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At least 19 recordsLinked to original sources
PubMed1987

Microtubules, intermediate filaments, and actin filaments in the odontoblast of rat incisor.

Actin filaments, intermediate filaments, and microtubules in the odontoblasts of rat incisors were investigated electron microscopically using heavy meromyosin and taxol. Actin filaments were abundant at the periphery of the odontoblast process in the form of a network or in bundles. In a branch of the odontoblast process, longitudinally oriented actin filament bundles were found. Most actin filaments were associated with the plasma membrane via electron-dense material which stained with tannic acid. The intermediate filaments had a diameter of 11 to 13 nm. They were distributed throughout the cytoplasm of odontoblasts. They ran lengthwise in the core of the odontoblast process, which showed a different distribution compared with that of actin filaments. Microtubules, which were disrupted after Triton X-100 but preserved by addition of taxol, tended to be associated with intermediate filaments. Such a relation was also seen in conventional preparations. Coated vesicles, which were abundant at the periphery of the odontoblast process, were often associated with actin filaments. Therefore, it is suggested that actin filaments, in the odontoblast process at least, play a role associated with the coated vesicles at the periphery of the process, and may be involved in coated vesicle transport.

Actins↗
PubMed1991

Analysis of the mechanism of assembly of mouse keratin 1/keratin 10 intermediate filaments in vitro suggests that intermediate filaments are built from multiple oligomeric units rather than a unique tetrameric building block.

The question as to whether keratin intermediate filaments (KIF) are built from a unique "building block" consisting of a pair of coiled-coil molecules has been studied by examining the earliest stages of reassembly of mouse K1/K10 KIF in vitro. Particles formed in protein solutions of about 45 micrograms/ml (near or below the critical concentration for assembly) or 0.5-1.65 mg/ml were monitored by turbidity, visualized by electron microscopy, and their structures resolved biochemically using crosslinking, limited proteolysis, and amino acid sequencing. The rate of KIF reassembly in vitro is limited by an initial slow step involving the formation of a three- or four-molecule oligomer. At 2 min, the particles in solution are about 65 nm long and consist of two molecules aligned antiparallel and staggered. A few minutes later, a three- and/or four-molecule species appears that may be the rate-limiting particle(s). It is also 65 nm long, but contains one or two additional molecules aligned in register but antiparallel with respect to one of the molecules on the two-molecule particle. The present data cannot establish whether the rate-limiting particle contains three or four molecules, or in fact consists of a mixture of both. Below the critical concentration for KIF assembly, it exists in solution in rapid exchange with particles containing one and two molecules. In solutions above the critical concentration for assembly, once this oligomer has formed in sufficient quantity, further assembly into KIF occurs rapidly; 90, 110, and 130-nm particles soon appear by apparent addition of a single molecule or oligomers containing two, three, four, or even several molecules. Within about 20 min short KIF about 200-500 nm long appear which later elongate to long (greater than 1 micron) KIF. These data suggest that KIF assembly requires the initial correct alignment of three or four molecules which, once formed, provides a template for further rapid addition of molecules leading to KIF assembly. Furthermore, the data establish that KIF are built from alternating rows of in-register and staggered antiparallel molecules. The present data confirm independently the observations of the previous paper and do not support earlier notions that IF are built from a tetrameric building block consisting of a pair of in-register molecules. Finally, the data suggest that the mechanism of assembly in vitro and the dynamic in vivo assembly-disassembly characteristics of KIF in particular and IF in general are mediated through a variety of small oligomeric species ranging in size from one to several molecules.

Animals↗
PubMed1988

Technical considerations in the immunohistochemical demonstration of intermediate filaments.

The intermediate filaments represent an important group of differentiation markers that have proven to be useful for the diagnosis and classification of human neoplasms. The immunohistochemical demonstration of these proteins is subject to numerous technical variables that include specificities and patterns of reactivities of polyclonal antisera and monoclonal antibodies, cross-reactivities of "specific" monoclonal antibodies with other intermediate filaments and other classes of antigens, masking of certain epitopes due to physiologic or pathologic changes in cells, effects of tissue fixation and subsequent tissue processing, and the sensitivity of the particular immunohistochemical detection system employed. Moreover, a wide variety of microenvironmental signals may affect the patterns of expression of intermediate filament proteins in different pathological conditions. For accurate interpretation of intermediate filament immunoreactivity, the pathologist must be familiar with the various sources of error relating to false positive or negative results. This requires the availability of optimally fixed tissues, a well-characterized set of antibodies to intermediate filament antigens and access to the results of extensive performance testing.

