The actin bundle within each microvillus of the intestinal brush border is laterally tethered to ... more The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, ll0-kD-calmodulin. Previous studies have shown that avian ll0-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the ll0-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells. The primary structure of the ll0-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxyterminal "tail" domain. The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC;
Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filament... more Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation.
Brush borders which are localized at the apical face of enterocytes, are composed of thousands of... more Brush borders which are localized at the apical face of enterocytes, are composed of thousands of stiff microvilli containing bundles of microfilaments made of actin. Their assembly occurs during terminal differentiation of the enterocytes when these cells migrate along the villus of the intestinal mucosa. The cell line HT 29 derived from a human colonic adenocareinoma whose differentiation can be induced, can also be used as a model to study in culture the assembly of the intestinal brush border. Villin is one of the actin binding proteins found in microvilli which compose brush borders. Villin is expressed in the adult and in the embryo before the appearance of the brush border. Villin can be used as a tissue-specific marker for normal diffentiated and undifferentiated cells derived from gastrointestinal tractus in the adult as well as in the embryo. Since villin is a good marker for intestinal cells and plays a structural role in the assembly of the brush border we have analysed its expression and its localization in HT 29 cells. In HT 29 cells, as in the tissue, villin is synthesized at low levels before the appearance of the brush border. The high rate of synthesis and the recruitement of villin at the apical pole of the cells can be correlated with the existence of a well developed brush border.
Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleu... more Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988)
HT29-18N2 (N2) cells, a subclone of the HT29 human colon carcinoma cell line, are shown in this r... more 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.
Villin is an actin-binding protein of the intestinal brush border that bundles, nucleates, caps, ... more 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.
Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endopla... more Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue . There were many unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles . These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane . The anti-RER antibodies labeled this structure alone at the light and electron microscopic levels . They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000 .
Biophotonics New Frontier: From Genome to Proteome, 2004
Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin-... more Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin-membrane linker undergoes a series of conformational changes that modulate its interactions with various partners and its localization in membrane or cytosolic pools. Its mobility and exchange rates within and between these two pools were assessed by two-photon fluorescence recovery after photobleaching in epithelial cell microvilli. Analysis of ezrin mutants with an altered actin-binding site revealed three ezrin membrane states of different mobilities and exchange properties, reflecting sequential association with membrane components and F-actin in the context of a fast overall turnover. E zrin is a member of the ezrin͞radixin͞moesin (ERM) family of actin-membrane linkers that play a key role in coupling signal transduction pathways with the maintenance of the cortical cytoskeleton architecture and its dynamic response to external stimuli (1, 2). ERM linkers concentrate in cell-surface structures rich in actin such as microvilli and filopodia, and their impaired expression or inactivation severely alters cell surface morphology, motility, and adhesion (1).
American Journal of Physiology - Gastrointestinal and Liver Physiology, 2002
Villin plays a key role in the maintenance of the brush border organization by bundling F-actin i... more Villin plays a key role in the maintenance of the brush border organization by bundling F-actin into a network of parallel filaments. Our previous in vivo data on villin knockout mice showed that, although this protein is not necessary for the bundling of F-actin, it is important for the reorganization of the actin cytoskeleton elicited by stress conditions. We further investigated villin property to initiate actin remodeling in cellular processes such as hepatocyte growth factor-induced motility, morphogenesis, and bacterial infection. Our data suggest that villin is involved in actin remodeling necessary for many cellular processes requiring the actin cytoskeleton plasticity.
The actin bundle within each microvillus of the intestinal brush border is laterally tethered to ... more The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, ll0-kD-calmodulin. Previous studies have shown that avian ll0-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the ll0-kD protein has been sequenced, providing direct evidence that this protein is a vertebrate homologue of the tail-less, single-headed myosin I first described in amoeboid cells. The primary structure of the ll0-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxyterminal "tail" domain. The head domain is homologous to the S1 domain of other known myosins, with highest homology observed between that of Acanthamoeba myosin IB and the S1 domain of the protein encoded by bovine myosin I heavy chain gene (MIHC;
Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filament... more Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation.
