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PubMed2003

Proteasome inhibition induces inclusion bodies associated with intermediate filaments and fragmentation of the Golgi apparatus.

The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.

Autophagy↗
PubMed1986

Intermediate filaments in alpha-keratins.

Previous x-ray diffraction studies on the alpha-keratins of hair and wool have revealed that the intermediate filaments (IF) have a helical structure rendered imperfect by a precisely defined dislocation. It has also been possible to deduce a surface lattice for the IF and to determine the number of IF molecules associated with each lattice point. In this work this information is combined with data on the ionic interactions between the coiled-coil rope segments of the IF molecules to provide a plausible model for the pattern of interactions that stabilize the framework of the IF in the "hard" alpha-keratins. Similar interaction studies of the proteins from the IF in the so-called "soft" alpha-keratin from the stratum corneum layer of the skin suggest that they are likely to have an essentially similar pattern.

Animals↗
PubMed1994

Intermediate filament reactivity in hyperplastic and neoplastic lesions from medaka (Oryzias latipes).

To determine if hyperplastic and neoplastic lesions from medaka showed similar immunoreactivity to intermediate filament antibodies as the tissues of origin, two week old medaka were exposed to 10 or 20 mg/L of methylazoxymethanol acetate for two hours and transferred to clean water for up to six months. Using a streptavidin peroxidase method, paraffin embedded Bouins fixed neoplasms were incubated with cytokeratin, vimentin, or neurofilament antibodies. Like their nonneoplastic cellular counterparts, hepatocellular carcinoma, pancreatic acinar carcinoma and mesenchymal neoplasms including hemangioma and hemangiopericytoma reacted negatively to cytokeratin antibodies. Cholangiocarcinoma, mesothelioma, and proliferative lesions containing biliary epithelial cells reacted positively to cytokeratin antibodies. All neoplasms and proliferative lesions were negative with vimentin and neurofilament antibodies. These data indicate that while some epithelial neoplasms showed cytokeratin reactivity similar to the parent tissues, additional markers are needed to identify mesenchymal tissues and neoplasms.

Adenoma, Liver Cell↗
PubMed1999

Gliarin and macrolin, two novel intermediate filament proteins specifically expressed in sets and subsets of glial cells in leech central nervous system.

Using monoclonal antibodies, we have identified two novel intermediate filament (IF) proteins, Gliarin and Macrolin, which are specifically expressed in the central nervous system of an invertebrate. The two proteins both contain the coiled-coil rod domain typical of the superfamily of IF proteins flanked by unique N- and C-terminal domains. Gliarin was found in all glial cells including macro- and microglial cells, whereas Macrolin was expressed in only a single pair of giant connective glial cells. The identification of Macrolin and Gliarin together with the characterization of the strictly neuronal IF protein Filarin in leech central nervous system demonstrate that multiple neuron- and glial-specific IFs are not unique to the vertebrate nervous system but are also found in invertebrates. Interestingly, phylogenetic analysis based on maximum parsimony indicated that the presence of neuron- and glial cell-specific IFs in coelomate protostomes as well as in vertebrates is not of monophyletic origin, but rather represents convergent evolution and appears to have arisen independently.

Amino Acid Sequence↗
PubMed2004

Great promises yet to be fulfilled: defining keratin intermediate filament function in vivo.

