PubMed Health⌕ Search

Biomedical subjects

M Arpin

Publications and source records attributed to M Arpin.

At least 19 recordsLinked to original sources
PubMed1998

Effect of hepatocyte growth factor on assembly of zonula occludens-1 protein at the plasma membrane.

Hepatocyte growth factor (HGF) is a paracrine cytokine that influences epithelial morphogenesis by modulating cell-cell adhesion and cell polarity. We have examined the role of HGF in the tight junction (TJ) formation. We followed the assembly and disassembly at the plasma membrane of the major component of the TJ, zonula occludens-1 (ZO-1) protein, after HGF treatment. We applied HGF to the basolateral compartment of MDCK cell monolayers grown on transwell filters to analyze the effect of HGF on polarized cells. Confocal laser scanning microscopy showed that HGF caused a marked reduction of ZO-1 at the lateral sites and a concomitant increase in the cytoplasm. We used the calcium switch assay to analyze the effect of HGF in early TJ development. In MDCK cells cultured in low calcium levels, ZO-1 is distributed intracellularly. The presence of HGF greatly retarded the movement of ZO-1 from the cytosol to the membrane after restoration of normal (1.8 mM) calcium levels for 1.5 and 3 hr. The presence of HGF during the calcium switch caused increased tyrosine phosphorylation of beta-catenin. The incubation of MDCK cells with vanadate, a potent tyrosine-specific phosphatase inhibitor, also affected the ZO-1 localization at the plasma membrane during the calcium switch. This was concomitant with increased tyrosine phosphorylation of beta-catenin. These results suggest that HGF affects the TJ assembly, and this phenomenon may be important in loosening of intercellular junctions and migration of epithelial cells during HGF-induced morphogenesis.

Animals↗
PubMed1998

Impaired interaction of naturally occurring mutant NF2 protein with actin-based cytoskeleton and membrane.

Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Transiently expressed wild-type isoforms were both observed underneath the plasma membrane. At this location they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrated identical properties. Three different truncated schwannomins, that are prototypic for most naturally occurring NF2 mutations, were affected neither in their location nor in their cytochalasin D sensitivity. However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain had lost the submembraneous localization and tended to accumulate in perinuclear patches that were unaffected by cytochalasin D treatment. A similar behavior was observed when the N-terminal domain was entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane.

Actins↗
PubMed1997

Ileal microvillar protein villin is tyrosine-phosphorylated and associates with PLC-gamma1. Role of cytoskeletal rearrangement in the carbachol-induced inhibition of ileal NaCl absorption.

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-gamma1 (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem. J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na+/H+ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-gamma1. Villin is present both in the Triton X-100-soluble and -insoluble fractions of the brush border. The Triton X-100-soluble pool is approximately 4-fold smaller than the brush border pool of villin that is present in the Triton X-100-insoluble fraction. Only the villin present in the Triton X-100-soluble fraction of ileal villus brush border associates with PLC-gamma1 and is tyrosine-phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-gamma1. These studies demonstrate that carbachol effects on NaCl absorption are accompanied by an increase in brush border PLC-gamma1 association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.

Actins↗
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↗
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

Differential addressing of 5-HT1A and 5-HT1B receptors in transfected LLC-PK1 epithelial cells: a model of receptor targeting in neurons.

Based on the finding that the mechanism of membrane protein sorting may be similar in neurons and epithelial cells, we have investigated the localization of the 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors in Eli Lilly and Company canine kidney (LLC-PK1) cells (epithelial cells from pig kidney) stably transfected with the corresponding cDNAs. Receptors were characterized by radioligand binding, measurement of coupled adenylate cyclase activity and immunofluorescene with specific antibodies. Confocal microscopy and cell surface biotinylation experiments showed that the 5-hydroxytryptamine1A receptor, which is at the somatodendritic level in neurons, was targeted to the basolateral surface whereas the 5-hydroxytryptamine1B receptor, which is localized on nerve endings, predominated intracellularly. Such a difference in the localization of these two receptors suggests that the signal for the differential addressing of 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors is contained within their primary sequence and confirms that epithelial cells can be used as a suitable model to study receptor targeting in neurons.

Animals↗
PubMed1995

Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella flexneri into HeLa cells.

Shigella flexneri is an enteroinvasive bacterium which causes bacillary dysentery in humans. A major feature of its pathogenic potential is the capacity to invade epithelial cells. Shigella entry into epithelial cells is considered a parasite-induced internalization process requiring polymerization of actin. Here we describe the cytoskeletal rearrangements during S. flexneri invasion of HeLa cells. After an initial contact of the bacterium with the cell surface, distinct nucleation zones of heavy chain actin polymerization appear in close proximity to the contact site underneath the parasite with long filaments being polymerized. These structures then push cellular protrusions that rise beside the entering bacterium, being sustained by tightly bundled long actin filaments organized in parallel orientation with their positive ends pointing to the cytoplasmic membrane. Finally, the cellular projections coalesce above the bacterial body, leading to its internalization. In addition, we found the actin-bundling protein plastin to be concentrated in these protrusions. Since plastin is known to bundle actin filaments in parallel orientation, colocalization of parallel actin filaments and plastin in the cellular protrusions strongly suggested a functional role of this protein in the architecture of parasite-induced cellular projections. Using transfection experiments, we show the differential recruitment of the two plastin isoforms (T- and L-) into Shigella entry zones. By transient expression of a truncated T-plastin which is deprived of one of its actin-binding sites, we also demonstrate the functional role of T-plastin in Shigella entry into HeLa cells.

