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PubMed1985

Differential expression of intermediate filament proteins distinguishes classic from variant small-cell lung cancer cell lines.

The expression of intermediate filament proteins in classic and variant-type small-cell lung carcinoma (SCLC) cell lines was studied using immunocytochemical techniques, two-dimensional gel electrophoresis and immunoblotting assays. Classic SCLC cell lines contain cytokeratin proteins but no neurofilaments. In contrast, variant cell lines do not contain detectable amounts of cytokeratins but partly express neurofilaments and vimentin. These results explain apparent discrepancies on the intermediate filament content of SCLC described in the recent literature. The application of antibodies to fresh biopsy specimens of SCLC may in the future allow the identification of the variant type of cells in clinical SCLC specimens and may have a major impact on therapeutic strategy and prognosis in these patients.

Carcinoma, Small Cell↗
PubMed1986

Interaction in vitro of nonepithelial intermediate filament proteins with total cellular lipids, individual phospholipids, and a phospholipid mixture.

The interaction of nonepithelial intermediate filament (IF) proteins with vesicles produced from total Ehrlich ascites tumor cell lipids results in the formation of complexes which in sucrose density gradient centrifugation attain positions distinctly different from those of the original reactants. In KBr density gradient equilibrium centrifugation, the IF protein-lipid adducts accumulate as thin proteolipid films on top of the KBr gradients, whereas in the absence of lipids the proteins remain distributed within the density gradients. Similar results were obtained with vesicles derived from individual phospholipids and a mixture thereof. The affinity of IF proteins for negatively charged phospholipids is greater than that for vesicles derived from uncharged phospholipids. Limited digestion of IF proteins with various proteinases demonstrated that for optimal association of the reactants IF proteins must carry an intact N terminus and that the isolated N-terminal polypeptide itself shows strong reactivity with lipid vesicles. Arginine-phosphate interactions between the N terminus and phospholipids seem to be partly responsible for this association. However, as shown by hydrophobic interaction chromatography on phenyl- and octyl-Sepharose 4B, IF proteins and their proteolytic derivatives also appear to have high affinities for aromatic and aliphatic substructures of biologically important molecules. The results are discussed in terms of a possible functional role of IF protein-lipid interactions in the association of nonepithelial intermediate filaments with intracellular membrane systems.

Animals↗
PubMed1992

Intermediate filamentous proteins in adult granulosa cell tumors. An immunohistochemical study of 25 cases.

Adult granulosa cell tumors (AGCTs) are classified as sex cord-stromal tumors of the ovary. However, they may be confused with other primary ovarian neoplasms. Intermediate filaments, specifically vimentin and cytokeratins, have been identified in AGCTs by immunohistochemistry performed on frozen and formalin-fixed, paraffin-embedded tissue and two-dimensional electrophoresis. Recently, however, immunohistochemical demonstration of cytokeratin has been used as supporting evidence of epithelial rather than sex cord-stromal differentiation in ovarian neoplasia. To investigate further intermediate filamentous proteins in AGCTs, 25 such tumors were studied by immunohistochemistry in formalin-fixed, paraffin-embedded sections. Cytoplasmic staining was observed, frequently in a distinct punctate, paranuclear pattern, in 14 of 25, 14 of 25, and seven of 17 tumors using monoclonal antibodies AE1/AE3, CAM 5.2, and 35BH11, respectively, which share the ability to detect low molecular weight cytokeratins. Staining for cytokeratin was not seen in any of the 17 tumors studied using the antibody 34BE12. Twenty-three of 25 tumors showed strong positivity for vimentin, characteristically seen as globoid paranuclear staining. Nine of 25 tumors contained desmin, which was restricted to the intermixed spindle cell, cortical type stromal component of the tumors. These patterns of immunoreactivity for intermediate filaments, particularly cytokeratins, are different than in common epithelial tumors of the ovary and may be useful in the differential diagnosis of ovarian neoplasia. Moreover, the immunohistochemical detection of cytokeratins should not be used as a criterion for excluding AGCT from the differential diagnosis of an ovarian neoplasm.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1989

Intermediate filaments and ubiquitin: a new thread in the understanding of chronic neurodegenerative diseases.

