scholarly journals Force measurements in E-cadherin–mediated cell doublets reveal rapid adhesion strengthened by actin cytoskeleton remodeling through Rac and Cdc42

2004 ◽  
Vol 167 (6) ◽  
pp. 1183-1194 ◽  
Author(s):  
Yeh-Shiu Chu ◽  
William A. Thomas ◽  
Olivier Eder ◽  
Frederic Pincet ◽  
Eric Perez ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Dominant Negative Form ◽  
Cytoskeleton Remodeling ◽  
Dependent Cell ◽  
Separation Force ◽  
E Cadherin

We have used a modified, dual pipette assay to quantify the strength of cadherin-dependent cell–cell adhesion. The force required to separate E-cadherin–expressing paired cells in suspension was measured as an index of intercellular adhesion. Separation force depended on the homophilic interaction of functional cadherins at the cell surface, increasing with the duration of contact and with cadherin levels. Severing the link between cadherin and the actin cytoskeleton or disrupting actin polymerization did not affect initiation of cadherin-mediated adhesion, but prevented it from developing and becoming stronger over time. Rac and Cdc42, the Rho-like small GTPases, were activated when E-cadherin–expressing cells formed aggregates in suspension. Overproduction of the dominant negative form of Rac or Cdc42 permitted initial E-cadherin–based adhesion but affected its later development; the dominant active forms prevented cell adhesion outright. Our findings highlight the crucial roles played by Rac, Cdc42, and actin cytoskeleton dynamics in the development and regulation of strong cell adhesion, defined in terms of mechanical forces.

scholarly journals Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

2011 ◽  
Vol 208 (5) ◽  
pp. 1055-1068 ◽  
Author(s):  
Bebhinn Treanor ◽  
David Depoil ◽  
Andreas Bruckbauer ◽  
Facundo D. Batista
Keyword(s):  
Actin Cytoskeleton ◽  
B Cell ◽  
Protein Function ◽  
Actin Polymerization ◽  
Lymphocyte Activation ◽  
Dominant Negative ◽  
Temporary Increase ◽  
Cortical Actin ◽  
Erm Proteins ◽  
Diffusion Dynamics

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.

Osmotic stress-induced remodeling of the cortical cytoskeleton

2002 ◽  
Vol 283 (3) ◽  
pp. C850-C865 ◽  
Author(s):  
Caterina Di Ciano ◽  
Zilin Nie ◽  
Katalin Szászi ◽  
Alison Lewis ◽  
Takehito Uruno ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
De Novo ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Signaling Mechanism ◽  
Actin Binding Domain ◽  
Cytoskeleton Remodeling

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

scholarly journals Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras

2015 ◽  
Vol 210 (7) ◽  
pp. 1065-1074 ◽  
Author(s):  
Julie M. Bianchini ◽  
Khameeka N. Kitt ◽  
Martijn Gloerich ◽  
Sabine Pokutta ◽  
William I. Weis ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Intercellular Adhesion ◽  
Dependent Cell ◽  
In Cells ◽  
E Cadherin ◽  
Cell Cell

As part of the E-cadherin–β-catenin–αE-catenin complex (CCC), mammalian αE-catenin binds F-actin weakly in the absence of force, whereas cytosolic αE-catenin forms a homodimer that interacts more strongly with F-actin. It has been concluded that cytosolic αE-catenin homodimer is not important for intercellular adhesion because E-cadherin/αE-catenin chimeras thought to mimic the CCC are sufficient to induce cell–cell adhesion. We show that, unlike αE-catenin in the CCC, these chimeras homodimerize, bind F-actin strongly, and inhibit the Arp2/3 complex, all of which are properties of the αE-catenin homodimer. To more accurately mimic the junctional CCC, we designed a constitutively monomeric chimera, and show that E-cadherin–dependent cell adhesion is weaker in cells expressing this chimera compared with cells in which αE-catenin homodimers are present. Our results demonstrate that E-cadherin/αE-catenin chimeras used previously do not mimic αE-catenin in the native CCC, and imply that both CCC-bound monomer and cytosolic homodimer αE-catenin are required for strong cell–cell adhesion.

