Tetraspanins in intercellular adhesion of polarized epithelial cells: spatial and functional relationship to integrins and cadherins

2001 ◽  
Vol 114 (3) ◽  
pp. 577-587 ◽  
Author(s):  
M. Yanez-Mo ◽  
R. Tejedor ◽  
P. Rousselle ◽  
F. Sanchez-Madrid
Keyword(s):  
Cell Adhesion ◽  
Functional Relationship ◽  
Chinese Hamster Ovary Cells ◽  
Basolateral Membrane ◽  
Chinese Hamster ◽  
Cell Contact ◽  
Similar Distribution ◽  
Spatial Cues ◽  
Independent Manner ◽  
Cell Cell

The subcellular distribution of tetraspanin molecules and their functional relationship with integrins in cell-cell adhesion was studied in detail in different polarized epithelial cell models. CD9, CD81 and CD151 tetraspanins were localized at lateral cell-cell contact sites in a similar distribution to E-cadherin. Interestingly, CD9 was partially localized at the apical microvillae of Madin-Darby canine kidney cells forming multimolecular complexes distinct from those found on the basolateral membrane, suggesting the coexistence of differential tetraspanin webs with different subcellular localization. We found that tetraspanin-associated beta1 integrins at cell-to-cell contacts were in a low-affinity conformational state, and that their localization at intercellular contacts was independent of cadherin expression and adhesion. Furthermore, integrin-tetraspanin complexes were functionally relevant in cell-cell adhesion in a cadherin-independent manner, without requiring a conformational change of the integrin moiety. Nevertheless, the integrin alpha3beta1 was ligand-binding competent and this binding did not disrupt association to tetraspanins. Moreover, Chinese hamster ovary cells treated with anti-tetraspanin mAbs or activatory anti-beta1 integrin mAbs were able to develop tubule-like structures. Together, these data support tetraspanin association as a new regulatory mechanism of integrin function and suggest a role for tetraspanins-integrin complexes in providing the cell with the spatial cues necessary for their proper polarization.

scholarly journals Actin-dependent regulation of the cardiac Na+/Ca2+ exchanger

2006 ◽  
Vol 290 (3) ◽  
pp. C691-C701 ◽  
Author(s):  
Madalina Condrescu ◽  
John P. Reeves
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytochalasin D ◽  
Lag Phase ◽  
Cell Contact ◽  
Wild Type ◽  
Filamentous Actin ◽  
Surface Distribution ◽  
Reverse Mode ◽  
Hydrophilic Domain

In the present study, the bovine cardiac Na+/Ca2+ exchanger (NCX1.1) was expressed in Chinese hamster ovary cells. The surface distribution of the exchanger protein, externally tagged with the hemagglutinin (HA) epitope, was associated with underlying actin filaments in regions of cell-to-cell contact and also along stress fibers. After we treated cells with cytochalasin D, NCX1.1 protein colocalized with patches of fragmented filamentous actin (F-actin). In contrast, an HA-tagged deletion mutant of NCX1.1 that was missing much of the exchanger's central hydrophilic domain Δ(241–680) did not associate with F-actin. In cells expressing the wild-type exchanger, cytochalasin D inhibited allosteric Ca2+ activation of NCX activity as shown by prolongation of the lag phase of low Ca2+ uptake after initiation of the reverse (i.e., Ca2+ influx) mode of NCX activity. Other agents that perturbed F-actin structure (methyl-β-cyclodextrin, latrunculin B, and jasplakinolide) also increased the duration of the lag phase. In contrast, when reverse-mode activity was initiated after allosteric Ca2+ activation, both cytochalasin D and methyl-β-cyclodextrin (Me-β-CD) stimulated NCX activity by ∼70%. The activity of the Δ(241–680) mutant, which does not require allosteric Ca2+ activation, was also stimulated by cytochalasin D and Me-β-CD. The increased activity after these treatments appeared to reflect an increased amount of exchanger protein at the cell surface. We conclude that wild-type NCX1.1 associates with the F-actin cytoskeleton, probably through interactions involving the exchanger's central hydrophilic domain, and that this association interferes with allosteric Ca2+ activation.