Keratins are abundant proteins in epithelial cells, in which they occur as a cytoplasmic network of 10 - 12 nm wide intermediate filaments (IFs). They are encoded by a large family of conserved genes in mammals, with more than 50 individual members partitioned into two sequence types. A strict requirement for the heteropolymerization of type I and type II keratin proteins during filament formation underlies the pairwise transcriptional regulation of keratin genes. In addition, individual pairs are regulated in a tissue-type and differentiation-specific manner. Elucidating the rationale behind the diversity and differential distribution of keratin proteins offers the promise of novel insight into epithelial biology. At present, we know that keratin IFs act as resilient yet pliable scaffolds that endow epithelial cells with the ability to sustain mechanical and non-mechanical stresses. Accordingly, inherited mutations altering the coding sequence of keratins underlie several epithelial fragility disorders. In addition, keratin IFs influence the cellular response to pro-apoptotic signals in specific settings, and the routing of membrane proteins in polarized epithelia. Here we review studies focused on a subset of keratin genes, K6, K16 and K17, showing a complex regulation in vivo, including a widely known upregulation during wound repair and in diseased skin. Progress in defining the function of these and other keratins through gene manipulation in mice has been hampered by functional redundancy within the family. Still, detailed studies of the phenotype exhibited by K6 and K17 null mice yielded novel insight into the properties and function of keratin IFs in vivo.

Animals↗
PubMed1996

Differential nuclear matrix-intermediate filament expression in human prostate cancer in respect to benign prostatic hyperplasia.

We have characterized the changes in composition of the nuclear matrix-intermediate filament complex (NM-IF) isolated from prostate cancer (PCa), compared with benign prostatic hyperplasia (BPH). We prepared the NM-IF from ten patients undergoing radical retropubic prostectomy; the benign hyperplastic tissue was obtained from the prostate lobe contralateral to the cancer zone. Several quantitative and qualitative changes have been identified. Three new proteins of molecular weight 48, 47 and 29 kDa and isoelectric point 6.0, 4.9 and 6.4, respectively, were detected in PCa, referred to here as P8, P5 and NM-1, P8 was present in all ten of the tumors examined, P5 was expressed in 9/10 PCa; conversely, they were present in only one and two BPH, respectively; NM-1 was found in eight tumors out of nine and never in BPH. These proteins are expressed in moderately differentiated malignant cells, suggesting that the proteins of the NM-IF complex can be interesting biomarkers for prostate cancer. Immunoblot analysis shows that P8 and P5 proteins belong to the IF superfamily. This observation, taken together with previous data obtained by our and other groups, suggests that the characterization of NM-IF protein changes could also shed light on mechanistic aspects of cancer progression.

Aged↗
PubMed1990

The two-chain coiled-coil molecule of native epidermal keratin intermediate filaments is a type I-type II heterodimer.

The composition of the two-chain coiled-coil molecule of murine epidermal keratin intermediate filaments (KIF) containing keratins 1 (type II) and 10 (type I) has been explored using native-type KIF as well as KIF reassembled in vitro from protein dissolved in urea solutions or from mixtures of 3H-labeled and unlabeled purified chains. By use of cross-linking, high resolution polyacrylamide gel electrophoresis and blotting for 3H-labeled keratins or with an anti-mouse keratin 10 antibody, it was found that individual keratin chains form type I or type II homodimers and homotetramers in solution that do not assemble into KIF in vitro. When mixed in urea solutions of 5 M or greater, such homo-oligomers rapidly rearrange into mostly heterodimers and heterotetramers that support filament assembly. On the other hand, prekeratin, isolated from newborn mouse epidermis with 0.1 M sodium citrate buffer, pH 2.6, under conditions that do not dissociate the native coiled-coil molecule, consists exclusively of type I-II heterodimers and heterotetramers. It is necessary to dissolve prekeratin in 8-9.5 M urea for several hours in order to dissociate the native heterodimer molecule and incorporate tracer amounts of a single 3H-labeled keratin chain. These data establish that native KIF consist of heterodimer coiled-coil molecules. Furthermore, heterodimers are much more stable than homodimers and are the favored form of association in solution. However, some homodimers (10-30% of total) always form after dissolution in concentrated urea and can be assimilated into KIF during reassembly in vitro. The isolation of alpha-helix-enriched dimer particles from the 2B region of the rod domains upon limited proteolysis confirmed the presence of mostly heterodimer and some homodimer molecules in reassembled KIF. These properties of keratin chains in urea solutions hereby clarify a number of conflicting reports in the literature concerning the composition of the coiled-coil molecule. The presence of some homo-oligomeric species in reassembled KIF correlates with earlier observations of polymorphism as well as stoichiometry.

