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

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.     

E-cadherin is required for gastrulation cell movements in zebrafish

E-cadherin is a member of the classical cadherin family and is known to be involved in cell-cell adhesion and the adhesion-dependent morphogenesis of various tissues. We isolated a zebrafish mutant (cdh1(rk3)) that has a mutation in the e-cadherin/cdh1 gene. The mutation rk3 is a hypomorphic allele, and the homozygous mutant embryos displayed variable phenotypes in gastrulation and tissue morphogenesis. The most severely affected embryos displayed epiboly delay, decreased convergence and extension movements, and the dissociation of cells from the embryos, resulting in early embryonic lethality. The less severely affected embryos survived through the pharyngula stage and showed flattened anterior neural tissue, abnormal positioning and morphology of the hatching gland, scattered trigeminal ganglia, and aberrant axon bundles from the trigeminal ganglia. Maternal-zygotic cdh1(rk3) embryos displayed epiboly arrest during gastrulation, in which the enveloping layer (EVL) and the yolk syncytial layer but not the deep cells (DC) completed epiboly. A similar phenotype was observed in embryos that received antisense morpholino oligonucleotides (cdh1MO) against E-cadherin, and in zebrafish epiboly mutants. Complementation analysis with the zebrafish epiboly mutant weg suggested that cdh1(rk3) is allelic to half baked/weg. Immunohistochemistry with an anti-beta-catenin antibody and electron microscopy revealed that adhesion between the DCs and the EVL was mostly disrupted but the adhesion between DCs was relatively unaffected in the MZcdh1(rk3) mutant and cdh1 morphant embryos. These data suggest that E-cadherin-mediated cell adhesion between the DC and EVL plays a role in the epiboly movement in zebrafish.     

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.     

A Highlights from MBoC Selection: Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.     

The mechanical basis of cell rearrangement. I. Epithelial morphogenesis during Fundulus epiboly

Many morphogenetic processes are accomplished by coordinated cell rearrangements. These rearrangements are accompanied by substantial shifts in the neighbor relationships between cells. Here we propose a model for studying morphogenesis in epithelial sheets by directed cell neighbor change. Our model describes cell rearrangements by accounting for the balance of forces between neighboring cells within an epithelium. Cell rearrangement and cell shape changes occur when these forces are not in mechanical equilibrium. We will show that cell rearrangement within the epidermal enveloping layer (EVL) of the teleost fish Fundulus during epiboly can be explained solely in terms of the balance of forces generated among constituent epithelial cells. Within a cell, we account for circumferential elastic forces and the force generated by hydrostatic and osmotic pressure. The model treats epithelial cells as two-dimensional polygons where the mechanical forces are applied to the polygonal nodes. A cell node protrudes or contracts when the nodal forces are not in mechanical equilibrium. In an epithelial sheet, adjacent cells share common boundary nodes; in this way, mechanical force is transmitted from cell to cell, mimicking junctional coupling. These junctional nodes can slide, and nodes may appear or disappear, so that the number of polygonal sides is variable. Computer graphics allows us to compare numerical simulations of the model with time-lapse cinemicroscopy of cell rearrangements in the living embryo, and data obtained from fixed and silver stained embryos. By manipulating the mechanical properties of the model cells we can study the conditions necessary to reproduce normal cell behavior during Fundulus epiboly. We find that simple stress relaxation is sufficient to account for cell rearrangements among interior cells of the EVL when they are isotropically contractile. Experimental observations show that the number of EVL marginal cells continuously decreases throughout epiboly. In order for the simulation to reproduce this behavior, cells at the EVL boundary must generate protrusive forces rather than contractile tension forces. Therefore, the simulation results suggest that the mechanical properties of EVL marginal cells at their leading edge must be quite different from EVL interior cells.     

Caveolae Mediate Growth Factor-induced Disassembly of Adherens Junctions to Support Tumor Cell Dissociation

Remodeling of cell–cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell–cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell–cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.     

Protein Kinase C Activation Upregulates Intercellular Adhesion of α-Catenin–negative Human Colon Cancer Cell Variants via Induction of Desmosomes

The α-catenin molecule links E-cadherin/ β-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. We studied several invasive human colon carcinoma cell lines lacking α-catenin. They showed a solitary and rounded morphotype that correlated with increased invasiveness. These round cell variants acquired a more normal epithelial phenotype upon transfection with an α-catenin expression plasmid, but also upon treatment with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations showed that the cells started to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect was not associated with α-catenin induction. Classical and confocal immunofluorescence showed only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins were dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell–cell contacts and exposition at the exterior cell surface. Electron microscopical observations revealed the TPA-induced appearance of typical desmosomal plaques. TPA-restored cell–cell adhesion was E-cadherin dependent as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti–E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect.

Our studies show that it is possible to bypass the need for normal α-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin remains essential for basic cell–cell adhesion, even in the absence of α-catenin.

