Villalobos, C.; Sadeghifar, A.; Maggiorani, J.; Delapena, J.; McDaniel, G.; Driscoll, T. P.

Cells sense the mechanical properties of their environment through physical engagement and spreading, with high stiffness driving nuclear translocation of the mechanosensitive transcription factor YAP. Restriction of cell spread area or environmental stiffness both inhibit YAP activation and nuclear translocation. The Arp2/3 complex plays a critical role in polymerization of branched actin networks that drive cell spreading, protrusion, and migration. While YAP activation has been closely linked to cellular spreading, the specific role of actin branching in force buildup and YAP activation is unclear. To assess the role of actin branching in this process, we measured cell spreading, YAP nuclear translocation, force on the adhesion adaptor protein Talin (FRET tension sensor), and extracellular forces (traction force microscopy, TFM) in 3T3 cells with and without inhibition of actin branching. The results indicate that YAP activation still occurs when actin branching and cell spreading is reduced. Interestingly, while actin de-branching resulted in decreased force on talin, relatively little change in average traction stress was observed, highlighting the distinct difference between molecular level and cellular level force regulation of YAP. While cell spreading is a driver of YAP nuclear translocation, this is likely through indirect effects. Changes in cell spreading induced by actin branching inhibition do not significantly perturb YAP activation. Additionally, this work provides evidence that focal adhesion molecular forces are not a direct regulator of YAP activation.