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PubMed1990

Modulation of keratin intermediate filament distribution in vivo by induced changes in cyclic AMP-dependent phosphorylation.

Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible dephosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24-30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma-32P-ATP in the presence or absence of cAMP-dependent kinase (A-kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X-100 extracted cells. Phosphorylation levels were increased by A-kinase but not by cAMP alone, indicating the presence of cAMP-dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP-dependent phosphorylation in acrylamide-induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8-bromo-cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8-bromo-cAMP, IBMX, and forskolin prevent acrylamide-induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP-dependent kinase and an endogenous, cAMP-independent kinase and 2) alteration of levels of cAMP-dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Polyribosomes are not associated with vimentin-type intermediate filaments in Ehrlich ascites tumor cells.

Polyribosomes were isolated from Mg2-stabilized, Triton X-100-resistant residual cell structures and from a postmitochondrial supernatant of hypotonically swollen and homogenized Ehrlich ascites tumor cells and analysed for their vimentin contents by polyacrylamide gradient slab gel electrophoresis. Whereas polyribosomes from detergent-resistant cell residues were accompanied by a small amount of vimentin on sucrose gradients, polyribosomes isolated from a hypotonic, postmitochondrial supernatant were free of vimentin. Since the presence of vimentin in cell residue-derived polyribosomes might be interpreted as the result of unspecific adsorption or cosedimentation of vimentin filament fragments, both due to the presence of high Mg2 concentration during cell extraction, the results suggest that there is no direct and stable interaction of polyribosomes with vimentin-type intermediate filaments in suspension-grown Ehrlich ascites tumor cells. This is in accordance with the electron microscopic observation that cytoplasmic regions rich in intermediate filaments are virtually devoid of polyribosomes. When the extraction of the same cells with Triton X-100 was carried out at low ionic strength and in the presence of small amounts of Ca2, residual cell structures were completely destabilized and clean nuclei without any attached cytoplasmic material were obtained. This is due to the degradation of the vimentin-type filaments by the Ca2-activated thiol proteinase highly specific for vimentin.

Animals↗
PubMed1990

Primitive neuroectodermal tumors of the central nervous system. Patterns of expression of neuroendocrine markers, and all classes of intermediate filament proteins.

Snap-frozen samples from 22 primitive neuroectodermal tumors (PNETs) primary in the central nervous system were studied with antibodies to synaptophysin, bombesin, somatostatin, substance P, vasoactive intestinal polypeptide, all classes of intermediate filaments, and desmoplakins I and II. Frozen sections were immunostained by the avidin-biotin peroxidase complex and indirect immunofluorescence microscopy methods. Selected cases were also studied by double and triple label immunofluorescence microscopy, and by two-dimensional gel electrophoresis and immunoblot analysis. We found that all 22 PNETs expressed synaptophysin extensively. Focal expression of 2 or more neuropeptides was noted in 10 samples studied. All PNETs expressed vimentin, 21 of 22 expressed glial filament protein (GFP), 16 of 22 expressed neurofilament proteins (NFP), 4 of 22 expressed desmin, and 3 of 22 expressed cytokeratins. In only one case were focal and questionable reactions with desmoplakin antibodies seen. Immunoblots confirmed the presence of desmin. Double and triple immunofluorescence revealed a number of antigenic coexpressions in individual cells including: synaptophysin with vimentin, GFP, NFP and desmin, vimentin-GFP, vimentin-NFP, vimentin-cytokeratin, vimentin-desmin and desmin-NFP; similarly, combinations of vimentin-GFP-NFP, vimentin-GFP-desmin, and vimentin-GFP-cytokeratin were found. The consistent expression of synaptophysin and 2 or more neuropeptides indicates that central nervous system PNETs have significant phenotypic features in common with neuroendocrine tumors. Their complex and variable intermediate filament complement patterns combined with their consistent expression of specific neuroendocrine differentiation markers, suggest that central nervous system PNETs comprise a distinct, albeit heterogeneous group of neoplasms.

Adolescent↗
PubMed1997

Transgenic mice carrying chimeric or mutated type III intermediate filament (IF) genes.

