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PubMed1989

A 48 kilodalton intermediate filament associated protein (IFAP) in reactive-like astrocytes induced by dibutyryl cyclic AMP in culture and in reactive astrocytes in situ.

In this paper we demonstrate that the 48 kilodalton (kDa) intermediate filament associated protein (IFAP), previously reported to be present in normal astrocytes, is also present in reactive astrocytes in situ and in reactive-like astrocytes induced by dibutyryl cyclic AMP in vitro. This IFAP is detectable by antibodies in normal rabbit serum (F2N) and is closely associated with glial fibrillary acidic protein-containing intermediate filaments (IF). The expression of 48 kDa IFAP is related to the acquisition of stellate shape by normal and reactive-like astrocytes in vitro. It is proposed that 48 kDa IFAP may be responsible for cross-linking IF into bundles and is thereby associated with cell process formation.

Animals↗
PubMed2004

Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development.

The efficient functioning of striated muscle is dependent upon the structure of several cytoskeletal networks including myofibrils, microtubules, and intermediate filaments. However, little is known about how these networks function together during muscle differentiation and maintenance. In vitro studies suggest that members of the muscle-specific RING finger protein family (MURF-1, 2, and 3) act as cytoskeletal adaptors and signaling molecules by associating with myofibril components (including the giant protein, titin), microtubules and/or nuclear factors. We investigated the role of MURF-2, the least-characterized family member, in primary cultures of embryonic chick skeletal and cardiac myocytes. MURF-2 is detected as two species (approximately 55 kDa and approximately 60 kDa) in embryonic muscle, which are down-regulated in adult muscle. Although predominantly located diffusely in the cytoplasm, MURF-2 also colocalizes with a sub-group of microtubules and the M-line region of titin. Reducing MURF-2 levels in cardiac myocytes using antisense oligonucleotides perturbed the structure of stable microtubule populations, the intermediate filament proteins desmin and vimentin, and the sarcomeric M-line region. In contrast, other sarcomeric regions and dynamic microtubules remained unaffected. MURF-2 knock-down studies in skeletal myoblasts also delayed myoblast fusion and myofibrillogenesis. Furthermore, contractile activity was also affected. We speculate that some of the roles of MURF-2 are modulated via titin-based mechanisms.

Animals↗
PubMed1995

Molecular characterization of a Brugia malayi intermediate filament protein which is an excretory-secretory product of adult worms.

Filarial parasites release macromolecules into their environment both in vitro and in vivo. These excretory-secretory products (E-S) have been studied with respect to function, vaccination potential, pathogenicity, and ability to serve as antigen targets for diagnostic tests. We have recently described monoclonal antibody OV-1 which binds to an intermediate filament in E-S and circulating antigens of Onchocerca volvulus. OV-1 also binds to cross-reactive antigens of Brugia malayi. Therefore, OV-1 was used to immunoscreen a B. malayi adult worm cDNA library in an attempt to clone a homologue (BMIF). BMIF is a 1664-bp full-length transcript which codes for 505 amino acids. BMIF has 95% sequence homology at the amino-acid level to OV1CF, an O. volvulus intermediate filament that was also selected with OV-1, and 75% homology to Ascaris intermediate filament A. Southern blot analysis suggests that BMIF is confined to a single location in the genomic DNA of B. malayi. Antibodies raised to BMIF identified native antigens in immunoblots of B. malayi adult worms, infective larvae and adult E-S. In addition, the antibody also bound to a 60-kDa antigen in immunoblots of poly(ethylene glycol)-precipitated immune complexes in sera from B. malayi infected patients. Localization studies showed that the antigen encoded by BMIF is present in the hypodermis, developing embryos and muscle of adult B. malayi. These studies show that BMIF is an E-S product of B. malayi adult worms which is detectable in sera from patients with brugian filariasis.

Amino Acid Sequence↗
PubMed1995

Direct involvement of a lamin-B-related (54 kDa) protein in the association of intermediate filaments with the postsynaptic membrane of the Torpedo marmorata electrocyte.

