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PubMed2006

GCP6 binds to intermediate filaments: a novel function of keratins in the organization of microtubules in epithelial cells.

In simple epithelial cells, attachment of microtubule-organizing centers (MTOCs) to intermediate filaments (IFs) enables their localization to the apical domain. It is released by cyclin-dependent kinase (Cdk)1 phosphorylation. Here, we identified a component of the gamma-tubulin ring complex, gamma-tubulin complex protein (GCP)6, as a keratin partner in yeast two-hybrid assays. This was validated by binding in vitro of both purified full-length HIS-tagged GCP6 and a GCP6(1397-1819) fragment to keratins, and pull-down with native IFs. Keratin binding was blocked by Cdk1-mediated phosphorylation of GCP6. GCP6 was apical in normal enterocytes but diffuse in K8-null cells. GCP6 knockdown with short hairpin RNAs (shRNAs) in CACO-2 cells resulted in gamma-tubulin signal scattered throughout the cytoplasm, microtubules (MTs) in the perinuclear and basal regions, and microtubule-nucleating activity localized deep in the cytoplasm. Expression of a small fragment GCP6(1397-1513) that competes binding to keratins in vitro displaced gamma-tubulin from the cytoskeleton and resulted in depolarization of gamma-tubulin and changes in the distribution of microtubules and microtubule nucleation sites. Expression of a full-length S1397D mutant in the Cdk1 phosphorylation site delocalized centrosomes. We conclude that GCP6 participates in the attachment of MTOCs to IFs in epithelial cells and is among the factors that determine the peculiar architecture of microtubules in polarized epithelia.

Animals↗
PubMed1998

Colocalization of single ribosomes with intermediate filaments in puromycin-treated and serum-starved mouse embryo fibroblasts.

Previous experiments have revealed a relatively weak electrostatic binding capacity of in vitro reconstituted intermediate filaments (IFs) as well as of natural IFs of whole cell mount preparations for purified ribosomal particles of mammalian origin. In order to demonstrate that such associations also occur in vivo, intact cells were subjected to double immunofluorescence microscopy using antibodies directed against vimentin and ribosomal protein S17. Since in proliferating cells the majority of the ribosomal particles are assembled into polyribosomes and these are to a great extent associated with microfilaments, in vitro cultured mouse embryo skin fibroblasts (MSF cells) were treated with puromycin to allow the formation of single ribosomes. Employing confocal laser scanning microscopy, the ribosomes were detected in colocalization with vimentin IFs. Disassembly of polyribosomes was also achieved by serum starvation of cultured cells. In this case, MSF cells of a low passage attained an extended and flattened appearance with the vimentin IFs being directly associated with the cell nuclei, radiating into the peripheral areas of the cells or showing a stress fiber-like distribution. In both cases, considerable quantities of ribosomal material were seen in close neighborhood to vimentin IFs. Frequently, these ribosome-IF associations were coaligned with microtubules and they also surrounded myosin I-decorated stress fibers. Double labeling with the vital, RNA-specific fluorochrome SYTO 14 produced a fluorescence pattern largely superimposable on that of ribosomal protein S17. Treatment of the starved cells with either demecolcine or cytochalasin D had an only moderately disturbing effect on vimentin IF distribution and the ribosomes stayed in contact with the vimentin IFs. On the basis of these results, it is conceivable that IFs play a role in the storage of ribonucleoprotein particles in general and non-translating ribosomes in particular in the cytoplasm of animal cells. In addition, the often seen coalignment of IFs with microtubules and microfilaments might serve facilitated and directional transport of ribonucleoprotein particles from the nucleus to peripheral areas of the cell.

Actin Cytoskeleton↗
PubMed1985

Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells.

Intermediate-sized filaments (IF) are among the most insoluble intracellular protein polymer structures. We have analyzed the small amounts of soluble vimentin, an IF protein, present in cytosol fractions obtained from lysis of cultured cells [rat RVF-SM cells, simian virus 40-transformed human fibroblasts, and human rhabdomyosarcoma (RD line) cells]. The molecular form of this soluble vimentin was determined by sucrose density gradient centrifugation, using vimentin-specific antibodies for subsequent ELISA and immunoblotting analyses. The majority of the soluble vimentin appeared in a distinct form indistinguishable in its sedimentation behavior from reconstituted tetrameric subunits of purified vimentin arrested at low ionic strength. The tetrameric coiled-coil nature of the soluble form of vimentin was indicated by the digestion pattern with chymotrypsin and by chemical crosslinking with copper-1,10-phenanthroline and dimethylsuberimidate. The competence of this soluble vimentin to assemble into IF at higher salt concentrations was demonstrated by electron microscopy. Pulse-chase experiments showed that the soluble form was not an exclusively posttranslational intermediate. We propose that in the living cell a small pool of a distinct soluble tetrameric form of vimentin exists which may exchange with polymeric IF vimentin.