     

E-cadherin is required for intestinal morphogenesis in the mouse

E-cadherin, the primary epithelial adherens junction protein, has been implicated as playing a critical role in nucleating formation of adherens junctions, tight junctions, and desmosomes. In addition to its role in maintaining structural tissue integrity, E-cadherin has also been suggested as an important modulator of cell signaling via interactions with its cytoplasmic binding partners, catenins, as well as with growth factor receptors. Therefore, we proposed that loss of E-cadherin from the developing mouse intestinal epithelium would disrupt intestinal epithelial morphogenesis and function. To test this hypothesis, we used a conditional knockout approach to eliminate E-cadherin specifically in the intestinal epithelium during embryonic development. We found that E-cadherin conditional knockout mice failed to survive, dying within the first 24 hours of birth. Examination of intestinal architecture at E18.5 demonstrated severe disruption to intestinal morphogenesis in animals lacking E-cadherin in the epithelium of the small intestine. We observed changes in epithelial cell shape as well as in the morphology of villi. Although junctional complexes were evident, junctions were abnormal, and barrier function was compromised in E-cadherin mutant intestine. We also identified changes in the epithelial cell populations present in E-cadherin conditional knockout animals. The number of proliferating cells was increased, whereas the number of enterocytes was decreased. Although Wnt/β-catenin target mRNAs were more abundant in mutants compared with controls, the amount of nuclear activated β-catenin protein was dramatically lower in mutants compared with controls. In summary, our data demonstrate that E-cadherin is essential for intestinal epithelial morphogenesis and homeostasis during embryonic development.     

Endothelial cell–cell adhesion during zebrafish vascular development

The vertebrate vasculature is an essential organ network with major roles in health and disease. The establishment of balanced cell–cell adhesion in the endothelium is crucial for the functionality of the vascular system. Furthermore, the correct patterning and integration of vascular endothelial cell–cell adhesion drives the morphogenesis of new vessels, and is thought to couple physical forces with signaling outcomes during development. Here, we review insights into this process that have come from studies in zebrafish. First, we describe mutants in which endothelial adhesion is perturbed, second we describe recent progress using in vivo cell biological approaches that allow the visualization of endothelial cell–cell junctions. These studies underline the profound potential of this model system to dissect in great detail the function of both known and novel regulators of endothelial cell–cell adhesion.     

Cadherin Cytoplasmic Domains Inhibit the Cell Surface Localization of Endogenous E-Cadherin,Blocking Desmosome and Tight Junction Formation and Inducing Cell Dissociation

The downregulation of E-cadherin function has fundamental consequences with respect to cancer progression, and occurs as part of the epithelial–mesenchymal transition (EMT). In this study, we show that the expression of the Discosoma sp. red fluorescent protein (DsRed)-tagged cadherin cytoplasmic domain in cells inhibited the cell surface localization of endogenous E-cadherin, leading to morphological changes, the inhibition of junctional assembly and cell dissociation. These changes were associated with increased cell migration, but were not accompanied by the down-regulation of epithelial markers and up-regulation of mesenchymal markers. Thus, these changes cannot be classified as EMT. The cadherin cytoplasmic domain interacted with β-catenin or plakoglobin, reducing the levels of β-catenin or plakoglobin associated with E-cadherin, and raising the possibility that β-catenin and plakoglobin sequestration by these constructs induced E-cadherin intracellular localization. Accordingly, a cytoplasmic domain construct bearing mutations that weakened the interactions with β-catenin or plakoglobin did not impair junction formation and adhesion, indicating that the interaction with β-catenin or plakoglobin was essential to the potential of the constructs. E-cadherin–α-catenin chimeras that did not require β-catenin or plakoglobin for their cell surface transport restored cell–cell adhesion and junction formation.     

Zebrafish Dynamin is required for maintenance of enveloping layer integrity and the progression of epiboly

Epiboly, the first morphogenetic cell movement that occurs in the zebrafish embryo, is the process by which the blastoderm thins and spreads to engulf the yolk cell. This process requires the concerted actions of the deep cells, the enveloping layer (EVL) and the extra-embryonic yolk syncytial layer (YSL). The EVL is mechanically coupled to the YSL which acts as an epiboly motor, generating the force necessary to draw the blastoderm towards the vegetal pole though actomyosin flow and contraction of the actomyosin ring. However, it has been proposed that the endocytic removal of yolk cell membrane just ahead of the advancing blastoderm may also play a role. To assess the contribution of yolk cell endocytosis in driving epiboly movements, we used a combination of drug- and dominant-negative-based approaches to inhibit Dynamin, a large GTPase with a well-characterized role in vesicle scission. We show that Dynamin-dependent endocytosis in the yolk cell is dispensable for epiboly of the blastoderm. However, global inhibition of Dynamin function revealed that Dynamin plays a fundamental role within the blastoderm during epiboly, where it maintains epithelial integrity and the transmission of tension across the EVL. The epithelial defects were associated with disrupted tight junctions and a striking reduction of cortically localized phosphorylated ezrin/radixin/moesin (P-ERM), key regulators of epithelial integrity in other systems. Furthermore, we show that Dynamin maintains EVL and promotes epiboly progression by antagonizing Rho A activity.     

