Thus both in Drosophila and

Thus, both in Drosophila and mammals, the Hippo pathway responds to cell–cell junctions via an apical NF2/Mer-containing complex. Indeed, Hippo signalling and NF2 are required for contact inhibition of growth in cell culture, which is thought to reflect an in vivo function as a sensor of tissue crowding [1].
The 6063 cytoskeleton and Hippo signalling As well as sensing neighbours through cell adhesion molecules, cell junctions are associated with a robust contractile acto-myosin network, which maintains epithelial integrity, receives mechanical inputs, and also regulates Yki/YAP [37, 38, 39, 40, 41]. The mechanism(s) linking actin/mechanotransduction to Yki/YAP activity remains a source of debate. A number of reports suggest that cytoskeletal tension promotes YAP activity independently of LATS [38, 42•, 43, 44]. On the other hand, F-actin disruption leads to increased LATS activity and YAP phosphorylation [41, 45•, 46]. Moreover, in flies, latrunculin-B treatment does not rescue Yki nuclear localisation in wts mutant cells [], and cytoskeletal tension blocks Wts activity via Ajuba and α-catenin, suggesting that acto-myosin may act upstream of Wts [19]. Actin/tension-mediated YAP modulation also involves AMOT, since binding of AMOT to F-actin prevents the AMOT:YAP interaction and releases YAP from inhibition [43, 48, 49, 50]. AMOT:F-actin association is antagonised by LATS-mediated phosphorylation of AMOT [48, 49, 50] and F-actin disruption [43]. Inhibition of the AMOT:YAP interaction by shear stress-induced actin polymerisation links blood flow to blood vessel maintenance by YAP in developing zebrafish []. Thus, LATS-dependent and LATS-independent modes of YAP regulation by actin appear to co-exist. However, actin cytoskeleton integrity is dominant in determining YAP activity, since for instance YAP mutants for the inhibitory LATS sites retain sensitivity to plating on soft substrates and treatment with actin depolymerisation drugs in cell culture [42•, 52•]. The relative contributions of these mechanisms under physiological mechanical environments and tissue architecture is an important area for further research, particularly in light of a recent report suggesting that eliminating the basement membrane (and thereby tension at a global scale) in developing Drosophila imaginal discs does not alter Yki activity [].
Compartmentalisation of Hippo signalling: basolateral membranes The basal polarity complex comprised of Scrib, Dlg and Lgl, which is known to antagonise the function of apical determinants such as Crb also regulates Hippo signalling (reviewed in [54]) (Figure 2). Par-1/MARK kinases are crucial basolateral polarity regulators that have recently been associated with Hippo signalling regulation [55]. In Drosophila, Par-1 activates Yki by phosphorylating Hpo at S30, restricting its activity [56]. This mechanism is conserved, at least in MDA-MB-231 cells, where MARK4 phosphorylates and inhibits MST and SAV, preventing interaction with LATS [57]. In addition, MARK3 negatively regulates MST in a DLG5-dependent manner [58], and in pre-implantation mouse embryos, MARK2/3 inhibit AMOT junctional localisation, thereby positively regulating YAP activity [35]. However, the role of MARK kinases is complex, since MARK1/3/4 have also been described as YAP negative regulators, acting downstream of LKB1 to regulate Scrib localisation and Hippo kinase activity [59]. As MARKs interact with both Scrib [59] and Dlg [58], perhaps their differential binding partners determine whether they stimulate or inhibit YAP. The integrin-containing focal adhesions (FAs) are the sites of cell attachment to the underlying extracellular matrix (ECM) and, as such, are key for the cell's ability to sense its mechanical environment. Integrin activation promotes YAP/TAZ nuclear accumulation and activation [60, 61•, 62]. YAP itself promotes FA gene expression, FA formation [], as well as ECM stiffening [64], thereby initiating a feed-forward loop that modulates the biophysical properties of tissues. Rho GTPases are downstream effectors of integrins that generally promote YAP/TAZ activity [62, 65, 66]. Integrins can also signal through Src family kinases to regulate YAP/TAZ [61•, 67•]. FAK/Src signalling can activate YAP both by direct phosphorylation of three tyrosine residues (Y341, Y357 and Y394) [] and indirectly via inhibitory phosphorylation of LATS [67•, 69•]. It is interesting to note that cell–cell junction signalling (YAP inhibition) and cell–ECM signalling (YAP activation) play antagonistic roles in YAP regulation, with the junctional protein αE-catenin acting as a mediator of this tug-of-war by antagonising integrin signalling [61•, 68•, 70]. Finally, integrins can inactivate Hippo signalling via integrin-linked kinase (ILK)-mediated phosphorylation of MYPT-PP1, which leads to NF2 inactivation [71]. The ECM proteoglycan Agrin has been reported to activate YAP both via ILK [], and by releasing YAP from an inhibitory complex with its receptor Dystroglycan [73••, 74••]. Interestingly, Agrin can promote YAP-dependent cardiac regeneration upon injury and its disappearance during the first week of life is thought to cause the loss of regenerative potential in the adult compared with the neonatal mouse heart [73••, 74••].