Animals↗
PubMed1993

Immunohistochemical study of intermediate filament proteins on routinely processed, celloidin-embedded human temporal bone sections by using a new technique for antigen retrieval.

Although immunohistochemical studies of intermediate filament proteins have been carried out on temporal bone sections by using modified fixation/embedding techniques to preserve antigenicity, there have been no light microscopic studies concerning immunohistochemical staining on routinely formalin-fixed celloidin-embedded human temporal bone sections. A method for immunostaining routinely processed celloidin-embedded tissues would be extremely valuable in that it would permit study of the extensive collections of formalin-celloidin temporal bone specimens that exist in major centers of otopathologic research. Recently, we have developed a new technique which can be used to retrieve the antigenicity masked by formalin fixation and decalcification. This method requires immersing slides for 30 min at room temperature in a solution of saturated sodium hydroxide in methanol before immunostaining. Using this method, 45 celloidin-embedded human temporal bone sections were stained with monoclonal antibodies to keratin, vimentin, neurofilament, glial fibrillary acidic protein and desmin as primary antibodies using a sensitive streptavidin-biotin procedure. The results obtained by using this technique are at least equivalent to those obtained with modified fixatives, cryosections or immuno-electron microscopy. This new method may provide a useful approach for studying routinely processed, celloidin-embedded human temporal bone sections and open a new field in immuno-otopathology.

Adult↗
PubMed1988

Vimentin and glial fibrillary acidic protein are codistributed in the same intermediate filament system of malignant glioma cells in vivo. A double-labeling immunoelectron-microscopical study.

Normal, reactive, and neoplastic astrocytes express two types of intermediate filament (IF) proteins, namely glial fibrillary acidic protein (GFAP) and vimentin. Their submicroscopical distribution in vivo is so far unknown. We therefore investigated four malignant gliomas by electron microscopy, applying postembedding double immunogold labeling. The IF proteins were randomly scattered over the same filament bundles, as in previous experiments on glioma cultures. No clustering or preferential intracytoplasmic location of either IF protein was visible. The demonstration of IF proteins within nuclei gives some support to the suggested intranuclear functions of IF proteins.

Astrocytes↗
PubMed1989

Myopathy associated with desmin type intermediate filaments. An immunoelectron microscopic study.

The muscle biopsy performed in a 58-year-old woman with a myopathy involving pelvic girdle and lower limbs displayed unusual intermediate filament aggregates by light and electron microscopy. No cardiac involvement was detected. The filamentous aggregates selective for type 1 fibers were found in subsarcolemmal and intermyofibrillar areas closely related to Z bands. Immunohistochemical study by light and electron microscopy using polyclonal and monoclonal antibodies and avidin-biotin peroxidase method revealed that aggregates consisted of desmin filaments. Desmin positive material was unstained with vimentine antibodies.

Cytoskeleton↗
PubMed1998

Properties of the novel intermediate filament protein synemin and its identification in mammalian muscle.

We examined specific properties of highly purified synemin (230 kDa), recently identified as a novel intermediate filament (IF) protein, from avian smooth muscle. Soluble synemin in 10 mM Tris-HCl, pH 8.5, appears as approximately 11-nm-diameter globular structures by negative-stain and low-angle shadow electron microscopy. Chemical crosslinking and SDS-PAGE analysis indicate that soluble synemin molecules contain two 230-kDa subunits. The pH- and ionic strength-dependent solubility properties of synemin are similar to those of the type III IF protein desmin, but under physiological-like conditions in which desmin self-assembles into long approximately 10-nm-diameter IFs, synemin self-associates into complex, approx 15- to 25-nm-diameter globular structures. Calpain digestion demonstrated that synemin is extremely proteolytically labile. Western blot analysis, with monospecific polyclonal antibodies against avian synemin, shows the presence of the reactive 230-kDa synemin band in samples of adult avian skeletal, cardiac, and smooth muscle and of two reactive bands at approximately 225 kDa (major) and approximately 195 kDa in adult porcine skeletal, cardiac, and smooth muscle. Partial purification of synemin from porcine smooth muscle also resulted in fractions highly enriched in the approximately 225- and approximately 195-kDa polypeptides. Conventional immunofluorescence and immunoconfocal microscopy of isolated myofibrils and of frozen sections also demonstrated, for the first time, that synemin is present in all three adult porcine muscle cell types and is colocalized with desmin in skeletal and cardiac muscle cells at the myofibrillar Z-lines.

