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PubMed1988

Local similarity in the amino acid sequence between the non-catalytic region of Rous sarcoma virus oncogene product p60v-src and intermediate filament proteins.

We found local sequence-similarity between the non-catalytic region of the Rous sarcoma virus oncogene product, p60v-src, and the core region of cytoskeletal keratin through an extensive similarity search of segments of proteins. The segments showing similarity in p60v-src were in the region that is important for morphological transformation, and corresponded to segments with unique structural features predicted for intermediate filament proteins. We suggest that cellular components related with intermediate filament proteins or the sequence shared by the two proteins may be involved in the regulation of the kinase activity or substrate specificity of p60v-src.

Amino Acid Sequence↗
PubMed2003

Molecular architecture of intermediate filaments.

Together with microtubules and actin microfilaments, approximately 11 nm wide intermediate filaments (IFs) constitute the integrated, dynamic filament network present in the cytoplasm of metazoan cells. This network is critically involved in division, motility and other cellular processes. While the structures of microtubules and microfilaments are known in atomic detail, IF architecture is presently much less understood. The elementary 'building block' of IFs is a highly elongated, rod-like dimer based on an alpha-helical coiled-coil structure. Assembly of cytoplasmic IF proteins, such as vimentin, begins with a lateral association of dimers into tetramers and gradually into the so-called unit-length filaments (ULFs). Subsequently ULFs start to anneal longitudinally, ultimately yielding mature IFs after a compaction step. For nuclear lamins, however, assembly starts with a head-to-tail association of dimers. Recently, X-ray crystallographic data were obtained for several fragments of the vimentin dimer. Based on the dimer structure, molecular models of the tetramer and the entire filament are now a possibility.

Amino Acid Sequence↗
PubMed1990

Immunocytochemistry of intermediate filaments in cultured arterial smooth muscle cells: differences in desmin and vimentin expression related to cell of origin and/or plating time.

The objective of this study was to determine whether intermediate filament expression, including desmin and vimentin, in cultured smooth muscle cells (SMCs) is related to cyto-differentiation or proliferation. Using antibodies to desmin and vimentin, we studied by immunoperoxidase technique the distribution of these proteins in subcultured SMCs derived from porcine aorta and coronary artery. In addition, the proliferative potentiality of the cells was estimated by the incorporation of [3H]thymidine into DNA. The frequency of desmin-positive cells in coronary arterial SMCs of 3 and 6 population doubling levels was significantly higher as compared to findings with the aortic SMCs and depended on the plating time. No difference was evident at the 12 population doubling level. In contrast, vimentin was present in the majority of both aortic and coronary arterial SMCs. With regard to the localization of vimentin, two cell types were observed, one had reaction products to vimentin in both perinuclear and cell-peripheral areas (type-I cell), the other only in the cell-peripheral region (type-II cell). The relative proportion of the type-I and -II cells varied with the period of culture. Most of the SMCs showed the type-I cell on the first day and the number of type-II cells was increased on the sixth day. Quiescent SMCs in serum-free media had the same percentage of desmin-positive cells and frequency distribution of type-I and -II cells as did the proliferating SMCs incubated in media containing 5% serum. These results suggest that intermediate filament expression, including desmin and vimentin in cultured SMCs, is related to cell origin and/or plating time, but not to the proliferating activity, per se.

Animals↗
PubMed1985

Intermediate filament forming ability of desmin derivatives lacking either the amino-terminal 67 or the carboxy-terminal 27 residues.