Mechanisms by which motor innervation induces postsynaptic membrane differentiation and functional compartmentalization of the subneural sarcoplasm in skeletal muscle fibres are still poorly understood. However, transmembrane control of cytoskeletal activities by the nerve terminal may be considered. Here, we examine several properties of a 54 kDa protein, previously identified in the postsynaptic membrane of the Torpedo marmorata electrocyte with anti-lamin B antibodies, in order to study its role in the assembly of the subneural intermediate filament meshwork. Using a ligand blot assay, we show that this protein binds desmin, a type III intermediate filaments protein, at micromolar concentrations. Moreover, purified acetylcholine receptor-rich membrane fragments are able to generate arrays of desmin filaments in vitro. Immunofluorescence experiments indicate that the 54 kDa protein becomes associated with the acetylcholine receptor-rich membrane at an early stage of development of the electrocyte, and that a polarized desmin network develops concomitantly from the postsynaptic membrane. Taken together, these data show that, like karyoskeletal lamin B, the 54 kDa protein is involved in the organization of the subneural intermediate filament meshwork. Control of the assembly of the subneural cytoskeleton by components of the postsynaptic membrane may thus be a prerequisite for the functional compartmentalization of the muscle fibre triggered by motor innervation.

Animals↗
PubMed1998

Taxol induces concomitant hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells.

Taxol, a microtubule stabilizing agent, has been extensively investigated for its antitumor activity. The cytotoxic effect of taxol is generally attributed to its antimicrotubule activity and is believed to be cell cycle dependent. Herein, we report that taxol induces hyperphosphorylation and reorganization of the vimentin intermediate filament in 9L rat brain tumor cells, in concentration- and time-dependent manner. Phosphorylation of vimentin was maximum at 10(-6) M of taxol treatment for 8 h and diminished at higher (10(-5) M) concentration. Enhanced phosphorylation of vimentin was detectable at 2 h treatment with 10(-6) M taxol and was maximum after 12 h of treatment. Taxol-induced phosphorylation of vimentin was largely abolished in cells pretreated with staurosporine and bisindolymaleimide but was unaffected by H-89, KT-5926, SB203580, genistein, and olomoucine. Thus, protein kinase C may be involved in this process. Hyperphosphorylation of vimentin was accompanied by rounding up of cells as revealed by scanning electron microscopy. Moreover, there was a concomitant reorganization of the vimentin intermediate filament in the taxol-treated cells, whereas the microtubules and the actin microfilaments were less affected. Taken together, our data demonstrate that taxol induces hyperphosphorylation of vimentin with concomitant reorganization of the vimentin intermediate filament and that this process may be mediated via a protein kinase C signaling pathway.

Animals↗
PubMed1991

Chromatin motion in neuronal interphase nuclei: changes induced by disruption of intermediate filaments.

Motion of nucleoli within interphase nuclei, known as nuclear rotation, may be used as a measure of motion of chromatin domains within the global confines of the nucleus. Mechanisms by which chromatin domains are transposed remain enigmatic. It has been established that nuclei are anchored by a network of intermediate filaments, structural proteins which share epitopes with nuclear lamins and possibly representing a constraint on nuclear rotation. It is postulated that selective removal of this constraint, by acrylamide, would result in increased chromatin motion. Mean rates of nucleolar displacement were quantified in neurons, in vitro. Nuclear rotation increased from a mean control rate of 0.102 +/- 0.002 micron/min (n = 52) to a maximum mean rate of 0.207 +/- 0.026 micron/min (n = 11), after 23 hr of exposure to 4 mM acrylamide. Despite this significant increase in motion of intranuclear domains, cytoplasmic structures in the immediate juxtanuclear area did not exhibit increases in rates of motion. Immunocytochemistry was used to visualize cytoskeletal structures and to assay selective disruption of neurofilaments by acrylamide. Increased rates of chromatin motion coincided with breakdown of the intermediate filament network. Ultrastructural analyses showed that the increase in chromatin motion induced by acrylamide was also associated with a significant (P less than 0.005) change in the thickness of the nuclear lamina, decreasing from 20.9 +/- 5.10 nm (n = 159) in controls to 18.9 +/- 3.1 nm (n = 148), to 19.5 +/- 3.6 nm (n = 240) and to 16.1 +/- 4.4 nm (n = 103) at 4, 8 and 22 hr exposure, respectively. Moreover, the number of mitochondria per unit area changed significantly (P less than 0.0001) with exposure to acrylamide, increasing from 9.1 +/- 2.2 mitochondrial profiles in controls to 16.5 +/- 5.3 profiles after 22 hr exposure to acrylamide. Distribution of other cytoskeletal components, actin and microtubules, was not altered and does not appear to play a significant role in the observed increase in rates of nuclear rotation. We conclude that the removal of the damping effects on chromatin motion normally imposed by the nuclear lamina and by intermediate filaments results in increased chromatin motion.

