Biomedical subjects

D Louvard

Publications and source records attributed to D Louvard.

At least 37 records · Page 2Linked to original sources

PubMed2000

LPP, an actin cytoskeleton protein related to zyxin, harbors a nuclear export signal and transcriptional activation capacity.

The LPP gene is the preferred translocation partner of the HMGIC gene in a subclass of human benign mesenchymal tumors known as lipomas. Here we have characterized the LPP gene product that shares 41% of sequence identity with the focal adhesion protein zyxin. LPP localizes in focal adhesions as well as in cell-to-cell contacts, and it binds VASP, a protein implicated in the control of actin organization. In addition, LPP accumulates in the nucleus of cells upon treatment with leptomycin B, an inhibitor of the export factor CRM1. The nuclear export of LPP depends on an N-terminally located leucine-rich sequence that shares sequence homology with well-defined nuclear export signals. Moreover, LPP displays transcriptional activation capacity, as measured by GAL4-based assays. Altogether, these results show that the LPP protein has multifunctional domains and may serve as a scaffold upon which distinct protein complexes are assembled in the cytoplasm and in the nucleus.

3T3 Cells↗

PubMed2000

Vimentin filaments in fibroblasts are a reservoir for SNAP23, a component of the membrane fusion machinery.

Soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) are core machinery for membrane fusion during intracellular vesicular transport. Synaptosome-associated protein of 23 kDa (SNAP23) is a target SNARE previously identified at the plasma membrane, where it is involved in exocytotic membrane fusion. Here we show that SNAP23 associates with vimentin filaments in a Triton X-100 insoluble fraction in fibroblasts in primary culture and HeLa cells. Upon treatment of human fibroblasts with N-ethyl-maleimide, SNAP23 dissociates from vimentin filaments and forms a protein complex with syntaxin 4, a plasma membrane SNARE. The vimentin-associated pool of SNAP23 can therefore be a reservoir, which would supply the plasma membrane fusion machinery, in fibroblasts. Our observation points to a yet unexplored role of intermediate filaments.

Animals↗

PubMed2000

Villin-like actin-binding proteins are expressed ubiquitously in Arabidopsis.

In an attempt to elucidate the biological function of villin-like actin-binding proteins in plants we have cloned several genes encoding Arabidopsis proteins with high homology to animal villin. We found that Arabidopsis contains at least four villin-like genes (AtVLNs) encoding four different VLN isoforms. Two AtVLN isoforms are more closely related to mammalian villin in their primary structure and are also antigenically related, whereas the other two contain significant changes in the C-terminal headpiece domain. RNA and promoter/beta-glucuronidase expression studies demonstrated that AtVLN genes are expressed in all organs, with elevated expression levels in certain types of cells. These results suggest that AtVLNs have less-specialized functions than mammalian villin, which is found only in the microvilli of brush border cells. Immunoblot experiments using a monoclonal antibody against pig villin showed that AtVLNs are widely distributed in a variety of plant tissues. Green fluorescent protein fused to full-length AtVLN and individual AtVLN headpiece domains can bind to both animal and plant actin filaments in vivo.

Amino Acid Sequence↗

PubMed1999

Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment.

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.

Animals↗

PubMed1999

Villin function in the organization of the actin cytoskeleton. Correlation of in vivo effects to its biochemical activities in vitro.

Villin is an actin-binding protein of the intestinal brush border that bundles, nucleates, caps, and severs actin in a Ca(2+)-dependent manner in vitro. Villin induces the growth of microvilli in transfected cells, an activity that requires a carboxyl-terminally located KKEK motif. By combining cell transfection and biochemical assays, we show that the capacity of villin to induce growth of microvilli in cells correlates with its ability to bundle F-actin in vitro but not with its nucleating activity. In agreement with its importance for microfilament bundling in cells, the KKEK motif of the carboxyl-terminal F-actin-binding site is crucial for bundling in vitro. In addition, substitutions of basic residues in a second site, located in the amino-terminal portion of villin, impaired its activity in cells and reduced its binding to F-actin in the absence of Ca(2+) as well as its bundling and severing activities in vitro. Altogether, these findings suggest that villin participates in the organization and stabilization of the brush border core bundle but does not initiate its assembly by nucleation of actin filaments.

