We study punctate adherens junctions (pAJs) to determine how short-lived cadherin clusters and relatively stable actin bundles interact despite differences in dynamics. We show that pAJ-linked bundles consist of two distinct regions-the bundle stalk (AJ-BS) and a tip (AJ-BT) positioned between cadherin clusters and the stalk. The tip differs from the stalk in a number of ways: it is devoid of the actin-bundling protein calponin, and exhibits a much faster F-actin turnover rate. While F-actin in the stalk displays centripetal movement, the F-actin in the tip is immobile. The F-actin turnover in both the tip and stalk is dependent on cadherin cluster stability, which in turn is regulated by F-actin. The close bidirectional coupling between the stability of cadherin and associated F-actin shows how pAJs, and perhaps other AJs, allow cells to sense and coordinate the dynamics of the actin cytoskeleton in neighboring cells-a mechanism we term "dynasensing."

Although Snail is essential for disassembly of adherens junctions during epithelial-mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin-rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell-cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT.

Higher serum levels of p-cresol in chronic kidney disease populations have been associated with increased cardiovascular mortality. However, studies on how p-cresol affects intercellular junctions between cardiomyocytes were limited. This study investigated the effect of p-cresol on adherens junction (AJ) of neonatal cultured cardiomyocytes and its underlying mechanism. A loss of N-cadherin and p120-catenin (p120ctn) immunostaining from cell-cell contact sites was noted by p-cresol treatment. In addition, p-cresol disrupted AJs by inducing formation of intercellular gaps. Our previous study has revealed that p-cresol increased intracellular calcium levels and activated protein kinase Cα (PKCα) by phosphorylation. The PKCα activation was involved in the p-cresol-mediated AJ disassembly, since pharmacological inhibition of PKCα abolished the above-mentioned p-cresol effect. This PKCα activation also led to the serine dephosphorylation of p120ctn and caused the dissociation of p120ctn from N-cadherin. This hypothesis was further confirmed in H9c2 cells by siRNA approach. SiRNA knockdown of PKCα prevented p-cresol-induced serine dephosphorylation of p120ctn and splitting of AJ. In conclusion, p-cresol caused PKCα-dependent AJ disassembly of cardiomyocytes, which might be related to asychronized contraction.

Adhesion molecules are known to play an important role in endothelial activation and angiogenesis. Here we determined the functional role of IQGAP1 in the regulation of endothelial adherens junctions. VE-cadherin is found to be associated with actin filaments and thus stable, but IQGAP1 at intercellular junctions is not bound to actin filaments and thus labile. Expression of GFP labeled VE-α-catenin is shown to increase the electrical resistance across HUVEC monolayers and diminishes endogenous labile IQGAP1 at the intercellular junctions. Knockdown of endogenous IQGAP1 enhances intercellular adhesion in HUVECs by increasing the association of VE-cadherin with P120 and β-catenin. IQGAP1 knockdown also decreases the interaction of N-cadherin with P120 and β-catenin. Together, these results suggest that a labile pool of IQGAP1 at intercellular junctions disassembles adherens junctions and thus impairs endothelial cell-cell adhesion.

In the natural and technological world, multi-agent systems strongly depend on how the interactions are ruled between their individual components, and the proper control of time-scales and synchronization is a key issue. This certainly applies to living tissues when multicellular assemblies such as epithelial cells achieve complex morphogenetic processes. In epithelia, because cells are known to individually generate actomyosin contractile stress, each individual intercellular adhesive junction line is subjected to the opposed stresses independently generated by its two partner cells. Contact lines should thus move unless their two partner cells mechanically match. The geometric homeostasis of mature epithelia observed at short enough time-scale thus raises the problem to understand how cells, if considered as noisy individual actuators, do adapt across individual intercellular contacts to locally balance their time-average contractile stress. Structural components of adherens junctions, cytoskeleton (F-actin) and homophilic bonds (E-cadherin) are quickly renewed at steady-state. These turnovers, if they depend on forces exerted at contacts, may play a key role in the mechanical adaptation of epithelia. Here we focus on E-cadherin as a force transducer, and we study the local regulation and the mechanosensitivity of its turnover in junctions. We show that E-cadherin turnover rates match remarkably well on either side of mature intercellular contacts, despite the fact that they exhibit large fluctuations in time and variations from one junction to another. Using local mechanical and biochemical perturbations, we find faster turnover rates with increased tension, and asymmetric rates at unbalanced junctions. Together, the observations that E-cadherin turnover, and its local symmetry or asymmetry at each side of the junction, are mechanosensitive, support the hypothesis that E-cadherin turnover could be involved in mechanical homeostasis of epithelia.

