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PubMed2001

A unique type I keratin intermediate filament gene family is abundantly expressed in the inner root sheaths of sheep and human hair follicles.

A unique type I keratin intermediate filament group, comprising three highly related proteins and expressed in the inner root sheath of hair follicles, has been identified in both sheep and human. The first members from these species are named oIRSa1 and hIRSa1 and each encodes a protein of 450 amino acids, with compositional characteristics intermediate between those of previously described hair keratin and epidermal cytokeratin type I intermediate filaments. Detection of abundant mRNA transcripts derived from the sheep and human genes by cRNA in situ hybridization only in the inner root sheath and not in the medulla concurs with the findings of earlier ultrastructural analyses that have reported intermediate filaments only in the inner root sheath. Clustering of the IRSa keratin genes is apparent in the genomes of both species. The three hIRSa genes, known to reside on human chromosome 17, are closely linked to three further type I keratin intermediate filament genes of unknown function. This new gene complex, contained almost entirely within a 156 kb BAC (hRPK.142_H_19), is likely to lie near the type I intermediate filament cytokeratin and hair keratin gene loci at 17q12-q21. A phylogenetic analysis including all known human type I intermediate filament cytokeratins, hHa keratins, hIRSa, and hIRSa-linked keratins suggests that origin of the IRSa keratin intermediate filament linkage group preceded origin of most of the epidermal cytokeratins and all hair keratins during emergence of the keratin intermediate filament genes.

Amino Acid Sequence↗
PubMed1998

Intermediate filament assembly: fibrillogenesis is driven by decisive dimer-dimer interactions.

Intermediate filaments are built from one to several members of a multigene family encoding fibrous proteins that share a highly conserved hierarchic assembly plan for the formation of multistranded filaments from distinctly structured extended coiled coils. Despite the rather low primary sequence identity, intermediate filaments form apparently similar filaments with regard to their spatial dimensions and physical properties. Over the past few years, substantial progress has been made in the elucidation of the complex expression patterns and clinically relevant phenotypes of intermediate filaments. The key question of how these filaments assemble and what the molecular architecture of their distinct assembly intermediates comprises, however, has still not been answered to the extent that has been achieved for microfilaments and microtubules.

Animals↗
PubMed1999

Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins.

Type IV neuronal intermediate filament proteins consist of alpha-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by replacing the head domain of alpha-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, alphaLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.

Amino Acid Sequence↗
PubMed1987

Intermediate filament expression in human fetal olfactory epithelium.

Antibodies to intermediate filaments and to desmoplakin were used to investigate the histogenetic origin of the olfactory neuroepithelium. Intermediate-filaments are tissue-specific molecular cytoskeletal markers; desmoplakin is the major desmosomal protein. The olfactory epithelia of eight human fetuses, aged 7 to 10 weeks (fertilization age) were immunofluorescently labeled with antibodies to the five classes of intermediate-filament proteins and to desmoplakin. Positive immunoreactivity to keratin and to desmoplakin was observed; both results indicate the epithelial nature of this tissue. The absence of neurofilaments and glial-fibrillary acidic protein in the tissue containing sensory neurons and gliallike supporting cells is a unique feature, and may be related to the fact that olfactory neuroepithelium, like other epithelia, undergoes continuous turnover.

Antibodies, Monoclonal↗
PubMed1987

Direct morphological demonstration of the coexistence of vimentin and desmin in the same intermediate filaments of vascular smooth muscle cells.

The stable coexistence of the intermediate filament proteins desmin and vimentin in vascular smooth muscle cells raises questions about the relative amounts of the two proteins in different individual cells, and the distribution of the two proteins in individual intermediate filaments within each cell. These questions have been explored by double immunofluorescence microscopy and double immunoelectron microscopy on semi-thin and ultrathin frozen sections of chicken aorta. The former studies indicate that there is a surprisingly wide variation in the desmin/vimentin ratio in adjacent smooth muscle cells. The latter studies show that both proteins are present in individual intermediate filaments, in clustered arrays rather than uniformly distributed. These findings extend earlier related results, and suggest that the turnover of intermediate filaments may involve the remodelling of existing filaments rather than their de novo polymerization.

