The variant senescence-associated secretory phenotype induced by centrosome amplification constitutes a pathway that activates hypoxia-inducible factor-1α.

The senescence-associated secretory phenotype (SASP) can promote paracrine invasion while suppressing tumour growth, thus generating complex phenotypic outcomes. Likewise, centrosome amplification can induce proliferation arrest yet also facilitate tumour invasion. However, the eventual fate of cells with centrosome amplification remains elusive. Here, we report that centrosome amplification induces a variant of SASP, which constitutes a pathway activating paracrine invasion. The centrosome amplification-induced SASP is non-canonical as it lacks the archetypal detectable DNA damage and prominent NF-κB activation, but involves Rac activation and production of reactive oxygen species. Consequently, it induces hypoxia-inducible factor 1α and associated genes, including pro-migratory factors such as ANGPTL4. Of note, cellular senescence can either induce tumourigenesis through paracrine signalling or conversely suppress tumourigenesis through p53 induction. By analogy, centrosome amplification-induced SASP may therefore be one reason why extra centrosomes promote malignancy in some experimental models but are neutral in others.

Spatio-Temporal Regulation of RhoGTPases Signaling by Myosin II.

RhoGTPase activation of non-muscle myosin II regulates cell division, extrusion, adhesion, migration, and tissue morphogenesis. However, the regulation of myosin II and mechanotransduction is not straightforward. Increasingly, the role of myosin II on the feedback regulation of RhoGTPase signaling is emerging. Indeed, myosin II controls RhoGTPase signaling through multiple mechanisms, namely contractility driven advection, scaffolding, and sequestration of signaling molecules. Here we discuss these mechanisms by which myosin II regulates RhoGTPase signaling in cell adhesion, migration, and tissue morphogenesis.

Mammalian Diaphanous 1 Mediates a Pathway for E-cadherin to Stabilize Epithelial Barriers through Junctional Contractility.

Formins are a diverse class of actin regulators that influence filament dynamics and organization. Several formins have been identified at epithelial adherens junctions, but their functional impact remains incompletely understood. Here, we tested the hypothesis that formins might affect epithelial interactions through junctional contractility. We focused on mDia1, which was recruited to the zonula adherens (ZA) of established Caco-2 monolayers in response to E-cadherin and RhoA. mDia1 was necessary for contractility at the ZA, measured by assays that include a FRET-based sensor that reports molecular-level tension across αE-catenin. This reflected a role in reorganizing F-actin networks to form stable bundles that resisted myosin-induced stress. Finally, we found that the impact of mDia1 ramified beyond adherens junctions to stabilize tight junctions and maintain the epithelial permeability barrier. Therefore, control of tissue barrier function constitutes a pathway for cadherin-based contractility to contribute to the physiology of established epithelia.

Mutations in SIPA1L3 cause eye defects through disruption of cell polarity and cytoskeleton organization.

Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1, with a de novo balanced translocation, 46,XY,t(2;19)(q37.3;q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1 and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3(-/-) mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition in the lens. Patient 2 with cataracts was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization.

Active contractility at E-cadherin junctions and its implications for cell extrusion in cancer.

Cellular contractility regulates tissue cohesion and morphogenesis. In epithelia, E-cadherin adhesion couples the contractile cortices of neighboring cells together to produce tension at junctions that can be transmitted across the epithelium in a planar fashion. We have recently demonstrated that contractility is also patterned in the apical-lateral axis within epithelial junctions. Our findings highlight the role that cytoskeletal regulation plays in controlling the levels of intra-junctional tension. Of note, dysregulation of this apicolateral pattern of tension can drive oncogenic cell extrusion. In this article, we provide a detailed description of the actomyosin cytoskeleton organization during oncogenic extrusion and discuss the implications of cell extrusion in cancer.

An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions.

Cell-cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell-cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha-Src family kinase-Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin-based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.

Self-organizing actomyosin patterns on the cell cortex at epithelial cell-cell junctions.

The behavior of actomyosin critically determines morphologically distinct patterns of contractility found at the interface between adherent cells. One such pattern is found at the apical region (zonula adherens) of cell-cell junctions in epithelia, where clusters of the adhesion molecule E-cadherin concentrate in a static pattern. Meanwhile, E-cadherin clusters throughout lateral cell-cell contacts display dynamic movements in the plane of the junctions. To gain insight into the principles that determine the nature and organization of these dynamic structures, we analyze this behavior by modeling the 2D actomyosin cell cortex as an active fluid medium. The numerical simulations show that the stability of the actin filaments influences the spatial structure and dynamics of the system. We find that in addition to static Turing-type patterns, persistent dynamic behavior occurs in a wide range of parameters. In the 2D model, mechanical stress-dependent actin breakdown is shown to produce a continuously changing network of actin bridges, whereas with a constant breakdown rate, more isolated clusters of actomyosin tend to form. The model qualitatively reproduces the dynamic and stable patterns experimentally observed at the junctions between epithelial cells.

