PubMed Health⌕ Search

Biomedical subjects

C Huet

Publications and source records attributed to C Huet.

At least 37 records · Page 2Linked to original sources
PubMed1992

An actin-binding site containing a conserved motif of charged amino acid residues is essential for the morphogenic effect of villin.

The actin-binding protein villin induces microvillus growth and reorganization of the cytoskeleton in cells that do not normally produce this protein. Transfection of mutagenized villin cDNAs into CV-1 cells was used to show that a conserved, COOH-terminally located cluster of charged amino acid residues (KKEK) is crucial for the morphogenic activity of villin in vivo. In vitro experiments with a 22 amino acid synthetic peptide corresponding to this region of villin provide evidence that this motif is part of an F-actin-binding site that induces G-actin to polymerize. Chemical cross-linking of actin to this peptide, the effects of amino acid substitutions in peptides, and the behavior of villin variants further corroborate the participation of the KKEK sequence in actin contacts.

Actins↗
PubMed1992

G- to F-actin modulation by a single amino acid substitution in the actin binding site of actobindin and thymosin beta 4.

The actin binding sites of actobindin and thymosin beta 4, two small polypeptides that inhibit actin polymerization by interacting with monomeric actin, have been localized using peptide mimetics. Both sites are functionally similar and extend over 20 residues and are located in the NH2-terminus of the polypeptides. They can be dissected into two functional entities: a conserved hexapeptide motif (LKHAET or LKKTET), which forms the major contact site through electrostatic interactions with actin, and a non-conserved NH2-terminal segment preceding the motif, which exerts the inhibitory activity on actin polymerization probably by steric hindrance. The introduction of a glutamic acid at the third position in the motif, creating LKEAET or LKETET sequences, which are similar to those found in some F-actin binding proteins, converts the peptide's inhibitory phenotype into an F-actin stimulatory property. These results allow the proposal of a simple model for G- to F-actin modulation.

Actins↗
PubMed1992

Amphibian intestinal villin: isolation and expression during embryonic and larval development.

An actin-binding protein of M(r) 105,000 has been isolated from anuran amphibian intestinal mucosa. Polyclonal antibodies directed against chicken and pig intestinal villins and anti-porcine villin headpiece monoclonal antibody crossreact with the amphibian M(r) 105,000 protein. Furthermore, the latter possesses an NH2-terminal sequence that is very homologous to those of avian and mammalian villins. In addition, polyclonal antibodies directed against amphibian intestinal M(r) 105,000 protein crossreact with chicken and mouse intestinal epithelial cell villins. These data indicate that the amphibian intestinal M(r) 105,000 protein is immunologically and structurally related to villin, an actin-binding protein expressed in specific epithelial tissues in vertebrates. Morphological, immunocytochemical and immunoblotting techniques were then used to investigate the expression of villin during embryonic and larval intestinal development of Xenopus laevis. Villin is not found in the egg or the endoderm of the early embryo. It is first detected just before hatching in the apical domain of endodermal cells at a time when few surface microvilli are visible by transmission electron microscopy. In the newly hatched larva, villin accumulates as these cells differentiate. These results provide a detailed developmental profile of Xenopus intestinal villin expression and demonstrate that this protein is a useful marker for the presumptive intestinal endoderm.

Actins↗
PubMed1990

Establishment of polarized endocytosis in differentiable intestinal HT29-18 subclones.

Subclones of the HT29-18 clone, derived from a human adenocarcinoma, are able to acquire an enterocyte-like phenotype depending on the culture conditions. To investigate fluid-phase and receptor-mediated endocytosis in the polarized subclone HT29-18-C1, we established culture conditions that allowed cell growth on permeable supports. HT29-18-C1 monolayers had an electrical resistance of 43 ohms.cm2 and developed a transepithelial potential of about 2 mV. Transferrin receptors were uniformly distributed on the entire cell surface of undifferentiated HT29-18 cells but were located on the basolateral membrane of differentiated cells. Transferrin had a high affinity (Kd = 2.5 x 10(-9) M) for its receptor independent of the state of differentiation. The number of transferrin receptors and the mRNA amounts encoding them were comparable in the undifferentiated and differentiated HT29-18 cells. Transferrin was quickly internalized and recycled back to the cell surface of undifferentiated HT29-18 cells. The same phenomenon also occurred in differentiated HT29-18 cells, but the receptors were limited to the basolateral membrane. In the presence of ammonium chloride, the process was slower but remained polarized. Fluid-phase uptake was also investigated with horseradish peroxidase (HRP) in differentiated HT29-18 C1 cells. HRP that was internalized in 1 hour from a given membrane domain preferentially recycled back to the same membrane domain. No significant accumulation of the enzyme in the late endosomes and lysosomes of the differentiated HT29-18-C1 cells was observed.

