Separation of wool intermediate filament protein and intermediate filament-associated protein on a Sepharose column.
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Amino Acids↗
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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↗
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↗
Intermediate filaments in motion: observations of intermediate filaments in cells using green fluorescent protein-vimentin.
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Animals↗
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↗
Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments.
We report here on the in vivo assembly of alpha-internexin, a type IV neuronal intermediate filament protein, in transfected cultured cells, comparing its assembly properties with those of the neurofilament triplet proteins (NF-L, NF-M, and NF-H). Like the neurofilament triplet proteins, alpha-internexin coassembles with vimentin into filaments. To study the assembly characteristics of these proteins in the absence of a preexisting filament network, transient transfection experiments were performed with a non-neuronal cell line lacking cytoplasmic intermediate filaments. The results showed that only alpha-internexin was able to self-assemble into extensive filamentous networks. In contrast, the neurofilament triplet proteins were incapable of homopolymeric assembly into filamentous arrays in vivo. NF-L coassembled with either NF-M or NF-H into filamentous structures in the transfected cells, but NF-M could not form filaments with NF-H. alpha-internexin could coassemble with each of the neurofilament triplet proteins in the transfected cells to form filaments. When all but 2 and 10 amino acid residues were removed from the tail domains of NF-L and NF-M, respectively, the resulting NF-L and NF-M deletion mutants retained the ability to coassemble with alpha-internexin into filamentous networks. These mutants were also capable of forming filaments with other wild-type neurofilament triplet protein subunits. These results suggest that the tail domains of NF-L and NF-M are dispensable for normal coassembly of each of these proteins with other type IV intermediate filament proteins to form filaments.
Amino Acid Sequence↗
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↗
Antibodies to neurofilament, glial filament, and fibroblast intermediate filament proteins bind to different cell types of the nervous system.
Antisera were raised to the 210,000-dalton and the 49,000-dalton proteins of a fraction enriched in intermediate (10 nm) filaments from human brain. Proteins of the filament preparation were separated by SDS-polyacrylamide gel electrophoresis and used for immunization and subsequent analysis of the reactions of the sera by rocket immunoelectrophoresis. Anti-210,000-dalton serum precipitated proteins of molecular weights 210,000, 160,000, and 68,000, and, thus, reacted with all the neurofilament triplet components. Anti-49,000-dalton serum did not react with the triplet proteins but precipitated the 49,000-dalton protein. By immunofluorescence on tissue sections, anti-210,000-dalton serum bound to neuronal axons in sciatic nerve and cerebellum. In dissociated cell cultures, rat dorsal root ganglion cells and their processes bound the serum, whereas nonneuronal cells did not. Some cultured cerebellar neurons were also positive, whereas astrocytes were not. At the ultrastructural level, anti-210,000-dalton serum bound to intermediate filaments inside axonal processes. Anti-49,000-dalton serum bound to astrocytes in sections of the cerebellum, and cultured astrocytes had filaments that stained, whereas other cell types did not. In sciatic nerve sections, elements stained with this serum, but cultured cells from newborn sciatic nerve were negative. An antiserum against the 58,000-dalton protein of the cytoskeleton of NIL-8 fibroblasts strongly stained sciatic nerve sections, binding to Schwann cells but not to axons or to myelin. In cerebellar sections, astrocytes were positive, as were blood vessels and cells in the pia. In cell cultures, anti-58,000-dalton serum stained filaments inside Schwann cells, fibroblasts, and astrocytes, but neurons were negative. Cells in the cultures and tissue sections of the nervous system failed to react with antiserum to the 58,000-dalton protein of skin intermediate filaments. In these studies, astrocytes in vivo and in culture were the only cells which had antigens related to two classes of intermediate filaments.
Astrocytes↗
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↗
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↗
Segregation of autoantibody to cytoskeletal filaments, actin and intermediate filaments with two types of chronic active hepatitis.
Regional differences in serological features of chronic active hepatitis (CAH) were assessed from study of unselected patients with CAH, 53 from Zagreb, Yugoslavia and 53 from Melbourne, Australia. A group of 29 patients from Zagreb with chronic persistent hepatitis (CPH) was also included. The serological marker studied was immunofluorescence reactivity to cytoskeletal components, actin cables (AC) and intermediate filaments (IF), these being mostly responsible for the smooth muscle antibody reactivity which occurs in CAH. The Zagreb cases of CAH differed from the Melbourne cases by having a significantly lower frequency of anti-AC (8% vs 55%) and a significantly higher frequency of anti-IF (34% vs 15%), and correspondingly had a higher frequency of markers of hepatitis B virus (HBV) infection (HBsAg, 53% vs 8% and anti-HBc, 72% vs 34%), and a differing HLA profile, with HLA BW35 higher (38% vs 14%) and B8 lower (18% vs 56%). This study illustrates the differences among populations in expression of CAH, in that HBsAg is held to specify the HBV-associated type, and antibody to actin is held to specify the autoimmune type. These differences would be determined in part by environmental influences, notably HBV, and genetic influences, notably HLA, and are reflected by the differing patterns of immunofluorescence reactivity with the cellular cytoskeleton.
Actins↗
Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments.