Animals↗
PubMed1987

Intermediate filament proteins as tissue specific markers in normal and neoplastic testicular tissue.

Normal testicular tissue and primary and metastatic testicular germ cell tumours were examined for their intermediate filament protein (IFP) expression. Seminomas were shown to react with antibodies to vimentin, while non-seminomatous germ cell tumours were strongly positive with antibodies to cytokeratin. In the case of teratocarcinoma, several components of the tumour can be distinguished using a combination of monoclonal and polyclonal antisera in the double-label immunofluorescence technique. We conclude that antibodies to cytokeratin and vimentin can be helpful in the diagnosis of testicular germ cell tumours, especially in the differentiation between seminomas and non-seminomatous testis tumours.

Fluorescent Antibody Technique↗
PubMed1992

Microinjection of IFA antibody induces intermediate filament aggregates in epithelial cell lines but perinuclear coils in fibroblast-like lines.

The murine monoclonal IFA antibody recognizes a conserved sequence present in almost all intermediate filament (IF) proteins. When IFA antibody was injected into 13 different primary or established cell lines, striking differences were detected between epithelial and fibroblastic cell lines. In epithelial cells keratin IFs were broken down within 4 h into numerous spheroid aggregates scattered throughout the cytoplasm. Keratin aggregates were first detected in the cytoplasmic periphery. In contrast, in fibroblastic cells, injection of IFA antibody led to the formation of perinuclear coils of vimentin. IFA antibody at a concentration of greater than 1 mg/ml had to be injected to initiate these transitions. When HeLa cells, which contain separate networks of vimentin and keratin filaments, were injected with IFA antibody, vimentin did not form perinuclear coils but was instead found together with keratin in aggregates. Electron micrographs of HeLa cells injected with IFA antibody showed that the aggregates have diameters between 0.5 and 2.6 microns and resembled the keratin aggregates observed in certain mitotic epithelial cells. Although the ultrastructural studies support an association of some aggregates with desmosomes, aggregates were, however, also induced by injection of IFA antibody into human keratinocytes in low calcium medium under conditions where desmosomes were not present.

3T3 Cells↗
PubMed1990

Intermediate filaments in smooth muscle from pregnant and non-pregnant human uterus.

The intermediate filament proteins desmin and vimentin from pregnant and non-pregnant uterine muscle and smooth-muscle cells in culture were analysed using SDS/PAGE. The desmin content in uterine muscle increases dramatically during pregnancy, whereas vimentin remains unchanged or changes very little. When muscle cells are kept in culture, a considerable increase in vimentin content is observed as compared with vimentin in freshly isolated non-pregnant uterine tissue. Our results strengthen the view that vimentin and desmin filaments have independent function and turnover, and point to a predominantly structural role for desmin filaments.

Blotting, Western↗
PubMed1978

Comparative electrophoretic study of Mallory body and intermediate filament protein.

To investigate the possibility that Mallory bodies (MBs) are composed of intermediate filaments (IFs), the electrophoretic patterns of MB proteins from human liver were compared with the IF protein extracted from chicken gizzard smooth muscle and extracts of mouse liver which contained MBs. The human MB had a protein component which had a similar mobility as that of the IF protein extracted from smooth muscle. A similar protein band was found in the extracts of mouse liver which contained MBs. No protein band corresponding to actin protein was found in human MB protein. The IF-like protein was also present in control mouse liver extract. The estimated molecular weight of the IF protein was 54,000. The evidence suggests that MBs are, in part, composed of IF protein. The IF protein is a normal constituent of liver and smooth muscle.

Animals↗
PubMed1982

Histogenesis of Ewing's sarcoma. An evaluation of intermediate filaments and endothelial cell markers.

Four cases of Ewing's sarcoma, three in bone and one from an extraskeletal site, were studied immunohistologically using monospecific antibodies against intermediate filament proteins of keratin, vimentin, desmin and neurofilament types. All cases were also evaluated for the presence of Factor VIII-related antigen (FVIIIR:Ag) and for the binding of Ulex europaeus I lectin (UEA I), both of which are endothelial markers. In all cases the tumor cells contained vimentin but not keratin, desmin or neurofilaments. The tumor cells could not be decorated with either anti-FVIIIR:Ag or UEA I, whereas the vascular endothelium was positive for both markers. The vimentin-positivity indicates a mesenchymal derivation of Ewing's sarcoma, while the lack of endothelial markers argues against the proposed endothelial origin of this tumor.

Adolescent↗
PubMed1993

Intermediate filaments and ATPase activity in the vascular wall of vertebrates.

