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PubMed2003

Activation of embryonic intermediate filaments contributes to glial scar formation after spinal cord injury in rats.

The expression of two intermediate filaments, nestin and vimentin, was studied in spinal cord injury (SCI) to elucidate their roles in the formation of glial scars. Rats were sacrificed 1, 4, and 7 days after induction of compression injury of the spinal cord using an aneurysm clip. The affected spinal cords were studied using antibodies against nestin and vimentin intermediate filaments. One day after spinal cord injury, some clusters of nestin-positive vessels were detected in the center of the injury, but few were seen in other cell types. Vimentin immunostaining was detected in some glial cells in the center and its level of immunoreactivity was enhanced in the ependymal cells of the central canal. On days 4 and 7 after spinal cord injury, astrocytes and some ependymal cells in the central canal were stained positively for nestin and increased expression of nestin was observed in vessels. Vimentin was detected in some macrophages and astrocytes in the lesions. Nestin was co-localized with glial fibrillary acidic protein in some glial cells in SCI. These findings imply that spinal cord cells in adult animals have embryonic capacity, and these cells are activated after injury, which in turn contributes to repair of spinal cord injury through formation of a glial scar.

Animals↗
PubMed1983

Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins.

Mammalian neurofilament triplet proteins (68 K, 160 K and 200 K) have been correlated by a biochemical, immunological and protein chemical study. The 160 K and 200 K triplet proteins are intermediate filament proteins in their own right, since they reveal the alpha-helical coiled-coil rod domain analyzed in detail for the 68 K protein. Triplet proteins display two distinct arrays. Their amino-terminal region built analogously to non-neuronal intermediate filament proteins should allow a co-polymerization process via the interaction of coiled-coil domains. The extra mass of all triplet proteins is allocated to carboxy-terminally located extensions of increasing size and unique amino acid sequences. These may provide highly charged scaffolds suitable for interactions with other neuronal components. Such a domain of 68 K reveals, in sequence analysis, 47 glutamic acids within 106 residues. The epitope recognized by a monoclonal antibody reacting probably with all intermediate filament proteins has been mapped. It is located within the last 20 residues of the rods, where six distinct intermediate filament proteins point to a consensus sequence.

Amino Acid Sequence↗
PubMed2006

Interactions of intermediate filament protein synemin with dystrophin and utrophin.

Synemin is a unique, very large intermediate filament (IF) protein present in all types of muscle cells, which forms heteropolymeric intermediate filaments (IFs) with the major IF proteins desmin and/or vimentin. We show herein that tissue-purified avian synemin directly interacts with both dystrophin and utrophin, and that specific expressed regions of both of the mammalian (human) synemin isoforms (alpha-synemin and beta-synemin) directly interact with specific expressed domains/regions of the dystrophin and utrophin molecules. Mammalian synemin is also shown to colocalize with dystrophin within muscle cell cultures. These results indicate that synemin is an important IF protein in muscle cells that helps fortify the linkage between the peripheral layer of cellular myofibrils and the costameric regions located along the sarcolemma and the sarcolemma region located within the neuromuscular and myotendinous junctions (NMJs and MTJs).

Animals↗
PubMed1982

Antibody to intermediate filaments of the cytoskeleton.

IgM antibodies against cultures of intermediate filaments (IMF) of the cytoskeleton were demonstrated by immunofluorescence in the sera of 94 (80%) of 118 patients with seropositive rheumatoid arthritis. These antibodies reacted with IMF in cultures of both human fetal fibroblasts and laryngeal carcinoma (HEp2) cells. Of 10 patients from whom paired synovial fluids were also available 8 had anti-IMF antibodies in both serum and fluid. In seronegative RA the incidence of anti-IMF was 40%, in ankylosing spondylitis 25%, in osteoarthrosis 16%, and in normal subjects 14%. Only a minority of RA sera positive for anti-IMF antibodies were also positive for smooth muscle antibody. Absorption experiments suggest that in RA anti-IMF is directed at the intermediate filament protein, vimentin.

Adult↗
PubMed1996

Intermediate filament protein domain interactions as revealed by two-hybrid screens.