The two major intermediate filament (IF) proteins from the esophagus epithelium of the snail Helix pomatia and the two major IF proteins from muscle tissue of the nematode Ascaris suum were investigated under a variety of assembly conditions. The lowest-order complexes from each of the four protostomic invertebrate (p-INV) IF proteins are parallel, unstaggered dimers involving two-stranded alpha-helical coiled coil formation of their approximately 350 amino acid residue central rod domain (i.e. long-rod). In the electron microscope these are readily recognized by their distinct approximately 56 nm long rod with two globular domains (i.e. representing the non-helical carboxy-terminal tail domain of the p-INV IF proteins) attached at one end, closely resembling vertebrate lamin dimers. The next-higher-order oligomers are tetramers, which are easily recognized by their two pairs of globular tail domains attached at either end of a approximately 72 nm long central rod portion. According to their size and shape, these tetramers are built from two dimers associated laterally in an antiparallel, approximately half-staggered fashion via the amino-terminal halves of their rod domains. This is similar to the NN-type tetramers found as the most abundant oligomer species in all types of vertebrate cytoplasmic IF proteins, which contain a approximately 310 amino acid residue central rod domain (i.e. short-rod). As a first step toward filament formation, the p-INV IF tetramers anneal longitudinally into protofilaments by antiparallel CC-type association of the carboxy-terminal halves of their dimer rods. The next step involves radial growth, occurring initially through lateral association of two four-chain protofilaments into octameric subfibrils, which then further associate into mature, full-width filaments. Head-to-tail polymers of dimers and paracrystalline fibers commonly observed with vertebrate lamins were only rarely seen with p-INV IF proteins. The globular domains residing at the carboxy-terminal end of p-INV IF dimers were studding the surface of the filaments at regular, approximately 24.5 nm intervals, thereby giving them a "beaded" appearance with an axial periodicity of about 24.5 nm, which is approximately 3 nm longer than the corresponding approximately 21.5 nm repeat pattern exhibited by short-rod vertebrate IFs.
Animals↗
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↗
Conservation of the structure of keratin intermediate filaments: molecular mechanism by which different keratin molecules integrate into preexisting keratin intermediate filaments during differentiation.
During development and differentiation, the intermediate filament component of the cytoskeleton of many cells and tissues is rebuilt by a dynamic exchange process in which one set of protein chains is replaced by another, without recourse to creation of a new network. One major example is the replacement of keratin 5/keratin 14 (K5/K14) keratin intermediate filaments (KIFs) by K1/K10 KIFs during terminal differentiation in the epidermis. The present work was undertaken to explore how this may occur. We have induced lysine-lysine cross-links with disulfosuccinimidyl tartrate in K5/K14 KIFs in order to determine the axial dimensions and relative axial alignments of the K5/K14 molecules. Many of the cross-links induced in subfilamentous oligomers containing one, two, or three molecules were also found in the intact KIF, indicating that the body of data thus generated provides physiologically relevant information on the structural organization in the KIF. A least-squares analysis using as data the positions of lysine residues involved in 23 induced cross-links has allowed the axial alignments of the various coiled-coil segments in the rod domain to be determined. Three modes of antiparallel alignment of two neighboring molecules were found: A11 (staggered by -16.7 nm), A22 (staggered by 28.8 nm), and A12 (almost in register; staggered by only 0.3 nm). Since the axial repeat length is about 1 nm less than the molecular length, the data require a fourth mode of molecule alignment, termed ACN, in which similarly directed molecules are overlapped by the equivalent of about 5-10 residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Plectin is a linker of intermediate filaments to Z-discs in skeletal muscle fibers.
Plectin is a versatile linker protein which is associated with various types of cytoskeletal components and/or filaments including intermediate filaments, and its deficiency causes the disruption of myofibrils, or muscular dystrophy. To better understand the functional role of plectin in skeletal muscle fibers, we have examined the topological and structural relationships of plectin to intermediate filaments and Z-discs in rat diaphragm muscles by confocal and immunoelectron microscopy. Immunofluorescence analysis revealed that plectin was colocalized with desmin at the periphery of Z-discs. This plectin localization around Z-discs was constantly maintained irrespective of the contracted or extended state of the muscle fibers, suggesting either direct or indirect association of plectin with Z-discs. Immunogold labeling in skinned muscle fibers clearly demonstrated that plectin-labeled fine threads linked desmin intermediate filaments to Z-discs and connected intermediate filaments to each other. These results indicate that through plectin threads desmin intermediate filaments form lateral linkages among adjacent Z-discs, preventing individual myofibrils from disruptive contraction and ensuring effective force generation.
Animals↗
Genetic skin diseases caused by mutations in keratin intermediate filaments.
Keratin intermediate filaments are the major differentiation products of epithelial cells such as the epidermis. The filaments are highly dynamic entities involved in the maintenance of the structural integrity of both the individual cells and the entire tissue. Recent biochemical studies suggest that the keratin proteins overlap each other in several key locations when packed together in filaments. Interestingly, mutations that introduce inappropriate amino acid substitutions in at least some of these overlap regions cause defective keratin filaments that result in at least three classes of autosomal dominant skin disease.
Amino Acid Sequence↗
Neurobehavioral characteristics of mice with modified intermediate filament genes.
Intermediate proteins comprise cytoskeletal elements that preserve the shape and structure of neurons. These proteins have been proposed to be involved in the onset and progression of amyotrophic lateral sclerosis (ALS), mainly characterized by motoneuron atrophy and paresis. In support of this hypothesis are the findings that genetically modified mice for intermediate filaments successfully mimic certain neuropathological aspects of ALS, such as reduced axonal caliber and retarded conduction speed in peripheral nerves, although often without leading to paresis. Nevertheless, even in those models with no overt phenotype, the involvement of intermediate proteins in motor function is underlined by the deficits in tests of balance and equilibrium revealed in mice containing transgenes for neurofilament of heavy molecular weight (NFH), alpha-internexin, peripherin, and vimentin. In addition, spatial learning was impaired in transgenic mice expressing transgenes for NFH and NFM, similar to the memory deficits reported in patients with ALS.
Amyotrophic Lateral Sclerosis↗
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↗