An Elmo–Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion

Cell–cell contact formation is a dynamic process requiring the coordination of cadherin-based cell–cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell–cell adhesion identified an Elmo–Dock complex. This was unexpected as Elmo–Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell–cell contacts in Madin–Darby canine kidney cells. At cell–cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell–cell adhesion. Upon completion of cell–cell adhesion, Elmo2 and Dock1 no longer localize to cell–cell contacts and are not required subsequently for the maintenance of cell–cell adhesion. These studies show that Elmo–Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell–cell adhesion.     

Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.     

The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity

Oligomerization of cadherins could provide the stability to ensure tissue cohesion. Cadherins mediate cell–cell adhesion by forming trans-interactions. They form cis-interactions whose role could be essential to stabilize intercellular junctions by shifting cadherin clusters from a fluid to an ordered phase. However, no evidence has been provided so far for cadherin oligomerization in cellulo and for its impact on cell–cell contact stability. Visualizing single cadherins within cell membrane at a nanometric resolution, we show that E-cadherins arrange in ordered clusters, providing the first demonstration of the existence of oligomeric cadherins at cell–cell contacts. Studying the consequences of the disruption of the cis-interface, we show that it is not essential for adherens junction formation. Its disruption, however, increased the mobility of junctional E-cadherin. This destabilization strongly affected E-cadherin anchoring to actin and cell–cell rearrangement during collective cell migration, indicating that the formation of oligomeric clusters controls the anchoring of cadherin to actin and cell–cell contact fluidity.     

A Highlights from MBoC Selection: Cdc42 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells through PAK4 and Par6B

Cdc42 has been implicated in numerous biochemical pathways during epithelial morphogenesis, including the control of spindle orientation during mitosis, the establishment of apical-basal polarity, the formation of apical cell–cell junctions, and polarized secretion. To investigate the signaling pathways through which Cdc42 mediates these diverse effects, we have screened an siRNA library corresponding to the 36 known Cdc42 target proteins, in a human bronchial epithelial cell line. Two targets, PAK4 and Par6B, were identified as necessary for the formation of apical junctions. PAK4 is recruited to nascent cell–cell contacts in a Cdc42-dependent manner, where it is required for the maturation of primordial junctions into apical junctions. PAK4 kinase activity is essential for junction maturation, but overexpression of an activated PAK4 mutant disrupts this process. Par6B, together with its binding partner aPKC, is necessary both for junction maturation and for the retention of PAK4 at sites of cell–cell contact. This study demonstrates that controlled regulation of PAK4 is required for apical junction formation in lung epithelial cells and highlights potential cross-talk between two Cdc42 targets, PAK4 and Par6B.     

Dynamic microtubules produce an asymmetric E-cadherin–Bazooka complex to maintain segment boundaries

Distributing junctional components around the cell periphery is key for epithelial tissue morphogenesis and homeostasis. We discovered that positioning of dynamic microtubules controls the asymmetric accumulation of E-cadherin. Microtubules are oriented preferentially along the dorso-ventral axis in Drosophila melanogaster embryonic epidermal cells, and thus more frequently contact E-cadherin at dorso-ventral cell–cell borders. This inhibits RhoGEF2, reducing membrane recruitment of Rho-kinase, and increasing a specific E-cadherin pool that is mobile when assayed by fluorescence recovery after photobleaching. This mobile E-cadherin is complexed with Bazooka/Par-3, which in turn is required for normal levels of mobile E-cadherin. Mobile E-cadherin–Bazooka prevents formation of multicellular rosette structures and cell motility across the segment border in Drosophila embryos. Altogether, the combined action of dynamic microtubules and Rho signaling determines the level and asymmetric distribution of a mobile E-cadherin–Bazooka complex, which regulates cell behavior during the generation of a patterned epithelium.     

Classical cadherins control nucleus and centrosome position and cell polarity

Control of cell polarity is crucial during tissue morphogenesis and renewal, and depends on spatial cues provided by the extracellular environment. Using micropatterned substrates to impose reproducible cell–cell interactions, we show that in the absence of other polarizing cues, cell–cell contacts are the main regulator of nucleus and centrosome positioning, and intracellular polarized organization. In a variety of cell types, including astrocytes, epithelial cells, and endothelial cells, calcium-dependent cadherin-mediated cell–cell interactions induce nucleus and centrosome off-centering toward cell–cell contacts, and promote orientation of the nucleus–centrosome axis toward free cell edges. Nucleus and centrosome off-centering is controlled by N-cadherin through the regulation of cell interactions with the extracellular matrix, whereas the orientation of the nucleus–centrosome axis is determined by the geometry of N-cadherin–mediated contacts. Our results demonstrate that in addition to the specific function of E-cadherin in regulating baso-apical epithelial polarity, classical cadherins control cell polarization in otherwise nonpolarized cells.