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D Louvard

Publications and source records attributed to D Louvard.

At least 55 records · Page 3Linked to original sources
PubMed1996

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Adenosine Triphosphate↗
PubMed1996

Critical centrifugal forces induce adhesion rupture or structural reorganization in cultured cells.

Cultured epithelial cells were exposed to accelerations ranging from 9,000 to 70,000g for time periods of 5, 15, or 60 min, by centrifugation in a direction tangential to their plastic substrate. Three regimes describe the cellular response: (1) Cell morphology and density remain unaltered at forces below a threshold of about 10(-7) N; (2) Between this critical force and a second threshold of about 1.5 10(-7)N, the number of adherent cells decreases exponentially with time and acceleration, with no alteration of cell morphology. This behavior can be modeled by a constant probability of detaching and by an exponential distribution of cell-to-substrate adhesive forces; (3) Past the second threshold, cells that are still adherent exhibit elongated morphologies, the degree of elongation increasing linearly with the force. The fact that cells lose their vinculin-rich focal contacts past the first threshold and that cells cultured on gelatin-coated plastic show an increased resistance to detachment suggests a rupture of cell-to-substrate adhesions upon centrifugation. Immunofluorescent labeling of cells for actin and tubulin shows a reorganization of the cytoskeleton upon centrifugation, and treatment of cells with the drugs cytochalasin D and nocodazole demonstrates that cytoskeletal elements are actively involved in the structural deformation of cells past the second acceleration threshold, microtubules and microfilaments paying antagonistic roles.

Breast↗
PubMed1996

Actin filaments facilitate two steps of endocytosis.

Recent reports have suggested a role for microtubules in the endocytic process and pointed out the role for actin filaments for the early steps of apical endocytosis in polarized epithelial cells. However, these studies do not address the respective contribution of these two types of filaments along the endocytic pathway. In addition, several studies failed to demonstrate the role for actin filaments in clathrin dependent endocytosis in non polarized cells. In the present study we have investigated the role for both filaments in the endocytosis of two ligands internalized via clathrin coated pits (transferrin and alpha 2-macroglobulin) in a mouse hepatoma cell line. By immunocytochemical analysis with confocal microscopy, and biochemical analysis using a temperature sensitive step at 18 degrees C, we have shown that actin filaments are involved in two steps of the degradative pathway, and that microtubules are required at a stage in between. Actin filaments increase first, the uptake of ligands and second, their delivery to the degradative compartment, whereas microtubules are required to maintain the distribution of the late endocytic compartment in its juxtanuclear position and facilitate the delivery of the ligands to the degradative compartment upstream of the actin filament requirement. Furthermore actin filaments facilitate the recycling of transferrin from the perinuclear region to the plasma membrane. Our data indicate for the first time the sequential involvement of actin filaments and microtubules along one intracellular membrane trafficking pathway.

Actin Cytoskeleton↗
PubMed1996

[Polarity of epithelial cells. Role of the actin microfilament system].

Polarized epithelial cells display a cell surface organization well adapted to their specialized functions in transports and exchanges with the external "milieu". The numerous microvilli that cover the apical plasma membrane arc supported by bundles of actin microfilaments and associated actin binding proteins. Three independent experimental approaches have been used to unravel the function(s) of villin: a) overexpression of the villin gene; b)inhibition of villin production with a villin antisense RNA; c) disruption of the villin gene by homologous recombination. The role of ezrine, a protein able to interact both with components of the plasma membrane and with actin microfilaments, in cellular morphogenesis and the signal transduction cascade elicited by the scatter factor (HGF) and its receptor, has also been investigated with molecular cell biology approaches.

Actin Cytoskeleton↗
PubMed1995

Targeting of Listeria monocytogenes ActA protein to the plasma membrane as a tool to dissect both actin-based cell morphogenesis and ActA function.

Actin assembly on the surface of Listeria monocytogenes in the cytoplasm of infected cells provides a model to study actin-based motility and changes in cell shape. We have shown previously that the ActA protein, exposed on the bacterial surface, is required for polarized nucleation of actin filaments. To investigate whether plasma membrane-associated ActA can control the organization of microfilaments and cell shape, variants of ActA, in which the bacterial membrane signal had been replaced by a plasma membrane anchor sequence, were produced in mammalian cells. While both cytoplasmic and membrane-bound forms of ActA increased the F-actin content, only membrane-associated ActA caused the formation of plasma membrane extensions. This finding suggests that ActA acts as an actin filament nucleator and shows that permanent association with the inner face of the plasma membrane is required for changes in cell shape. Based on the observation that the amino-terminal segment of ActA and the remaining portion which includes the proline-rich repeats cause distinct phenotypic modifications in transfected cells, we propose a model in which two functional domains of ActA cooperate in the nucleation and dynamic turnover of actin filaments. The present approach is a new model system to dissect the mechanism of action of ActA and to further investigate interactions of the plasma membrane and the actin cytoskeleton during dynamic changes of cell shape.