scholarly journals Changes in E-cadherin rigidity sensing regulate cell adhesion

2017 ◽  
Vol 114 (29) ◽  
pp. E5835-E5844 ◽  
Author(s):  
Caitlin Collins ◽  
Aleksandra K. Denisin ◽  
Beth L. Pruitt ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Signaling Molecules ◽  
Membrane Dynamics ◽  
Cell Contact ◽  
Adhesion Assay ◽  
Cell Periphery ◽  
Actin Organization ◽  
Rigidity Sensing ◽  
E Cadherin ◽  
Cell Cell

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion.

scholarly journals A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity

2007 ◽  
Vol 178 (2) ◽  
pp. 323-335 ◽  
Author(s):  
Lene N. Nejsum ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Basolateral Membrane ◽  
Surface Polarity ◽  
Cell Contacts ◽  
Protein Receptors ◽  
Epithelial Cell Surface ◽  
E Cadherin ◽  
Cell Cell

Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin–mediated epithelial cell–cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell–cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide–sensitive factor attachment protein receptors) did not inhibit cell–cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell–cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell–cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell–cell contacts and immediately initiate cell surface polarity.

Antisense RNA inactivation of gene expression of a cell-cell adhesion protein (gp64) in the cellular slime mold Polysphondylium pallidum

1996 ◽  
Vol 109 (5) ◽  
pp. 1009-1016
Author(s):  
S. Funamoto ◽  
H. Ochiai
Keyword(s):  
Gene Expression ◽  
Cell Adhesion ◽  
Antisense Rna ◽  
Resistant Cell ◽  
Cell Contact ◽  
Cellular Slime Mold ◽  
Adhesion Protein ◽  
Cell Adhesion Protein ◽  
Polysphondylium Pallidum ◽  
Cell Cell

The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cell-cell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into P. pallidum cells and the transformants were characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to re-form in aggregates smaller in size than did the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hour starved L3mc2 cells remained considerably dissociated from each other after 5 minutes gyrating, although aggregation gradually increased by 50% during a further 55 minutes gyrating in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cell-cell adhesion protein, gp64. We therefore conclude that the gp64 protein is involved in forming the EDTA-resistant cell-cell contact. In spite of the absence of gp64, L3mc2 exhibited normal developmental processes, a fact which demonstrates that another cell-cell adhesion system exists in the development of Polysphondylium. This is the first report in which an antisense RNA technique was successfully applied to Polysphondylium.

E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton

1998 ◽  
Vol 111 (8) ◽  
pp. 1071-1080 ◽  
Author(s):  
S.M. Reuver ◽  
C.C. Garner
Keyword(s):  
Cell Adhesion ◽  
Model Systems ◽  
Molecular Organization ◽  
Cell Contact ◽  
Adhesion Sites ◽  
Associated Proteins ◽  
Adhesion Complex ◽  
Cortical Cytoskeleton ◽  
E Cadherin ◽  
Cell Cell

Members of the SAP family of synapse-associated proteins have recently emerged as central players in the molecular organization of synapses. In this study, we have examined the mechanism that localizes one member, SAP97, to sites of cell-cell contact. Utilizing epithelial CACO-2 cells and fibroblast L-cells as model systems, we demonstrate that SAP97 is associated with the submembranous cortical cytoskeleton at cell-cell adhesion sites. Furthermore, we show that its localization into this structure is triggered by E-cadherin. Although SAP97 can be found in an E-cadherin/catenin adhesion complex, this interaction seems to be mediated by the attachment of SAP97 to the cortical cytoskeleton. Our results are consistent with a model in which SAP97 is recruited to sites of cell-cell contact via an E-cadherin induced assembly of the cortical cytoskeleton.

Analysis of Cell‐Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules

2001 ◽  
Vol 11 (1) ◽  
Author(s):  
Peter Sonderegger ◽  
Stefan Kunz ◽  
Christoph Rader ◽  
Daniel M. Suter ◽  
Esther T. Stoeckli
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecules ◽  
Cell Adhesion Molecules ◽  
Cell Contact ◽  
Ig Superfamily ◽  
Cell Cell

scholarly journals Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion

2009 ◽  
Vol 135 (1) ◽  
pp. 43-58 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Owen M. Woodward ◽  
Damian B. van Rossum ◽  
Nicholas C. Zachos ◽  
Linxi Chen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Chinese Hamster Ovary Cells ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Chinese Hamster ◽  
T84 Cells ◽  
Cl Secretion ◽  
Intestinal Chloride Secretion ◽  
Kinase A

Intestinal Cl− secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl− secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl− secretion. FSK-stimulated Cl− secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl− secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl− secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl− conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl−>Br−>I− permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl− secretion, which is carried by a novel, previously undescribed Cl− channel.

scholarly journals Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis.