Acrylamide↗
PubMedDate not supplied

Peptides from the conserved ends of the rod domain of desmin disassemble intermediate filaments and reveal unexpected structural features: a circular dichroism, Fourier transform infrared, and electron microscopic study.

Synthetic peptides representing the conserved ends of the rod domain of desmin are shown to disassemble preformed desmin filaments when added in moderate molar excess. This argues for a similar importance of both ends of the rod for filament stability. Recent structural models of intermediate filaments suggest close proximity of the ends and perhaps even an interaction (N. Geisler, J. Schünemann, and K. Weber, 1992, Eur. J. Biochem. 206, 841-852; P. M. Steinert, L. N. Marekov, R. D. B. Fraser, and D. A. D. Parry, 1993, J. Mol. Biol. 230, 436-452). Since the disassembling activity of the peptides, in addition to their sequences, should be related in some way to their secondary structure, we have investigated the structures of a number of related peptides which all arise from the ends of the rod using electron microscopic and spectroscopic methods. All peptides showed the expected alpha-helical structure at low concentrations in the presence of trifluoroethanol, as revealed by circular dichroism. At higher concentrations the peptides showed extensive self-aggregation into various types of filaments. The filaments contain the peptides in beta-sheet conformation as shown by Fourier transform infrared spectroscopy.

Amino Acid Sequence↗
PubMed2001

Development- and differentiation-dependent reorganization of intermediate filaments in fiber cells.

PURPOSE: To define the remodeling of lens fiber cell intermediate filaments (IF) that occurs with both development and differentiation. METHODS: Prenatal and postnatal mice were probed for the IF proteins phakosin, filensin, and vimentin, using light microscope immunocytochemical methodology. RESULTS: The pattern of vimentin accumulation in elongating fiber cells changed with development. Early in development vimentin first emerged predominantly as focal accumulations in the basal region of both epithelial and primary fiber cells. A light diffuse cytoplasmic staining was also noted. Later in embryonic development, and through maturity, vimentin in fiber cells was predominantly associated with the plasma membrane with no anterior-posterior polarity. Phakosin and filensin were first detected in the very latest stages of primary fiber elongation and continued to accumulate well after cells had completed elongation. Initially, these proteins accumulated in the anterior half of the fiber cells and were cytoplasmic in distribution. After P13, the pattern of initial distribution in differentiating fiber cells changed to a predominantly plasma membrane localization. Neither beaded filament protein showed focal basal accumulations. In mature lenses, all three proteins ultimately disappeared from the nuclear fiber cells. CONCLUSIONS: Beaded filament protein accumulation lags significantly behind both primary and secondary fiber cell elongation, suggesting a functional role subsequent to elongation. The subcellular distribution of vimentin and the beaded filament proteins showed marked differences within the cell, with differentiation, and with development. The differences in time of initial synthesis and in distribution of these IF proteins may bear on hypotheses about the role of IFs in fiber cell elongation and in structural-functional polarity of the fiber cell.

Animals↗
PubMed1998

A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases.

