YAP Nuclear Localization in the Absence of Cell-Cell Contact Is Mediated by a Filamentous Actin-dependent, Myosin II- and Phospho-YAP-independent Pathway during Extracellular Matrix Mechanosensing

Abstract

Cell-cell contact inhibition and the mechanical environment of cells have both been shown to regulate YAP nuclear localization to modulate cell proliferation. Changes in cellular contractility by genetic, pharmacological, and matrix stiffness perturbations regulate YAP nuclear localization. However, because contractility and F-actin organization are interconnected cytoskeletal properties, it remains unclear which of these distinctly regulates YAP localization. Here we show that in the absence of cell-cell contact, actomyosin contractility suppresses YAP phosphorylation at Ser 112 , however, neither loss of contractility nor increase in YAP phosphorylation is sufficient for its nuclear exclusion. We find that actin cytoskeletal integrity is essential for YAP nuclear localization, and can override phosphoregulation or contractility-mediated regulation of YAP nuclear localization. This actin-mediated regulation is conserved during mechanotransduction, as substrate compliance increased YAP phosphorylation and reduced cytoskeletal integrity leading to nuclear exclusion of both YAP and Ser(P) 112 -YAP. These data provide evidence for two actin-mediated pathways for YAP regulation; one in which actomyosin contractility regulates YAP phosphorylation, and a second that involves cytoskeletal integrity-mediated regulation of YAP nuclear localization independent of contractility. We suggest that in non-contact inhibited cells, this latter mechanism may be important in low stiffness regimes, such as may be encountered in physiological environments.

Fluid shear stress activates YAP1 to promote cancer cell motility

“…It is possible that incomplete inhibition of FAK by PF-562271 could allow WSS to partially stimulate YAP1 nuclear localization; however, expression of pFAK Y397 following PF-562271 treatment resembled cells in suspension, which contain negligible levels of FAK activity. Alternatively, WSS may activate YAP1 via FAK-independent mechanisms, consistent with previous studies suggesting that focal adhesions are not required for YAP1 nuclear localization741.…”

Mutual dependence of the MRTF–SRF and YAP–TEAD pathways in cancer-associated fibroblasts is indirect and mediated by cytoskeletal dynamics

“…Mechanical cues, including substrate stiffness, were shown previously to promote nuclear localization of YAP and TAZ (Wada et al 2011;Calvo et al 2013;Das et al 2016;Dupont 2016). Application of force to integrins induces MRTF-A nuclear accumulation (Zhao et al 2007), and substrate stiffness can activate it in lung fibroblasts and mesenchymal stem cells (Huang et al 2012;Buxboim et al 2014).…”

“…YAP and TAZ, which bind DNA in association with members of the TEAD family of DNA-binding cofactors, were first characterized as effectors of the Hippo growth control pathway (for review, see Meng et al 2016). In addition, they also respond to Rho-GTPase signaling, accumulating in the nucleus in response to high cytoskeletal tension induced by mechanical cues (Dupont et al 2011;Wada et al 2011;Das et al 2016). Previous studies have shown that YAP is activated in CAFs and that YAP-TEAD signaling maintains their contractile and proinvasive properties (Calvo et al 2013;Dupont 2016).…”

Hydrogel-based microchannels to measure confinement- and stiffness-sensitive Yes-associated-protein activity in epithelial clusters

“…We have also shown that nuclear YAP localization increases in sparser epithelial clusters cultured on 2D substrates, which is in agreement with another recent study. [26] Notably, we report that denser epithelial clusters exhibit lower YAP activity even on stiff ECM. However, our measurements for epithelial clusters inside channels of varying width show that the densitydependent YAP activity does not hold true in confined ECM settings.…”

“…Note that the final cell densities on soft and stiff ECMs are different because we found a sparser distribution of cells on the stiffer ECM, which could be due to increased mechanosensitive spreading and EMT. [24,26,27] . These results indicate that the density of cells in an epithelial cluster significantly influenced the mechanosensitive YAP localization when epithelial cells were grown on stiff substrates.…”