The vascular wall of aorta and vena cava was examined for adenosine triphosphatase (ATPase) activity and cytoskeletal intermediate filaments (IF) in different representatives of vertebrates. Enzyme activity was studied by the modified method of Padykula and Herman. A streptavididin-biotin immunohistochemical method was applied to reveal desmin (D) and vimentin (V) IF. Endothelial cells of all vessels were V-positive and D-negative and exhibit high ATPase activity. Vascular smooth muscle cells (SMC) in lower vertebrates (pisces and amphibia) were also V-positive and D-negative, but showed low ATPase activity. SMC were D-positive and V-negative and possessed high enzyme activity in aves and mammals, similar to that of the endothelium. In cow vascular wall D-reactivity and high ATPase activity were mostly expressed in bundles of mosaically arranged thick SMC fibres of the outer aortic media as well as in the longitudinal fibres in the inferior vena cava. In higher vertebrates SMC of vasa vasorum were both V- and D-positive and showed high enzyme activity. The results demonstrate that D-immunoreactivity is mostly expressed in SMC of layers of high functional activity, which correlates with the intense ATPase reaction in these cells.

Adenosine Triphosphatases↗
PubMed1992

Structure of desmoplakin and its association with intermediate filaments.

Desmoplakins (DPs) I and II are two major related proteins located in the desmosomal plaque where they have been proposed to play a role in attaching intermediate filaments (IF) to the inner cell surface. The predicted amino acid sequence of DP was obtained by analysis of overlapping cDNA clones. Computer-aided analysis suggests that DPI will form a dumbbell-shaped homodimer, with a central alpha-helical coiled coil rod domain of 132 nm and two globular end domains. The DPII molecule is missing 599 residues from the central domain, resulting in a rod about one third the length of DPI. The carboxyl terminus comprises three subdomains each containing almost 5 repeats of a 38 residue repeating motif with a periodicity in acidic and basic residues similar to that found in the rod domain of IF proteins. This suggests a possible mechanism by which these proteins might interact. The amino terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical rich bundles. A series of c-myc-tagged mammalian expression vectors encoding specific predicted domains of DPI were transiently expressed in COS-7 cells. Light and electron microscopical observations revealed that DP polypeptides including the 90 kDa carboxyl terminal globular domain of DPI specifically colocalized with and ultimately resulted in the complete disruption of keratin and vimentin IF. This effect was specific for the carboxyl terminus, as the expression of the 95 kDa rod domain of DPI did not visibly alter IF networks.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion Molecules↗
PubMed1990

Tailless keratins assemble into regular intermediate filaments in vitro.

To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.

Amino Acid Sequence↗
PubMed1996

Characterization of pinin, a novel protein associated with the desmosome-intermediate filament complex.

We have identified a protein named pinin that is associated with the mature desmosomes of the epithelia (Ouyang, P., and S.P. Sugrue. 1992. J. Cell Biol. 118:1477-1488). We suggest that the function of pinin is to pin intermediate filaments to the desmosome. Therefore, pinin may play a significant role in reinforcing the intermediate filament-desmosome complex. cDNA clones coding for pinin were identified, using degenerative oligonucleotide probes that were based on the internal amino acid sequence of pinin for the screening of a cDNA library. Immunoblotting of expressed recombinant proteins with the monoclonal 08L antibody localized the 08L epitope to the carboxyl end of the protein. Polyclonal antibodies directed against fusion proteins immunoidentified the 140-kD protein in tissue extracts. Immunofluorescence analysis, using the antifusion protein antibody, demonstrated pinin at lateral epithelial boundaries, which is consistent with desmosomal localization. The conceptual translation product of the cDNA clones contained three unique domains: (a) a serine-rich domain; (b) a glutamine-proline, glutamine-leucine repeat domain; and (c) an acidic domain rich in glutamic acid. Although the 3' end of the open reading frame of the clone for pinin showed near identity to a partial cDNA isolated for a pig neutrophil phosphoprotein (Bellavite, P., F. Bazzoni, et al. 1990. Biochem. Biophys. Res. Commun. 170:915-922), the remaining sequence demonstrated little homology to known protein sequences. Northern blots of mRNA from chicken corneal epithelium, MDCK cells, and various human tissues indicated that pinin messages exhibit tissue-specific variation in size, ranging from 3.2 to 4.1 kb. Genomic Southern blots revealed the existence of one gene for pinin, suggesting alternative splicing of the mRNA. Expression of the full-length cDNA clones in human 293 cells and monkey COS-7 cells demonstrated that a 140-kD immunoreactive species on Western blots corresponded to pinin. Pinin cDNA transfected into the transformed 293 cells resulted in enhanced cell-cell adhesion. Immunofluorescence staining revealed that the expressed pinin protein was assembled to the lateral boundaries of the cells in contact, which is consistent with the staining pattern of pinin in epithelial cells.