Actins↗

PubMed1999

In vivo, villin is required for Ca(2+)-dependent F-actin disruption in intestinal brush borders.

Villin is an actin-binding protein localized in intestinal and kidney brush borders. In vitro, villin has been demonstrated to bundle and sever F-actin in a Ca(2+)-dependent manner. We generated knockout mice to study the role of villin in vivo. In villin-null mice, no noticeable changes were observed in the ultrastructure of the microvilli or in the localization and expression of the actin-binding and membrane proteins of the intestine. Interestingly, the response to elevated intracellular Ca(2+) differed significantly between mutant and normal mice. In wild-type animals, isolated brush borders were disrupted by the addition of Ca(2+), whereas Ca(2+) had no effect in villin-null isolates. Moreover, increase in intracellular Ca(2+) by serosal carbachol or mucosal Ca(2+) ionophore A23187 application abolished the F-actin labeling only in the brush border of wild-type animals. This F-actin disruption was also observed in physiological fasting/refeeding experiments. Oral administration of dextran sulfate sodium, an agent that causes colonic epithelial injury, induced large mucosal lesions resulting in a higher death probability in mice lacking villin, 36 +/- 9.6%, compared with wild-type mice, 70 +/- 8.8%, at day 13. These results suggest that in vivo, villin is not necessary for the bundling of F-actin microfilaments, whereas it is necessary for the reorganization elicited by various signals. We postulate that this property might be involved in cellular plasticity related to cell injury.

Actin Cytoskeleton↗

PubMed1999

Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway.

ERM (Ezrin-Radixin-Moesin) proteins function as plasma membrane-actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. We have investigated ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1. Here we show that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, we found that ezrin interacts with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway.

Actin Cytoskeleton↗

PubMed1999

Raft association of SNAP receptors acting in apical trafficking in Madin-Darby canine kidney cells.

We have investigated the relationships between the apical sorting mechanism using lipid rafts and the soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE) machinery, which is involved in membrane docking and fusion. We first confirmed that anti-alpha-SNAP antibodies inhibit the apical pathway in Madin- Darby canine kidney (MDCK) cells; in addition, we report that a recombinant SNAP protein stimulates the apical transport whereas a SNAP mutant inhibits this transport step. Based on t-SNARE overexpression experiments and the effect of botulinum neurotoxin E, syntaxin 3 and SNAP-23 have been implicated in apical membrane trafficking. Here, we show in permeabilized MDCK cells that antisyntaxin 3 and anti-SNAP-23 antibodies lower surface delivery of an apical reporter protein. Moreover, using a similar approach, we show that tetanus toxin-insensitive, vesicle-associated membrane protein (TI-VAMP; also called VAMP7), a recently described apical v-SNARE, is involved. Furthermore, we show the presence of syntaxin 3 and TI-VAMP in isolated apical carriers. Polarized apical sorting has been postulated to be mediated by the clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We provide evidence that syntaxin 3 and TI-VAMP are raft-associated. These data support a raft-based mechanism for the sorting of not only apically destined cargo but also of SNAREs having functions in apical membrane-docking and fusion events.

Amino Acid Sequence↗

PubMed1999

Regulatory sequences of the mouse villin gene that efficiently drive transgenic expression in immature and differentiated epithelial cells of small and large intestines.

Villin is an early marker of epithelial cells from the digestive and urogenital tracts. Indeed villin is expressed in the stem cells and the proliferative cells of the intestinal crypts. To investigate the underlying molecular mechanisms and particularly those responsible for the restricted tissue specificity, a large genomic region of the mouse villin gene has been analyzed. A 9-kilobase (kb) regulatory region of the mouse villin gene (harboring 3.5 kb upstream the transcription start site and 5.5 kb of the first intron) was able to promote transcription of the LacZ reporter gene in the small and large intestines of transgenic mice, in a transmissible manner, and thus efficiently directed subsequent beta-galactosidase expression in epithelial cells along the entire crypt-villus axis. In the kidney, the transgene was also expressed in the epithelial cells of the proximal tubules but is likely sensitive to the site of integration. A construct lacking the first intron restricted beta-galactosidase expression to the small intestine. Thus, the 9-kb genomic region contains the necessary cis-acting elements to recapitulate the tissue-specific expression pattern of the endogenous villin gene. Hence, these regulatory sequences can be used to target heterologous genes in immature and differentiated epithelial cells of the small and/or large intestinal mucosa.