Animals↗
PubMed1984

Differential effect of arginine modification with 1,2-cyclohexanedione on the capacity of vimentin and desmin to assemble into intermediate filaments and to bind to nucleic acids.

When the intermediate filament proteins vimentin and desmin were reacted for a short period of time with the arginine-specific reagent 1,2-cyclohexanedione, the modification had a severe, inhibitory effect on the assembly of intermediate filaments and on the susceptibility of the basic, amino-terminal polypeptide of both proteins to degradation by the intermediate filament-specific, Ca2+-activated proteinase. However, it had only a slightly inhibitory effect on the binding of vimentin and desmin to ribosomal RNA from Ehrlich ascites tumour cells. Since the Ca2+-activated proteinase is very likely to be a trypsin-like enzyme, with a preference for arginyl and lysyl peptide bonds, the results indicate that the arginine residues of the amino-terminal polypeptide of vimentin and desmin are highly essential for filament assembly but largely dispensable for the binding of both proteins to nucleic acids. This was supported by the observation that two breakdown products of vimentin lacking a 5 X 10(3) Mr and an 8 X 10(3) Mr polypeptide from the amino terminus, respectively, did not assemble into intermediate filaments but were still capable of binding to rRNA. Both polypeptides also bound to single-stranded DNA-cellulose under non-denaturing conditions, but passed the affinity column in the presence of 6 M-urea. Thus, the binding of vimentin to nucleic acids appears to be based on two components: a non-specific electrostatic interaction mediated by the positively charged arginine residues of the amino-terminal polypeptide that is insensitive to denaturation by urea, and a specific interaction that is sensitive to denaturation by urea.

Arginine↗
PubMedDate not supplied

Monoclonal antibodies to intermediate filament proteins: diagnostic specificity in orbital pathology.

Intermediate filaments derived from different cell types are antigenically distinct. Monoclonal antibodies to human intermediate filament proteins can, therefore, be used as tissue-specific reagents capable of distinguishing cell type in poorly differentiated neoplasms. We report a case demonstrating the specificity of antiintermediate filament protein antibodies in establishing a difficult orbital diagnosis of esthesioneuroblastoma.

Antibodies, Monoclonal↗
PubMed1982

In vitro differentiation of mouse teratocarcinoma cells monitored by intermediate filament expression.

Teratocarcinoma differentiation has been studied using sera specific for each of the five intermediate filament (IF) classes. These antibodies distinguish cells of epithelial, muscle, neural, astrocytic, and mesenchymal origin. In embryoid bodies, derived from embryo transplants and obtained in the ascitic fluid by transplantation of teratocarcinoma, the cells of the inner cellular mass did not express any of these intermediate filament types while the outer cells expressed cytokeratin. Intermediate filament expression in the embryoid body thus appears analogous to that in the blastocyst and differs from that in embryonal carcinoma (EC) lines. Twelve EC lines have now been shown to express vimentin although in some EC lines not all cells express vimentin. Other established permanent differentiated cell lines, derived from EC lines in vitro or from tumors in vivo, have been characterized with respect to the type of IF they contain. The distribution of different IF types has been examined in EC cells induced to differentiate by addition of retinoic acid. The proportion of cells expressing each type of intermediate filament appears to depend on the EC cell line used, on the inducing agent, and on the length of treatment. Thus, for instance, F9 cells express cytokeratin, PCC3 derivatives express vimentin, many 1009 derivatives express either glial fibrillar acidic protein (GFA) or neurofilament proteins. Overall the results obtained are in excellent agreement with emerging principles of intermediate filament expression during embryonic differentiation, thus emphasizing the potential use of the various EC lines to study differentiation in culture.

