Recruitment and activation of Rac1 by the formation of E-cadherin-mediated cell-cell adhesion sites

2001 ◽  
Vol 114 (10) ◽  
pp. 1829-1838 ◽  
Author(s):  
M. Nakagawa ◽  
M. Fukata ◽  
M. Yamaga ◽  
N. Itoh ◽  
K. Kaibuchi
Keyword(s):  
Cell Adhesion ◽  
Specific Binding ◽  
Green Fluorescence Protein ◽  
Neutralizing Antibody ◽  
Small Gtpases ◽  
Cell Contact ◽  
Adhesion Sites ◽  
P21 Activated Kinase ◽  
E Cadherin ◽  
Cell Cell

Rac1, a member of the Ρ family small GTPases, regulates E-cadherin-mediated cell-cell adhesion. However, it remains to be clarified how the localization and activation of Rac1 are regulated at sites of cell-cell contact. Here, using enhanced green fluorescence protein (EGFP)-tagged Rac1, we demonstrate that EGFP-Rac1 is colocalized with E-cadherin at sites of cell-cell contact and translocates to the cytosol during disruption of E-cadherin-mediated cell-cell adhesion by Ca(2+) chelation. Re-establishment of cell-cell adhesion by restoration of Ca(2)(+) caused EGFP-Rac1 to become relocalized, together with E-cadherin, at sites of cell-cell contact. Engagement of E-cadherin to the apical membrane by anti-E-cadherin antibody (ECCD-2) recruited EGFP-Rac1. We also investigated whether E-cadherin-mediated cell-cell adhesion induced Rac1 activation by measuring the amounts of GTP-bound Rac1 based on its specific binding to the Cdc42/Rac1 interactive binding region of p21-activated kinase. The formation of E-cadherin-mediated cell-cell adhesion induced Rac1 activation. This activation was inhibited by treatment of cells with a neutralizing antibody (DECMA-1) against E-cadherin, or with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). IQGAP1, an effector of Rac1, and EGFP-Rac1 behaved in a similar manner during the formation of E-cadherin-mediated cell-cell adhesion. Rac1 activation was also confirmed by measuring the amounts of coimmunoprecipitated Rac1 with IQGAP1 during the establishment of cell-cell adhesion. Taken together, these results suggest that Rac1 is recruited at sites of E-cadherin-mediated cell-cell adhesion and then activated, possibly through PI 3-kinase. http://www/biologists.com/JCS/movies/jcs2094.html

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.

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 Recruitment of E-cadherin associated with α- and β-catenins and p120ctn to the nectin-based cell-cell adhesion sites by the action of 12-O-tetradecanoylphorbol-13-acetate in MDCK cells

2005 ◽  
Vol 10 (5) ◽  
pp. 435-445 ◽  
Author(s):  
Ryoko Okamoto ◽  
Kenji Irie ◽  
Akio Yamada ◽  
Tatsuo Katata ◽  
Atsunori Fukuhara ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Mdck Cells ◽  
Adhesion Sites ◽  
E Cadherin ◽  
Cell Cell

scholarly journals Two Cell Adhesion Molecules, Nectin and Cadherin, Interact through Their Cytoplasmic Domain–Associated Proteins

2000 ◽  
Vol 150 (5) ◽  
pp. 1161-1176 ◽  
Author(s):  
Kouichi Tachibana ◽  
Hiroyuki Nakanishi ◽  
Kenji Mandai ◽  
Kumi Ozaki ◽  
Wataru Ikeda ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Lines ◽  
Cell Adhesion Molecules ◽  
Cytoplasmic Domain ◽  
Adhesion Sites ◽  
Adhesion System ◽  
Associated Proteins ◽  
Mechanism Of Interaction ◽  
E Cadherin ◽  
Cell Cell

We have found a new cell–cell adhesion system at cadherin-based cell–cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca2+-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell–cell adhesion systems at AJs by the use of α-catenin–deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of α-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and α- and β-catenins was recruited to nectin-based cell–cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain–associated proteins and suggest that these two cell–cell adhesion systems cooperatively organize cell–cell AJs.

scholarly journals Association of ARVCF with Zonula Occludens (ZO)-1 and ZO-2: Binding to PDZ-Domain Proteins and Cell-Cell Adhesion Regulate Plasma Membrane and Nuclear Localization of ARVCF

2004 ◽  
Vol 15 (12) ◽  
pp. 5503-5515 ◽  
Author(s):  
P. Jaya Kausalya ◽  
Dominic C.Y. Phua ◽  
Walter Hunziker
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Nuclear Localization ◽  
Pdz Domain ◽  
Cell Contact ◽  
Binding Motif ◽  
Zonula Occludens ◽  
Pdz Domain Proteins ◽  
E Cadherin ◽  
Cell Cell