Animals↗

PubMed1999

Association of myosin I alpha with endosomes and lysosomes in mammalian cells.

Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIalpha) is associated with endosomes and lysosomes. We show that the overproduction of MMIalpha or the production of nonfunctional truncated MMIalpha affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIalpha, promote the dissociation of MMIalpha from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIalpha might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

Actins↗

PubMed1998

The rod cGMP phosphodiesterase delta subunit dissociates the small GTPase Rab13 from membranes.

Small Rab GTPases are involved in the regulation of membrane trafficking. They cycle between cytosolic and membrane-bound forms. These membrane association/dissociation are tightly controlled by regulatory proteins. To search for proteins interacting with Rab13, a small GTPase associated with vesicles in fibroblasts and predominantly with tight junctions in epithelial cells, we screened a HeLa two-hybrid cDNA library and isolated a clone encoding a protein of 17.4 kDa. This protein, almost identical to the bovine rod cGMP phosphodiesterase delta subunit, was named human delta-PDE. The delta-PDE binds specifically to Rab13. It exhibits two putative C-terminal sequences necessary for the interaction with PDZ (PSD95, Dlg, ZO-1) domains contained in many proteins localized to specific plasma membrane microdomains. Immunofluorescence microscopic studies revealed that the vesicular stomatitis virus (VSV)-tagged delta-PDE is localized in vesicular structures accumulated near the plasma membrane in epithelial cells. Deletion of the PDZ binding motifs impair VSV-delta-PDE subcellular distribution. Purified recombinant delta-PDE had the capacity to dissociate Rab13 from cellular membranes. Our data support the proposal that delta-PDE, but not GDP dissociation inhibitor, may serve to control the dynamic of the association of Rab13 with cellular membranes.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1998

A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells.

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

Amino Acid Sequence↗

PubMed1998

I-SceI-induced gene replacement at a natural locus in embryonic stem cells.

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 x 10[-6]) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.

Animals↗

PubMed1997

Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells.

The dissociation, migration, and remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) entail modifications in cell adhesion and in the actin cytoskeleton through unknown mechanisms. Here we report that ezrin, a membrane-cytoskeleton linker, is crucial to HGF-mediated morphogenesis in a polarized kidney-derived epithelial cell line, LLC-PK1. Ezrin is a substrate for the tyrosine kinase HGF receptor both in vitro and in vivo. HGF stimulation causes enrichment of ezrin recovered in the detergent-insoluble cytoskeleton fraction. Overproduction of wild-type ezrin, by stable transfection in LLC-PK1 cells, enhances cell migration and tubulogenesis induced by HGF stimulation. Overproduction of a truncated variant of ezrin causes mislocalization of endogenous ezrin from microvilli into lateral surfaces. This is concomitant with altered cell shape, characterized by loss of microvilli and cell flattening. Moreover, the truncated variant of ezrin impairs the morphogenic and motogenic response to HGF, thus suggesting a dominant-negative mechanism of action. Site-directed mutagenesis of ezrin codons Y145 and Y353 to phenylalanine does not affect the localization of ezrin at microvilli, but perturbs the motogenic and morphogenic responses to HGF. These results provide evidence that ezrin displays activities that can control cell shape and signaling.

Animals↗

PubMed1997

The role of actin binding proteins in epithelial morphogenesis: models based upon Listeria movement.

