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PubMed1992

Colchicine-sensitive and colchicine-insensitive intermediate filament systems distinguished by a new intermediate filament-associated protein, IFAP-70/280 kD.

A monoclonal antibody was produced, using as antigen a BHK-21 cytoskeletal preparation enriched in intermediate filaments (IF) and their associated proteins. This antibody reacted exclusively with a reproducible set of 70-280 kD polypeptides present in minor quantities in this preparation, as detected by immunoblot analysis. Based upon several criteria, this immunologically related group of polypeptides was designated as IFAP-70/280 kD (IF-Associated Protein): (1) it co-isolated with IF in vitro, (2) it co-localized (by both immunofluorescence and immunoelectron microscopy) with IF in situ in all stages of cell spreading, and (3) it segregated in vitro with the 54/55 kD (desmin/vimentin) structural IF subunit proteins of BHK cells through two cycles of in vitro disassembly/assembly. Immunogold labeling further localized IFAP-70/280 kD to regions of parallel or loosely bundled IF in situ, suggesting a role in regulating the supramolecular organization of IF. When this monoclonal antibody was used for double-label immunofluorescence observations of colchicine-treated BHK cells, it demonstrated the presence of colchicine-sensitive and colchicine-insensitive IF. Anti-IFAP-70/280 kD localized entirely to the drug-induced juxtanuclear IF cap, while a polyclonal antibody directed against the desmin/vimentin structural IF subunits and the previously characterized monoclonal anti-IFAP-300 kD [Yang et al., 1985; J. Cell Biol. 100:620] localized to both the juxtanuclear IF cap and a colchicine-insensitive IF network peripheral to the cap in the same cells. The colchicine-insensitive IF pattern often exhibited similarities to that observed for the actin-based stress fiber system, suggesting that stress fiber association may be an additional factor in IF organization.

Animals↗

PubMed1985

Intermediate filaments with novel protein composition from certain goldfish cells.

Using the conditions for vimentin filament recycling, intermediate filaments (approximately 10 nm) were prepared from the cytoskeleton of a goldfish tumor cell line (erythrophoroma or xanthophoroma). 2-D analysis showed unusual protein composition, with four proteins of molecular weights of 60, 45, 56 and 51 kilodaltons in ratios of approximately 4:4:1:1. These correspond to four of the major cytoskeletal proteins of both the tumor cells and normal xanthophores.

Animals↗

PubMedDate not supplied

Prenatal low-dose gamma irradiation of the inner ear induces changes in the expression of intermediate filaments.

The expression of intermediate filaments (1F) was analysed in the inner ear in normally developed adult CBA/CBA mice and in mice of the same age which had been gamma irradiated in utero with a low dose 1-2 Gy single exposure. Well characterized monoclonal antibodies (mAbs) against all classes of intermediate filament proteins (cytokeratins-Cks, vimentin, neurofilaments, desmin and glial fibrillar acidic protein) were used. With the exception of neurofilament proteins, the expression of intermediate filament proteins was the same in adult normal and irradiated inner ears, irrespective of gestational age at exposure. A complex Ck pattern occurred in the various cell types comprising the membranous labyrinth. In spite of the differences in cell shape and internal organization of organelles, epithelia actively involved in inner ear fluid homeostasis (stria vascularis, dark cell epithelium, endolymphatic duct and sac) revealed, according to our mAbs, the same expression of Cks, except for the mouse counterpart of human Ck 7, which was found exclusively in the stria vascularis and the endolymphatic duct and sac. The pattern of intermediate filament composition in the labyrinth was the same in the mouse as in man. Irradiation on gestational days 12 or 13 (the otocyst stage)--but not at more advanced embryonic age--induced immunoreactivity for neurofilament proteins in vestibular hair cells (HC) and to a minor extent also in cochlear HC. No such positivity was found in the control material.

Abnormalities, Radiation-Induced↗

PubMed1988

[Intermediate filament proteins as markers in tumor diagnosis].