Animals↗
PubMed1991

Intermediate filaments formed de novo from tail-less cytokeratins in the cytoplasm and in the nucleus.

The roles of the different molecular domains of intermediate filament (IF) proteins in the assembly and higher order organization of IF structures have recently been studied by various groups but with partially controversial results. To examine the requirement of the aminoterminal (head) and the carboxyterminal (tail) domain of cytokeratins (CKs) for de novo IF formation in the living cell, we have constructed cDNAs coding for intact as well as head- and/or tail-less human CKs 8 and 18 and the naturally tail-less human CK 19, all under the control of the human beta-actin promoter. After transient and stable transfections of mouse 3T3-L1 cells, which are devoid of any CKs, we have studied, with such constructs, the resulting gene products by gel electrophoresis and immunolocalization techniques. By light and electron microscopy we show that extended cytoplasmic IF meshworks are formed from pairs of the type II CK 8 with the type I CKs 18 or 19 as well as from pairs of tail-less CK 8 with tail-less CKs 18 or 19 in the transfected cells, proving that the absence of the tail domain in both types of CKs does not prevent the de novo formation of regular IFs. Most surprisingly, however, we have observed spectacular alterations in the nucleocytoplasmic distribution of the IFs formed from tail-less CKs. In many of the transfected cells, a large part, or all, of the detectable CKs was found to occur in extensive IF bundles in the nucleoplasm. Intranuclear accumulations of CK deposits, however mostly nonfibrillar, were also observed when the cells had been transfected with cDNAs encoding tail-less CKs also lacking their head domains, whereas CKs deleted only in the head domain were found exclusively in the cytoplasm. The specific domain requirements for the assembly of cytoplasmic IF bundles are discussed and possible mechanisms of intranuclear accumulation of IFs are proposed.

3T3 Cells↗
PubMed1994

Coiled-coil stutter and link segments in keratin and other intermediate filament molecules: a computer modeling study.

Structural discontinuities have previously been identified in four regions of the coiled-coil rod domain structure present in intermediate filament (IF) protein molecules. These include a point at which a phase shift occurs in the heptad periodicity characteristic of the sequence of polar and apolar residues in alpha-helical coiled-coils, and three links that lack a heptad substructure. We have studied these regions by computer-based molecular modeling and comparative sequence analysis and conclude that the phasing discontinuity can be accommodated without significant distortion of the overall double-helical chain conformation; the L2 link has a similar conformation in all different types of IF molecules, a favorable conformation being one in which the two strands wrap tightly around each other; the L12 links vary in length between different IF types but contain important sequence similarities suggestive of a partial beta structure; the L1 links show larger variations in length, a lower degree of similarity, and probably diverse structures. Variations in the overall charges of the different links suggest that ionic interactions may play a significant role in filament assembly. The results also have general significance for other alpha-fibrous proteins in which either the characteristic heptad phasing undergoes a discontinuity or where a short non-coiled-coil sequence occurs within a coiled-coil rod domain structure.

Amino Acid Sequence↗
PubMed1994

Structural elements of the amino-terminal head domain of vimentin essential for intermediate filament formation in vivo and in vitro.

