipid, as a precursor for synthesis of larger glycolipids. Vimentin- SW13 cells and embryonic fibroblasts glycosylated [14C]C8-Glc-S-Cer less extensively than their vimentin+ counterparts. Second, we used chloroquine to inhibit the hydrolysis of sphingolipids in endosomes and lysosomes. Chloroquine markedly decreased the incorporation of sugars into glycolipids larger than glucosylceramide. The defect in glycolipid synthesis in vimentin- cells probably results from impaired intracellular transport of glycolipids and sphingoid bases between the endosomal/lysosomal pathway and the Golgi apparatus and endoplasmic reticulum. Intermediate filaments may accomplish this function by contributing to the organization of subcellular organelles and/or by binding proteins that participate in transport processes.

Animals↗
PubMed1984

Tumor promoters induce a specific morphological signature in the nuclear matrix-intermediate filament scaffold of Madin-Darby canine kidney (MDCK) cell colonies.

Tumor promoters such as phorbol 12-tetradecanoate 13-acetate (TPA), mezerein, teleocidin, aplysiatoxin, and benzoyl peroxide, although structurally unrelated, induce similar, profound changes in morphology in differentiated epithelial Madin-Darby canine kidney (MDCK) cell colonies. The alteration is evident in the organization of intermediate filaments in intact cells and in whole mounts of the nuclear matrix-intermediate filament (NM-IF) scaffold of the epithelial sheet. This substructure, obtained by salt extraction of the cytoskeletal framework, represents only 5% of the total cell protein but contains all of the intermediate filaments, nuclear matrix, and desmosomal core proteins arranged essentially as in the intact cell. The NM-IF is profoundly reorganized after exposure to TPA and retains the morphological changes observed in intact cells. These include bundling of the intermediate filaments, disruption of cell-cell borders, and marked deformation of the polygonal geometry of epithelia. Thus, TPA and all other complete or second-stage tumor promoters examined have a characteristic morphological signature that is not induced by mitogens, metabolic inhibitors, or agents known to disrupt microtubules or microfilaments. This signature, characteristic of tumor promoters, occurs in the absence of both protein and RNA synthesis. These results suggest that this response is prior to and independent of other biochemical markers for tumor promoters. Of the major filament systems, the cytokeratin network is implicated as an early or possibly primary site of tumor-promoter action because characteristics of the promoted cytoskeletal signature are observed in epithelial colonies after prior exposure to colchicine or cytochalasin D. Despite the massive reorganization of cytoskeletal morphology induced by TPA, the distribution of prelabeled proteins into structural fractions (i.e., cytoskeletal, chromatin, and the NM-IF) remains essentially unchanged. The sensitivity and specificity of the epithelial cell response suggest its possible use as a screen for promoting compounds.

Alkaloids↗
PubMed1983

Functional changes of intermediate filaments in fibroblastic cells revealed by a monoclonal antibody.

We describe reversible changes of intermediate filaments of fibroblastic cells associated with changes in the functional state of the cells. The changes are revealed by comparing the immunofluorescence patterns given by a monoclonal antibody and a polyclonal serum, both recognizing vimentin. The state of the filaments depends on culture density; this effect cannot be attributed to the nutritional state of the cells, their growth rate, or substances released into the medium. It seems to depend mainly on the aggregation of filaments during strong cell movements. The possible significance of these findings for the functional role of intermediate filaments is discussed.

Animals↗
PubMed1988

Intermediate filaments: a review of the basic biology.

This review presents the current knowledge of the basic biology of intermediate filaments, including their phylogenetic distribution and their distribution within mammalian cells. The current understanding of their structure, recently described using recombinant DNA, peptide sequencing, and immunologic methods, is discussed in terms of a revised chemical classification that not only includes the acidic and neutral basic keratins, vimentin, glial fibrillary protein, desmin, and neurofilament, but also includes the more recently related nuclear lamins. This new structural knowledge allows a more rational approach to the diagnostic use and development of polyclonal and monoclonal anti-intermediate filament antibodies. Many of the problems encountered with fixation, cross-reactivity, and epitope masking can be related to these structural concepts.