mRNA localization is an evolutionarily widespread phenomenon that can facilitate subcellular protein targeting. Extensive work has focused on mRNA targeting through 'zip-codes' within untranslated regions (UTRs), whereas much less is known about translation-dependent cues. Here, we examine mRNA localization in Caenorhabditis elegans embryonic epithelia. From an smFISH-based survey, we identified mRNAs associated with the cell membrane or cortex, and with apical junctions in a stage- and cell type-specific manner. Mutational analyses for one of these transcripts, dlg-1/discs large, revealed that it relied on a translation-dependent process and did not require its 5' or 3' UTRs. We suggest a model in which dlg-1 transcripts are co-translationally localized with the nascent protein: first the translating complex goes to the cell membrane using sequences located at the C-terminal/3' end, and then apically using N-terminal/5' sequences. These studies identify a translation-based process for mRNA localization within developing epithelia and determine the necessary cis-acting sequences for dlg-1 mRNA targeting.

Endothelial cell-cell junctions maintain endothelial integrity and regulate vascular morphogenesis and homeostasis. Cell-cell junctions are usually depicted with a linear morphology along the boundaries between adjacent cells and in contact with cortical F-actin. However, in the endothelium, cell-cell junctions are highly dynamic and morphologically heterogeneous.

Squamous epithelial cells have both adherens junctions and desmosomes. The ability of these cells to organize the desmosomal proteins into a functional structure depends upon their ability first to organize an adherens junction. Since the adherens junction and the desmosome are separate structures with different molecular make up, it is not immediately obvious why formation of an adherens junction is a prerequisite for the formation of a desmosome. The adherens junction is composed of a transmembrane classical cadherin (E-cadherin and/or P-cadherin in squamous epithelial cells) linked to either beta-catenin or plakoglobin, which is linked to alpha-catenin, which is linked to the actin cytoskeleton. The desmosome is composed of transmembrane proteins of the broad cadherin family (desmogleins and desmocollins) that are linked to the intermediate filament cytoskeleton, presumably through plakoglobin and desmoplakin. To begin to study the role of adherens junctions in the assembly of desmosomes, we produced an epithelial cell line that does not express classical cadherins and hence is unable to organize desmosomes, even though it retains the requisite desmosomal components. Transfection of E-cadherin and/or P-cadherin into this cell line did not restore the ability to organize desmosomes; however, overexpression of plakoglobin, along with E-cadherin, did permit desmosome organization. These data suggest that plakoglobin, which is the only known common component to both adherens junctions and desmosomes, must be linked to E-cadherin in the adherens junction before the cell can begin to assemble desmosomal components at regions of cell-cell contact. Although adherens junctions can form in the absence of plakoglobin, making use only of beta-catenin, such junctions cannot support the formation of desmosomes. Thus, we speculate that plakoglobin plays a signaling role in desmosome organization.

Proper blood vessel formation requires coordinated changes in endothelial cell polarity and rearrangement of cell-cell junctions to form a functional lumen. One important regulator of cell polarity is the centrosome, which acts as a microtubule organizing center. Excess centrosomes perturb aspects of endothelial cell polarity linked to migration, but whether centrosome number influences apical-basal polarity and cell-cell junctions is unknown. Here, we show that excess centrosomes alter the apical-basal polarity of endothelial cells in angiogenic sprouts and disrupt endothelial cell-cell adherens junctions. Endothelial cells with excess centrosomes had narrower lumens in a 3D sprouting angiogenesis model, and zebrafish intersegmental vessels had reduced perfusion following centrosome overduplication. These results indicate that endothelial cell centrosome number regulates proper lumenization downstream of effects on apical-basal polarity and cell-cell junctions. Endothelial cells with excess centrosomes are prevalent in tumor vessels, suggesting how centrosomes may contribute to tumor vessel dysfunction.