Pulsatile contractility of actomyosin networks organizes the cellular cortex at lateral cadherin junctions.

The physical properties of cells reflect how the structure and dynamics of the actomyosin cortex are coupled to the plasma membrane. In epithelia, adhesive E-cadherin clusters associate with the cell cortex to assemble the junctional actomyosin that participates in epithelial morphogenesis. E-cadherin is present not only at the apical zonula adherens (ZA), but also distributed throughout the lateral adherens junction (LAJ) below the ZA. However, the organizational dynamics of the actomyosin network at the LAJs remains elusive. To address this, we used quantitative real-time imaging to characterize the dynamics of actomyosin contractility at lateral cadherin contacts. Here, we report that contractility is coordinated into smaller actomyosin rings that link cadherin clusters together within the larger cortical network at the lateral junctions. We conclude that Myosin II activity determines the contractility of actomyosin cables between cadherin clusters to propagate pulsatility across lateral cell-cell contacts.

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.

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

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.

Cortical F-actin stabilization generates apical-lateral patterns of junctional contractility that integrate cells into epithelia.

E-cadherin cell-cell junctions couple the contractile cortices of epithelial cells together, generating tension within junctions that influences tissue organization. Although junctional tension is commonly studied at the apical zonula adherens, we now report that E-cadherin adhesions induce the contractile actomyosin cortex throughout the apical-lateral axis of junctions. However, cells establish distinct regions of contractile activity even within individual contacts, producing high tension at the zonula adherens but substantially lower tension elsewhere. We demonstrate that N-WASP (also known as WASL) enhances apical junctional tension by stabilizing local F-actin networks, which otherwise undergo stress-induced turnover. Further, we find that cells are extruded from monolayers when this pattern of intra-junctional contractility is disturbed, either when N-WASP redistributes into lateral junctions in H-Ras(V12)-expressing cells or on mosaic redistribution of active N-WASP itself. We propose that local control of actin filament stability regulates the landscape of intra-junctional contractility to determine whether or not cells integrate into epithelial populations.

Multicomponent analysis of junctional movements regulated by myosin II isoforms at the epithelial zonula adherens.

The zonula adherens (ZA) of epithelial cells is a site of cell-cell adhesion where cellular forces are exerted and resisted. Increasing evidence indicates that E-cadherin adhesion molecules at the ZA serve to sense force applied on the junctions and coordinate cytoskeletal responses to those forces. Efforts to understand the role that cadherins play in mechanotransduction have been limited by the lack of assays to measure the impact of forces on the ZA. In this study we used 4D imaging of GFP-tagged E-cadherin to analyse the movement of the ZA. Junctions in confluent epithelial monolayers displayed prominent movements oriented orthogonal (perpendicular) to the ZA itself. Two components were identified in these movements: a relatively slow unidirectional (translational) component that could be readily fitted by least-squares regression analysis, upon which were superimposed more rapid oscillatory movements. Myosin IIB was a dominant factor responsible for driving the unilateral translational movements. In contrast, frequency spectrum analysis revealed that depletion of Myosin IIA increased the power of the oscillatory movements. This implies that Myosin IIA may serve to dampen oscillatory movements of the ZA. This extends our recent analysis of Myosin II at the ZA to demonstrate that Myosin IIA and Myosin IIB make distinct contributions to junctional movement at the ZA.

Hepatocyte growth factor acutely perturbs actin filament anchorage at the epithelial zonula adherens.

Cadherin adhesion molecules function in close cooperation with the actin cytoskeleton. At the zonula adherens (ZA) of polarized epithelial cells, E-cadherin adhesion induces the cortical recruitment of many key cytoskeletal regulators, which act in a dynamic integrated system to regulate junctional integrity and cell-cell interactions. This capacity for the cytoskeleton to support the ZA carries the implication that regulators of the junctional cytoskeleton might also be targeted to perturb junctional integrity. In this report, we now provide evidence for this hypothesis. We show that hepatocyte growth factor (HGF), which is well-known to disrupt cell-cell interactions, acutely perturbs ZA integrity much more rapidly than generally appreciated. This is accompanied by significant loss of junctional F-actin, a process that reflects loss of filament anchorage at the junctions. We demonstrate that this involves uncoupling of the unconventional motor myosin VI from junctional E-cadherin, a novel effect of HGF that is mediated by intracellular calcium. We conclude that regulators of the junctional cytoskeleton are likely to be major targets for cadherin junctions to be acutely modulated in development and perturbed in disease.