We have recently shown that there is a previously unsuspected link between the intracellular inclusions seen in several major chronic human degenerative diseases, including neurodegenerative diseases: the inclusions showing ubiquitin immunoreactivity. The conditions include Parkinson's disease, motor neurone disease, Alzheimer's disease, Pick's disease, and alcoholic liver disease as well as cerebellar astrocytomas and a myopathy. The inclusions found in these diseases are reported to contain intermediate filaments: neurofilaments are associated with Lewy bodies in Parkinson's disease, Pick's bodies in Pick's disease and neurofibrillary tangles in Alzheimer's disease, cytokeratins are found in Mallory bodies in alcoholic liver disease, glial fibrillary acidic proteins and vimentin are found in Rosenthal fibres in astrocytomas, and desmin is found in cytoplasmic bodies in cytoplasmic body myopathy. Therefore five classes of intermediate filaments are found in inclusions which also contain ubiquitin immunoreactivity; we have also shown that ubiquitin immunoreactivity is present in vesicles in some areas of granulovacuolar degeneration in Alzheimer's disease. Protein ubiquitination is considered a signal for extralysosomal protein degradation, (although ubiquitination may have several other important functions). We have recently shown that intermediate filaments are involved in protein sequestration before degradation by lysosomally mediated autophagy: therefore intermediate filament-containing ubiquitinated inclusions may be the hallmarks of cellular attempts to eliminate pathogenic insults by the activation of both extralysosomal and lysosomal mechanisms of intracellular protein degradation. We have recently been able to reproduce, at least in part, some of the clinical observations in tissue culture cells. Ubiquitinated protein conjugates accumulate in lysosomes in fibroblasts treated with the lysosomal cysteine protease inhibitor E-64, which may mimic aspects of granulovacuolar degeneration.

Chronic Disease↗
PubMed1990

Cyclic hair-loss and regrowth in transgenic mice overexpressing an intermediate filament gene.

We have produced transgenic mice containing up to 250 copies of a sheep wool intermediate filament keratin gene to study the effect of its expression on hair structure and development. Several transgenic lines expressed the gene and in the one containing 250 transgenes, a pattern of hair-loss and regrowth was stably established. Successive waves of hair growth follow periods of denuding like the natural progression of hairs in the mouse hair cycle. By in situ hybridization we have shown that the sheep transgenes are expressed at the correct stage in mouse hair development and at a high level. The transgenic hairs contain not only an elevated level of intermediate filament keratin protein but also a decreased level of the filament-associated proteins. This imbalance disrupts the normal ordered array of these proteins in the cells of the hair cortex and leads to weakened fibres which are prematurely lost.

Amino Acids↗
PubMed1989

Malignant small-cell epithelial tumor of the peritoneum coexpressing mesenchymal-type intermediate filaments.

A 28-year-old man with a large abdominal mass was found to have a tumor involving the peritoneum that had no other apparent primary origin. By light microscopy, the histologic pattern of the tumor was that of a small-cell epithelial neoplasm. Electron-microscopic examination demonstrated desmosomes and paranuclear aggregates of intermediate filaments. Immunohistochemical studies revealed reactivity for keratin, desmin, and vimentin. The globoid staining observed for the last two markers correlated with the paranuclear concentration of intermediate filaments observed by electron microscopy. We believe this case represents an unusual small-cell epithelial tumor, expressing mesenchymal-type intermediate filaments, that should be distinguished from other small-cell neoplasms.

Abdominal Neoplasms↗
PubMed1981

Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells.