Classification of tumors is based on histogenesis and on determination of malignancy. In well differentiated neoplasias the tumor tissue reveals a similar morphological pattern similar to that of the normal tissue from which they have originated. In contrast less differentiated neoplasias do not show such similarities to normal tissue in conventional stains and special procedures such as electron microscopy or immunohistology have to be performed in order to detect cell specific products. In many undifferentiated tumors this is not possible because loss of differentiation and organisation in tumor cells do not allow the production of cell specific substances. A new possibility for determining the histogenesis of tumors is the use of antibodies which are specific for one type of intermediate filaments. Intermediate filaments are structures, which together with microtubules and microfilaments form the cytoskeleton. Intermediate filaments are composed of different polypeptides, which show a cell type specificity. Keratins are the intermediate filaments characteristically found in keratinizing and nonkeratinizing epithelia. Desmin is the specific intermediate filament type of sarcomeric, visceral and some type of vascular smooth muscle tissue. Vimentin filaments are characteristic of endothelial cells, fibroblasts, macrophages, chondrocytes and most but not all lymphatic cells and the only intermediate filament type present in these cells. Neurofilaments are composed of three different polypeptides, which form the so called neurofilament triplet and are characteristically found in central and peripheral neurons. Glial fibrillary acidic protein (GFAP) forms the intermediate filament system of normal and reactive astrocytes and also some ependymal cells contain GFAP. Thus cells and tissues can be divided into five different types by the use of appropriate polyclonal or monoclonal antibodies. In the current study we were interested to determine with a large number of specimens, whether primary tumors or metastases continue to express the intermediate type characteristic of the normal tissue. The following results demonstrate, there is abundant evidence that intermediate filaments can be used as cell type specific markers both for normal tissue and for tumors. 1. To exclude wrong negative results by intermediate filament typing, a reliable detection of intermediate filaments should be performed on cryostat sections or on material, which has been recently ethanol fixed and paraffin embedded. With many antibodies fixation of the tissue in formalin results in a decrease of reactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Biomarkers, Tumor↗

PubMed1989

Intermediate filaments in biology and disease.

Intermediate filaments comprise a large heterogenous family of proteins in animal cells. Distinct from microfilaments and microtubules, they are a major component of the cytoskeleton and nuclear envelope. The expression of intermediate filament protein types is developmentally regulated and relatively cell type specific. Although there are at least five distinct classes of intermediate filament types, all the subunit proteins have similar structural features and appear to have envolved from some common gene ancestor. The cytoplasmic intermediate filaments exhibit a complex organization, forming associations with components of the nucleus, plasma membrane, and potentially other cytoplasmic structures such as microtubules. The specific function of this prominent cytoplasmic structure is currently unknown. However, the organization or expression of intermediate filaments is known to be altered in association with a variety of human diseases. Currently, specific antibodies to individual intermediate filament proteins are being used as an immunohistochemical aid for tumor typing in diagnostic pathology.

Animals↗

PubMed1986

The teleost cone cytoskeleton. Localization of actin, microtubules, and intermediate filaments.

This laboratory has been using the teleost retinal cone as a model for studying the mechanisms and regulation of retinal cell motility. In previous inhibitor studies, the authors have shown that dark-induced cone elongation requires microtubules, whereas light-induced contraction requires actin filaments. This study examines the distributions of actin filaments, microtubules, and intermediate filaments in the cone cytoskeleton. Actin filaments have been localized in isolated cones by labeling with fluorescent derivatives of phalloidin; microtubules were localized by immunofluorescent labeling with anti-tubulin. Actin, microtubule, and intermediate filament distributions have also been examined in detergent-lysed motile cell models of cones fixed with a new method that enhances preservation of the cytoskeleton. Longitudinal bundles of actin filaments extend from the cone's calycal processes through the ellipsoid and into the myoid. No actin filaments are detectable in the perinuclear region and axon, but filaments are present in both pre- and post-synaptic components of the synapse. Intermediate filaments are numerous in the perinuclear region and cone axon but relatively sparse in the myoid. In contrast, microtubule distribution is more uniform: numerous longitudinally oriented microtubules are present throughout the length of the cell. Thus the cone cytoskeleton reflects the highly polarized shape and function of the cell, with actin filaments localized to the distal movable part of the cell and intermediate filaments localized to the proximal part of the cell, which is anchored in the retina.

Actins↗

PubMed1992

Intermediate filament dynamics.

The view of intermediate filaments as static cytoskeletal elements is changing. Studies of exogenous intermediate filament proteins, either microinjected or expressed from transfected genes, have demonstrated that a continuous incorporation of subunits into the polymerized filaments is taking place. This incorporation appears to be required for maintaining normal cytoplasmic networks of intermediate filaments. At the post-translational level, phosphorylation is an important factor in regulating dynamic aspects of intermediate filament organization and structure.

Animals↗

PubMed1990

Cellular intermediate filament networks and their derangement in alcoholic hepatitis.