All intermediate filament proteins possess three distinct domains: heads, rod and tail, and subdomains within the rod called helices 1A, 1B, 2A, and 2B. Subunit packing within a filament is a consequence of interactions among these domains. Several such interactions are known, but probably many more contribute to stabilizing filament structure. We examined a number of such potential interactions using the yeast two-hybrid system. Domains or subdomains of murine vimentin, a Type III intermediate filament protein, were fused with either the DNA-binding or trans-activating domain of GAL4, a transcription factor. Interaction between the vimentin domains/subdomains functionally reconstituted GAL4, thereby activating transcription of a GAL1-LacZ reporter gene. The oligomeric state at which the interactions took place, i.e. whether the domains/subdomains were dimeric or tetrameric as they interacted, was also determined. These studies revealed a number of interesting interactions, among which was a strong homotypic binding to helix 2B to form tetramers. They also demonstrated a lack of interaction among others expected to do so based on current structural models. From these results we deduced which of the candidates for interactions, suggested by current models, were true protein-protein interactions and which represented nearest-neighbors only. Thus, the A11 and A22 modes of molecular alignment identified by Steinert et al. (Steinert, P. M., Marekov, L. N., Fraser, R. D. B., and Parry, D. A. D. (1993) J. Mol. Biol. 230, 436-452) are probably true interactions, whereas the A12 and ACN modes may describe adjacent but non-interacting molecules.

Animals↗
PubMed1983

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

We studied the localization of desmin (skeletin), the major subunit of muscle-type intermediate filaments, by high resolution immunoelectron microscopy in adult chicken skeletal muscle. Immunoferritin labeling of ultrathin frozen sections of intact fixed sartorius muscle showed the presence of desmin between adjacent Z-bands and as strands peripheral to Z-bands, forming apparent connections between the Z-bands with adjacent sarcolemma, mitochondria, and nuclei. We observed no desmin labeling, however, in the vicinity of the T-tubules. In addition, intermediate filaments were morphologically discernible at the level of the Z-bands in plastic sections of glycerol-extracted muscle that had been infused with unlabeled antidesmin antibodies. Our results indicate that the desmin present in adult skeletal muscle, that had previously been detected by immunofluorescence light microscopy, is largely if not entirely in the form of intermediate filaments. The results provide evidence that these filaments serve to interconnect myofibrils at the level of their Z-bands, and to connect Z-bands with other specific structures and organelles in the myotube, but not with the T-tubule system.

Animals↗
PubMedDate not supplied

Intermediate filament typing of tumor cells in fine needle aspirates by means of monoclonal antibodies.

Well-characterized monoclonal antibodies directed against different intermediate filament proteins were used in the typing of tumor cells in 30 fine needle aspiration (FNA) biopsy specimens received for routine cytologic examination to assess the value of intermediate filament typing in FNA cytology. Tumors from sites that included liver, large bowel, pancreas, breast, skin, thyroid, thigh and kidney were examined with monoclonal antibodies specific for either all keratins or keratin subsets, vimentin, desmin or specific neurofilament polypeptides. Intermediate filament typing helped to confirm, revise or refine the diagnoses made by light microscopy and provided information of value in the classification of tumors of uncertain origin.

Antibodies, Monoclonal↗
PubMed1994

Making heads and tails of intermediate filament assembly, dynamics and networks.

Thus far, intermediate filaments (IFs) have been the least understood of the three cytoskeletal filament systems with regard to their structure, assembly, network formation, and dynamics. This picture is now slowly but definitely changing, as recent in vivo and in vitro experiments, including generation of transgenic animals, have yielded important new data shedding light on the following areas: the molecular architecture of IFs; the role of the highly variable end domains during IF assembly and network formation; the factors that govern whether IF proteins are involved in de novo filament formation or are incorporated into a pre-existing IF network; and the effects of post-translational modifications, such as phosphorylation and glycosylation of IF polypeptides, on filament assembly, dynamics and turnover.

Animals↗
PubMedDate not supplied

Cytoplasmic intermediate filaments in cultured glial cells.

Monolayer cultures of rat glial cells derived from 12 to 14 day fetal spinal cords and rat C6 glioma cells were examined for immunofluorescent reactivity with human or rabbit autoantibodies to intermediate filaments and with rabbit antibody to GFAP. The embryonic glial cells and C6 glima cells reacted identically with both. Protoplasmic astrocytes gave immunofluorescent staining of an intricate network of cytoplasmic filaments while fibrous astrocytes and C6 glioma cells gave diffuse cytoplasmic staining of the cell body and cell processes. Cells, pretreated with colchicine or vinblastine, showed staining of thick coils of peri-nuclear, circum-nuclear, or juxtanuclear filaments. The observations suggest that intermediate filaments in glial cells contain unique antigenic determinants as well as antigenic determinants shared with intermediate filaments in other cell types,

Animals↗
PubMed1985

Efficient detection of intermediate filament proteins using a panspecific monoclonal antibody: anti-IFA.

Anti-IFA, a panspecific monoclonal antibody which was raised against human glial fibrillary acidic protein (GFAP), recognizes a determinant common to GFAP and all other intermediate filament proteins. This antibody can be used to identify intermediate filament proteins from both vertebrate and invertebrate tissues. Its utility in immunoblot studies of intermediate filament proteins is enhanced by using cytoskeleton extracts and protease treatment to facilitate the transfer of high molecular weight (greater than 70 000) proteins from gels to nitrocellulose membranes.