Monospecific antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a BALB/c mouse immunized with rat skeletal myofibrils. After cloning 3 times on agarose, two stable clones were obtained and chosen for further characterization. The first clone, JLB1, produced an antibody that recognizing an antigen distributed in the M-line region and on either site of the Z line of myofibrils. The second clone, JLB7, produced an antibody reacting only with an antigen located at the M-line region of myofibrils. Both JLB1 and JLB7 antibodies decorate the typical intermediate filaments of a variety of cultured cells. Colcemid treatment of cells before reaction with both antibodies resulted in the coiling or capping (or both) of the fibers around the nucleus. Brief treatment of cells with cytochalasin B did not affect the integrity of the fibers stained by both antibodies whereas, under the same conditions, microfilament bundles visualized by another monoclonal antibody (JLA20) against actin were disassembled into many aggregates in the cytoplasm. Identical staining patterns of the intermediate filaments are obtained by double-label immunofluorescence microscopy of the same cell stained with these monoclonal antibodies and rabbit autoimmune serum (which has been shown to react with the components of the intermediate filaments). By using immunoprecipitation, protein bands at 210,000 and 95,000 daltons from chicken embryo fibroblasts were identified as the potential antigens recognized by JLB1 and JLB7 monoclonal antibodies, respectively. The widespread occurrence of these antigenic determinants in different cultured cells suggests the highly conservative property of these intermediate-filament components.

Animals↗
PubMed1999

Amphioxus type I keratin cDNA and the evolution of intermediate filament genes.

We report the cloning of an intermediate filament (IF) cDNA from the cephalochordate amphioxus that encodes a protein assignable to the type I keratin group. This is the first type I keratin reported from an invertebrate. Molecular phylogenetic analyses reveal that amphioxus also possesses a type II keratin, and that the genes encoding short-rod IF proteins underwent different patterns of duplication in vertebrates and their closest relatives, the cephalochordates. Extensive IF gene duplication and divergence may have facilitated the origin of new specialised cell types in vertebrates.

Amino Acid Sequence↗
PubMed1994

A conserved region in the tail domain of vimentin is involved in its assembly into intermediate filaments.

Although the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg-asp-gly (RDG), of the tail domain incorporated into existing IF networks in vimentin-expressing (vim+) cells, but were assembly-incompetent in cells that did not express IF proteins (vim-). The failure of the RDG mutant vimentins to assemble into filament arrays in vim- cells was reversible by re-introducing a wild-type vimentin cDNA, whereupon both wild-type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process.

Adrenal Gland Neoplasms↗
PubMed1982

Differential diagnosis of human carcinomas, sarcomas and their metastases using antibodies to intermediate-sized filaments.

Intermediate filaments (IF) are tissue-specific in so far that epithelial, mesenchymal, muscle and neural tissue types can be distinguished by the use of specific antibodies to keratin, vimentin, desmin and neurofilaments or glial filaments respectively. We have examined the possibility of using these sera in the differential diagnosis of human malignant tumors. Using antisera to human skin keratin and bovine lens vimentin we could differentiate between carcinomas (keratin +) and sarcomas (vimentin +). Furthermore, we could show that when cells become malignant and metastasize they retain their original IF and do not develop additional IF systems. We conclude that antibodies to IF proteins are powerful tools in the hands of a pathologist as an additional method to improve identification of tumors and their metastases.

Antibodies, Neoplasm↗
PubMed1991

Expression of intermediate filament proteins in the mature inner ear of the rat and guinea pig.

The expression of intermediate filament proteins was studied in the mature inner ear of the rat and guinea pig, using a panel of polyclonal and monoclonal antibodies directed against cytokeratins, desmin, neurofilament proteins and glial fibrillary acidic protein (GFAP). The epithelial lining of the endolymphatic space displayed a complex expression pattern of cytokeratin filament proteins, suggesting greater cell diversity than was known sofar from morphological studies. The cytokeratin antibodies when applied to the inner ear tissues revealed the presence of only cytokeratin polypeptides which are typical of simple epithelia (i.e. nos. 7, 8, 18, and 19). Profound differences in cytokeratin expression patterns were, however, found in the various cell types of both the cochlear and vestibular partition. Remarkably, the sensory cells appeared to be devoid of both cytokeratins and neurofilament proteins. Staining with a 200 kDa neurofilament antibody displayed the presence of different populations of ganglion cells in the spiral ganglion and the vestibular ganglion. There was no reaction with antibodies directed against desmin and GFAP. The great resemblance of the intermediate filament protein expression patterns in the inner ear of the rat and guinea pig indicates a close similarity between the different epitopes.