The zonula adherens (ZA), a type of adherens junction (AJ), plays a major role in epithelial cell-cell adhesions. It remains unknown how the ZA is remodeled during epithelial reorganization. Here we found that the ZA was converted to another type of AJ with punctate morphology (pAJ) at the margins of epithelial colonies. The F-actin-stabilizing protein EPLIN (epithelial protein lost in neoplasm), which functions to maintain the ZA via its association with αE-catenin, was lost in the pAJs. Consistently, a fusion of αE-catenin and EPLIN contributed to the formation of ZA but not pAJs. We show that junctional tension was important for retaining EPLIN at AJs, and another force derived from actin fibers laterally attached to the pAJs inhibited EPLIN-AJ association. Vinculin was required for general AJ formation, and it cooperated with EPLIN to maintain the ZA. These findings suggest that epithelial cells remodel their junctional architecture by responding to mechanical forces, and the αE-catenin-bound EPLIN acts as a mechanosensitive regulator for this process.

Keratinocytes form the main protective barrier in the skin to separate the underlying tissue from the external environment. In order to maintain this barrier, keratinocytes form robust junctions between neighbouring cells as well as with the underlying extracellular matrix. Cell-cell adhesions are mediated primarily through cadherin receptors, whereas the integrin family of transmembrane receptors is predominantly associated with assembly of matrix adhesions. Integrins have been shown to also localise to cell-cell adhesions, but their role at these sites remains unclear.

The transcriptional co-activator YAP controls cell proliferation, survival, and tissue regeneration in response to changes in the mechanical environment. It is not known how mechanical stimuli such as tension are sensed and how the signal is transduced to control YAP activity. Here, we show that the LIM domain protein TRIP6 acts as part of a mechanotransduction pathway at adherens junctions to promote YAP activity by inhibiting the LATS1/2 kinases. Previous studies showed that vinculin at adherens junctions becomes activated by mechanical tension. We show that vinculin inhibits Hippo signaling by recruiting TRIP6 to adherens junctions and stimulating its binding to and inhibition of LATS1/2 in response to tension. TRIP6 competes with MOB1 for binding to LATS1/2 thereby blocking MOB1 from recruiting the LATS1/2 activating kinases MST1/2. Together, these findings reveal a novel pathway that responds to tension at adherens junctions to control Hippo pathway signaling.

The Notch pathway mediates cell-to-cell communication in a variety of tissues, developmental stages and organisms. Pathway activation relies on the interaction between transmembrane ligands and receptors on adjacent cells. As such, pathway activity could be influenced by the size, composition or dynamics of contacts between membranes. The initiation of Notch signalling in the Drosophila embryo occurs during cellularization, when lateral cell membranes and adherens junctions are first being deposited, allowing us to investigate the importance of membrane architecture and specific junctional domains for signalling. By measuring Notch-dependent transcription in live embryos, we established that it initiates while lateral membranes are growing and that signalling onset correlates with a specific phase in their formation. However, the length of the lateral membranes per se was not limiting. Rather, the adherens junctions, which assemble concurrently with membrane deposition, contributed to the high levels of signalling required for transcription, as indicated by the consequences of α-Catenin depletion. Together, these results demonstrate that the establishment of lateral membrane contacts can be limiting for Notch trans-activation and suggest that adherens junctions play an important role in modulating Notch activity.