Animals↗
PubMed2000

Plasticin, a type III neuronal intermediate filament protein, assembles as an obligate heteropolymer: implications for axonal flexibility.

The assembly characteristics of the neuronal intermediate filament protein plasticin were studied in SW13 cells in the presence and absence of a cytoplasmic filament network. Full-length plasticin cannot polymerize into homopolymers in filament-less SW13c1.2Vim(-) cells but efficiently coassembles with vimentin in SW13c1.1Vim(-) cells. By cotransfecting plasticin and vimentin in SW13c1.1Vim(-) cells, we show that plasticin assembly requires vimentin in noncatalytic amounts. Differing effects on assembly were seen with point mutations of plasticin monomers that were analogous to the keratin mutations that cause epidermolysis bullosa simplex (EBS). In particular, plasticin monomers with point mutations analogous to those in EBS do not uniformly inhibit neurofilament (NF) network formation. A point mutation in the helix termination sequence resulted in complete filament aggregation when coexpressed with vimentin but showed limited coassembly with low- and medium-molecular-weight NF proteins (NF-L and NF-M, respectively). In transfected SW13c1.1Vim(+) cells, a point mutation in the first heptad of the alpha-helical coil region formed equal amounts of filaments, aggregates, and a mixture of filaments and aggregates. Furthermore, coexpression of this point mutation with NF-L and NF-M was associated with a shift toward increased numbers of aggregates. These results suggest that there are important structural differences in assembly properties between homologous fish and mammalian intermediate filament proteins. These structural differences may contribute to the distinctive growth characteristics of the teleost visual pathway.

Amino Acid Substitution↗
PubMed1994

Expression of the neural intermediate filament proteins peripherin and neurofilament-66/alpha-internexin in neuroblastoma.

BACKGROUND: Peripherin and neurofilament (NF)-66/alpha-internexin are recently characterized, neuron-specific intermediate filament proteins that are expressed in the developing peripheral nervous system. Peripherin, in particular, is highly enriched in neuronal derivatives of the neural crest. We speculated that these intermediate filament proteins would be expressed in neuroblastoma (NB), a neural crest-derived tumor with many neuronal features. EXPERIMENTAL DESIGN: By use of antibodies specific to peripherin and NF-66/alpha-internexin, we detected these proteins on Western blots of NB tissue extracts and in paraffin sections of NBs. RESULTS: Western blotting indicated that NB tumor extracts contained immunoreactive proteins that co-migrated with rat peripherin and human NF-66/alpha-internexin from normal tissues, thus establishing the specificity of the antibodies for these proteins in tumors. The antibody specific for peripherin labeled all NBs, including immature NBs, composite ganglioneuroblastomas and ganglioneuromas. In contrast, the NF-66/alpha-internexin antibody labeled only 50% of NBs, and only weakly labeled most ganglioneuroblastomas and ganglioneuromas. Neither antibody labeled other small blue cell tumors such as lymphomas, rhabdomyosarcomas, Wilms' tumors, and Ewing sarcomas. CONCLUSIONS: The specificity of the peripherin labeling of NB and the ability of the peripherin antibody to label the entire spectrum of NBs, including ganglioneuroblastomas and ganglioneuromas, indicate that this intermediate filament protein has potential as a diagnostic marker for these related neural crest neoplasms.

Animals↗
PubMed1992

Intermediate filament antigens of 60 and 65 kDa in the nuclear matrix of plants: their detection and localization.

Although the presence of a matrix in plant nuclei has been reported, major questions remain about its structural and biochemical features. We have used an intermediate filament antibody of broad specificity to explore whether Daucus carota (carrot) nuclei and nuclear matrices contain intermediate filament/lamin antigens and, if so, where specifically they are localized. SDS-PAGE and Western blotting revealed two bands, at 60 and 65 kDa, that were highly immunoreactive with the intermediate filament antibody (IFA) of Pruss et al. (1981, Cell 27, 419-428). This pattern was observed consistently, not only with carrot cell-free nuclei and nuclear matrices, but also with nuclear preparations from Vicia faba (broad bean) and Pisum sativum (pea). Immunofluorescence studies with whole carrot nuclei localized the IFA antigens to the nucleoplasm and disclosed no accentuated peripheral labeling. Agarose-embedded nuclear matrices showed not only fluorescence throughout the nucleoplasm but also heavy labeling surrounding the nucleoli and suggestions of peripheral labeling. At the ultrastructural level, immunogold results from pre- and postembedment treatments supported the conclusion that IFA antigens occur throughout the nucleoplasm, with possibly a slight concentration at the periphery. These combined results provide substantial evidence that plant nuclei and their matrices possess at least two major intermediate filament antigens with molecular weights characteristic of animal lamins. Whether or not these antigens represent plant lamins, their nonperipheral localization hints at significant differences among the eukaryotic kingdoms in nuclear organization.