scholarly journals Role of Nectin in Formation of E-Cadherin–based Adherens Junctions in Keratinocytes: Analysis with the N-Cadherin Dominant Negative Mutant

2003 ◽  
Vol 14 (4) ◽  
pp. 1597-1609 ◽  
Author(s):  
Yoshinari Tanaka ◽  
Hiroyuki Nakanishi ◽  
Shigeki Kakunaga ◽  
Noriko Okabe ◽  
Tomomi Kawakatsu ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Adherens Junctions ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Adhesion Activity ◽  
Negative Mutant ◽  
E Cadherin ◽  
Cell Cell

E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.

scholarly journals Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response

2009 ◽  
Vol 296 (3) ◽  
pp. C463-C475 ◽  
Author(s):  
Ana C. P. Thirone ◽  
Pam Speight ◽  
Matthew Zulys ◽  
Ori D. Rotstein ◽  
Katalin Szászi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
De Novo ◽  
Rho Kinase ◽  
Small Gtpases ◽  
Hyperosmotic Stress ◽  
Dominant Negative ◽  
Lim Kinase ◽  
Tubular Cells ◽  
Cofilin Phosphorylation ◽  
Underlying Mechanisms

Hyperosmotic stress induces cytoskeleton reorganization and a net increase in cellular F-actin, but the underlying mechanisms are incompletely understood. Whereas de novo F-actin polymerization likely contributes to the actin response, the role of F-actin severing is unknown. To address this problem, we investigated whether hyperosmolarity regulates cofilin, a key actin-severing protein, the activity of which is inhibited by phosphorylation. Since the small GTPases Rho and Rac are sensitive to cell volume changes and can regulate cofilin phosphorylation, we also asked whether they might link osmostress to cofilin. Here we show that hyperosmolarity induced rapid, sustained, and reversible phosphorylation of cofilin in kidney tubular (LLC-PK1 and Madin-Darby canine kidney) cells. Hyperosmolarity-provoked cofilin phosphorylation was mediated by the Rho/Rho kinase (ROCK)/LIM kinase (LIMK) but not the Rac/PAK/LIMK pathway, because 1) dominant negative (DN) Rho and DN-ROCK but not DN-Rac and DN-PAK inhibited cofilin phosphorylation; 2) constitutively active (CA) Rho and CA-ROCK but not CA-Rac and CA-PAK induced cofilin phosphorylation; 3) hyperosmolarity induced LIMK-2 phosphorylation, and 4) inhibition of ROCK by Y-27632 suppressed the hypertonicity-triggered LIMK-2 and cofilin phosphorylation.We thenexamined whether cofilin and its phosphorylation play a role in the hypertonicity-triggered F-actin changes. Downregulation of cofilin by small interfering RNA increased the resting F-actin level and eliminated any further rise upon hypertonic treatment. Inhibition of cofilin phosphorylation by Y-27632 prevented the hyperosmolarity-provoked F-actin increase. Taken together, cofilin is necessary for maintaining the osmotic responsiveness of the cytoskeleton in tubular cells, and the Rho/ROCK/LIMK-mediated cofilin phosphorylation is a key mechanism in the hyperosmotic stress-induced F-actin increase.

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes

1996 ◽  
Vol 109 (13) ◽  
pp. 3013-3023 ◽  
Author(s):  
A.J. Zhu ◽  
F.M. Watt
Keyword(s):  
Cell Adhesion ◽  
Terminal Differentiation ◽  
Intercellular Adhesion ◽  
Dominant Negative ◽  
Epidermal Keratinocytes ◽  
Dominant Negative Mutant ◽  
Beta Catenin ◽  
Human Epidermal Keratinocytes ◽  
Negative Mutant ◽  
E Cadherin

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.

