Suzie Verma

Cortactin is necessary for E-cadherin–mediated contact formation and actin reorganization

Classical cadherin adhesion molecules are key determinants of cell–cell recognition during development and in post-embryonic life. A decisive step in productive cadherin-based recognition is the conversion of nascent adhesions into stable zones of contact. It is increasingly clear that such contact zone extension entails active cooperation between cadherin adhesion and the force-generating capacity of the actin cytoskeleton. Cortactin has recently emerged as an important regulator of actin dynamics in several forms of cell motility. We now report that cortactin is recruited to cell–cell adhesive contacts in response to homophilic cadherin ligation. Notably, cortactin accumulates preferentially, with Arp2/3, at cell margins where adhesive contacts are being extended. Recruitment of cortactin is accompanied by a ligation-dependent biochemical interaction between cortactin and the cadherin adhesive complex. Inhibition of cortactin activity in cells blocked Arp2/3-dependent actin assembly at cadherin adhesive contacts, significantly reduced cadherin adhesive contact zone extension, and perturbed both cell morphology and junctional accumulation of cadherins in polarized epithelia. Together, our findings identify a necessary role for cortactin in the cadherin–actin cooperation that supports productive contact formation.

N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway

N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WASP WH1 domain. We conclude that N-WASP–WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity.

Tension-Sensitive Actin Assembly Supports Contractility at the Epithelial Zonula Adherens

BACKGROUND Actomyosin-based contractility acts on cadherin junctions to support tissue integrity and morphogenesis. The actomyosin apparatus of the epithelial zonula adherens (ZA) is built by coordinating junctional actin assembly with Myosin II activation. However, the physical interaction between Myosin and actin filaments that is necessary for contractility can induce actin filament turnover, potentially compromising the contractile apparatus itself. RESULTS We now identify tension-sensitive actin assembly as one cellular solution to this design paradox. We show that junctional actin assembly is maintained by contractility in established junctions and increases when contractility is stimulated. The underlying mechanism entails the tension-sensitive recruitment of vinculin to the ZA. Vinculin, in turn, directly recruits Mena/VASP proteins to support junctional actin assembly. By combining strategies that uncouple Mena/VASP from vinculin or ectopically target Mena/VASP to junctions, we show that tension-sensitive actin assembly is necessary for junctional integrity and effective contractility at the ZA. CONCLUSIONS We conclude that tension-sensitive regulation of actin assembly represents a mechanism for epithelial cells to resolve potential design contradictions that are inherent in the way that the junctional actomyosin system is assembled. This emphasizes that maintenance and regulation of the actin scaffolds themselves influence how cells generate contractile tension.

A WAVE2–Arp2/3 actin nucleator apparatus supports junctional tension at the epithelial zonula adherens

WAVE2–Arp2/3 is a major nucleator of actin assembly at the zonula adherens and likely acts in response to junctional Rac signaling. It supports myosin II recruitment to, and tension generation at, the junction.

Cortactin scaffolds Arp2/3 and WAVE2 at the epithelial zonula adherens.

Background: Productive epithelial interactions require actin filament assembly at E-cadherin adhesions. Results: Cortactin localizes to the zonula adherens through interactions with E-cadherin and N-WASP; there it recruits Arp2/3 and WAVE2 by separate mechanisms to support actin nucleation. Conclusion: Cortactin acts as a coincident scaffold. Significance: Cortactin can regulate the dynamic integration of cadherin adhesion with the actin cytoskeleton. Cadherin junctions arise from the integrated action of cell adhesion, signaling, and the cytoskeleton. At the zonula adherens (ZA), a WAVE2-Arp2/3 actin nucleation apparatus is necessary for junctional tension and integrity. But how this is coordinated with cadherin adhesion is not known. We now identify cortactin as a key scaffold for actin regulation at the ZA, which localizes to the ZA through influences from both E-cadherin and N-WASP. Using cell-free protein expression and fluorescent single molecule coincidence assays, we demonstrate that cortactin binds directly to the cadherin cytoplasmic tail. However, its concentration with cadherin at the apical ZA also requires N-WASP. Cortactin is known to bind Arp2/3 directly (Weed, S. A., Karginov, A. V., Schafer, D. A., Weaver, A. M., Kinley, A. W., Cooper, J. A., and Parsons, J. T. (2000) J. Cell Biol. 151, 29–40). We further show that cortactin can directly bind WAVE2, as well as Arp2/3, and both these interactions are necessary for actin assembly at the ZA. We propose that cortactin serves as a platform that integrates regulators of junctional actin assembly at the ZA.