Adenocarcinoma↗
PubMed1989

Villin induces microvilli growth and actin redistribution in transfected fibroblasts.

The function of villin, an actin-binding protein, has been investigated by transfecting fibroblasts with cloned human cDNAs encoding wild-type villin or functional villin domains. Synthesis of large amounts of villin induced the growth of numerous long microvilli on cell surfaces together with the redistribution of F-actin. These microvilli contained a cytoskeleton of F-actin, and their appearance was frequently accompanied by the disappearance of stress fibers. The complete villin gene sequence was required to exert its morphogenic effect. Villin lacking one actin-binding domain (113 amino acids), located at its carboxyterminal end, did not induce growth if microvilli or stress fiber disruption. Our results indicate that villin plays a key role in vivo in the morphogenesis of microvilli.

Actins↗
PubMed1988

Villin expression in the visceral endoderm and in the gut anlage during early mouse embryogenesis.

Villin is an evolutionarily well conserved, Ca2+ regulated actin-binding protein, and a major structural component of the brush border of specialized absorptive cells. Using paraffin sections and an affinity purified polyclonal anti-villin antibody, we have investigated the early expression of villin during mouse embryogenesis. Villin is first detectable at the early post-implantation stage in visceral endodermal cells at the periphery of the egg cylinder. In this extra embryonic layer, the expression of villin increases and then persists until full term gestation. In the embryo, villin first appears in gut anlage during the axial rotation. Using the same methodology, villin expression is also demonstrated in differentiating embryoid bodies from a teratocarcinoma. Both in extra embryonic and embryonic extracts, villin expression is confirmed by immunoblot and Northern blot analysis which reveal, respectively, a single polypeptide of 93 kd and an mRNA of 3.4 kb in length, two well defined parameters for adult mouse villin gene expression. The results presented here show that paraffin sections allow very sensitive and highly resolutive detection of antigens in early embryogenesis. They provide a detailed developmental profile of villin expression and demonstrate the usefulness of villin as a marker for epithelial cells involved in absorptive processes.

Animals↗
PubMed1988

Human intestinal goblet cells in monolayer culture: characterization of a mucus-secreting subclone derived from the HT29 colon adenocarcinoma cell line.

HT29-18N2 (N2) cells, a subclone of the HT29 human colon carcinoma cell line, are shown in this report to be a model system for the study of human goblet cell differentiation and mucin secretion. Grown in the absence of glucose, these cells formed homogeneous epithelial monolayers of columnar cells with typical goblet cell morphology. Differentiation occurred on uncoated glass; laminin, fibronectin, or collagen type I or IV did not enhance differentiation. HT29-18N2 cells grown on uncoated or matrix-coated permeable filters formed differentiated monolayers, but mucin granules within some of these cells polarized along intraepithelial lumens. Polyclonal antibodies raised against purified human colonic mucin, and also a monoclonal antibody against a protease-sensitive epitope of human colonic mucin, stained secretory granules of all differentiated goblet cells within N2 cell monolayers but did not stain predifferentiated goblet cells lacking large secretory granules. Monoclonal antibodies against specific carbohydrate sequences of human mucins also failed to stain N2 cells before differentiation, but recognized varying fractions of differentiated N2 goblet cells. Autoradiographic visualization of radiolabeled glycoproteins demonstrated transport and secretion of N2 cell mucin granules. Cholinergic stimulation of differentiated N2 cell monolayers resulted in depletion of intracellular mucin granules.

Adenocarcinoma↗
PubMed1988

Differentiation of a clone isolated from the HT29 cell line: polarized distribution of histocompatibility antigens (HLA) and of transferrin receptors.

The HT29 cell line, derived from a human colon adenocarcinoma, is able to differentiate if galactose replaces glucose in the culture medium. We have isolated a clone (HT29-18) from this cell line which displays differentiated properties of the parent cell line. HT29-18 cells grown in glucose-containing medium form multiple layers of round cells without specific cell-cell adhesion. In contrast, when grown in galactose-containing medium, they form a monolayer with tight junctions and exhibit a well differentiated brush border at their apical membrane, which faces the culture medium. The polarized properties of HT29-18 cells grown in galactose-containing medium were demonstrated by immunofluorescent techniques with antibodies against 2 plasma membrane proteins. Class I histocompatibility antigens (HLA) and transferrin receptors, 2 well characterized integral membrane proteins, are uniformly distributed on the cell surface of undifferentiated HT29-18 cells, but acquire a polarized distribution during differentiation, localized on the basolateral membranes and absent from the apical surface. Binding of 125I-labeled transferrin was used to determine transferrin receptor distribution on apical and basolateral membranes. Functional tight junctions in the differentiated cultures were demonstrated, as the monolayer was impermeable to a permeation dye (ruthenium red) as well as to antibodies. The sealing of these tight junctions is, as in vivo, Ca++-dependent as they could be opened by a short incubation in Ca++-free medium.