Brush borders which are localized at the apical face of enterocytes, are composed of thousands of... more Brush borders which are localized at the apical face of enterocytes, are composed of thousands of stiff microvilli containing bundles of microfilaments made of actin. Their assembly occurs during terminal differentiation of the enterocytes when these cells migrate along the villus of the intestinal mucosa. The cell line HT 29 derived from a human colonic adenocareinoma whose differentiation can be induced, can also be used as a model to study in culture the assembly of the intestinal brush border. Villin is one of the actin binding proteins found in microvilli which compose brush borders. Villin is expressed in the adult and in the embryo before the appearance of the brush border. Villin can be used as a tissue-specific marker for normal diffentiated and undifferentiated cells derived from gastrointestinal tractus in the adult as well as in the embryo. Since villin is a good marker for intestinal cells and plays a structural role in the assembly of the brush border we have analysed its expression and its localization in HT 29 cells. In HT 29 cells, as in the tissue, villin is synthesized at low levels before the appearance of the brush border. The high rate of synthesis and the recruitement of villin at the apical pole of the cells can be correlated with the existence of a well developed brush border.
Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleu... more Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988)
HT29-18N2 (N2) cells, a subclone of the HT29 human colon carcinoma cell line, are shown in this r... more 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.
Villin is an actin-binding protein of the intestinal brush border that bundles, nucleates, caps, ... more 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.
Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endopla... more Rabbits were immunized with membrane fractions from either the Golgi complex or the rough endoplasmic reticulum (RER) by injection into the popliteal lymph nodes. The antisera were then tested by indirect immunofluorescence on tissue culture cells or frozen, thin sections of tissue . There were many unwanted antibodies to cell components other than the RER or the Golgi complex, and these were removed by suitable absorption steps. These steps were carried out until the pattern of fluorescent labeling was that expected for the Golgi complex or RER. Electron microscopic studies, using immunoperoxidase labeling of normal rat kidney (NRK) cells, showed that the anti-Golgi antibodies labeled the stacks of flattened cisternae that comprise the central feature of the Golgi complex, many of the smooth vesicles around the stacks, and a few coated vesicles . These antibodies were directed, almost entirely, against a single polypeptide with an apparent molecular weight of 135,000. The endoplasmic reticulum (ER) in NRK cells is an extensive, reticular network that pervades the entire cell cytoplasm and includes the nuclear membrane . The anti-RER antibodies labeled this structure alone at the light and electron microscopic levels . They were largely directed against four polypeptides with apparent molecular weights of 29,000, 58,000, 66,000, and 91,000 .
Biophotonics New Frontier: From Genome to Proteome, 2004
Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin-... more Ezrin plays a key role in coupling signal transduction to cortical cell organization. This actin-membrane linker undergoes a series of conformational changes that modulate its interactions with various partners and its localization in membrane or cytosolic pools. Its mobility and exchange rates within and between these two pools were assessed by two-photon fluorescence recovery after photobleaching in epithelial cell microvilli. Analysis of ezrin mutants with an altered actin-binding site revealed three ezrin membrane states of different mobilities and exchange properties, reflecting sequential association with membrane components and F-actin in the context of a fast overall turnover. E zrin is a member of the ezrin͞radixin͞moesin (ERM) family of actin-membrane linkers that play a key role in coupling signal transduction pathways with the maintenance of the cortical cytoskeleton architecture and its dynamic response to external stimuli (1, 2). ERM linkers concentrate in cell-surface structures rich in actin such as microvilli and filopodia, and their impaired expression or inactivation severely alters cell surface morphology, motility, and adhesion (1).
American Journal of Physiology - Gastrointestinal and Liver Physiology, 2002
Villin plays a key role in the maintenance of the brush border organization by bundling F-actin i... more Villin plays a key role in the maintenance of the brush border organization by bundling F-actin into a network of parallel filaments. Our previous in vivo data on villin knockout mice showed that, although this protein is not necessary for the bundling of F-actin, it is important for the reorganization of the actin cytoskeleton elicited by stress conditions. We further investigated villin property to initiate actin remodeling in cellular processes such as hepatocyte growth factor-induced motility, morphogenesis, and bacterial infection. Our data suggest that villin is involved in actin remodeling necessary for many cellular processes requiring the actin cytoskeleton plasticity.
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Papers by Daniel Louvard