Antibodies, Monoclonal↗
PubMed1987

Epithelioid leiomyosarcoma of the stomach. A case study of the intermediate filaments.

The intermediate filament typing of skeletal and smooth muscle tumors has shown that these neoplasms are characterized by the combined expression of desmin and vimentin intermediate filaments. A case of epithelioid leiomyosarcoma of the stomach was studied by conventional light microscopy and by indirect immunofluorescence using tissue-specific antibodies against intermediate filaments. The tumor cells labeled strongly with vimentin antibodies and were negative for desmin and prekeratin. This peculiar staining pattern may be the result of poor differentiation of the tumor cells with resultant loss of expression of desmin, or may be due to origin from a distinctive smooth muscle cell characterized by the exclusive expression of vimentin intermediate filaments.

Cytoskeleton↗
PubMed1991

Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network.

Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.

Cell Line↗
PubMed1988

Analysis of Triton X-100 insoluble proteins of human chorionic and amniotic tissues with special emphasis on intermediate filaments.

The intermediate filament composition of the chorionic villi of the human full-term placenta and that of the amniotic sac was analysed. The analysis of the protein pattern of the Triton X-100 resistant tissue residues on SDS-polyacrylamide gel did not help us in intermediate filament identification because of the protein heterogeneity of the samples. The in vitro polymerization-depolymerization of the detergent resistant tissue proteins did not result in considerable enrichment of the intermediate filament proteins either. The direct immunological identification done by immunoblotting electrophoresis on the detergent resistant tissue residues, revealed that vimentin and cytokeratins were synthetized in detectable quantities in the human extraembryonal tissues.

Amnion↗
PubMed1991

Expression of intermediate filament proteins in fetal and adult human kidney: modulations of intermediate filament patterns during development and in damaged tissue.

The expression of intermediate filament proteins, particularly individual cytokeratins (CKs), vimentin, and glial filament protein, was immunohistochemically investigated using frozen sections and Carnoy-fixed, paraffin-embedded tissue from normal fetal and adult human kidneys as well as from pathologically altered kidneys. In fetal kidneys, the co-expression of CKs and vimentin was detected in the visceral and parietal epithelium of the glomerulus, the proximal tubules, the thin loops of Henle, and the collecting ducts. In contrast, in the tubules of normal adult kidneys, the presence of vimentin and CKs was nearly always mutually exclusive. While CKs 8 and 18 were present in all tubular epithelia, CKs 19 and 7 each exhibited a distinctive distribution pattern, there being a striking alteration between positive and negative segments and, not infrequently, intratubular heterogeneities. In certain segments, particular cell types (e.g., "plica cells," intercalated cells) could thus be recognized. In tubular epithelia altered by various injurious conditions, novel or enhanced expression of vimentin, CK 19 and CK 7, and, less frequently, CK 17 and glial filament protein was noted in certain segments. The increase in intermediate filament protein expression in altered (particularly proximal) tubules appeared to parallel the reduction in the degree of differentiation. Vimentin was never detected in distal tubules. The present results reveal a considerable similarity between the intermediate filament patterns in non-neoplastic proximal tubules of fetal and damaged kidney tissue and those in clear-cell and chromophilic renal cell carcinomas. They also serve to illustrate that the analysis of both fetal development and reactive cell changes may significantly contribute to our understanding of differentiation phenomena in malignant tumors.

Antibodies, Monoclonal↗
PubMedDate not supplied

Control of germ cell nuclear behaviour at fertilization by Tetrahymena intermediate filament protein.

Intermediate filament protein [relative molecular mass (Mr) 49,000 (49K)] from the ciliated protozoan Tetrahymena has been shown to resemble intermediate filament proteins from mammalian cells in several respects, and to have a possible role in the oral morphogenesis preceding binary fission in Tetrahymena. Here, based on immunofluorescence localization of the 49K protein in Tetrahymena during the early stages of conjugation, we suggest that the protein is involved in some nuclear events, such as the production of four haploid nuclei by prezygotic divisions (meiosis), selection of one of the four meiotic products, formation of the gametic pronucleus by the mitotic division of the selected meiotic product, transfer of the gametic pronucleus across a cell-cell junction, and zygote formation by pronuclear fusion.

