Timing Mechanotransduction: Mechanically Dynamic Biomaterials Reveal the Temporal Hierarchy of YAP/TAZ Control Nodes.

Mechanotransduction is a cardinal regulator of cell behavior, yet its temporal unfolding and hierarchy remain poorly defined. Here, we develop dynamically softening polyacrylamide hydrogels that enable in situ modulation of substrate stiffness across physiological ranges while preserving integrin-mediated adhesion. Time-resolved analyses reveal a biphasic response to extracellular softening. YAP/TAZ are abruptly inactivated at an early stiffness threshold, coincident with rapid collapse of the subnuclear adhesion-F-actin-LINC nucleo-cytoskeletal continuum. At this step, peripheral focal adhesions remain unexpectedly resilient, persisting while undergoing centripetal remodeling. Disrupting SUN2 lowers the mechanosensitive threshold, whereas increased contractility raises it, still in LINC-dependent manner. Early YAP/TAZ shutoff is accompanied by rapid microtubule reorganization away from a centrosomal aster, and by AMOT accumulation. Changes in nuclear flattening, cell rounding, and peripheral adhesion collapse emerge later at lower stiffness thresholds. Mechanotransduction is directionally asymmetric when cells are challenged in situ: YAP/TAZ switch off abruptly at a defined softness threshold, whereas reactivation is efficiently achieved only by cyclic (not static) strain, consistent with ratchet-like temporal integration. Together, these findings establish a spatiotemporal framework for dynamic mechanotransduction and prioritize the nodes that operate on physiologically relevant timescales, providing timing-based constraints to distinguish initiating events from downstream adaptations.