Adenocarcinoma↗
PubMed1987

Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT-29) provide new models for cell polarity and terminal differentiation.

A clone HT29-18 has been isolated from the parent cell line HT-29, which derived from a human colon adenocarcinoma (Fogh, J., and G. Trempe, 1975, Human Tumor Cells in Vitro, J. Fogh, editor, Plenum Publishing Corp., New York, 115-141). This clone is able to differentiate as the parent cell line does. Differentiation occurs when glucose is replaced by galactose in the culture medium (Pinto, M., M.D. Appay, P. Simon-Assman, G. Chevalier, N. Dracopoli, J. Fogh, and A. Zweibaum, 1982, Biol. Cell., 44:193-196). We demonstrate here that the differentiated cloned population HT29-18/gal is heterogenous: although 90% of the cells show morphological characteristics of "absorptive cells", only 20-30% of them display sucrase-isomaltase in their apical microvillar membranes. About 10% of the entire cell population consists of cells containing mucous granules similar to intestinal goblet cells. We have isolated two subclones, HT29-18-C1 and HT29-18-N2, from the differentiated HT29-18/gal cells. HT29-18-C1 cells show morphological characteristics of polarized absorptive cells, when growing either in glucose- or in galactose-containing media, but the sucrase-isomaltase is not expressed in the cells grown in glucose-containing medium. The clone HT29-18-N2 is also polarized in both culture conditions and is similar to globlet cells in vivo. It grows as a monolayer, exhibits tight junctions, and contains numerous mucous granules whose exocytosis can be triggered by carbachol, a parasympathomimetic drug. We conclude that the clone HT29-18 first isolated was a multipotent cell population from which we isolated several subclones that differentiate either as absorptive (HT29-18-C1) or as mucous (HT29-18-N2) cells. In contrast to the parent HT-29 cell line, the subclones retain most of their differentiated properties in glucose-containing medium.

Adenocarcinoma↗
PubMed1987

Changes in villin synthesis and subcellular distribution during intestinal differentiation of HT29-18 clones.

Brush border in enterocytes is a cell surface specialization intimately associated with terminal differentiation of these cells. HT29-18, a clone derived from the HT-29 human colonic adenocarcinoma cell line, and HT29-18-C1, a subclone from HT29-18 described in the companion paper (Huet, C., C. Sahuquillo-Merino, E. Coudrier, and D. Louvard, 1987, J. Cell Biol., 105:345-357), undergo terminal differentiation with brush borders in the absence of glucose or upon replacement of glucose by galactose in the medium. Taking advantage of this clone and its subclone which can be manipulated in vitro, we have studied the synthesis and subcellular distribution of villin, one major protein in the microvillus core of the brush border. For this study, a monoclonal antibody against villin (BDID2C3) has been isolated and characterized in detail. In addition an ELISA has been set up to measure villin accurately in total cell extracts. Villin content in differentiated HT29-18 cells is close to that seen in normal human colonic cells but 10 times lower in undifferentiated HT29-18 cells. The rate of villin synthesis is dramatically increased in the course of enterocytic differentiation, while villin is remarkably stable after synthesis. We have recently shown, using a cDNA probe for villin, that this change is controlled either at the transcription level or by RNA stabilization (Pringault, E., M. Arpin, A. Garcia, J. Finidori, and D. Louvard, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:3119-3124). As shown by immunofluorescence and immunogold labelings, villin is targeted to the brush border area of differentiated HT29-18 cells but remains diffusely distributed in undifferentiated ones.

Adenocarcinoma↗
PubMed1985

Can villin be used to identify malignant and undifferentiated normal digestive epithelial cells?

We have investigated the presence of villin (a Ca2+-regulated actin binding protein) in various tissues (normal or malignant) and in established cell lines by using sensitive immunochemical techniques on cell extracts and immunofluorescence analysis on frozen sections. Our results show that villin is a marker that can be used to distinguish normal differentiated epithelial cells from the simple epithelia lining the gastrointestinal tract and renal tubules. Villin is found in the absorptive cells of the small and large intestines, in the duct cells of pancreas and biliary system, and in the cells of kidney proximal tubules. Furthermore, undifferentiated normal and tumoral cells of intestinal origin in vivo and in cell culture express villin. Therefore, expression of villin is seen in cells that do not necessarily display the morphological features characteristic of their terminally differentiated state, such as the microvilli-lined brush border. We suggest the possible clinical implications of using villin as a marker in the diagnosis of metastatic adenocarcinomas.