Actins↗
PubMed1995

Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells.

We have used an antisense RNA strategy to investigate the role of the actin-associated protein, villin, in the brush-border morphogenesis of human intestinal CaCO2 cells. Stable expression of a cDNA encoding antisense villin RNA resulted in the permanent down-regulation of the endogenous villin message and dramatically affected brush-border assembly. Ultrastructural and immunolocalization studies revealed that epithelial cell polarity was largely maintained. However, in contrast to brush-border markers such as dipeptidyl-peptidase IV, the apical localization of sucrase-isomaltase was specifically impaired. Retransfection of the villin antisense-expressing cell line with a cDNA encoding a partial sense villin RNA restored both brush-border assembly and sucrase-isomaltase apical expression. The suggestion that brush-border morphogenesis may be important for the trafficking of certain proteins is discussed.

Actins↗
PubMed1995

Conformational behaviour of a synthetic peptide of the C-terminus of villin that interacts with actin: an NMR, CD and stimulated annealing study.

The solution structure of a synthetic 22-amino acid peptide (P1) corresponding to the extreme C-terminal end and one of the F-actin binding sites of villin has been determined by 1H NMR and CD spectroscopy. The structure of this peptide was compared to that of a peptide in which lysine to glutamic acid substitutions were introduced at positions 17 and 19 (P11), abolishing F-actin binding. Both peptides are largely unstructured in aqueous solution. Changes observed in the NMR and CD spectra of both peptides are consistent with alpha-helix formation in trifluoroethanol/water mixtures. A set of 189 interproton distances derived from nuclear Overhauser enhancement (NOE) measurements, 17 phi-angle constraints obtained from 3JNH alpha coupling constants, as well as about 10 N ... O distance restraints deduced from amide proton exchange kinetics with deuterium, were used for the structure determination. The three-dimensional structure of P1 and P11 is characterized by two helical regions, one extending from residues 2 to 5 and a second covering residues 7 to 17. The central fragment, ranging from Leu-7 to Leu-15, is more stable. The C-terminal residues are less structured, particularly within peptide P11. The significance of these structural results is discussed in relation to the biological activity of villin.

Actins↗
PubMed1994

CNRS defended.

Explore the source record for details and available documents.

France↗
PubMed1994

Membrane-actin microfilament connections: an increasing diversity of players related to band 4.1.

The cortical actin cytoskeleton participates in various membrane-based processes which necessitate a large amount of plasticity in the molecular components involved in these interactions. A family of proteins homologous to band 4.1 is involved in the reorganization of the actin cytoskeleton in response to various stimuli, and probably plays a role in transmembrane signalling. This family includes tyrosine phosphatases, substrates of tyrosine kinases and a candidate for a tumor-suppressor gene.

Actin Cytoskeleton↗
PubMed1994

Comparative analysis of neutral endopeptidase (NEP) and villin gene expression during mouse embryogenesis and enterocyte maturation.

Neutral endopeptidase (Endopeptidase 24.11; NEP; neprilysin), an integral membrane protein, and villin, a major microvillar cytoskeletal actin-binding protein, are both typically associated with brush border epithelia. In this study, cRNA probes were hybridized in situ to investigate the expression of NEP and villin genes in embryo and adult mouse enterocytes. During development, villin mRNAs were easily detected in the immature digestive tract well before establishment of the brush border. In 17-day-old embryos, a transient elevation of villin mRNA occurred just prior to a dramatic increase in microvilli length and density. NEP only appeared by day 17 as the embryonic gut began to become functional. It therefore appears that the onset of transcription of specialized cytoskeletal proteins from the brush border preceded that of intrinsic membrane-bound enzyme from microvilli. In the adult intestinal fold, both mRNAs were expressed along the whole length of the villus with maximal expression at its base. In contrast, both proteins were uniformly expressed along the whole crypt-villus axis. Quantitative analysis revealed an asymmetric intracellular distribution of both mRNAs that were differentially polarized in the apical cytoplasm of enterocytes.

Age Factors↗
PubMed1994

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

Amino Acid Sequence↗
PubMed1994

Functional differences between L- and T-plastin isoforms.