Animals↗
PubMed2002

Effects of Charcot-Marie-Tooth-linked mutations of the neurofilament light subunit on intermediate filament formation.

Neurofilaments (NFs) are the major intermediate filaments (IFs) of mature neurons. They play important roles in the structure and function of axons. Recently, two mutations in the neurofilament light (NFL) subunit have been identified in families affected by Charcot-Marie-Tooth (CMT) neuropathy type 2. We have characterized the effects of these NFL mutations on the formation of IF networks using a transient transfection system. Both mutations disrupted the self-assembly of human NFL. The Q333P mutant in the rod domain of NFL also disrupted the formation of rat and human NFL/NFM heteropolymers. The phenotypes produced by the P8R mutation in the head domain of NFL were less severe. The P8R mutant NFL co-polymerized with NFM to form bundled filaments and, less often, aggregates. Our results suggest that alterations in the formation of a normal IF network in neurons elicited by these NFL mutations may contribute to the development of Charcot-Marie-Tooth neuropathy.

Amino Acid Sequence↗
PubMed1989

Ca2+-dependent deimination-induced disassembly of intermediate filaments involves specific modification of the amino-terminal head domain.

Peptidylarginine deiminase (proteinarginine iminohydrolase, EC 3.5.3.15) converted some arginine residues to citrulline residues in soluble vimentin, in a micromolar Ca2+-dependent manner and resulted in the loss of polymerization competence of the intermediate filament protein. When about 8 mol of residues/mol of vimentin were deiminated, there was a complete loss of filament forming ability. This enzyme also deiminated vimentin filaments which had been polymerized, and deimination of vimentin filaments resulted in filament disassembly. Similar results were obtained with other intermediate filaments such as desmin and glial filaments. High performance liquid chromatography and amino acid analyses of lysine-specific protease-generated fragments from deiminated vimentin (about 8 mol of citrulline/mol of vimentin) showed a differential deimination of three structural domains. The head domain was predominant. These observations suggest that the head domain strongly influences integrity of the intermediate filament.

Amino Acids↗
PubMed1988

Relationship between the nerve growth factor-regulated clone 73 gene product and the 58-kilodalton neuronal intermediate filament protein (peripherin).

Exposure of PC12 cells to nerve growth factor (NGF) has been shown to induce an mRNA that encodes a novel neuronal intermediate filament protein. The findings presented here concern the identity of this filament protein. The major protein in NGF-treated PC12 cell cytoskeletons derived by extraction with 1% Triton X-100 is of apparent Mr = 58,000, focuses by isoelectric focusing as several closely spaced spots of pl 5.6-5.8, and is elevated relative to non-NGF-treated cells. Partial microsequencing of this material reveals 2 internal sequences that are identical to a 14-residue sequence encoded by the NGF-regulated clone 73 mRNA, but not to sequences of other known proteins. An antiserum raised against a 19-residue synthetic peptide corresponding to the deduced C-terminus of the protein encoded by the NGF-regulated clone 73 mRNA specifically recognizes the 58,000-Mr protein. Properties of the 58-kilodalton protein strongly suggest that it corresponds to an intermediate filament protein (peripherin) previously identified in PC12 cells and in peripheral and certain CNS neurons. Identification of the intermediate filament protein encoded by an NGF-induced message should facilitate studies of its regulation and function.

Adrenal Gland Neoplasms↗
PubMed1997

To bead or not to bead? Lens-specific intermediate filaments revisited.

For nearly three decades cytoplasmic intermediate filaments (IFs) have been described as 10 nm thick, unbranched ropes radiating from the cell nucleus and extending to the plasma membrane. This stereotype is now being challenged by the discovery and molecular characterization of the beaded filaments (BFs), a novel class of IFs composed of the lens-specific proteins filensin and phakinin. In contrast to 'mainstream' IFs, BFs have a distinctly nodular appearance and form a meshwork underneath the plasma membrane of the lens fiber cells. In vitro assembly studies, expression of filensin and phakinin in cultured cells, and analysis of the corresponding genes reveal that these proteins have evolved from two different subfamilies of IF proteins, thus yielding a unique structure. The new information provides a basis for understanding how the various forms of tissue-specific IF proteins might have developed adopting to the constraints of a specialized environment.

Animals↗