Animals↗
PubMed1984

Characterization and regulation in the expression of a gene coding for the intermediate filament protein desmin.

Using synthetic oligonucleotide probes, we have isolated chicken cDNA clones for the intermediate filament protein desmin. We show that the gene for this protein probably exists as a single copy in the haploid chicken genome and is transcribed into one mature mRNA species of approximately equal to 2.4 kilobases. Expression of this mRNA is tissue-specific, as it is present in high abundance in smooth and skeletal muscle but is absent from erythrocytes, spinal cord, and lens cells. A 10- to 20-fold increase in desmin mRNA is observed in myogenic cells upon fusion, which suggests that the level of expression of the desmin gene and the accumulation of desmin filaments during muscle differentiation is regulated at the transcriptional and/or posttranscriptional level but not at the translational level. Hybridization studies and nucleotide sequence comparison of the cDNAs specific for desmin and for other intermediate filament subunits reveal a region that is highly conserved (80%) among different members of the intermediate filament protein gene superfamily, with the exception of the chicken vimentin gene; this gene appears to be less homologous to the genes for the other chicken intermediate filament subunits than the mammalian vimentin gene is to the genes for other mammalian intermediate filament proteins and to the chicken desmin gene.

Animals↗
PubMed1986

Differential expression of intermediate filament proteins in metastatic and nonmetastatic variants of the BSp73 tumor.

The pattern of intermediate filament protein expression was studied in tumor cell variants of the BSp73 spontaneous rat adenocarcinoma of the pancreas exhibiting distinct morphology and metastatic phenotype. The non-metastasizing AS cells which adhere and spread on a solid substrate express only the vimentin type mesenchymal intermediate filament protein. The ASML metastatic cell variant which adheres but does not spread on the substrate expresses a complex pattern of cytokeratins characteristic of the adenocarcinoma of the pancreas and a low level of vimentin. The differences in the expression of the intermediate filament proteins between the variants were also reflected at the level of the corresponding mRNAs as revealed by RNA blot analysis with complementary DNA clones specific to vimentin and the acidic as well as the basic cytokeratin subfamily. When the two cell variants were cultured for 72 h in suspension culture on nonadhesive substrata the AS cells responded with a marked reduction in their vimentin synthesis. The ASML variant cells which are characterized by a round configuration in both monolayer and suspension culture continue to synthesize the same intermediate filament proteins under both culture conditions. The relationships among environmental conditions that affect cell shape and contacts, the shifts in the expression of intermediate filaments, and the metastatic property of tumor cells are discussed.

Adenocarcinoma↗
PubMed1981

All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody.

We have produced a monoclonal antibody that reacts with all classes of intermediate filaments in immunofluorescence assays, including glial filaments in astrocytes, neurofilaments in axons, tonofilaments in epithelial PtK2 cells and intermediate filaments in fibroblasts. It also binds to Z lines in skeletal muscle. In SDS-polyacrylamide gels, the antibody binds to most and perhaps all of the major intermediate filament proteins that have been previously defined, including glial fibrillary acidic protein, the three vertebrate neurofilament proteins (the "neurofilament triplet"), vimentin, desmin, several cytokeratins and the neurofilament proteins of squid and the marine worm Myxicola. In addition, the antibody binds to a protein with an approximate molecular weight of 66,000 that may be a component of all intermediate filaments. These findings suggest that all vertebrate and invertebrate intermediate filament proteins share a common antigenic determinant and raise the possibility that all intermediate filaments contain a 66,000 molecular weight protein.

Animals↗
PubMed2004

The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton.

Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.

Adaptor Protein Complex 3↗
PubMedDate not supplied

On-grid immunogold labeling of glial intermediate filaments in epoxy-embedded tissue.