Amino acid sequence data and results from limited proteolytic digestion have been used to define the three-domain structure of intermediate filament proteins. A centrally located highly alpha-helical domain of about 310 residues well-conserved in sequence principles and length is flanked by the highly variable sequences of the non-alpha-helical headpiece and tailpiece. A direct involvement in filament formation of one or both terminal domains was previously proposed for desmin since chymotryptic removal of head and tailpiece provided a derivative unable to form filaments. In order to evaluate directly the importance of these regions we have prepared desmin derivatives lacking either the amino-terminal 67 (T-desmin) or carboxy-terminal 27 residues (L-desmin). Whereas the latter derivative is fully polymerization-competent the fragment lacking only the basic and arginine-rich headpiece cannot form filaments on its own and remains in a protofilamentous stage. These structures of T-desmin are not incorporated into filaments when mixed with protofilaments of desmin. If, however, the two proteins are mixed in 7 M-urea subsequent dialysis provides morphologically normal filaments containing T-desmin. The results suggest that at least certain hybrid protofilaments containing less than four headpieces are accepted in the filament. The removal of the 27 carboxy-terminal residues in L-desmin, although not interfering with filament formation, leads to a change in surface since filaments show lateral aggregation at 170 mM but not at 50 mM salt. The results are discussed in relation to current models of intermediate filament structure.

Amino Acid Sequence↗
PubMed1991

Some neural intermediate filaments contain both peripherin and the neurofilament proteins.

Mammalian neurons and neuron-like cultured cells express the neural intermediate filament (IF) proteins neurofilament (NF)-L, NF-M, NF-H, and peripherin. To determine whether these proteins are found within the same 10-nm filament, light and electron microscope immunocytochemistry using peripherin and NF-specific antibodies was performed on PC12 cells, nervous tissue, and isolated neural filaments from the cauda equina. Double-label immunofluorescence showed that peripherin and NF-L, -M, and -H were found in identical filamentous patterns in interphase and mitotic PC12 cells. Furthermore, expression of mutant peripherin in PC12 cells disrupted not only the peripherin network but also NF-containing filaments. Immunoelectron microscopy of PC12 cell cytoskeletons showed that peripherin and NF subunit proteins were found in the same filament. In situ, in the sciatic nerve, peripherin/NF-L or peripherin/NF-M/-H double-label immunofluorescence illustrates at least three types of nerve fibers: those containing NF only, those labeled predominantly for peripherin, and fibers in which peripherin and NF subunits were colocalized. Immunoelectron microscopy of filaments isolated from nerve roots comprising the sciatic nerve also showed the same three labeling patterns seen by light microscopy. Some neural IF appear to contain predominantly NF proteins or peripherin, but in others, both proteins are found within the same IF.

Amino Acid Sequence↗
PubMed1991

A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

Animals↗
PubMed1990

Cytoskeletal dynamics in rabbit synovial fibroblasts: I. Effects of acrylamide on intermediate filaments and microfilaments.

Rabbit synovial fibroblasts respond to changes in cell shape and cytoskeletal architecture by altering specific gene expression. We have tested the ability of acrylamide, a neurotoxin that alters the distribution of intermediate filaments in cultured PtK1 cells, to induce metalloprotease expression in synovial fibroblasts. Cells treated with 2-20 mM acrylamide for 5 to 24 h underwent shape changes similar to cells treated with the tumor promoter phorbol myristate acetate. Intermediate filaments visualized with anti-vimentin antibodies did not collapse into a perinuclear cap in these rounded cells, but were still present in the extended cell processes. Unexpectedly, when actin was visualized in acrylamide-treated cells, extensive dissociation and clumping of microfilaments was observed. Concentrations of acrylamide greater than 10 mM were cytotoxic, but cells recovered completely after 24 h incubation with 5 mM acrylamide. Like other agents that alter cell shape and actin distribution in synovial fibroblasts, acrylamide also induced expression of the secreted metalloprotease collagenase. Although some recent evidence suggests that acrylamide may be able to exert its collagenase-inducing effects extracellularly, perhaps through transmembrane matrix receptors, our observation that this neurotoxin dramatically alters protein synthesis in synovial fibroblasts suggests that direct effects on cell metabolism may also play a role in acute acrylamide intoxication.

Acrylamide↗
PubMed1983

Intermediate filaments in malignant melanomas. Identification and use as marker in surgical pathology.