We have addressed the question of how keratin intermediate filaments are associated with the cell envelope at the periphery of cornified epidermal cells. Many peptides from human epidermal cell envelopes containing isopeptide crosslinks inserted by transglutaminases in vivo have been characterized. A major subset involves the type II keratin chains keratin 1, 2e, 5, or 6 crosslinked to several protein partners through a lysine residue located in a conserved region of the V1 subdomain of their head domains. This sequence specificity was confirmed in in vitro crosslinking experiments. Previously the causative mutation in a family with diffuse nonepidermolytic palmar-plantar keratoderma was shown to be the loss in one allele of the same lysine residue of the keratin 1 chain. Ultrastructural studies of affected palm epidermis have revealed abnormalities in the organization of keratin filaments subjacent to the cell envelope and in the shape of the cornified cells. Together, these data suggest a mechanism for the coordination of cornified cell structure by permanent covalent attachment of the keratin intermediate filament cytoskeleton to the cell envelope by transglutaminase crosslinking. Furthermore, these studies identify the essential role of a conserved lysine residue on the head domains of type II keratins in the supramolecular organization of keratin filaments in cells.

Amino Acid Sequence↗
PubMedDate not supplied

Cytokeratin intermediate filaments in oral and odontogenic epithelia.

With the development of monoclonal antibodies, the intermediate filaments of the cytoskeleton have attracted a great deal of interest in the last twenty years. Because they are highly antigenic, they could be easily identified using immunohistochemical methods and their specificity for one type of cell offered possibilities in the field of diagnostic pathology. The intermediate filaments most specific for epithelial cells are the cytokeratins (CK). After CK were classified, and certain "rules" regarding CK defined, research proceeded a pace to investigate epithelia in a vast array of anatomical and pathological situations. However, much of the immunohistochemical data that was generated during the 1980's was difficult to digest and some difficult to interpret. Reasons for this include identification of CK by molecular weight rather than CK number, or confusion as to which CK were being labelled by a particular antibody clone known to detect more than one CK. The aim of this article is therefore to present a digest of current knowledge of the CK present within the epithelium of the oral cavity (including the odontogenic epithelium) and its glandular component, and to highlight the significance of the CK phenotype in our understanding of pathological change.

Animals↗
PubMed2000

Localization of transitin mRNA, a nestin-like intermediate filament family member, in chicken radial glia processes.

We have examined the gene expression of two radial glia intermediate filament proteins, transitin and vimentin, in the developing chick CNS. Despite global similarities in their mRNA distributions, marked regional differences are observed. Most notably, we show that transitin mRNA is localized along radial glial processes and is localized to radial glia endfeet, whereas vimentin mRNA is not localized in radial glia. Localization of transitin mRNA is best shown in the diencephalic radial glia, as well as cerebellar Bergmann glia. In addition, in the early embryonic optic tectum, telencephalon, and retina, transitin mRNA is highly localized to radial glia endfeet, which is suggestive of its transport in these cells. These in vivo demonstrations of transitin mRNA localization are confirmed by in situ hybridization analysis of cultured chick brain radial glia, which demonstrates the presence of granular staining for transitin mRNA in glial processes. Transitin mRNA distribution in developing muscle also shows a highly regulated expression pattern, especially along the Z-lines of myofibrils. As further support for the transport and localization of transitin mRNA in radial glia and muscle, we have identified a consensus RNA transport signal in transitin mRNA that is absent from vimentin. These data suggest that the local regulation of transitin protein synthesis may contribute to its function as an intermediate filament protein in radial glia.

Animals↗
PubMed1988

Immunocytochemical study of intermediate filaments in cultured human trabecular cells.

In order to study which classes of intermediate filaments (IF) comprise the IF of human trabecular cells, we undertook immunocytochemical investigations on cultured human trabecular cells using anti-keratin, anti-vimentin, anti-desmin and anti-glial fibrillar acidic protein (GFAP) rabbit sera. Immunostaining with the keratin antibody and the GFAP antibody only produced nonspecific staining. Immunostaining with the vimentin antibody and the desmin antibody produced intracytoplasmic filamentous staining. Pretreatment of the cells with colcemid to induce the perinuclear concentration of IF resulted in the demonstration of a characteristic perinuclear immunofluorescence, confirming the existence of vimentin and desmin. Some cells showed particularly strong positivity to desmin. The desmin antibody used in this study only faintly labeled the IF of cultured human skin fibroblasts, known to contain vimentin but not desmin. These results suggest that the IF of human trabecular cells are composed of vimentin and desmin, and that human trabecular cells possess muscle cell-like functions.