Intermediate filaments are major components of most eukaryotic cells that form from the polymerization of protein subunits that are expressed in tissue and development specific fashions. The interactions of intermediate filaments with a myriad of other cellular proteins and structures give rise to a complex overall cellular architecture that is likely responsible for cellular well-being. The mature 10-nm filaments are relatively stable cellular structures, but the intermediate filaments undergo major morphological and biochemical changes, especially during mitosis, differentiation, and in response to certain drugs. Evidence exists that hepatocyte intermediate filaments (keratin filaments) are deranged in alcoholic hepatitis, an inflammatory liver disease of alcoholics and heavy spree drinkers. The classical and characteristic pathological hepatocyte inclusion bodies of alcoholic hepatitis, Mallory bodies, are composed in part of normal keratins that likely derive from the pre-existing hepatocyte intermediate filament network. It is unclear if intermediate filament network derangement in alcoholic hepatitis is directly caused by the actions of ethanol or its metabolites on intermediate filaments or their associated structures, or whether alcohol causes a cellular insult or injury elsewhere and a subsequent response (e.g., immune) causes intermediate filament network derangement. The precise mechanisms responsible for intermediate filament derangement remain to be elucidated; however, experimental data exist that support and refute several hypotheses. Hopefully, further studies will help determine a better overall understanding of the abnormalities of intermediate filaments and their relationship to the pathophysiology of alcoholic hepatitis and other diseases.

Animals↗

PubMed1992

The 53kDa polypeptide component of the bovine fibre cell cytoskeleton is derived from the 115kDa beaded filament protein: evidence for a fibre cell specific intermediate filament protein.

The 115kDa protein found enriched in the PMCC (plasma membrane-cytoskeleton complex) fraction of the cortex in bovine lens fibre cells is proteolytically processed to a stable 53kDa product. The 115 kDa protein and the 53kDa polypeptide have been purified by a combination of ion exchange and hydroxyapatite chromatography. Tryptic peptide mapping using reverse phase HPLC and subsequent peptide sequencing confirmed that the 53kDa polypeptide is derived from the 115kDa protein. The 53kDa fragment is also a component of the PMCC as well as being a major component of the urea soluble fraction of lens plasma membranes which have been extracted with buffers containing 1M KC1. The 53kDa polypeptide has escaped identification as a breakdown product of the 115kDa protein because it is not recognised by a commonly used monoclonal antibody, R2D2, specific for the bovine 115kDa protein. This result suggests that proteolysis is important in determining the function(s) of the 115kDa protein, and that part of this function is satisfied by the 53kDa protein core. Both the purified 115kDa protein and the 53kDa polypeptide were unable to form either beaded or intermediate filaments on their own but they were able to form short 10nm rods indicative of an intermediate stage in intermediate filament assembly. Comparison ot the assembly properties of the 53 and 115kDa proteins indicate that there are sequences in the 115kDa protein which inhibit in vitro assembly. This is similar to the situation with neurofilament proteins. We suggest that the 115kDa protein is a lens-specific intermediate filament protein.

Amino Acid Sequence↗

PubMed1991

Coalignment of vimentin intermediate filaments with microtubules depends on kinesin.

Intermediate filaments in most types of cultured cells coalign with microtubules. Depolymerization of microtubules results in collapse of vimentin and desmin intermediate filaments to the nucleus where they form a perinuclear cap. Collapse can also be induced by microinjection of antibodies against intermediate filament or microtubule proteins. Thus, two filament systems interact with each other. But the molecules mediating this interaction are unknown. One of the candidates for this role is a microtubule motor kinesin. Recent data showed that kinesin is involved in the plus end-directed movement of the membranous organelles along microtubules such as radial extension of lysosomes in macrophages and centrifugal movement of pigment in melanophores. Here we report that injection of the anti-kinesin antibody into human fibroblasts results in the redistribution of intermediate filaments to a tight perinuclear aggregate but had no effect on the distribution of microtubules. Thus, kinesin is involved not only in organelle movement but also in interaction of the two major cytoskeletal systems, intermediate filaments and microtubules.

Adenosine Triphosphatases↗

PubMed1992

Intermediate filament molecular biology.

Epidermal keratin intermediate filaments appear to have a structural function. The functions of other intermediate filaments are being elucidated using a combination of molecular genetic methods, including the expression of dominant negative mutant proteins and gene targeting. The differential expression of intermediate filament genes is regulated by both the accessibility of multiple regulatory elements and the activity or level of multiple positive and negative transcription factors.