ARVCF, an armadillo-repeat protein of the p120ctnfamily, associates with classical cadherins and is present in adherens junctions, but its function is poorly understood. Here, we show that ARVCF interacts via a C-terminal PDZ-binding motif with zonula occludens (ZO)-1 and ZO-2. ARVCF and ZO-1 partially colocalize in the vicinity of the apical adhesion complex in polarized epithelial Madin-Darby canine kidney cells. ARVCF, ZO-1, and E-cadherin form a complex and are recruited to sites of initial cell-cell contact in sparse cell cultures. E-cadherin binding and plasma membrane localization of ARVCF require the PDZ-binding motif. Disruption of cell-cell adhesion releases ARVCF from the plasma membrane and an increased fraction of the protein localizes to the nucleus. Nuclear localization of ARVCF also requires the PDZ-binding motif and can be mediated by the PDZ domains of ZO-2. Thus, the interaction of ARVCF with distinct PDZ-domain proteins determines its subcellular localization. Interactions with ZO-1 and ZO-2, in particular, may mediate recruitment of ARVCF to the plasma membrane and the nucleus, respectively, possibly in response to cell-cell adhesion cues.

scholarly journals Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes

2000 ◽  
Vol 113 (10) ◽  
pp. 1803-1811
Author(s):  
Y. Hanakawa ◽  
M. Amagai ◽  
Y. Shirakata ◽  
K. Sayama ◽  
K. Hashimoto
Keyword(s):  
Cell Adhesion ◽  
Adherens Junctions ◽  
Dominant Negative ◽  
Cell Contact ◽  
Beta Catenin ◽  
Hacat Cells ◽  
Subsequent Formation ◽  
Contact Sites ◽  
E Cadherin ◽  
Cell Cell

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.

scholarly journals Positive Role of IQGAP1, an Effector of Rac1, in Actin-Meshwork Formation at Sites of Cell-Cell Contact

2004 ◽  
Vol 15 (3) ◽  
pp. 1065-1076 ◽  
Author(s):  
Jun Noritake ◽  
Masaki Fukata ◽  
Kazumasa Sato ◽  
Masato Nakagawa ◽  
Takashi Watanabe ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Filaments ◽  
Actin Binding ◽  
Cell Contact ◽  
Positive Role ◽  
Rac1 Activity ◽  
Guanosine Triphosphatase ◽  
Interfering Rna ◽  
E Cadherin ◽  
Cell Cell

The small guanosine triphosphatase Rac1 is activated by E-cadherin-mediated cell-cell adhesion and is required for the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact. However, the modes of activation and action of Rac1 remain to be clarified. We here found that suppression of IQGAP1, an actin-binding protein and an effector of Rac1, by small interfering RNA apparently reduced the accumulation of actin filaments, E-cadherin, and β-catenin at sites of cell-cell contact in Madin-Darby canine kidney II epithelial cells under the conditions in which knockdown of Rac1 reduced them. Knockdown of Rac1 did not affect the localization of these junctional components in cells expressing a constitutively active IQGAP1 mutant defective in Rac1/Cdc42 binding. Knockdown of either Rac1 or IQGAP1 accelerated the 12-O-tetradecanoylphorbol-13-acetate-induced cell-cell dissociation. The basal Rac1 activity, which was maintained by E-cadherin-mediated cell-cell adhesion, was inhibited in the IQGAP1-knocked down cells, whereas the Rac1 activity was increased in the cells overexpressing IQGAP1. Together, these results indicate that Rac1 enhances the accumulation of actin filaments, E-cadherin, and β-catenin by acting on IQGAP1 and suggest that there exists a positive feedback loop comprised of “E-cadherin-mediated cell-cell adhesion→Rac1 activation→actin-meshwork formation by IQGAP1→increasing E-cadherin-mediated cell-cell adhesion.”

scholarly journals Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion.

1996 ◽  
Vol 135 (6) ◽  
pp. 1899-1911 ◽  
Author(s):  
C L Adams ◽  
W J Nelson ◽  
S J Smith
Keyword(s):  
Cell Adhesion ◽  
Time Lapse ◽  
Cell Contact ◽  
Beta Catenin ◽  
Actin Reorganization ◽  
Cell Contacts ◽  
Spatial Ordering ◽  
Triton X ◽  
E Cadherin ◽  
Cell Cell

Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta-catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549-557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E-cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.

scholarly journals Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells

1997 ◽  
Vol 139 (4) ◽  
pp. 1047-1059 ◽  
Author(s):  
Kenji Takaishi ◽  
Takuya Sasaki ◽  
Hirokazu Kotani ◽  
Hideo Nishioka ◽  
Yoshimi Takai
Keyword(s):  
Cell Adhesion ◽  
Tight Junction ◽  
Cell Lines ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Wild Type ◽  
Cell Functions ◽  
Adhesion Sites ◽  
E Cadherin ◽  
Cell Cell

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.

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