Drosophila trachea is a classical model for analyzing epithelial, especially tubular epithelial biology. We identify lateral E-cadherin mediated junctions that encircle the cells just basal to the zonula adherens in the larval trachea. The lateral junction is associated with downstream adapters, including catenins, and has a distinct junctional actin cortex. The lateral cortex contributes to the development of a supracellular actomyosin mesh in the late larvae. Establishment of this cytoskeletal structure depends on lateral junction associated Rho1 and Cdc42 GTPases, and Arp and WASP pathways. The supracellular network takes the character of stress fibers along the AP axis in the early hours of pupation. It contributes to the shortening of the epithelial tube albeit in a manner redundant to ECM-mediated compression mechanism. In conclusion, we show the in vivo existence of functional lateral adherens junction and suggest a role for it in mediating dynamic cytoskeletal events during tissue scale morphogenesis.

Adherens junctions (AJs) are membrane-anchored structures composed of E-cadherin and associated proteins, including catenins and actin. The unique plasticity of AJs mediates both the rigidity and flexibility of cell-cell contacts essential for embryonic morphogenesis and adult tissue remodeling. We identified the SUMO protease ULP-2 as a regulator of AJ assembly and show that dysregulated ULP-2 activity impairs epidermal morphogenesis in Caenorhabditis elegans embryos. The conserved cytoplasmic tail of HMR-1/E-cadherin is sumoylated and is a target of ULP-2 desumoylation activity. Coupled sumoylation and desumoylation of HMR-1 are required for its recruitment to the subapical membrane during AJ assembly and the formation of the linkages between AJs and the apical actin cytoskeleton. Sumoylation weakens HMR-1 binding to HMP-2/β-catenin. Our study provides a mechanistic link between the dynamic nature of the SUMO machinery and AJ plasticity and highlight sumoylation as a molecular switch that modulates the binding of E-cadherin to the actin cytoskeleton.

Proper epithelial structure requires adherens junction (AJ) assembly. In the early Drosophila embryo, AJ assembly depends on Bazooka (Baz; PAR-3), but it is unclear how Baz affects AJ assembly and what precursors are involved. To understand this process at the molecular level, we counted the number of core AJ proteins and Baz proteins at an average spot AJ (SAJ) and determined their dynamics with fluorescence recovery after photobleaching experiments. These data reveal that SAJs are subdivided into Baz clusters and cadherin-catenin clusters with independent protein numbers and dynamics. This independence suggests that precursory cadherin-catenin clusters might form before SAJ assembly. We identify cadherin-catenin clusters forming between apical microvilli. Further analyses show that they form independently of Baz and that Baz functions in repositioning them to apicolateral sites for full SAJ assembly. Our data implicate cell protrusions in initial cadherin-catenin clustering in the Drosophila melanogaster embryo. Then, independent Baz clusters appear to engage the cadherin-catenin clusters to assemble SAJs.

Epithelial cell-cell adhesion is controlled by multiprotein complexes that include E-cadherin-mediated adherens junctions (AJs) and ZO-1-containing tight junctions (TJs). Previously, we reported that reduction of E-cadherin N-glycosylation in normal and cancer cells promoted stabilization of AJs through changes in the composition and cytoskeletal association of E-cadherin scaffolds. Here, we show that enhanced interaction of hypoglycosylated E-cadherin-containing AJs with protein phosphatase 2A (PP2A) represents a mechanism for promoting TJ assembly. In MDCK cells, attenuation of cellular N-glycosylation with siRNA to DPAGT1, the first gene in the N-glycosylation pathway, reduced N-glycosylation of surface E-cadherin and resulted in increased recruitment of stabilizing proteins gamma-catenin, alpha-catenin, vinculin and PP2A to AJs. Greater association of PP2A with AJs correlated with diminished binding of PP2A to ZO-1 and claudin-1 and with increased pools of serine-phosphorylated ZO-1 and claudin-1. More ZO-1 was found in complexes with occludin and claudin-1, and this corresponded to enhanced transepithelial resistance (TER), indicating physiological assembly of TJs. Similar maturation of AJs and TJs was detected after transfection of MDCK cells with the hypoglycosylated E-cadherin variant, V13. Our data indicate that E-cadherin N-glycans coordinate the maturity of AJs with the assembly of TJs by affecting the association of PP2A with these junctional complexes.