Animals↗
PubMed1986

The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments.

Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming subgroups of this class. The significant homologies between types I, II and III occur almost exclusively in segments of the chain that have been identified as having a coiled-coil structure together with the relatively short sections connecting these segments. The non-coiled-coil segments at the C- and N-termini show no significant homology between types, nor is homology in these segments apparent in all members of one type. Component 8c-1 does not show homology in its terminal segments with the known sequence of any other filamentin.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

A synthetic peptide representing the consensus sequence motif at the carboxy-terminal end of the rod domain inhibits intermediate filament assembly and disassembles preformed filaments.

All intermediate filament (IF) proteins share a highly conserved sequence motif at the COOH-terminal end of their rod domains. We have studied the influence of a 20-residue peptide, representing the consensus motif on filament formation and stability. Addition of the peptide at a 10-20-fold molar excess over keratins K8 plus K18 had a severe effect on subsequent IF assembly. Filaments displayed a rough surface and variable diameters with a substantial amount present in unravelled form. At higher peptide concentration (50-100-fold molar excess), IF formation was completely inhibited and instead only loose aggregates of "globular" particles were formed. The peptide also influenced performed keratin IF in a dose-dependent manner. While a three-fold molar excess was sufficient to cause partial fragmentation of IF, a 50-fold molar excess caused complete disassembly within 5 min. Loosely associated protofibrils, short needlelike IF fragments, and aggregates of globular particles were detected. The motif peptide also caused the disassembly of filaments formed by desmin, a type III IF protein. Peptide concentrations and incubation times required for complete disassembly were somewhat higher than for the filaments containing K8 plus K18. A 50-fold molar excess was sufficient to cause complete disassembly within 1 h. Peptides unrelated in sequence to the motif did not interfere with filament formation or stability even when present for more than 12 h at a 100-fold molar excess. The results suggest that the motif sequence normally binds to a specific acceptor site for which the motif peptide can successfully compete. Taken together with current models of IF structure the results indicate that normal binding of the motif sequence to its acceptor must play an essential role in IF formation, possibly by directing the proper alignment of neighboring tetramers or protofilaments. Finally we show that in vitro formed IF are much more sensitive and dynamic strutures than previously thought.

Amino Acid Sequence↗
PubMed1990

Fluorescent measurement of desmin intermediate filament assembly.

Intermediate filaments (IF) are cytoskeletal elements that are believed to play a major role in the specification and maintenance of cell form. Although previously thought to be stable and static because of their relative insolubility in physiological solvents, IF have recently been shown to have dynamic properties not unlike those of other cytoskeletal elements. The methodology for measuring this dynamic behavior, however, has been mostly borrowed from studies of other filament proteins and are poorly suited to IF because of their unusual physicochemical properties. In this report we introduce a fluorescence assay for quantifying in vitro IF assembly. Desmin subunits labeled with iodoacetamidofluorescein (IAF) to approximately 0.4 mol/mol retain the ability to polymerize into filaments indistinguishable from unlabeled IF in the electron microscope. By spectrophotometry, however, up to 90% of the starting fluorescence is quenched upon maximal IF assembly from IAF-desmin subunits. This quench is proportional to the total concentration of desmin subunits and is a sensitive measure of the assembly process. The critical concentration of assembly, measured at 170 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.0, is 0.2 microM. This indicates that a significant level of unpolymerized desmin exists in steady-state equilibrium with polymerized filaments under these conditions and suggests that IF subunit-filament equilibria may play a role in cytoskeletal dynamics.

Animals↗
PubMed1989

Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.

The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.

Amino Acid Sequence↗
PubMed1985

Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

Amino Acids↗
PubMed1988

Filamentous cross-bridges link intermediate filaments to the nuclear pore complexes.

Intermediate filament-nuclear matrix interactions were studied in cultured rat ventral prostate cells and isolated rat uterine epithelial cells. Cytokeratin filaments were identified by immunoelectron microscopy. In addition to conventional thin section of Triton X-100 treated cells, subcellular residues composed of intermediate filaments and nuclear matrix were critical-point dried and platinum-carbon replicated. The results demonstrate the presence of a previously unrecognized type of filamentous cross-bridges that link intermediate filaments to the nuclear pore complexes.

Animals↗