On-grid immunogold labeling of structures like intermediate filaments has been difficult to achieve. Presumably this is because such structures are thinner than the thin sections themselves and because gold-labeled reagents remain on the surface and do not penetrate epoxy resins. Many pathologic and other tissues, however, are primarily available as epoxy-embedded blocks, and a postembedding gold procedure capable of detecting such thin structures would be useful. This study aimed to investigate the astrocytic intermediate filament antigen glial fibrillary acidic protein (GFAP) in glutaraldehyde-fixed, epoxy-embedded brain biopsy tissue from a child with Alexander's disease. A protocol was developed for performing on-grid immunogold labeling which minimized nonspecific deposition of gold reagent. The method utilized ovalbumin and skim milk in the washes and diluent for the gold reagent and the same solution with added Tween-20 and high sodium chloride in the diluent for antibodies and normal serum. In grids etched with metaperiodate and hydrogen peroxide, the astrocytic intermediate filaments were only occasionally and sparsely labeled. When an etching procedure with sodium ethoxide was employed, however, extensive labeling was obtained on the astrocytic intermediate filaments. In contrast, the larger, pathological Rosenthal fibers characteristic of Alexander's disease were labeled after both etching procedures, but labeling was enhanced after ethoxide etching. Postosmicated material showed much less labeling. The findings demonstrate that postembedding procedures can be used with epoxy-embedded material to immunolabel thin structures like intermediate filaments.

Cytoskeleton↗
PubMed1996

Intermediate filaments in the nervous system: implications in cancer.

In this review, we describe the different intermediate filament (IF) proteins, their assembly into IFs, the functions of IFs and their relation to disease with a particular emphasis on the intermediate filaments expressed in the nervous system. In the mammalian nervous system, seven intermediate filament proteins are known to be expressed in neurons or neuroblasts. These include the three neurofilament triplet proteins, which are present in both central and peripheral neurons; alpha-internexin, which is the first neuronal intermediate filament protein expressed in the developing mammalian nervous system and present primarily in CNS neurons in the adult nervous system; peripherin, which is most abundant in the PNS; vimentin, which is expressed in neuronal progenitor cells along with nestin, as well as in a few adult neurons. In contrast to these neuron-specific IF proteins, the glial fibrillary acidic protein (GFAP) is glial specific and expressed in mature astrocytes. Vimentin and nestin are also expressed in glial progenitor cells and vimentin is expressed along with GFAP in some mature astrocytes. As a whole, the expression of IF proteins is tissue specific and developmentally regulated. As a result, IF proteins are good markers for determining the cell origin and differentiation status of tumor cells. For example, peripherin is expressed in neuroblastomas, GFAP in astrocytomas and neurofilaments in tumors of neuronal origin. However, tumor cells may express IF patterns which are irrelevant to their cell origin. Therefore, one has to be very careful in using IF patterns as sole indicators of cell origin and differentiation status of tumors.

Animals↗
PubMed1996

Effects of intermediate filament disruption on the early development of the peripheral nervous system of Xenopus laevis.

The principal function of intermediate filaments is to strengthen cells. Their developmentally regulated, tissue-specific patterns of expression further suggest that they modulate cellular structural properties during development. To explore the role of intermediate filaments in development, we injected RNA encoding a truncated form of the Xenopus laevis middle-molecular-weight neurofilament protein (NF-M) into embryonic frog blastomeres at the 2-cell stage. A similar truncated form of mammalian NF-M disrupts neurofilaments (Type IV) and vimentin (Type III) intermediate filaments in transfected fibroblasts. In cultures made from dissociated neural tubes and their adjacent myotomes, the resultant protein disrupted both desmin filaments in muscle cells and neurofilaments in neurons during the first day of culture, which corresponds to stage 35/36 in the intact embryo. We next examined the effects of this truncated neurofilament protein on development of the nervous system. The greatest effects were seen on development of cranial and primary motor nerves, which were severely stunted as late as stage 37/38. In addition to these effects, ectopic neurons also appeared immediately beneath the epidermis along the flank of tadpoles expressing the truncated neurofilament protein. Whereas the former effects on peripheral nerve development were nearly identical to effects obtained with injected neurofilament antibodies, the ectopic neurons were novel, suggesting they resulted from the disruption of intermediate filaments other than the neurofilaments. These experiments thus implicate intermediate filaments in several functions important for normal neural development.