Fimbrins/plastins are a family of highly conserved actin-bundling proteins. They are present in all eukaryotic cells including yeast, but each isoform displays a remarkable tissue specificity. T-plastin is normally found in epithelial and mesenchymal cells while L-plastin is present in hematopoietic cells. However, L-plastin has been also found in tumor cells of non-hematopoietic origin (Lin, C.-S., R. H. Aebersold, S. B. Kent, M. Varma, and J. Leavitt. 1988. Mol. Cell. Biol. 8:4659-4668; Lin, C.-S., R. H. Aebersold, and J. Leavitt. 1990. Mol. Cell. Biol. 10: 1818-1821). To learn more about the biological significance of their tissue specificity, we have overproduced the T- and L-plastin isoforms in a fibroblast-like cell line, CV-1, and in a polarized epithelial cell line, LLC-PK1. In CV-1 cells, overproduction of T- and L-plastins induces cell rounding and a concomitant reorganization of actin stress fibers into geodesic structures. L-plastin remains associated with microfilaments while T-plastin is almost completely extracted after treatment of the cells with non-ionic detergent. In LLC-PK1 cells, T-plastin induces shape changes in microvilli and remains associated with microvillar actin filaments after detergent extraction while L-plastin has no effect on these structures and is completely extracted. The effect of T-plastin on the organization of microvilli differs from that of villin, another actin-bundling protein. Our experiments indicate that these two isoforms play differing roles in actin filament organization, and do so in a cell type-specific fashion. Thus it is likely that these plastin isoforms play fundamentally different roles in cell function.

Actin Cytoskeleton↗
PubMed1993

Regulatory sequences on the human villin gene trigger the expression of a reporter gene in a differentiating HT29 intestinal cell line.

To develop a molecular tool for tissue-specific targeting of gene expression in immature and differentiated epithelial cells of the small and large intestinal mucosa, we have isolated the 2-kb 5'-flanking region of the human villin gene. This region contains numerous short sequences that are conserved among other tissue-specific promoters of genes expressed in differentiated enterocytes. This DNA fragment promotes the transcription and expression of the luciferase reporter gene in villin-positive intestinal, renal, and hepatoma cell lines but not in a villin-negative keratinocyte cell line. The pattern of expression corresponds that of the endogenous gene, indicating that this sequence can direct intestine-specific transcription. In the differentiating HT29 intestinal cell line, expression of the reporter gene is already detectable in undifferentiated cells, and dramatically increases when terminal differentiation is induced. Thus, as previously reported for the endogenous gene the isolated 5'-flanking region of the villin gene responds positively to conditions known to stimulate terminal differentiation of these cultured epithelial intestinal cells. The reported results indicate that this genomic fragment contains sufficient regulatory elements to recapitulate the expression pattern of the villin promoter during intestinal differentiation.

Adenocarcinoma↗
PubMed1993

Water handling in Caco-2 cells: effects of acidification of the medium.

Caco-2 cells were cultured on permeable supports. At confluence the minute-by-minute net water movement (Jw) was automatically recorded. Simultaneously, unidirectional [14C]mannitol, 22Na+, and/or 36Cl- fluxes and transepithelial resistances were measured. The water and mannitol permeabilities went progressively down between 9 and 16 days after seeding and then stabilized. In this last condition the hydrostatic permeability coefficient (Phydr) was 2.67 +/- 0.31 cm s-1 while the osmotic permeability coefficient (Posm) was 0.0017 +/- 0.0004 cm s-1. Phydr but not Posm was dependent on the temperature and on the presence of Na+ in the medium. A net secretory Jw was observed 16 days after seeding, in the absence of any osmotic, hydrostatic or chemical gradient. This secretory Jw was associated with net Cl- (1.43 +/- 0.43 muequiv h-1 cm-2) and Na+ (1.05 +/- 0.35 muequiv h-1 cm-2) secretions. Amiloride reduced, in open-circuit conditions, both Na+ and Cl- apical to basal fluxes, thus enhancing the net Na+ and Cl- exit. Acidification of the medium (pH 6.2) reversibly increased water and mannitol permeabilities in 10-day-old cultures. In 16-day-old cultures the same shift in medium pH did not change mannitol permeability, while stimulating water secretion. These results, obtained in the absence of supracellular structures (villae, crypts) and subepithelial components (muscular, vascular and conjunctive tissues) indicate that paracellular and transport-associated water pathways are sensitive to changes in the pH of the medium in Caco-2 cell layers.

Adenocarcinoma↗
PubMed1993

Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker.

Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlström, and A. Vaheri. 1989. J. Biol. Chem. 264:16727-16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy-terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin-containing structures and is partially detergent insoluble. The amino-terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.

Amino Acid Sequence↗
PubMed1993

Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D.

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the 'random' nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins.

Actins↗
PubMed1992

Do unconventional myosins exert functions in dynamics of membrane compartments?

Unconventional myosins have now been identified in amoeba as well as in higher eucaryotic cells. Their cellular localization, their ability to bind membrane vesicles and their ability to produce in vitro movement suggest that they can generate forces on the plasma membrane relative to actin filaments as well as on membrane compartments relative to actin. Genetic approaches and biochemical analysis of cells over-producing nonfunctional domains of unconventional myosins have provided direct evidence for a role of unconventional myosins in movement of intracellular vesicles and have allowed us to formulate hypotheses about the possible mechanisms by which unconventional myosins could participate in the intracellular transport of membrane proteins and secretory proteins.

Animals↗