Vascular endothelial (VE)-cadherin forms homotypic adherens junctions (AJs) in the endothelium, whereas N-cadherin forms heterotypic adhesion between endothelial cells and surrounding vascular smooth muscle cells and pericytes. Here we addressed the question whether both cadherin adhesion complexes communicate through intracellular signaling and contribute to the integrity of the endothelial barrier. We demonstrated that deletion of N-cadherin (Cdh2) in either endothelial cells or pericytes increases junctional endothelial permeability in lung and brain secondary to reduced accumulation of VE-cadherin at AJs. N-cadherin functions by increasing the rate of VE-cadherin recruitment to AJs and induces the assembly of VE-cadherin junctions. We identified the dual Rac1/RhoA Rho guanine nucleotide exchange factor (GEF) Trio as a critical component of the N-cadherin adhesion complex, which activates both Rac1 and RhoA signaling pathways at AJs. Trio GEF1-mediated Rac1 activation induces the recruitment of VE-cadherin to AJs, whereas Trio GEF2-mediated RhoA activation increases intracellular tension and reinforces Rac1 activation to promote assembly of VE-cadherin junctions and thereby establish the characteristic restrictive endothelial barrier.

Endothelial cell-cell junctions control efflux of small molecules and leukocyte transendothelial migration (TEM) between blood and tissues. Inhibitors of phosphoinositide 3-kinases (PI3Ks) increase endothelial barrier function, but the roles of different PI3K isoforms have not been addressed. In this study, we determine the contribution of each of the four class I PI3K isoforms (p110alpha, -beta, -gamma, and -delta) to endothelial permeability and leukocyte TEM. We find that depletion of p110alpha but not other p110 isoforms decreases TNF-induced endothelial permeability, Tyr phosphorylation of the adherens junction protein vascular endothelial cadherin (VE-cadherin), and leukocyte TEM. p110alpha selectively mediates activation of the Tyr kinase Pyk2 and GTPase Rac1 to regulate barrier function. Additionally, p110alpha mediates the association of VE-cadherin with Pyk2, the Rac guanine nucleotide exchange factor Tiam-1 and the p85 regulatory subunit of PI3K. We propose that p110alpha regulates endothelial barrier function by inducing the formation of a VE-cadherin-associated protein complex that coordinates changes to adherens junctions with the actin cytoskeleton.

The extracellular matrix (ECM) plays crucial roles in tissue homeostasis. Abnormalities in ECM composition are associated with pathological conditions, such as fibrosis and cancer. These ECM alterations are sensed by the epithelium and can influence its behavior through crosstalk with other mechanosensitive complexes, including the adherens junctions (AJs). We have previously shown that the AJs, through their component PLEKHA7, recruit the RNAi machinery to regulate miRNA levels and function. We have particularly shown that the junctional localization of RNAi components is critical for their function. Here, we investigated whether different ECM substrates can influence the junctional localization of RNAi complexes. To do this, we plated colon epithelial Caco2 cells on four key ECM substrates found in the colon under normal or pathogenic conditions, namely laminin, fibronectin, collagen I, and collagen IV, and we examined the subcellular distribution of PLEKHA7, and of the key RNAi components AGO2 and DROSHA. Fibronectin and collagen I negatively impacted the junctional localization of PLEKHA7, AGO2, and DROSHA when compared to laminin. Furthermore, fibronectin, collagen I, and collagen IV disrupted interactions of AGO2 and DROSHA with their essential partners GW182 and DGCR8, respectively, both at AJs and throughout the cell. Combinations of all substrates with fibronectin also negatively impacted junctional localization of PLEKHA7 and AGO2. Additionally, collagen I triggered accumulation of DROSHA at tri-cellular junctions, while both collagen I and collagen IV resulted in DROSHA accumulation at basal areas of cell-cell contact. Altogether, fibronectin and collagens I and IV, which are elevated in the stroma of fibrotic and cancerous tissues, altered localization patterns and disrupted complex formation of PLEKHA7 and RNAi components. Combined with our prior studies showing that apical junctional localization of the PLEKHA7-RNAi complex is critical for regulating tumor-suppressing miRNAs, this work points to a yet unstudied mechanism that could contribute to epithelial cell transformation.