Animals↗
PubMed2002

Tissue-specific co-expression and in vitro heteropolymer formation of the two small branchiostoma intermediate filament proteins A3 and B2.

The two small intermediate filament (IF) proteins A3 and B2 of the cephalochordate Amphioxus were investigated. Blot overlays indicated a heterotypic interaction pattern of the recombinant proteins. While the individual proteins formed only aggregates, the stoichiometric mixture formed obligatory heteropolymeric filaments. Mutant proteins with a single cysteine residue in equivalent positions gave rise to filaments that oxidize to the disulfide-linked heterodimer, which can again form IF. Thus the A3/B2 filaments, which are expressed in the intestinal epithelium, are based on a hetero coiled coil. This keratin-like assembly process of A3 plus B2 was unexpected, since previous evolutionary tree calculations performed by two laboratories on the various Amphioxus IF proteins identified keratin I and II orthologs but left the A/B group as a separate branch. We discuss obvious evolutionary aspects of the Amphioxus IF multigene family, including the previously made observation that B1, the closest relative of B2, forms homopolymeric IF in vitro and is, like vertebrate type III proteins, expressed in mesodermally derived tissues.

Aging↗
PubMed1990

Age-dependent pattern of intermediate filament protein expression in the human pineal gland.

Intermediate filament distribution was studied in children and adult human pineal glands by using immunocytochemistry and immunoblotting. The demonstration of cytokeratin positive cell clusters in the infant pineal gland may be related to ependymal cells arising from the subcommissural organ. The SCO ependymal cells and the ependymal cells in the pineal parenchyma express the neuro-ectoderm specific individual cytokeratins no. 8 and 18. Cytokeratin positive ependymocytes additionally exhibited vimentin and were serotonin negative as revealed by double immunofluorescence labelling. By contrast, in adult pineal glands cytokeratin positive cells were not clustered. An interesting but unexplained increase of neurofilament 68 KD positive nerve fibres was detectable.

Adult↗
PubMed1992

Specific types of prosomes are associated to subnetworks of the intermediate filaments in PtK1 cells.

Prosomes are small ribonucleoprotein (RNP) particles of unique morphology in the electron microscope but of variable protein and RNA composition, depending on the differentiation state of the cells studied. They were initially observed as subcomplexes of untranslated mRNP. In previous studies, we found that prosomes are associated to the intermediate filaments (IF) of cytokeratin type in HeLa and PtK1 cells. Here we have studied in detail the association of prosomal antigens with the IF networks in PtK1 cells. Contrary to our earlier conclusions, in these cells the vimentin fibers also carry prosomes which, thus, distribute in between the two types of networks. During the selective collapse of the IF induced by acrylamide, and upon recovery after the withdrawal of the drug, no dissociation of the prosome and IF networks of cytokeratin- and vimentin-type could be observed. These data show that even in a dynamic situation, prosome and IF antigens do not dissociate, indicating strongly that they are located on one and the same structure. Furthermore, the differential distribution of specific prosomal antigens between both types of intermediate filament networks indicates that prosomes do not ubiquitously populate the intermediate filaments but occupy subnetworks of either vimentin or cytokeratin type.

Acrylamide↗
PubMed1991

Effect of human immunodeficiency virus type 1 protease on the intermediate filament subunit protein vimentin: cleavage, in vitro assembly and altered distribution of filaments in vivo following microinjection of protease.