Adult↗
PubMed1988

Expression of intermediate filaments and synaptophysin show neuronal properties and lack of glial characteristics in Y79 retinoblastoma cells.

The expression of intermediate filaments and synaptophysin in Y79 retinoblastoma cells was studied. The cells grew normally in suspension as floating aggregates. Sodium butyrate and dibutyryl cyclic AMP induced adherence and rapid spreading of Y79 cells on laminin-coated substratum and many of the cells presented long cellular extensions. As judged by immunostaining with monoclonal and polyclonal antibodies, most untreated Y79 cells expressed synaptophysin, whereas only vimentin intermediate filaments were detected in a few cells. A more bright fibrillar vimentin-positivity, which responded by coiling to disruption of microtubules, could be seen in the spread cells. Furthermore, especially when exposed to butyrate, a distinct fibrillar positivity could be revealed in some of the spread cells with antibodies recognizing a phosphorylated epitope in neurofilaments. Western blotting results showed the presence of the molecular weight 57,000 vimentin polypeptide and the molecular weight 38,000 synaptophysin polypeptide in both undifferentiated and spread Y79 cells, and a phosphorylated molecular weight 200,000 neurofilament polypeptide in spread cells only. In contrast, both immunostaining and Western blotting results with several antibodies indicated lack of glial fibrillary acidic protein in Y79 cells suggesting hence, a lack of glial differentiation. The present results do not support the hypothesis that retinoblastomas would originate from a common precursor cell of neurons and glia, and show the presence of mainly neuronal features in Y79 retinoblastoma cells.

Blotting, Western↗
PubMed1992

Primary structure of tektin A1: comparison with intermediate-filament proteins and a model for its association with tubulin.

Tektins are proteins that form filamentous polymers in the walls of ciliary and flagellar microtubules and that have biochemical and immunological properties similar to those of intermediate-filament proteins. We report here the sequence of a cDNA for tektin A1, one of the main tektins from Strongylocentrotus purpuratus sea urchin embryos. By hybridization analysis, tektin A mRNA appears maximally at ciliogenesis. The predicted structure of tektin A1 (M(r) 52,955) is a series of alpha-helical rod segments separated by nonhelical linkers. The two halves of the rod appear homologous and are probably related by gene duplication. Comparison of tektin A1 with intermediate-filament proteins, including nuclear lamins, reveals a low amino acid homology but similar molecular motif, i.e., pattern of helical and nonhelical domains. This study indicates that tektins are unique proteins but may be evolutionarily related to intermediate-filament proteins, and suggests a structural basis for the interaction of tektins and tubulin in microtubules.

Amino Acid Sequence↗
PubMed1989

Cell type heterogeneity of intermediate filament expression in epithelia of the human pituitary gland.

In the present study we have localized immunohistochemically the intermediate filament proteins of the human pituitary gland (adenohypophysis, pars intermedia and pars tuberalis) by an indirect immunoperoxidase technique or by double immunofluorescence methods and analysed the individual cytokeratin polypeptides using two-dimensional gel electrophoresis. We found that the expression of cytokeratins in different epithelial cells of the human anterior pituitary gland was heterogeneous. Whereas the endocrine cells only expressed cytokeratins 8 and 18, the folliculo-stellate cells exhibited a reactivity for cytokeratins 7, 8, 18 and 19 as well as for GFAP and vimentin. The squamous epithelial cells of the pars tuberalis and the Ratke's cysts showed a more complex cytokeratin pattern of both squamous and simple type. Whereas in may cystic epithelial cells including the "pseudo-follicles" a triple expression of cytokeratin, vimentin and GFAP could be observed, only some basal cells of squamous epithelial nests coexpressed cytokeratin and vimentin. The differences in the intermediate filament protein distribution are discussed in the light of embryological relationships of the different parts of the human pituitary gland.

Desmin↗
PubMed2002

p34cdc2-mediated phosphorylation mobilizes microtubule-organizing centers from the apical intermediate filament scaffold in CACO-2 epithelial cells.