Antigens↗
PubMed1987

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits.

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.

Animals↗
PubMed1996

Specific recognition of coiled coils by infrared spectroscopy: analysis of the three structural domains of type III intermediate filament proteins.

The central domain of cytoplasmic intermediate filament (IF) proteins from vertebrates contains some 310 residues and forms a double-stranded coiled coil (rod) with a length of about 46 nm. The flanking terminal domains show a high cell type specific variability both in sequence and in length. Using Fourier transform infrared (FTIR) spectroscopy we measured secondary structures of isolated domains of type III and IV IF proteins and of the soluble tetramers and the filaments formed by type III IF proteins. The amide I spectrum of the desmin rod is virtually identical to the spectra of other coiled-coil proteins such as tropomyosin and the myosin rod. All these double-stranded coiled coils reveal spectra distinctly different from classical alpha-helical spectra. The spectrum of coiled coils is a triplet of approximately equally strong bands. One band occurs at normal alpha-helix position, while the other two are found at lower wavenumbers. Theoretical aspects of these findings are discussed in the accompanying paper by W. C. Reisdorf and S. Krimm [(1996) Biochemistry 35, 1383-1386]. The amino-terminal head domain of desmin has a multicomponent spectrum with major fractions of beta-sheet. The carboxy-terminal tail domains of desmin and the neurofilament proteins L and H, the latter in the phosphorylated and in the dephosphorylated forms, have very similar FTIR spectra, indicating mostly random structure. The spectrum of desmin type III protofilaments is very similar to the sum of the spectra of the three isolated domains. Polymerization into filaments seems to induce a small change in secondary structure.

Desmin↗
PubMed1979

Viral infections and IgM autoantibodies to cytoplasmic intermediate filaments.

Seventy-four out of 113 sera from patients with infectious hepatitis, chickenpox, measles and mumps reacted with both smooth muscle and cytoplasmic filaments in cultured fibroblasts and neuroblastoma. Five out of eighty-five control sera also reacted in this way. That the cytoplasmic structures are intermediate filaments was suggested by their rearrangement into coils of perinuclear filaments in colchicine- or vinblastine-treated fibroblasts, but not in cytochalasin B-treated cells. The idenity of these structures was confirmed by the demonstration that the same structures reacted with the post-viral sera and a rabbit and human anti-intermediate filament antibody. Immunoabsorption studies showed that twenty-seven out of thirty-two positive sera were neutralised by skeletin, the intermediate filament protein from smooth muscle. In all but one of the sera, the antibody was IgM. Antibody titres fell in the second specimen in eleven out of fourteen pairs of acute and convalescent sera. The association between viral infections and autoantibodies suggest that production of antibodies suggests that production of antibody to intermediate filaments may be initiated by viruses.

Adolescent↗
PubMed1988

Application of monoclonal antibodies to intermediate filament proteins in surgical pathology of head and neck tumours. An overview.

Intermediate filament proteins are distributed in a tissue specific manner throughout human tissues. Using monoclonal or polyclonal antibodies to cytokeratins, vimentin, desmin, neurofilament proteins or the glial fibrillary acidic protein, epithelial, mesenchymal, myogenic, nervous and glial tissues, respectively, can be distinguished by immunohistochemical techniques. Since tumour cells generally retain the intermediate filament proteins typical for their cells of origin, such antibodies can also be used to discriminate between different types of neoplasma, i.e. carcinoma, lymphoma, myosarcoma, etc. Furthermore, monoclonal antibodies to individual cytokeratin proteins can be used to distinguish between several types of epithelial tissues and different types of carcinomas. The application of such antibodies in the histopathology of head and neck tumours can be of great help in the characterization of tumours that cannot be identified on the basis of routine histological techniques.

Antibodies, Monoclonal↗
PubMed1988

Alterations in intermediate filament proteins in rat kidney proximal tubule epithelial cells.

Changes in the intermediate filament composition of rat kidney proximal tubule cells in culture have been investigated. The data suggest that differentiated tubular epithelial cells do not express vimentin, but vimentin expression is induced when the cells begin to proliferate in culture. The cultured cells are positive for both cytokeratins and vimentin by immunofluorescence microscopy. The data support the concept that the intermediate filament composition of proximal tubule epithelial cells can be altered during proliferation induced by nephrotoxic chemicals or by neoplastic transformation.

Animals↗