Involvement of the small GTPases XRhoA and XRnd1 in cell adhesion and head formation in early Xenopus development

Development ◽  
1999 ◽  
Vol 126 (23) ◽  
pp. 5339-5351 ◽  
Author(s):  
K. Wunnenberg-Stapleton ◽  
I.L. Blitz ◽  
C. Hashimoto ◽  
K.W. Cho
Keyword(s):  
Cell Adhesion ◽  
Membrane Trafficking ◽  
Ventral Side ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Axis Formation ◽  
Adhesion Properties ◽  
Bmp Receptor ◽  
Xenopus Development ◽  
Head Formation

The Rho family of small GTPases regulates a variety of cellular functions, including the dynamics of the actin cytoskeleton, cell adhesion, transcription, cell growth and membrane trafficking. We have isolated the first Xenopus homologs of the Rho-like GTPases RhoA and Rnd1 and examined their potential roles in early Xenopus development. We found that Xenopus Rnd1 (XRnd1) is expressed in tissues undergoing extensive morphogenetic changes, such as marginal zone cells involuting through the blastopore, somitogenic mesoderm during somite formation and neural crest cells. XRnd1 also causes a severe loss of cell adhesion in overexpression experiments. These data and the expression pattern suggest that XRnd1 regulates morphogenetic movements by modulating cell adhesion in early embryos. Xenopus RhoA (XRhoA) is a potential XRnd1 antagonist, since overexpression of XRhoA increases cell adhesion in the embryo and reverses the disruption of cell adhesion caused by XRnd1. In addition to the potential roles of XRnd1 and XRhoA in the regulation of cell adhesion, we find a role for XRhoA in axis formation. When coinjected with dominant-negative BMP receptor (tBR) in the ventral side of the embryo, XRhoA causes the formation of head structures resembling the phenotype seen after coinjection of wnt inhibitors with dominant-negative BMP receptor. Since dominant-negative XRhoA is able to reduce the formation of head structures, we propose that XRhoA activity is essential for head formation. Thus, XRhoA may have a dual role in the embryo by regulating cell adhesion properties and pattern formation.

scholarly journals LI-Cadherin–mediated Cell–Cell Adhesion Does Not Require Cytoplasmic Interactions

1997 ◽  
Vol 136 (5) ◽  
pp. 1109-1121 ◽  
Author(s):  
Bertolt Kreft ◽  
Dietmar Berndorff ◽  
Anja Böttinger ◽  
Silvia Finnemann ◽  
Doris Wedlich ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Adhesive Properties ◽  
Glycosyl Phosphatidylinositol ◽  
Cytoplasmic Proteins ◽  
Classical Cadherins ◽  
Drosophila S2 Cells ◽  
Detergent Extraction ◽  
Dependent Cell ◽  
Cell Cell

The adhesive function of classical cadherins depends on the association with cytoplasmic proteins, termed catenins, which serve as a link between cadherins and the actin cytoskeleton. LI-cadherin, a structurally different member of the cadherin family, mediates Ca2+-dependent cell–cell adhesion, although its markedly short cytoplasmic domain exhibits no homology to this highly conserved region of classical cadherins. We now examined whether the adhesive function of LI-cadherin depends on the interaction with catenins, the actin cytoskeleton or other cytoplasmic components. In contrast to classical cadherins, LI-cadherin, when expressed in mouse L cells, was neither associated with catenins nor did it induce an upregulation of β-catenin. Consistent with these findings, LI-cadherin was not resistant to detergent extraction and did not induce a reorganization of the actin cytoskeleton. However, LI-cadherin was still able to mediate Ca2+dependent cell–cell adhesion. To analyze whether this function requires any interaction with proteins other than catenins, a glycosyl phosphatidylinositol–anchored form of LI-cadherin (LI-cadherinGPI) was constructed and expressed in Drosophila S2 cells. The mutant protein was able to induce Ca2+-dependent, homophilic cell–cell adhesion, and its adhesive properties were indistinguishable from those of wild type LI-cadherin. These findings indicate that the adhesive function of LI-cadherin is independent of any interaction with cytoplasmic components, and consequently should not be sensitive to regulatory mechanisms affecting the binding of classical cadherins to catenins and to the cytoskeleton. Thus, we postulate that the adhesive function of LI-cadherin is complementary to that of coexpressed classical cadherins ensuring cell–cell contacts even under conditions that downregulate the function of classical cadherins.

Sign in / Sign up

Export Citation Format

Share Document