Animals↗

PubMed1989

Malignant fibrous histiocytoma. Heterogeneous patterns of intermediate filament proteins by immunohistochemistry.

Patterns of intermediate filament expression of 10 malignant fibrous histiocytomas (MFHs) were immunohistochemically evaluated using acetone-fixed frozen sections. Seven cases represented the storiform-pleomorphic subtype, 2 were of myxoid type, and 1 was of giant-cell type. All cases had been studied by electron microscopy, and no proof for the diagnoses of liposarcoma, rhabdomyosarcoma, and leiomyosarcoma could be obtained. All tumors showed prominent vimentin immunoreactivity in the tumor cells. Cytokeratin-positive neoplastic cells were found in 2 cases, and in the majority of tumor cells in 1 of these. The 68k neurofilament-positive cells were found in 2 cases. Desmin was not found beyond doubt in the neoplastic cells in any cases, and all cases were negative for glial fibrillary acidic protein. The expression of several types of intermediate filament indicates divergent differentiation properties in MFH and may suggest the heterogeneity of this entity, but more cases should be studied to elaborate any possible consistent patterns of intermediate filament expression in different types of MFH. The expression of multiple types of intermediate filament proteins in MFH can alternatively signify random activation of the corresponding genes in the primitive tumor cells. The complex patterns of intermediate filament proteins in morphologically defined MFHs should be taken into account in the practical immunohistologic analysis of tumors.

Cell Differentiation↗

PubMed1986

Evidence for an interaction between the cell surface and intermediate filaments in cultured fibroblasts.

Intermediate filaments (IF) were found in close proximity to the plasma membrane in substrate attached baby hamster kidney cells (BHK-21) and chick embryo fibroblasts (CEF) as well as cells removed from their substrate in the absence of trypsin. However, in cells removed with trypsin, it appeared that IF had retracted away from the membrane. In cells with abundant extracellular matrix (ECM), colchicine induced massive cables of IF, which appeared to interact with specialized areas of the inner plasma membrane. In cells lysed to extract most microfilaments and cytoplasmic constituents, the intact IF network which remained was closely associated with the ECM. From these ultrastructural observations it was concluded that IF interact in some way with a "cell membrane complex" defined as comprising the plasma membrane and molecules attached to its inner and outer surfaces. In order to investigate the possibility that components of the membrane complex may co-isolate with IF, native intermediate filaments (NIF) were prepared. In addition to the structural subunits and other associated polypeptides, a approximately 220 kd species which reacted specifically with antibodies directed against the ECM protein fibronectin (FN) was observed; 220 kd was still present after NIF were isolated under pH conditions where FN is more soluble, suggesting that its presence was not simply due to the coprecipitation of two insoluble proteins. Immunofluorescence and immunogold localization confirmed that FN is a component of the cell membrane complex with which IF appeared to interact.

Animals↗

PubMed1990

Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro.

To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

Amino Acid Sequence↗

PubMed1987

Assemblies of psoriatic keratin and their relation to normal intermediate filament structures.

Protein extracts from normal human epidermis reassemble in vitro into 8-10 nm diameter filaments characteristic of intermediate filaments, whereas extracts from psoriatic epidermal scales reassemble, under identical conditions, into a variety of paracrystalline bundles. Optical diffraction and image analysis of these paracrystalline bundles reveal an axial repeat of 16.5 nm, which subdivides into three bands of 5.5 nm, and a lateral spacing of 5.1 nm. This information, together with available sequence studies of intermediate filaments and biochemical data, suggests that the subunit of psoriatic keratin is made up essentially from the coiled-coil alpha-helical rod domain of the normal keratin subunits, whereas the random coil domains are missing or greatly reduced in size.

Cytoskeleton↗

PubMed1991

Selective alteration of cytokeratin intermediate filament by cyclosporine A is a lethal toxicity in PTK2 cell cultures.