Mice carrying chimeric, truncated or mutated genes encoding intermediate filament (IF) proteins type III do not show any detectable severe pathology. However, upon (over)expression of the transgene in the eye lens all animals develop lens opacification (cataract). At the cellular level the loss of visual acuity is preceded by interference with the terminal differentiation of lens fibre cells, plasma membrane damage, distorted assembly of the IF cytoskeleton and perturbation of the cytoskeleton-membrane complex. The degree of expression is paralleled by the extent of the damages.

Animals↗
PubMed1984

Anti-intermediate filament antibody in mice infected with Plasmodium berghei.

Fifteen mice (CBA) were infected with lethal Plasmodium berghei and the development of anti-intermediate filament antibody (anti-IF) studied. Sera from all the infected animals reacted with the cytoplasmic network of intermediate filaments (IF) in the human epidermal laryngeal carcinoma (HEp2) cell line, as demonstrated by indirect immunofluorescence. Sera from 5 control animals injected with 10(4) unparasitized red cells showed no anti-IF reactivity. Anti-IF of IgM class was first detectable at day 11 in infected animals. Later, IgG as well and IgM anti-IF was found. A steady increase in anti-IF titre was observed during infection and a relationship between anti-IF titre and parasitaemia rate was noted.

Animals↗
PubMed1985

Intermediate filament and cross-linked envelope expression in human lung tumor cell lines.

Human lung tumor cell lines established from the major histological types of lung cancer were examined by immunofluorescent staining techniques for their patterns of intermediate filament (keratin, vimentin, and neurofilament triplet protein) expression. All cell lines examined, both small cell lung carcinoma (SCLC) and non-SCLC (squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and mesothelioma) contained keratin, consistent with their epithelial derivation. These lung carcinoma cell lines also expressed vimentin, the characteristic intermediate filament of mesenchymal cells in vivo. In light of the proposed neuroectodermal origin of SCLC, cell lines were also studied for neurofilament expression. Two of four SCLC tumor cell lines, as well as non-SCLC cell lines, showed no reactivity with antibodies to neurofilament triplet protein. Two of the SCLC cell lines stained weakly with anti-neurofilament antibody. Examination of specific keratin patterns in human lung tumor cell lines by selective immunoprecipitation with keratin antiserum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that small-sized keratin proteins (Mr 44,000 to 52,000) were present in cell lines derived from SCLC and non-SCLC types of lung cancer. Tumor cell lines exhibiting squamous differentiation by light microscopic criteria (i.e., intracellular keratin, intercellular bridging, "pearl" formation, and/or individual cell keratinization) also displayed a preponderance of intermediate-sized keratins (Mr 57,000 and 59,000) and exhibited another feature of terminal keratinocyte differentiation (cross-linked envelope formation). Mesothelioma cell lines had varying keratin profiles. The presence of keratin proteins in all SCLC cell lines examined argues against a neuroectodermal origin for these tumors and is consistent with the notion that these tumors arise from a common bronchial "stem cell," similar to that from which other types of bronchogenic carcinomas arise.

Animals↗
PubMed2005

Neuropathological changes in ten cases of neuronal intermediate filament inclusion disease (NIFID): a study using alpha-internexin immunohistochemistry and principal components analysis (PCA).

Ten cases of neuronal intermediate filament inclusion disease (NIFID) were studied quantitatively. The alpha-internexin positive neurofilament inclusions (NI) were most abundant in the motor cortex and CA sectors of the hippocampus. The densities of the NI and the swollen achromatic neurons (SN) were similar in laminae II/III and V/VI but glial cell density was greater in V/VI. The density of the NI was positively correlated with the SN and the glial cells. Principal components analysis (PCA) suggested that PC1 was associated with variation in neuronal loss in the frontal/temporal lobes and PC2 with neuronal loss in the frontal lobe and NI density in the parahippocampal gyrus. The data suggest: 1) frontal and temporal lobe degeneration in NIFID is associated with the widespread formation of NI and SN, 2) NI and SN affect cortical laminae II/III and V/VI, 3) the NI and SN affect closely related neuronal populations, and 4) variations in neuronal loss and in the density of NI were the most important sources of pathological heterogeneity.

Adult↗
PubMed1997

Circular dichroism of model peptides emulating the amphipathic alpha-helical regions of intermediate filaments.