The intermediate filament (IF) subunit protein vimentin is efficiently cleaved in vitro by purified human immunodeficiency virus type 1 (HIV-1) protease. Immunological data confirm that identical sites are cleaved when vimentin is polymerized into filaments or occurs as protofilaments. Preformed filaments require 10 times more protease to achieve the same extent of cleavage seen with protofilaments, suggesting that the cleavage sites are partially masked in IFs. The primary cleavage gives rise to molecule lacking most of the tail domain and which not only remains in preformed filaments, but also is capable of polymerizing into essentially normal 10 nm filaments. However, these filaments of the vimentin primary cleavage product show a propensity to form large lateral aggregates. The three secondary cleavage products of vimentin additionally lack portions of the head domain, are almost quantitatively released from preformed filaments and are not capable of forming filaments de novo. These results confirm and extend previous data obtained with desmin and provide a limit for that portion of the tail domain of type III IF subunit proteins that play a role in IF formation and stability. Microinjection of HIV-1 protease into cultured human skin fibroblasts resulted in a large increase in the percentage of cells with an altered and abnormal distribution of vimentin IFs. Most commonly, the IFs were observed to have collapsed into a clump with a juxtanuclear localization. The efficient cleavage of vimentin observed in vitro and the ability of microinjected HIV-1 protease to alter IF distribution in vivo suggest that IF proteins may serve as substrates within HIV-1 infected cells and may play a role in viral infection.

Amino Acid Sequence↗
PubMed1994

Multiple roles of intermediate filaments.

The widespread distribution, diversity of types, and highly conserved features of intermediate filaments (IFs) and the diversity of associated proteins indicate multiple roles for IFs in the nucleus and the cytoplasm of eukaryotic cells. Traditional functions include maintenance of cell shape, cell locomotion, movement and positioning of organelles and guiding cell development. Recent studies indicate some of these functions can now be expanded. Currently available information shows that IFs are important to nuclear function and cell division, to transport between the nucleus and cytoplasm, to the mechanical integrity of cells and tissues, to reorganization of the cytoplasm and chromatin in response to intercellular signals, and to embryonic and cellular differentiation.

Animals↗
PubMed1996

Differentiation pathways in primary invasive breast carcinoma as suggested by intermediate filament and biopathological marker expression.

The expression of intermediate filament proteins (IFPs) in 65 primary breast carcinomas was analysed by a panel of specific antibodies. Results were integrated with the oestrogen and progesterone receptor (ER and PGR) status, Ki-67 marking, and epidermal growth factor receptor (EGFr) expression. Invasive breast carcinomas could be divided into three main groups: group 1 revealed positivity only for 'simple epithelial' cytokeratins (CKs 7, 8, 18, and 19); group 2 also stained with the antibodies K8.12 and 34 beta E12; while group 3 showed co-expression of CKs 14 and 17, vimentin, and alpha-smooth muscle actin. Group 3 consistently comprised tumours with the highest Ki-67 levels, EGFr positivity, and ER-PGR negative status. On the other hand, groups 1 and 2 usually exhibited a positive hormonal status, lower proliferative activity, and EGFr negativity. The results of this study indicate that the determination of IFPs can significantly contribute to the identification of groups of patients with different biopathological settings and possibly different clinical behaviour.

Biomarkers, Tumor↗
PubMed1986

Protein-chemical identification of the major cleavage sites of the Ca2+ proteinase on murine vimentin, the mesenchymal intermediate filament protein.

Neutral thiol proteinases (calpains), activated by calcium are involved in the intracellular turnover of intermediate filaments but the precise position of the cleavage points has remained unknown. Here we identify by direct sequence analysis the major cleavage sites found when murine vimentin is digested by limited proteolysis in vitro with calpain purified from porcine kidney. Contrary to some previous suggestions, no absolute sequence specifity could be detected although 10 specific sites have been identified. This result is in line with the cDNA derived amino-acid sequence of a calpain, which pointed to a similarity of the catalytic site with the active sites in papain, cathepsin and actinidin. However, all major cleavage sites are located within regions of the vimentin molecule, which in current models of intermediate filament structure are thought to be non-helical: the amino-terminal headpiece, the carboxy-terminal tailpiece and the spacer separating the two major coiled-coil domains. The sequence information about the cleavage sites was extended to provide the amino-terminal 119 residues of murine vimentin.

Amino Acid Sequence↗