Animals↗
PubMed1993

[The study of intermediate filament organization of highly and poorly differentiated nasopharyngeal carcinoma cell lines].

By employing cytochemistry, immunocytochemistry, selective double extraction, whole mount electron microscopy and electrophoretic scanning quantitative analysis, the intermediate filaments (IF) of in vitro cultured nasopharyngeal carcinoma cell lines CNE-1 (highly differentiated) and CNE-2Z (poorly differentiated) have been studied. The results demonstrated that although the keratin-type IF were organized in a network pattern in both kinds of cells, significant differences could be observed between the two kinds of cells regarding IF organization, subtypes and quantity of keratin. The poorly differentiated cell line CNE-2Z exhibited a far more irregular and confused IF organization structure, and the content of keratin was lower while the variety of keratin subtypes was increased.

Carcinoma↗
PubMedDate not supplied

Cytoskeleton architecture of C6 rat glioma cell subclones differing in intermediate filament protein expression.

Whole-mount electron microscopy was used in conjunction with immunogold labeling to characterize the cytoskeleton architecture of C6 rat glioma cell subclones. These subclones differ in intermediate filament (IF) protein composition and either contain vimentin (subclone C6D8) or do not express any of the known cytoplasmic IF proteins (subclone C6D10) (Röser et al., 1991). In C6D8 cells short thin (3 nm) connecting filaments frequently linked vimentin to actin filaments and, in addition, connected vimentin filaments to each other. Occasionally, direct contacts were noticed between actin and vimentin filaments. Thin connecting filaments were present at a significantly higher number in IF-deficient C6D10 cells, forming a dense cytoplasmic network in conjunction with actin filament bundles as the dominating structure. The data indicate that thin connecting filaments are present in C6 cells independent of the expression of cytoplasmic IF proteins. They suggest that structural linkages between vimentin and actin filaments mediated by thin connecting filaments could play a major role in determining the cytoskeleton architecture of these cells.

Actins↗
PubMed1982

Identification of Langerhans cells: simultaneous use of sera to intermediate filaments, T6 and HLA-DR antigens on oral mucosa, human epidermis and their tumours.

Langerhans cells in oral mucosa and epidermis have been identified using antibodies to intermediate filament proteins in conjunction with antibodies to T6 and HLA-DR antigens. Langerhans cells, lymphocytes and melanocytes are positive when tested with antibodies to vimentin, and negative with antibodies to prekeratin. Langerhans cells are also positive with antibodies directed against HLA-DR and T6 antigens. In contrast keratinocytes are positive for prekeratin and negative for vimentin, HLA-DR and T6. These methods provide a useful tool for the identification of Langerhans cells at the level of the light microscope, and have been used to show changes in the number and arrangement of these cells in squamous cell carcinomas of skin and oral mucosa, and in inflammatory conditions.

Antibodies, Monoclonal↗
PubMed2000

AlphaB-crystallin regulates intermediate filament organization in situ.

AlphaB-crystallin is a small heat shock protein (hsp) and molecular chaperone that can interact with a wide spectrum of cellular components including intermediate filaments (IF). The significance of these interactions is not currently known. We have tested whether increased alphaB-crystallin expression effects changes in the IF systems in situ. Adenoviral-mediated gene transfer was used to overexpress alphaB-crystallin in primary astrocytes. A positive correlation was observed between overexpression of alphaB-crystallin and diffuse, filigree IF. AlphaB-crystallin did not appear to alter the polymerization state of IF proteins. These data show that an increase in alphaB-crystallin expression in the absence of stress can modify the organizational state of IF and that alphaB-crystallin can function as an IF debundling protein.

Adenoviridae↗
PubMed1986

Interaction of frog virus 3 with the cytomatrix. IV. Phosphorylation of vimentin precedes the reorganization of intermediate filaments around the virus assembly sites.

Frog virus 3 (FV3) assembles in morphologically distinct assembly sites in the cytoplasm of infected cells. As the assembly sites form, the intermediate filaments (IF) aggregate, delimit the assembly sites, and remain so throughout infection. To determine the molecular basis of reorganization of IF, we analysed the vimentin of uninfected and FV3-infected cells by two-dimensional gel electrophoresis. The results showed that (i) the vimentin was more acidic in FV3-infected cells than in uninfected cells, (ii) the acidification of vimentin in FV3-infected cells was possibly due to a fourfold increase in phosphorylation, and (iii) the phosphorylation of vimentin preceded the reorganization of IF around virus assembly sites. A temperature-sensitive mutant of FV3 (ts9467), which at the non-permissive temperature neither reorganized IF nor formed assembly sites, failed to increase the phosphorylation of vimentin. Together, the above results suggest that changes in phosphorylation may modulate IF organization and that changes in IF organization are required for FV3 assembly site formation.

Animals↗