The a and d positions of the heptad repeats (abcdefg) found in the alpha-helical sections of intermediate-filament proteins are hydrophobic, and the remaining locations are almost exclusively hydrophilic and often charged. Two synthetic peptides that maximize these features were designed, synthesized, and investigated by circular dichroism for alpha-helix formation in water and in 50% trifluoroethanol (TFE). A 14-residue peptide, AcNLEELKKKLEELKGNH2 (NLEKG14), had mean residue ellipticities at 222 nm ([theta]222) of -18400 +/- 1000 and -37,200 +/- 1900 deg cm2 dmol(-1), in water at 2 degrees C and in 50% TFE at 2 degrees C, respectively. A longer version of NLEKG14, AcNLEELKKKLEELKQQLEELKKKLEELKQQNH2 (NLEKQ29), had [theta]222 of -43,000 +/- 2200 deg cm2 dmol(-1) in water and in 50% TFE at 2 degrees C. Using -43,000 deg cm2 dmol(-1) as [theta]222 for a 100% helix, NLEKG14 in 50% TFE at 25 degrees C was estimated to be 77% helix. This estimate was confirmed by two-dimensional 1H NMR studies of NLEKG14 in 50% TFE. Comparison with the sequences and conformations found in IF proteins indicates that the alpha-helical regions in the proteins may be exceptionally stable, but the high values for the ellipticity of alpha-helices now revealed allow for significant portions of the protein rod regions to be occupied by conformations other than alpha-helix.

Circular Dichroism↗
PubMed1989

[Intermediate filament patterns of the nephron and the urothelium].

The expression of intermediate filament (IF) proteins (vimentin and individual cytokeratins [CKs]) has been studied in the normal human kidney using immunohistochemical methods. For CKs 19 and 7, a conspicuously alternating pattern of positive and negative tubule segments was found, suggesting an inverse relationship between epithelial specialization and extent of the CK cytoskeleton. Coexpression of CKs and vimentin (typical of the common types of renal cell carcinomas) was found in fetal but not in adult proximal tubules. The surface epithelium of the adult renal papilla was characterized by the presence of basal cells expressing certain stratification-related CKs. These data underline the correlation of IF protein expression not only with the epithelial cell type but also with its developmental and functional state.

Cytoskeleton↗
PubMed2005

[The role of intermedial filament nestin in malignant melanoma progression].

UNLABELLED: Nestin is one of intermedial filaments exprimed in proliferating progenitor cells of the CNS and PNS (central and peripheral nervous system). Postnatal reexpression of the protein occures mainly in CNS tumors and correlates with a high grade of malignancy. The aim of our study is assessment of the nestin expression in benign and malignant skin melanocytic lesions with respect to presume a prognostic role of this protein. We examined 127 bioptic specimens, including 42 nodular melanomas (NM), 32 superficial spreading melanomas (SSM), 10 dysplastic nevi and 43 common intradermal or dermoepidermal nevi. We proved significant increase in nestin expression in melanoma groups, especially in nodular melanomas, where nestin was localized mainly in the peripheral, invasive areas of the tumor mass. CONCLUSION: Detection of nestin expression might be used as an additional melanocytic tumour marker.

Disease Progression↗
PubMed2000

The 'ins' and 'outs' of intermediate filament organization.

A major function shared by several types of cytoplasmic intermediate filaments (IFs) is to stabilize cellular architecture against the mechanical forces it is subjected to. As for other fibrous cytoskeletal arrays, a crucial determinant of this function is the spatial organization of IFs in the cytoplasm. However, very few crossbridging proteins are specific for IFs - most IF-associated proteins known to exert a structural role act to tether IFs to other major cytoskeletal elements, such as F-actin, microtubules or adhesion complexes. In addition, IFs are endowed with the ability to participate in their own organization. This intriguing property is probably connected to the unusual degree of sequence diversity and sequence-specific regulation that characterize IF genes and their proteins. This dependence upon a combination of extrinsic and intrinsic determinants contributes to distinguish IFs from other fibrous cytoskeletal polymers and is key to their function.

Eukaryotic Cells↗
PubMed1984

Immunocytochemical demonstration of intermediate filaments in a granular cell ameloblastoma.