We summarize recent findings on the organization of the protein actin in eucaryotic cells. In particular we focus on how actin can be used to generate a vectorial force that is required for cell movement. These forces arise from protein molecules that recruit actin to the plasma membrane in such a manner that actin filaments extend outward from the cell body. This type of actin dependent force generation has been described in a nucleation-release model, which is one of several models currently being tested to explain actin dependent cell movement. Data in support of this model has arisen unexpectedly from studies of an intracellular bacteria, Listeria monocytogenes. This bacteria uses actin to propel itself during infection of eucaryotic cells. By studying Listeria movement, the roles of several eucaryotic actin interacting proteins have been identified. One of these is zyxin, a human protein that shares important structural and possibly functional properties with ActA, an actin dependent force generating protein of Listeria. We intend to test the function of these and other actin interacting proteins in a simplified system that should facilitate precise measurement of their properties of force generation in vitro.

Actins↗

PubMed1997

Epithelial cell growth and differentiation. IV. Controlled spatiotemporal expression of transgenes: new tools to study normal and pathological states.

The gut epithelium represents a dynamic, well-organized developmental system for examining self-renewal, differentiation, repair, and tumorigenesis. The apical pole of the enterocytes, the brush border, is composed of an array of well-organized actin microfilaments that support the plasma membrane. Villin, one actin-binding protein that contributes to the assembly and dynamics of the microvillus bundle, exhibits special features such as restricted tissue specificity and early expression in the immature crypt cells. The regulatory elements of the villin gene are suitable to control the expression of transgenes in intestinal cells. Engineering genetically modified animals by classic transgenesis using the villin promoter or by gene targeting in the villin locus will allow the establishment of animal models that may recapitulate human intestinal disorders.

Animals↗

PubMed1996

The junction-associated protein, zonula occludens-1, localizes to the nucleus before the maturation and during the remodeling of cell-cell contacts.

The junction-associated protein zonula occludens-1 (ZO-1) is a member of a family of membrane-associated guanylate kinase homologues thought to be important in signal transduction at sites of cell-cell contact. We present evidence that under certain conditions of cell growth, ZO-1 can be detected in the nucleus. Two different antibodies against distinct portions of the ZO-1 polypeptide reveal nuclear staining in subconfluent, but not confluent, cell cultures. An exogenously expressed, epitope-tagged ZO-1 can also be detected in the nuclei of transfected cells. Nuclear accumulation can be stimulated at sites of wounding in cultured epithelial cells, and immunoperoxidase detection of ZO-1 in tissue sections of intestinal epithelial cells reveals nuclear labeling only along the outer tip of the villus. These results suggest that the nuclear localization of ZO-1 is inversely related to the extent and/or maturity of cell contact. Since cell-cell contacts are specialized sites for signaling pathways implicated in growth and differentiation, we suggest that the nuclear accumulation of ZO-1 may be relevant for its suggested role in membrane-associated guanylate kinase homologue signal transduction.

Animals↗

PubMed1996

Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix.

The differentiation of small intestinal epithelial cells may require stimulation by microenvironmental factors in vivo. In this study, the effects of mesenchymal and luminal elements in nonmalignant epithelia] cells isolated from the human fetus were studied in vitro. Enterocytes from the human fetus were cultured and microenvironmental factors were added in stages, each stage more closely approximating the microenvironment in vivo. Four stages were examined: epithelial cells derived on plastic from intestinal culture and grown as a cell clone, the same cells grown on connective tissue support, primary epithelial explants grown on fibroblasts with a laminin base, and primary epithelial explants grown on fibroblasts and laminin with n-butyrate added to the incubation medium. The epithelial cell clone dedifferentiated when grown on plastic; however, the cells expressed cytokeratins and villin as evidence of their epithelial cell origin. Human connective tissue matrix from Engelbreth-Holm-Swarm sarcoma cells (Matrigel) modulated their phenotype: alkaline phosphatase activity increased, microvilli developed on their apical surface, and the profile of insulin-like growth factor binding proteins resembled that secreted by differentiated enterocytes. Epithelial cells taken directly from the human fetus as primary cultures and grown as explants on fibroblasts and laminin expressed greater specific enzyme activities in brush border membrane fractions than the cell clone. These activities were enhanced by the luminal molecule sodium butyrate. Thus the sequential addition of connective tissue and luminal molecules to nonmalignant epithelia] cells in vitro induces a spectrum of changes in the epithelial cell phenotype toward full differentiation.

Alkaline Phosphatase↗