The biological functions of the non-alpha-helical, N- and C-terminal head and tail domains of intermediate filament (IF) proteins are still ill-defined. Previously, it has been shown that the basic, N-terminal head piece of the type III IF protein vimentin is essential for regular IF assembly and that arginine residues within the N-terminus may be involved. In order to identify particular regions within this domain essential for filament formation and stabilization, N-terminally truncated and arginine substitution forms of vimentin were constructed via site-directed in vitro mutagenesis of murine vimentin cDNA. The de novo filament assembly properties of these modified forms were compared with those of wild-type vimentin after transient expression in vimentin-free, cultured cells. In order to investigate their filament assembly competence in vitro, they were also produced in an E. coli expression system. It could be demonstrated that deletion of the first 10, 13, 17, and 32 amino acid residues, respectively, from the N-terminus of vimentin has an increasingly deleterious effect on filament assembly in vitro and network formation in vivo and that, thus, the highly conserved sequence motif, SSYRRXFGG, located in the N-terminus of various IF proteins and partially or totally removed by the above deletions plays a particularly important role in both activities. These results were confirmed and extended by arginine point mutations in the N-terminal head region, which showed that only one of the two adjacent arginine residues located within the conserved sequence motif is essential for filament assembly and stability in vitro as well as network formation in vivo. The neighboring arginine residues could be replaced by lysine residues without severe effects on the assembly properties of the respective mutant proteins. Distinction between the assembly-promoting potentials of the two arginine residues of the N-terminal doublet was considerably facilitated by a Val389-->Asp substitution toward the carboxy-end of the 2B segment of the vimentin rod domain. The synergistic effect of point mutations in this and the N-terminal region of the vimentin molecule implies the interaction of both protein domains in the process of filament assembly. Mutant vimentin proteins that were characterized by distinct incompetence to assemble into IFs caused a massive collapse of the endogenous vimentin filament system when expressed in mouse skin fibroblasts.

Amino Acid Sequence↗
PubMed1990

Characterization by monoclonal antibody of a highly conserved antigenic determinant expressed on human platelet membranes and intermediate filament type III.

The murine monoclonal antibody (MoAb) CB21, raised after immunization with sonicated extracts of human platelets, has been shown to react with a line-restricted surface molecule and also a cytoplasmic structure displaying no restriction in terms of lineage and species. The surface structure recognized by the CB21 MoAb is exclusively expressed on the surface membrane of human platelets, being undetectable on other cells or lines so far tested. After permeabilization, the majority of the cells and lines tested with the CB21 MoAb displayed strong cytoplasmic reactivity with a constant typical filamentous distribution. Biochemical and morphological analyses showed that the cytoplasmic counterpart recognized by the CB21 MoAb is the intermediate filament type III.

Animals↗
PubMed1992

An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.

Actin Cytoskeleton↗
PubMed1985

Three-dimensional observations on intermediate filaments of the squamous epithelium of the uterine cervix.

The following conclusions have been drawn from electron-microscopic three-dimentional observations on the intermediate filaments (IFs) of the squamous epithelium from the uterine cervix. (1) IFs form a three-dimensional reticular network and wrap around the intracellular periphery of cells extending from the basal to the superficial layers. (2) In basal and parabasal cells, the IFs are distributed in a pattern radiating from the perinuclear location toward the cellular periphery. (3) The use of PEG-embedding method after detergent-extraction with Triton X-100 and saponin is suitable for the three-dimensional observations on IFs of stratified squamous epithelium from the uterine cervix.

Cervix Uteri↗
PubMed1983

Structural homology between hard alpha-keratin and the intermediate filament proteins desmin and vimentin.

Although it has been assumed that the microfibrils in hard -keratin are members of the class of structures known as intermediate filaments (IF), no firm chemical evidence relating the low-sulfur proteins in hard -keratin to other IF proteins has yet been published. We now present primary sequence data for two components from wool keratin which show striking similarities with two IF proteins, desmin and vimentin. The sequences show marked homology, a heptad repeat and a 9.5-residue periodicity in the linear disposition of the acidic and the basic residues. These data thus provide the first evidence that the low-sulfur proteins in hard -keratin and the other IF proteins do indeed have both a similar structure and a common evolutionary origin.

Amino Acid Sequence↗