The nature and location of intermediate filament proteins (IFP) may provide new insights into the origin and differentiation of neoplastic cells. An immunofluorescent study of these IFP in a case of a granular cell ameloblastoma revealed that all tumor cells contained the IFP keratin. Some granular cells, however, also contained the IFP vimentin, which is considered specific for mesenchymal tissues only. The implications of these observations are discussed. Study with monoclonal antibodies indicated the origin of the ameloblastoma from non-keratinized squamous epithelium. A comparison of the anti-keratin immunofluorescence pattern of the ameloblast-like cells in the present tumor with ameloblasts in the tooth germ revealed no similarities, indicating that despite some resemblance of the peripheral columnar cells to ameloblasts, these cells differ in other aspects.

Aged↗
PubMed1991

Expression of intermediate filament proteins in the adult human cochlea.

The immunohistochemical localization of intermediate filament proteins was studied in frozen sections of chemically fixed, nondecalcified adult human cochleas. Cytokeratins were found in all epithelial cells lining the cochlear duct (including most supporting cells of the organ of Corti) but were absent in the hair cells. Neurofilament proteins were present in the nerve endings at the hair cells, in the neural bundles, and in the ganglion cells. Vimentin staining occurred in most of the supporting structures and was roughly complementary to the regions showing cytokeratin staining and neurofilament staining. However, the region of the spiral prominence and outer sulcus, as well as the pillar cells and Deiters' cells in the organ of Corti, showed coexpression of vimentin and cytokeratins. No definite immunostaining was observed with antibodies to desmin and glial fibrillary acidic protein.

Adult↗
PubMed2002

Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function.

The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear matrix elements, the nuclei of a series of cultured cells were subjected to the Li-diiodosalicylate (LIS) extraction protocol developed for the preparation of nuclear matrices and analyzed by immunofluorescence microscopy and immunoblotting with antibodies directed against lamin B and cIF proteins. When nuclei released from hypotonically swollen L929 suspension cells in the presence of digitonin or Triton X-100 were exposed to such strong shearing forces that a considerable number were totally disrupted, a thin, discontinuous layer of vimentin IFs remained tenaciously adhering to still intact nuclei, in apparent coalignment with the nuclear lamina. Even in broken nuclei, the distribution of vimentin followed that of lamin B in areas where the lamina still appeared intact. The same retention of vimentin together with desmin and glial IFs was observed on the nuclei isolated from differentiating C2C12 myoblast and U333 glioma cells, respectively. Nuclei from epithelial cells shed their residual perinuclear IF layers as coherent cytoskeletal ghosts, except for small fractions of vimentin and cytokeratin IFs, which remained in a dot-to cap-like arrangement on the nuclear surface, in apparent codistribution with lamin B. LIS extraction did not bring about a reduction in the cIF protein contents of such nuclei upon their transformation into nuclear matrices. Moreover, in whole mount preparations of mouse embryo fibroblasts, DNA/chromatin emerging from nuclei during LIS extraction mechanically and chemically cleaned the nuclear surface and perinuclear area from loosely anchored cytoplasmic material with the production of broad, IF-free annular spaces, but left substantial fractions of the vimentin IFs in tight association with the nuclear surface. Accordingly, double-immunogold electron microscopy of fixed and permeabilized fibroblasts disclosed a close neighborhood of vimentin IFs and lamin B, with a minimal distance between the nanogold particles of ca. 30 nm. These data indicate an extremely solid interconnection of cIFs with structural elements of the nuclear matrix, and make them, together with their susceptibility to crosslinkage to MARs and other genomic DNA sequences under native conditions, complementary or even integral constituents of the karyoskeleton.

Animals↗
PubMed2002

The body margin of the planarian Dugesia japonica: characterization by the expression of an intermediate filament gene.

We have cloned and sequenced a cDNA encoding an intermediate filament protein (IF) from the planarian Dugesia japonica named DjIFb. The deduced amino acid sequence of DjIFb has similarity to those of protostomic IFs and lamins, supporting a previous hypothesis that the protostomic IFs, including DjIFb, are evolutionarily closer to lamins than to vertebrate cytoplasmic IFs. In addition, analysis of the exon/intron organization revealed that 8 out of 10 introns of DjIFb were coincident in their position, even in the codon phase, with those of the non-neuronal IF of the snail Helix aspersa. This suggests that the Platyhelminthes are not the most primitive Bilateria but instead are evolutionarily close to the Mollusca. The DjIFb gene was expressed in particular cells, probably a kind of adhesive gland cell, which were present in the marginal region encircling the planarian body. The localization of DjIFb protein suggests that it plays an important role in the secretion of an adhesive substance. The specific expression pattern of the DjIFb gene enabled us to monitor how the body margin forms during planarian regeneration.