Cortactin Is a Functional Target of E-cadherin-activated Src Family Kinases in MCF7 Epithelial Monolayers

Src family kinases (SFKs) signal in response to E-cadherin to support cadherin adhesion and the integrity of cell-cell contacts (McLachlan, R. W., Kraemer, A., Helwani, F. M., Kovacs, E. M., and Yap, A. S. (2007) Mol. Biol. Cell 18, 3214–3223). We now identify the actin-regulatory protein, cortactin, as a target of E-cadherin-activated SFK signaling. Tyr-phosphorylated cortactin was found at cell-cell contacts in established epithelial monolayers, and cortactin became acutely tyrosine-phosphorylated when E-cadherin adhesion was engaged. In all circumstances, cortactin tyrosine phosphorylation was blocked by inhibiting SFK signaling. Importantly, Tyr-phosphorylated cortactin was necessary to preserve the integrity of cadherin contacts and the perijunctional actin cytoskeleton. Moreover, expression of a phosphomimetic cortactin mutant could prevent SFK blockade from disrupting cadherin organization, thereby placing cortactin functionally downstream of SFK signaling at cadherin adhesions. We conclude that SFK and cortactin constitute an important signaling pathway that functionally links E-cadherin adhesion and the actin cytoskeleton.

Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions

In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur.

Tuba and N-WASP function cooperatively to position the central lumen during epithelial cyst morphogenesis.

The process of epithelial lumenogenesis requires coordination of a network of signaling machinery communicated to each cell through subsequent cell divisions. Formation of a single hollow lumen has previously been shown to require Tuba, a Cdc42 GEF, for Cdc42 activation and correct spindle orientation. Using a Caco-2 model of lumenogenesis, we show that knockdown (KD) of the actin regulator N-WASP, causes a multilumen phenotype similar to Tuba KD. Defects in lumenogenesis in Tuba KD and N-WASP KD cells are observed at the two cell stage with inappropriate marking of the pre-apical patch (PAP) – the precursor to lumen formation. Strikingly, both Tuba and N-WASP depend on each other for localization to the PAP. We conclude that N-WASP functions cooperatively with Tuba to facilitate lumenogenesis and this requires the polyproline region of N-WASP.

An RPTPα/Src Family Kinase /Rap1 signaling module recruits Myosin IIB to support contractile tension at apical E-cadherin junctions.

The role of myosin IIB in junctional contractility and its mode of regulation are not well understood. It is demonstrated that junctional recruitment of myosin IIB requires the activation of a receptor-type protein tyrosine phosphatase alpha–Src family kinase–Rap1 pathway. This reinforces the concept that E-cadherin–based signaling recruits distinct myosin II paralogues to generate contractile tension.

Tyrosine dephosphorylated cortactin downregulates contractility at the epithelial zonula adherens through SRGAP1

Contractile adherens junctions support cell−cell adhesion, epithelial integrity, and morphogenesis. Much effort has been devoted to understanding how contractility is established; however, less is known about whether contractility can be actively downregulated at junctions nor what function this might serve. We now identify such an inhibitory pathway that is mediated by the cytoskeletal scaffold, cortactin. Mutations of cortactin that prevent its tyrosine phosphorylation downregulate RhoA signaling and compromise the ability of epithelial cells to generate a contractile zonula adherens. This is mediated by the RhoA antagonist, SRGAP1. We further demonstrate that this mechanism is co-opted by hepatocyte growth factor to promote junctional relaxation and motility in epithelial collectives. Together, our findings identify a novel function of cortactin as a regulator of RhoA signaling that can be utilized by morphogenetic regulators for the active downregulation of junctional contractility.Epithelial cell-cell adhesions are contractile junctions, but whether contractility can be down-regulated is not known. Here the authors report how tyrosine dephosphorylation of the cytoskeletal scaffold, cortactin, recruits the RhoA antagonist SRGAP1 to relax adherens junctions in response to HGF.