The zonula adherens matura redefines the apical junction of intestinal epithelia.

Cell-cell apical junctions of epithelia consist of multiprotein complexes that organize as belts regulating cell-cell adhesion, permeability, and mechanical tension: the tight junction (zonula occludens), the zonula adherens (ZA), and the macula adherens. The prevailing dogma is that at the ZA, E-cadherin and catenins are lined with F-actin bundles that support and transmit mechanical tension between cells. Using super-resolution microscopy on human intestinal biopsies and Caco-2 cells, we show that two distinct multiprotein belts are basal of the tight junctions as the intestinal epithelia mature. The most apical is populated with nectins/afadin and lined with F-actin; the second is populated with E-cad/catenins. We name this dual-belt architecture the zonula adherens matura. We find that the apical contraction apparatus and the dual-belt organization rely on afadin expression. Our study provides a revised description of epithelial cell-cell junctions and identifies a module regulating the mechanics of epithelia.

Interplay of MPP5a with Rab11 synergistically builds epithelial apical polarity and zonula adherens.

Adherens junction remodeling regulated by apical polarity proteins constitutes a major driving force for tissue morphogenesis, although the precise mechanism remains inconclusive. Here, we report that, in zebrafish, the Crumbs complex component MPP5a interacts with small GTPase Rab11 in Golgi to transport cadherin and Crumbs components synergistically to the apical domain, thus establishing apical epithelial polarity and adherens junctions. In contrast, Par complex recruited by MPP5a is incapable of interacting with Rab11 but might assemble cytoskeleton to facilitate cadherin exocytosis. In accordance, dysfunction of MPP5a induces an invasive migration of epithelial cells. This adherens junction remodeling pattern is frequently observed in zebrafish lens epithelial cells and neuroepithelial cells. The data identify an unrecognized MPP5a-Rab11 complex and describe its essential role in guiding apical polarization and zonula adherens formation in epithelial cells.

The extracellular and intracellular regions of Crb2a play distinct roles in guiding the formation of the apical zonula adherens.

The transmembrane protein Crumbs (Crb), a key regulator of apical polarity, has a known involvement in establishment of the apical zonula adherens in epithelia, although the precise mechanism remains elusive. The zonula adherens are required to maintain the integrity and orderly arrangement of epithelia. Loss of the zonula adherens leads to morphogenetic defects in the tissues derived from epithelium. In this study, we revealed that the intracellular tail of Crb2a promoted the apical distribution of adherens junctions (AJs) in zebrafish retinal and lens epithelia, but caused assembly into unstable punctum adherens-like adhesion plaques. The extracellular region of Crb2a guided the transformation of AJs from the punctum adherens into stable zonula adherens. Accordingly, a truncated form of Crb2a lacking the extracellular region (Crb2aΔEX) could only partially rescue the retinal patterning defects in crb2a null mutant zebrafish (crb2am289). By contrast, constitutive over-expression of Crb2aΔEX disrupted the integrity of the outer limiting membrane in photoreceptors, which is derived from the zonula adherens of the retinal neuroepithelium. This study demonstrated that both the extracellular region and the intracellular tail of Crb2a are required to guide the formation of the apical zonula adherens.

Rap1, Canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor.

In Drosophila epithelial cells, apical exclusion of Bazooka (the Drosophila Par3 protein) defines the position of the zonula adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. Here, we show that the small GTPase Rap1, its effector Canoe (Cno) and the Cdc42 effector kinase Mushroom bodies tiny (Mbt), converge in regulating epithelial morphogenesis by coupling stabilization of the adherens junction (AJ) protein E-Cadherin and Bazooka retention at the ZA. Furthermore, our results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating that their functions during ZA morphogenesis are interlinked. In this context, we find the Rap1-GEF Dizzy is enriched at the ZA and our results suggest that it promotes Rap1 activity during ZA morphogenesis. Altogether, we propose the Dizzy, Rap1 and Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis.

PLEKHA7 defines an apical junctional complex with cytoskeletal associations and miRNA-mediated growth implications.

E-cadherin-p120 catenin complexes are essential for adherens junction (AJ) formation and for the maintenance of the normal epithelial phenotype. PLEKHA7 was originally identified as a member of this complex that tethers microtubules to the AJs and supports their overall integrity. Recently, we revealed that PLEKHA7 regulates cellular behavior via miRNAs by associating with the microprocessor complex at the apical zonula adherens (ZA). We have also identified a new set of PLEKHA7 interacting partners at the apical ZA, via proteomics. Our analysis shows that the main groups of proteins associating with PLEKHA7 are cytoskeletal-related and RNA-binding proteins. Here, we provide extended evidence for association of PLEKHA7 with several of these proteins. We also show that PLEKHA7 loss activates the actin regulator cofilin in a p120-dependent manner, providing an explanation for the effects of PLEKHA7 on the cortical actin ring. Interestingly, PLEKHA7 regulates the levels and associates with PP1α, a phosphatase responsible for cofilin activation. Finally, we clarify the mode of regulation of the oncogenic miR-19a by PLEKHA7. Overall, our findings support a multi-layered role of PLEKHA7 in converging cytoskeletal dynamics and miRNA-mediated growth regulation at the ZA, with potentially critical implications in cancer that warrant further investigation.

Tropomyosin isoforms support actomyosin biogenesis to generate contractile tension at the epithelial zonula adherens.

Epithelial cells generate contractile forces at their cell-cell contacts. These are concentrated at the specialized apical junction of the zonula adherens (ZA), where a ring of stabilized E-cadherin lies adjacent to prominent actomyosin bundles. Coupling of adhesion and actomyosin contractility yields tension in the junction. The biogenesis of junctional contractility requires actin assembly at the ZA as well as the recruitment of nonmuscle myosin II, but the molecular regulators of these processes are not yet fully understood. We now report a role for tropomyosins 5NM1 (Tm5NM1) and 5NM2 (Tm5NM2) in their generation. Both these tropomyosin isoforms were found at the ZA and their depletion by RNAi or pharmacological inhibition reduced both F-actin and myosin II content at the junction. Photoactivation analysis revealed that the loss of F-actin was attributable to a decrease in filament stability. These changes were accompanied by a decrease in E-cadherin content at junctions. Ultimately, both long-term depletion of Tm5NM1/2 and acute inhibition with drugs caused junctional tension to be reduced. Thus these tropomyosin isoforms are novel contributors to junctional contractility and integrity.

The interdependence of the Rho GTPases and apicobasal cell polarity.

Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease.

E-cadherin supports steady-state Rho signaling at the epithelial zonula adherens.

In simple polarized epithelial cells, the Rho GTPase commonly localizes to E-cadherin-based cell-cell junctions, such as the zonula adherens (ZA), where it regulates the actomyosin cytoskeleton to support junctional integrity and tension. An important question is how E-cadherin contributes to Rho signaling, notably whether junctional Rho may depend on cadherin adhesion. We sought to investigate this by assessing Rho localization and activity in epithelial monolayers depleted of E-cadherin by RNAi. We report that E-cadherin depletion reduced both Rho and Rho-GTP at the ZA, an effect that was rescued by expressing a RNAi-resistant full-length E-cadherin transgene. This impact on Rho signaling was accompanied by reduced junctional localization of the Rho GEF ECT2 and the centralspindlin complex that recruits ECT2. Further, the Rho signaling pathway contributes to the selective stabilization of E-cadherin molecules in the apical zone of the cells compared with E-cadherin at the lateral surface, thereby creating a more defined and restricted pool of E-cadherin that forms the ZA. Thus, E-cadherin and Rho signaling cooperate to ensure proper ZA architecture and function.