Animals↗
PubMed1987

Human lung tumours: does intermediate filament co-expression correlate with other morphological or immunocytochemical features?

Co-expression of intermediate filaments is being increasingly reported for many human tumours including carcinoma of the lung. However, it is unclear whether such findings are unusual or restricted to a group of highly atypical tumours. In the present study the pattern of co-expression of intermediate filaments in 94 human lung tumours has been correlated with light and electron microscopical features which are thought to be atypical for particular tumour types. These same aberrant patterns of intermediate filament co-expression have also been compared with the proliferative rate of these tumours as determined by labelling with the monoclonal antibody Ki67. The results of this study have shown that the aberrant expression of intermediate filaments is not a feature unique to a group of highly unusual tumours but is found throughout the spectrum of lung cancer. The implications of these findings for the use of anti-intermediate filament antibodies in pulmonary pathology are discussed with suggestions for future directions which might be taken in this field.

Adenocarcinoma↗
PubMed1996

Role of intermediate filaments in migration, invasion and metastasis.

The expression of intermediate filament proteins is remarkably tissue-specific which suggests that the intermediate filament (IF) type(s) present in cells is somehow related to their biological function. However, in some cancers-particularly malignant melanoma and breast carcinoma, there is a strong indication that vimentin and keratin IFs are coexpressed, thus presenting as a dedifferentiated or interconverted (between epithelial and mesenchymal) phenotype. In this review, two in vitro models are presented which recapitulate the interconverted phenotype in human melanoma and breast carcinoma, and allow, for the first time, unique observations to be made with respect to the role of IFs in cancer progression. These studies have provided direct evidence linking overexpression of keratin IFs in human melanoma with increased migratory and invasive activity in vitro, which can be down-regulated by substituting dominant-negative keratin mutants. Overexpression of vimentin IFs in the breast carcinoma model leads to augmentation of motility and invasiveness in vitro, which can be transiently down-regulated by treatment with antisense oligonucleotides to vimentin. Additional experimental evidence suggests that the mechanism(s) responsible for the differential expression of metastatic properties associated with the interconverted phenotype rest(s) in the unique interaction, either direct or indirect, of IFs with specific integrins interacting with the extracellular matrix. In this review, we discuss the observations derived from the human melanoma and breast carcinoma models to address the hypothesis that the ability to coexpress vimentin and keratins confers a selective advantage to tumor cells in their interpretation of and response to signaling cues from the extracellular matrix. The ramifications of these observations are discussed with respect to the patholophysiology of the respective in situ tumors.

Animals↗
PubMed1987

Intermediate filaments and their organization in human corneal endothelium.

The native intermediate filament network within human corneal endothelium was identified by a monoclonal antibody to vimentin intermediate filament protein. Human corneal endothelial cells in tissue culture were shown to react positively to this monoclonal antibody by indirect immunofluorescence microscopy. In cryostat sections of human cornea, only the endothelial cells and keratocytes stained for vimentin. Fluorescent staining patterns of the cultured endothelial cells demonstrated that vimentin forms arrays of cytoplasmic filaments which encapsulate the nucleus and anchor in the apical junctions. These results reveal a previously undescribed cytoskeleton in human corneal endothelium and suggest that the intermediate filament vimentin comprises this structural network which is, in part, responsible for nuclear centration and cell-to-cell contacts. In addition, these results may indicate the possible embryogenesis of the corneal endothelium, since vimentin is a marker for cells of mesenchymal origin.

Adult↗