Intermediate-sized filaments have been studied in human malignant melanomas and in normal melanocytes by immunofluorescence microscopy with antibodies directed against keratin, vimentin, desmin, neurofilament protein, and glial filament protein. Both human melanotic and amelanotic tumor cells and tumor metastases as well as normal melanocytes in human skin and in the rat eye contain exclusively intermediate filaments of the vimentin type. No reaction was seen with antibodies to keratin, desmin, neurofilaments, or glial filaments. These latter four antisera, however, gave strong reactions in epidermis and other epithelial tissues, muscle, or neural tissues, respectively. The results favor a mesenchymal character of melanocytes, although a neuroectodermal origin in an early developmental stage is possible. The finding that melanomas contain exclusively vimentin intermediate filaments may prove useful in differential diagnosis of melanomas from other tumor types.

Fluorescent Antibody Technique↗
PubMed1985

Intermediate filament proteins in human testis and testicular germ-cell tumors.

Normal testicular tissue and 76 testicular germ-cell tumors of various types were immunohistochemically evaluated for the expression of intermediate filament proteins of different types. In normal testes, the rete testis epithelium was positive to cytokeratin, and the Sertoli cells, stromal cells, and Leydig cells were positive for vimentin. Cytokeratin-positive cells were also found lining atrophic seminiferous tubules and were occasionally seen within nonatrophic seminiferous tubules. The classical seminomas showed vimentin positivity, but this was usually observed in a small number of tumor cells. In addition, nearly half the seminomas contained single cytokeratin-positive cells, some of which were multinucleated and appeared to represent syncytiotrophoblastic giant cells. The tumor cells in embryonal carcinomas, endodermal sinus tumors, and choriocarcinomas displayed cytokeratin positivity. In some embryonal carcinomas vimentin-positive tumor cells were also found, probably representing attempts at further differentiation of the tumor cells. In immature teratomas, both the immature and the mature epithelial structures showed cytokeratin positivity. The stromal components, including cartilage, contained vimentin, and the smooth-muscle elements, desmin. Neural tissue positive for neurofilaments and glial tissue positive for glial fibrillary acidic protein, were observed in 5 and 3 of 15 cases, respectively. It is considered that antibodies to intermediate filaments are suitable tools to characterize the differentiation patterns of testicular germ-cell tumors and have the potential to aid in the differential diagnosis especially between seminoma and embryonal carcinoma.

Antibodies, Monoclonal↗
PubMedDate not supplied

[Changes in the prekeratin set and in the intracellular distribution of intermediate filaments during the embryonic development of the liver in the rat].

Monoclonals against three individual proteins of the epithelial intermediate filaments, prekeratins (PK40, PK49, PK55), were used for immunofluorescence studies of the cryostat sections of the rat embryonic liver. The dynamics of expression and intracellular distribution of prekeratins reflects that of morphological rearrangement of the liver. The development of the system of liver beams was accompanied by changes in the expression and intracellular distribution of PK49 and PK55 and the development of the system of bile ducts by changes in all three PKs. From day 20-21 of embryogenesis all three PKs are expressed in cholangiocytes, while PK49 and PK55 in hepatocytes only.

Animals↗
PubMed1995

Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments.