1997 ◽  
Vol 8 (7) ◽  
pp. 1329-1341 ◽  
Author(s):  
N Sheibani ◽  
P J Newman ◽  
W A Frazier
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Cell Contact ◽  
Platelet Endothelial Cell Adhesion ◽  
Cell Adhesion Molecule 1 ◽  
Endothelial Cell Adhesion ◽  
Cell Cell

Expression of thrombospondin-1 (TS1) in polyoma middle-sized T (tumor)-transformed mouse brain endothelial cells (bEND.3) restores a normal phenotype and suppresses their ability to form hemangiomas in mice. We show that TS1 expression results in complete suppression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression and altered cell-cell interactions in bEND.3 cells. To further investigate the role of PECAM-1 in regulation of endothelial cell-cell interactions and morphogenesis, we expressed human (full length) or murine (delta 15) PECAM-1 isoforms in TS1-transfected bEND.3 (bEND/TS) cells. Expression of either human or murine PECAM-1 resulted in an enhanced ability to organize and form networks of cords on Matrigel, an effect that was specifically blocked by antibodies to PECAM-1. Anti-PECAM-1 antibodies also inhibited tube formation in Matrigel by normal human umbilical vein endothelial cells. However, PECAM-1-transfected bEND/TS cells did not regain the ability to form hemangiomas in mice and the expressed PECAM-1, unlike the endogenous PECAM-1 expressed in bEND.3 cells, failed to localize to sites of cell-cell contact. This may be, in part, attributed to the different isoforms of PECAM-1 expressed in bEND.3 cells. Using reverse transcription-polymerase chain reaction, we determined that bEND.3 cells express mRNA encoding six different PECAM-1 isoforms, the isoform lacking both exons 14 and 15 (delta 14&15) being most abundant. Expression of the murine delta 14&15 PECAM-1 isoform in bEND/TS cells resulted in a similar phenotype to that described for the full-length human or murine delta 15 PECAM-1 isoform. The delta 14&15 isoform, despite the lack of exon 14, failed to localize to sites of cell-cell contact even in clones that expressed it at very high levels. Thus, contrary to recent reports, lack of exon 14 is not sufficient to result in junctional localization of PECAM-1 isoforms in bEND/TS cells.

scholarly journals N-Cadherin Association with Lipid Rafts Regulates Its Dynamic Assembly at Cell-Cell Junctions in C2C12 Myoblasts

2005 ◽  
Vol 16 (5) ◽  
pp. 2168-2180 ◽  
Author(s):  
Marie Causeret ◽  
Nicolas Taulet ◽  
Franck Comunale ◽  
Cyril Favard ◽  
Cécile Gauthier-Rouvière
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Lipid Rafts ◽  
Membrane Lipid ◽  
Cell Junctions ◽  
Cell Contact ◽  
C2c12 Myoblasts ◽  
Cell Contacts ◽  
Dynamic Assembly ◽  
Cell Cell

Cadherins are homophilic cell-cell adhesion molecules implicated in cell growth, differentiation, and organization into tissues during embryonic development. They accumulate at cell-cell contact sites and act as adhesion-activated signaling receptors. Here, we show that the dynamic assembly of N-cadherin at cell-cell contacts involves lipid rafts. In C2C12 myoblasts, immunofluorescence and biochemical experiments demonstrate that N-cadherin present at cell-cell contacts is colocalized with lipid rafts. Disruption of lipid rafts leads to the inhibition of cell-cell adhesion and disorganization of N-cadherin–dependent cell-cell contacts without modifying the association of N-cadherin with catenins and its availability at the plasma membrane. Fluorescent recovery after photobleaching experiments demonstrate that at the dorsal plasma membrane, lipid rafts are not directly involved in the diffusional mobility of N-cadherin. In contrast, at cell-cell junctions N-cadherin association with lipid rafts allows its stabilization enabling the formation of a functional adhesive complex. We show that lipid rafts, as homophilic interaction and F-actin association, stabilize cadherin-dependent adhesive complexes. Homophilic interactions and F-actin association of N-cadherin are both required for its association to lipid rafts. We thus identify lipid rafts as new regulators of cadherin-mediated cell adhesion.

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