We have shown previously that centrosomes and other microtubule-organizing centers (MTOCs) attach to the apical intermediate filament (IF) network in CACO-2 cells. In this cell line, intermediate filaments do not disorganize during mitosis. Therefore, we speculated that the trigger of the G(2)-M boundary may also detach MTOCs from their IF anchor. If that was the case, at least one of the proteins involved in the attachment must be phosphorylated by p34(cdc2) (cdk1). Using confocal microscopy and standard biochemical analysis, we found that p34(cdc2)-mediated phosphorylation indeed released MTOCs from IFs in permeabilized cells. In isolated, immunoprecipitated multiprotein complexes containing both gamma-tubulin and cytokeratin 19, p34(cdc2) phosphorylated only one protein, and phosphorylation released cytokeratin 19 from the complexes. We conclude that this as yet unidentified protein is a part of the molecular mechanism that attaches MTOCs to IFs in interphase.

CDC2 Protein Kinase↗
PubMed1991

Caprine peripheral neuroblastomas: structural and ultrastructural features, intermediate filaments profile and neuro-endocrine characterization.

In this report the morphological and immunohistochemical features with respect to intermediate filament proteins and the neuroendocrine nature of bilateral neuroblastomas possibly originating from the adrenal medulla in a goat kid are presented. Histologically, the tumours were composed of small, round, blue cells organized in highly cellular nests and sheets separated by fibrovascular septa. Isolated cells mimicking the morphological features of neurones were observed in both tumours. Ultrastructurally, dense-core neurosecretory granules, about 100 nm in diameter, and irregularly organized neurotubular networks were seen. The small tumour cells were only labelled by vimentin, while the neurone-like cells were labelled by both neurone-specific enolase and synaptophysin. The lack of staining of the tumour cells by the neurofilament proteins antiserum, which was also observed in a human neuroblastoma (used as positive control) has been previously reported and is probably the result of the tissue processing and/or the poor differentiation stage of the tumours. The vimentin labelling of tumour cells could be explained by the poor differentiation stage of the tumours, since vimentin is the only intermediate filament protein in presumptive neuroblasts, being replaced by neurofilament proteins in later stages of neurogenesis.

Animals↗
PubMed1997

Protein phosphatases maintain the organization and structural interactions of hepatic keratin intermediate filaments.

The importance of protein phosphatases in the maintenance of cytoskeletal structure is supported by the serious liver injury caused by microcystin-LR, a hepatotoxic inhibitor of type-1 and type-2A serine/threonine protein phosphatases. We used the microcystin-LR-induced cell injury as a model to study the roles of protein dephosphorylation in maintaining cytoskeletal structure and cellular interactions in primary rat hepatocyte cultures. Confocal microscopy revealed that the first visible effect of microcystin-LR is disruption of desmoplakin organization at the cell surface, indicating dissociation of desmosomes. This effect is followed by a dramatic reorganization of both the intermediate filament (keratins 8 and 18) and microfilament networks, resulting in a merged structure in which the intermediate filaments are organized around a condensed actin core. Keratin 8, keratin 18 and desmoplakin I/II are the major cytoskeleton-associated targets for microcystin-LR-induced phosphorylation. Hyperphosphorylation of keratin 8 and 18 is accompanied by an increased keratin solubility, which correlates with the observed morphological effects. Phosphopeptide mapping shows that four specific tryptic phosphopeptides are highly phosphorylated predominantly in the soluble pool of keratin 18, whereas keratin 8 shows no indications of such assembly state-specific sites. Phosphopeptide maps of keratins phosphorylated in vivo and in vitro indicate that Ca2+/calmodulin-dependent kinase may be involved in regulating the serine-specific phosphorylation of both keratin 8 and keratin 18, while cAMP-dependent protein kinase does not seem to play a major role in this context. Taken together, our results show that the interactions between keratin intermediate filaments and desmosomes as well as the assembly states of their main constituent proteins, are directly regulated by serine/threonine kinase/phosphatase equilibria.

Actin Cytoskeleton↗