Amino Acid Sequence↗
PubMed2006

Regulatory mechanisms and functions of intermediate filaments: a study using site- and phosphorylation state-specific antibodies.

Intermediate filaments (IF) form the structural framework of the cytoskeleton. Although histopathological detection of IF proteins is utilized for examining cancer specimens as reliable markers, the molecular mechanisms by which IF are involved in the biology of cancer cells are still unclear. We found that site-specific phosphorylation of IF proteins induces the disassembly of filament structures. To further dissect the in vivo spatiotemporal dynamics of IF phosphorylation, we developed site- and phosphorylation state-specific antibodies. Using these antibodies, we detected kinase activities that specifically phosphorylate type III IF, including vimentin, glial fibrillary acidic protein and desmin, during mitosis. Cdk1 phosphorylates vimentin-Ser55 from prometaphase to metaphase, leading to the recruitment of Polo-like kinase 1 (Plk1) to vimentin. Upon binding to Phospho-Ser55 of vimentin, Plk1 is activated, and then phosphorylates vimentin-Ser82. During cytokinesis, Rho-kinase and Aurora-B specifically phosphorylate IF at the cleavage furrow. IF phosphorylation by Cdk1, Plk1, Rho-kinase and Aurora-B plays an important role in the local IF breakdown, and is essential for the efficient segregation of IF networks into daughter cells. As another part of our research on IF, we have set out to find the binding partners with simple epithelial keratin 8/18. We identified tumor necrosis factor receptor type 1-associated death domain protein (TRADD) as a keratin 18-binding protein. Together with data from other laboratories, it is proposed that simple epithelial keratins may play a role in modulating the response to some apoptotic signals. Elucidation of the precise molecular functions of IF is expected to improve our understanding of tumor development, invasion and metastasis.

Animals↗
PubMed1985

Immunologic properties of enzymatically degraded human keratin intermediate filaments.

In order to gain insight into the metabolism of keratin intermediate filaments (KIF) as well as the ability of KIF degradation products to interact with the immune system, we performed enzymatic degradation of purified KIF and examined their interaction with anti-KIF autoantibodies and their ability to act as immunogens. Aliquots of KIF aggregates were exposed to 3 different enzymes, that is, alpha-chymotrypsin, plasmin, and trypsin, in dose- and time-dependent experiments. The effect of the digestion was monitored sequentially by polyacrylamide gel electrophoresis and simultaneously by transmission electron microscopy. Furthermore, the KIF degradation proteins were then examined for their ability to bind anti-KIF autoantibodies by immunoblot and for their immunogenicity. In addition, preincubation of KIF with anti-KIF autoantibodies prior to the digestion procedure was performed to investigate a possible protective effect of this treatment against proteolytic degradation. The experiments demonstrated that: (1) KIF are degraded by serine proteinases, (2) with prolonged incubation time intact KIF are progressively replaced by more granular-amorphous material in transmission electron microscopy, (3) anti-KIF autoantibodies bind to KIF degradation proteins, (4) preincubation of KIF with anti-KIF autoantibodies does not exert any major protective effect for KIF against proteolytic degradation, and (5) the enzymatic degradation products of KIF can function as effective immunogens causing the formation of high-titer anti-KIF antibodies.

Autoantibodies↗
PubMed1990

Disruption of keratin intermediate filaments by ultraviolet radiation in cultured human keratinocytes.

Fluorescence microscopy has been utilized to investigate the effects of UV irradiation on the organization of keratin intermediate filaments in normal human epidermal keratinocytes. Sun lamp irradiation induced the condensation of keratin intermediate filaments into the perinuclear region and inhibited the reorganization of keratin filaments normally induced by Ca2+. Exposure to UVC appeared to disrupt keratin filaments similarly, whereas UVA had no discernible effect.

Cells, Cultured↗