The passive tension-sarcomere length relation of rat cardiac muscle was investigated by studying passive (or not activated) single myocytes and trabeculae. The contribution of collagen, titin, microtubules, and intermediate filaments to tension and stiffness was investigated by measuring (1) the effects of KCl/KI extraction on both trabeculae and single myocytes, (2) the effect of trypsin digestion on single myocytes, and (3) the effect of colchicine on single myocytes. It was found that over the working range of sarcomeres in the heart (lengths approximately 1.9-2.2 microns), collagen and titin are the most important contributors to passive tension with titin dominating at the shorter end of the working range and collagen at longer lengths. Microtubules made a modest contribution to passive tension in some cells, but on average their contribution was not significant. Finally, intermediate filaments contributed about 10% to passive tension of trabeculae at sarcomere lengths from approximately 1.9 to 2.1 microns, and their contribution dropped to only a few percent at longer lengths. At physiological sarcomere lengths of the heart, cardiac titin developed much higher tensions (> 20-fold) than did skeletal muscle titin at comparable lengths. This might be related to the finding that cardiac titin has a molecular mass of 2.5 MDa, 0.3-0.5 MDa smaller than titin of mammalian skeletal muscle, which is predicted to result in a much shorter extensible titin segment in the I-band of cardiac muscle. Passive stress plotted versus the strain of the extensible titin segment showed that the stress-strain relationships are similar in cardiac and skeletal muscle. The difference in passive stress between cardiac and skeletal muscle at the sarcomere level predominantly resulted from much higher strains of the I-segment of cardiac titin at a given sarcomere length. By expressing a smaller titin isoform, without changing the properties of the molecule itself, cardiac muscle is able to develop significant levels of passive tension at physiological sarcomere lengths.

Animals↗
PubMed1999

Characterization of distinct early assembly units of different intermediate filament proteins.

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

Amino Acid Sequence↗
PubMed1988

Immunohistochemical localization of intermediate filament proteins in calcifying epithelial odontogenic tumors.

Three calcifying epithelial odontogenic tumors (CEOT) were examined immunohistochemically. The localization of intermediate filaments was characterized through the use of polyclonal anti-keratin antiserum (TK which detects 41-65 kd keratins), 2 monoclonal keratin antibodies (PKK1 specific for the 44, 46, 52 & 53 kd keratins and KL1, specific for the 55-57 kd keratins) and monoclonal antibodies for vimentin and desmin. The tumor epithelial cells were slightly positive or negative for PKK1 detectable keratins, but slightly to strongly positive for KL1 and TK antibodies. Tumor epithelium was slightly positive for vimentin but negative for desmin. The tumor foci were composed of both dark-staining and pale-staining cells; the former had a more intense reaction with KL1 and TK antibodies than the latter. Homogeneous acellular material was either PAS-positive or negative, with or without calcification, and keratin-negative.

Adult↗
PubMed1988

Squamous differentiation in human breast epithelial cells in culture, with comparative observations on intermediate filaments from intact epithelium.

Intact, grossly normal human mammary tissue and cultured breast epithelial cells were examined by transmission electron microscopy for indicators of squamous differentiation. Intact epithelium contained 7-10 nm filaments which we interpreted as cytokeratin intermediate type filaments; many formed bundles which lacked conspicuous electron density. Two types of cell process were present: one was luminal and covered in a glycocalyx, whereas the other lacked a coat and was confined to lateral membranes. In culture, mammary epithelial cells contained intermediate filament bundles which were conspicuous by their enhanced electron density compared with those of intact epithelium. These were acceptable as dense tonofibrils i.e. indicators of squamous differentiation. Cultured cells also possessed cell processes broadly comparable to those of intact epithelium. This study confirms the phenomenon of squamous differentiation consequent upon culture, in a system which differed methodologically from previous studies, and makes hitherto neglected comparisons between intact and cultured breast epithelium.

Breast↗
PubMedDate not supplied

[Expression of intermediate filament and oncogene proteins in squamous cell carcinomas of the oral cavity. Correlation of differentiation].

We investigated normal oral epithelium and 20 oral squamous cell carcinomas with antibodies against oncogene products (p21, EGF-R) and intermediate filaments (cytokeratins 4, -10/11, -13, -18, Vimentin). Antigen expressions in oral epithelium and squamous cell carcinomas were associated in different patterns with epithelial maturation. Dedifferentiation of the squamous cell carcinomas was accompanied by quantitative changes in the oncogene products and a more complex pattern of the cellular intermediate filament equipment.

Antigens, Neoplasm↗
PubMed1986

Intermediate filament proteins in choroid plexus and ependyma and their tumors.