The cytoplasm of eukaryotic cells contain a series of three filamentous structures, microtubules, microfilaments, and intermediate filaments that are termed the cytoskeleton. Cytokeratin, one type of intermediate filament, has no known physiological function, yet, can comprise up to 30% of the total cytoplasmic protein content. As there are no selective toxins to cytokeratins, it is not known if alterations to these hydrophobic filaments is a lethal event. Cyclosporine A, a novel hydrophobic immunosuppressant compound used to prevent allograft rejection, may show a selective toxicity to the cytokeratin filaments. This effect is seen in PtK2 cell cultures as a single large perinuclear aggregate of collapsed cytokeratin filaments (5 mM, 72 hr). Microtubules and microfilaments are not affected in PtK2 cell cultures (5 mM, 72 hr). Increased LDH levels into cell culturing media occur soon after cyclosporine exposure to PtK2 cell cultures (5 mM, 2 hr). Cytokeratin filaments show no changes at 12 hr exposure but show thickening, decreased plasma membrane attachments and some peri-nuclear ring formations at 24 hr (5 mM, 24 hr). Cyclosporine G, an analog of cyclosporine A, does not exhibit the cytokeratin filament collapse (5 mM, 72 hr). The effect of cyclosporine A on DNA binding protein (Mr 64 kd), believed to be a nuclear scaffolding protein related to intermediate filaments, exhibited an early invagination and folding of the nuclear membrane (5 mM, 4 hr). Due to a hydrophobic bonding potential between cyclosporine A and cytokeratin and cytokeratin-like intermediate filaments, cyclosporin A may be a selective cytokeratin toxin. Alteration of the cytokeratin filaments in PtK2 cell cultures may be a lethal event.

Animals↗

PubMed1985

Monoclonal antibody to a 43 000 Mr surface protein of a human leukaemia cell line (THP-1) crossreacts with the fibroblast intermediate filament protein vimentin.

Monoclonal antibodies were produced against surface antigens of live cells from a human acute monocytic leukaemia cell line (THP-1). One clone, VIC-C2, when assayed by immunofluorescence microscopy, brightly stained the surface of THP-1 cells and the cytoplasm of Langerhans cells, fibroblasts and melanocytes in sections of human skin. The immunoreactive cytoplasmic structures were filamentous and resembled intermediate filaments. By double immunofluorescence microscopy using VIC-C2 and polyclonal antibodies to vimentin, the VIC-C2 antigen was shown to be located on intermediate filaments of cultured fibroblasts and to follow these filaments during various drug-induced rearrangements. As demonstrated by immunoprecipitation, antibody gel overlay and immunoblotting of two-dimensional polyacrylamide gels, VIC-C2 recognized two different antigens in extracts of THP-1 cells: one of Mr = 43 000 and pI = 7, the other of Mr = 57 000. In extracts from various cultured fibroblast cells only the 57 000 Mr antigen was detected. This 57 000 Mr protein was identified as vimentin by immunoblotting of rat glioma C6 cytoskeletons on two-dimensional gels. When vimentin was digested with chymotrypsin, only fragments containing parts of both helical rod pieces and the connecting non-helical spacer-region were strongly antigenic, whereas the helical rods alone were only weakly crossreactive. Moreover, immunoprecipitation revealed that VIC-C2 preferentially reacted with native compared to denatured vimentin.

Antibodies, Monoclonal↗

PubMed1989

Flow cytometric analysis of epidermal subpopulations from normal and psoriatic skin using monoclonal antibodies against intermediate filaments.

Keratin-type intermediate filament proteins show characteristic expression in normal and pathologic epidermis. Some keratins are restricted to the basal cell layers, and others occur exclusively in the suprabasal compartment. SDS-gel-electrophoresis and immunohistochemistry are generally used for the assessment of keratin profiles and their localizations. In the present investigation, flow cytometric analysis of four different monoclonal antibodies (MAb) against intermediate filament-type proteins, in addition to measurement of relative DNA content, was performed on cell suspensions derived from lesional and clinically uninvolved skin of psoriatic patients and from skin of healthy controls. MAb Ks8.12, reacting with keratins 13 and 16, was used as a marker for hyperproliferation. Pab601 recognizes the basal cell layer(s) of human epidermis. Keratin 10 expression as a marker of keratinization was quantified with RKSE60 and the anti-vimentin MAb MVI was used as a marker for non-keratinocytes. Psoriatic skin showed significantly reduced numbers of RKSE60-positive cells and MVI-positive cells compared with normal skin. In contrast to normal skin and uninvolved skin of psoriatic patients in which only a minority of the cells were Ks8.12 positive, up to 60% of the cell population in psoriatic lesions bound with MAb. Simultaneous measurement of relative DNA content and MAb binding showed that Pab601 binding was associated with cells in S-phase and G2M-phase of the cell cycle, whereas RKSE60 and Ks8.12 binding were associated with diploid cells. Multiparameter flow cytometry allows quantitative population analysis that could lead to a better understanding of the complex mechanisms of epidermal growth control under normal and pathologic conditions.

Adult↗