Animals↗
PubMed1985

Assembled and unassembled pools of clathrin: a quantitative study using an enzyme immunoassay.

Using polyclonal antibodies raised against clathrin, we have developed an enzyme-linked immunoassay that can specifically measure the quantity of clathrin in crude cell extracts. We found that the quantity (weight percent of total protein) of clathrin was similar in cell types that exhibit large differences in their levels of endocytosis and exocytosis (lymphoid cells, 0.11%; liver cells, 0.07%, fibroblasts, 0.18%; myeloma cells, 0.16%). However, the quantity of clathrin was found to be significantly higher in brain cortex (0.75%). Cellular clathrin was separated by high-speed centrifugation into two fractions: an unassembled form present in high-speed supernatants and an assembled form (clathrin coats) present in the pellets. We show that the fraction of clathrin in the unassembled state varies considerably depending on the cell type studied (14% in brain cortex to 70% in lymphocytes). Our data support the view that the amount of clathrin (relative to total cell protein) in eucaryotic cells is not related to the extent of receptor-mediated endocytosis and intracellular membrane traffic. However, the fraction of assembled clathrin seems to be higher in endocytically and/or exocytically active cells.

Animals↗
PubMed1984

Independent variation in the number of coated pits and of coated vesicles in cultured fibroblasts.

When tissue culture cells were maintained at 37 degrees C in a serum-free medium for 4 hr no change in the number of coated pits could be detected using ultrastructural techniques. However, the number of coated vesicles was highly significantly increased, being 179% more than in the control cultures. If the cells were put back into a medium supplemented with 5% calf serum, the number of coated pits was unchanged, but the number of coated vesicles decreased and returned to the control level within a few minutes. The same results were obtained when using ligands such as Low Density Lipoprotein or alpha-2-macroglobulin which are known to be internalized via coated structures. It is concluded that coated pits appear and disappear at equal rates and that coated vesicles can accumulate independently. It is suggested that this could be due to the presence of a large reserve of soluble clathrin. This pool would have a low turnover rate because cycloheximide did not block coated vesicle accumulation over the period studied.

Animals↗
PubMed1983

I--isolation and electron microscope studies of a potent platelet-aggregating glycoprotein from the venom of Crotalus durissus cascavella.

A potent acid-soluble platelet-aggregating glycoprotein was purified from the venom of Crotalus durissus cascavella by molecular sieve chromatography on Sephadex G-75 and by adsorption onto Sepharose 4B gels at acidic pH. This new protein with an apparent Mr of 300,000 at acidic pH and containing a low amount of sugars is non-toxic for mice. Electron microscope studies showed that the platelet-aggregating glycoprotein appeared as regular rosettes of 150 A in diameter at acidic pH and underwent polymerization in rod-like particles in the presence of sodium chloride. This glycoprotein, probably hydrophobic, is dissociated into an active molecular form whose apparent Mr was 144,000; however, it is believed to still be a not totally dissociated molecule.

Animals↗
PubMed1980

Specific glycolipid antigen(s) in SV40-transformed cell membranes.

A glycolipid extract was prepared from an SV40-transformed hamster cell line (EH-SV) according to the Folch partition procedure. The glycolipids from the aqueous layer were incorporated in liposomal membranes composed of lecithin/sphingomyelin/cholesterol (1:1:2 by weight). This liposomal preparation was inoculated in Syrian hamsters to raise immune sera. The sera were absorbed with trypsinized "normal" hamster cells (EH-N) and tested on various cell lines by the indirect immunofluorescence technique. When used for staining living cells, the immune serum produced a distinct cell-surface fluorescence with SV40-transformed cell lines regardless of the cell origin (e.g., rat or hamster). No reaction was observed with heterologous Py-transformed cell lines, spontaneously transformed cells, or sera from non-immunized hamsters. When used for staining acetone-fixed cells, the antiglycolipid serum reacted specifically with a thermostable antigen in the nuclear envelope and the cytoplasm of SV40-transformed cells. The sera lack interfering SV40 T reactivity. The results indicate the presence of related SV40-specific glycolipid antigen(s) in the plasma membrane, the nuclear membrane and probably other endomembranes of SV40-transformed cells.

Animals↗