Actins↗
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

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

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↗
PubMed1992

Unipolar reorganization of F-actin layer at bacterial division and bundling of actin filaments by plastin correlate with movement of Shigella flexneri within HeLa cells.

Shigella flexneri causes bacillary dysentery, an invasive disease of colonic epithelial cells in humans. The capacity of bacteria, once they have entered into a cell and escaped the phagocytic vacuole, to spread intracellularly and directly to adjacent cells without further extracellular passage is a key factor in invasion of the epithelial layer. Movement of intracellular bacteria is dependent upon the polymerization of actin; concentration of the formed filaments to one end of the bacterium is associated with initiation of movement. This movement may lead to the formation of a protrusion at the cell surface through which the bacterium passes to an adjacent cell. Development of these protrusions in infected HeLa cells is described, with emphasis on two critical observations. First, initiation of movement is coupled with bacterial division since elongation of the bacterial body is associated with relocalization of the previously uniformly distributed layer of actin to one pole of the bacterium. Second, the actin-bundling protein plastin appears to bundle the actin filaments just posterior to the bacterium, producing an ongoing contraction of the cylindrical actin tail that may be associated with forward movement of the bacterium within the protrusion.

Actins↗
PubMed1990

From the structure to the function of villin, an actin-binding protein of the brush border.

Villin, a calcium-regulated actin-binding protein, modulates the structure and assembly of actin filaments in vitro. It is organized into three domains, the first two of which are homologous. Villin is mainly produced in epithelial cells that develop a brush border and which are responsible for nutrient uptake. Expression of the villin structural gene is precisely regulated during mouse embryogenesis and is restricted in adults, to certain epithelia of the gastrointestinal and urogenital tracts. The function of villin has been assessed by transfecting CV1 cells with a human cDNA encoding wild-type villin or mutant villin. Synthesis of large amounts of villin in cells which do not normally produce this protein induces the growth of microvilli on the cell surface and the redistribution of F-actin, concomitant with the disappearance of stress fibers. The complete villin sequence is required for the morphogenic effect. These results suggest that villin plays a key role in the morphogenesis of microvilli.

Actins↗
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↗
PubMed1989

Partial deduced sequence of the 110-kD-calmodulin complex of the avian intestinal microvillus shows that this mechanoenzyme is a member of the myosin I family.

The actin bundle within each microvillus of the intestinal brush border is laterally tethered to the membrane by bridges composed of the protein complex, 110-kD-calmodulin. Previous studies have shown that avian 110-kD-calmodulin shares many properties with myosins including mechanochemical activity. In the present study, a cDNA molecule encoding 1,000 amino acids of the 110-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 110-kD protein (or brush border myosin I heavy chain) consists of two domains, an amino-terminal "head" domain and a 35-kD carboxy-terminal "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; Hoshimaru, M., and S. Nakanishi. 1987. J. Biol. Chem. 262:14625-14632). The carboxy-terminal domain shows no significant homology with any other known myosins except that of the bovine MIHC. This demonstrates that the bovine MIHC gene most probably encodes the heavy chain of bovine brush border myosin I (BBMI). A bacterially expressed fusion protein encoded by the brush border 110-kD cDNA binds calmodulin. Proteolytic removal of the carboxy-terminal domain of the fusion protein results in loss of calmodulin binding activity, a result consistent with previous studies on the domain structure of the 110-kD protein. No hydrophobic sequence is present in the molecule indicating that chicken BBMI heavy chain is probably not an integral membrane protein. Northern blot analysis of various chicken tissue indicates that BBMI heavy chain is preferentially expressed in the intestine.

Amino Acid Sequence↗
PubMed1988

Localization of the villin gene on human chromosome 2q35-q36 and on mouse chromosome 1.

A partial cDNA clone coding for the 110 carboxyterminal amino acids of human villin was used for mapping the human villin gene. In situ hybridization experiments on human chromosomes with tritiated probe allowed the regional localization of the villin locus to chromosome 2 at q35-36. Data obtained from restriction fragment length polymorphism analysis of two mouse species demonstrated the assignment of the villin gene to mouse chromosome 1 by assessment of linkage with the fast skeletal isoform of the myosin light-chain gene. These villin gene localizations add a fourth locus to the conserved gene cluster encoding the fast skeletal muscle isoform of the myosin light chain, isocitrate dehydrogenase, and the gamma crystallins and confirm the partial homology of the human chromosome 2 long arm and mouse chromosome 1.

Animals↗