The intermediate filament protein types of normal choroid plexus and ependymal tissue and their putative tumors were investigated. In normal human choroid plexus tissue, but not in ependyma, keratin could be demonstrated immunohistochemically. By immunoblotting, keratins 8, 18, and 19 were found, but glial fibrillary acidic protein (GFAP) was absent. In mouse and rat, choroid plexus epithelium and ependymal lining cells were keratin-positive. In addition, many ependymal cells were vimentin-positive. Keratin was immunohistochemically found in three of four choroid plexus papillomas, two of two choroid plexus carcinomas, and the lining cells of three neuroepithelial cysts. GFAP-positive cells were present in some choroid plexus tumors. In contrast, none of the eight ependymomas contained keratin, but all were strongly positive for GFAP. The results show that choroid plexus lining cells and choroid plexus tumors have true epithelial characteristics in their cytoskeleton, in contrast to ependymomas, which do not show keratin positivity but show glial filaments, as would be seen in astrocytic tumors.

Adult↗
PubMed1993

Phosphorylation and disruption of intermediate filament proteins in oligodendrocyte precursor cultures treated with calyculin A.

Treatment of primary cultures of oligodendrocyte precursors with calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, caused the phosphorylation of two intermediate filament components, nestin and vimentin. Phosphoamino acid analysis demonstrated that phosphorylation took place mainly on serine and to a lesser extent on threonine residues. In addition, calyculin A treatment caused a shift in the distribution of the two proteins from the Triton-X-100 insoluble fraction to the detergent soluble fraction as demonstrated by immunoblotting. This redistribution, which was evident within 15 min after treatment and was nearly completed by 90 min, was accompanied by a disruption of the intermediate filament network. Thus, both nestin and vimentin retracted from the cytoplasmic processes to form a large perikaryal ring as shown by immunocytochemical analysis. Both morphological and biochemical changes were reversed 2-5 hr after removal of calyculin A from the culture medium.

Amino Acids↗
PubMed2002

Oxysterol-binding-protein (OSBP)-related protein 4 binds 25-hydroxycholesterol and interacts with vimentin intermediate filaments.

Oxysterol-binding protein (OSBP) is the prototypical member of a class of phospholipid and oxysterol-binding proteins that interacts with the Golgi apparatus and regulates lipid and cholesterol metabolism. As a result of recent sequencing efforts, eleven other OSBP-related proteins (ORPs) have been identified in humans. We have investigated the structure, oxysterol-binding activity, cellular localization and function of ORP4 (also designated OSBP2 or HLM), a homologue that shares the highest degree of similarity with OSBP. Two ORP4 cDNAs were identified: a full-length ORP4 containing a pleckstrin homology (PH) domain and an oxysterol-binding region (designated ORP4-L), and a splice variant in which the PH domain and part of the oxysterol-binding domain were deleted (designated ORP4-S). ORP4 mRNA and protein expression overlapped partially with OSBP and were restricted to brain, heart, muscle and kidney. Like OSBP, ORP4-L bound [3H]25-hydroxycholesterol with high affinity and specificity. In contrast, ORP4-S did not bind [3H]25-hydroxycholesterol or [3H]7-ketocholesterol. Immunofluorescence localization in stably transfected Chinese hamster ovary cells showed that ORP4-S co-localized with vimentin and caused the intermediate filament network to bundle or aggregate. ORP4-L displayed a diffuse staining pattern that did not overlap with vimentin except when the microtubule network was disrupted with nocodazole. Oxysterols had no effect on the localization of either ORP4. Cells overexpressing ORP4-S had a 40% reduction in the esterification of low-density-lipoprotein-derived cholesterol, demonstrating that ORP4 interaction with intermediate filaments inhibits an intracellular cholesterol-transport pathway mediated by vimentin. These studies elucidate a hitherto unknown relationship between OSBPs and the intermediate filament network that influences cholesterol transport.

Alternative Splicing↗