Transient Calcium Signaling Reprograms YAP Mechanosensitivity through Actin Remodeling

YAP is a central regulator of cell fate, proliferation, and tissue homeostasis that integrates physical cues from the extracellular matrix (ECM). While stiff environments canonically drive YAP nuclear localization and soft environments promote cytoplasmic sequestration, the logic by which cells integrate mechanical inputs with transient biochemical signals remains poorly defined. Here, we examine how intracellular calcium transients interact with substrate compliance to regulate YAP dynamics and transcriptional outputs across epithelial, myoblast, and fibroblast lineages. Using collagen-coated polyacrylamide hydrogels with tunable mechanics, we show that substrate compliance shapes the kinetics of calcium signaling, calcium-mediated actin remodeling, and YAP nuclear shuttling. We demonstrate that calcium signaling can transiently relax baseline mechanical constraints on YAP activity, triggering nuclear translocation and transcriptional activation even on compliant or non-adhesive substrates where YAP is classically suppressed. We identify substrate compliance as a biophysical regulator that filters the transduction of calcium transients into gene-specific programs. Targets such as CYR61 are induced across all mechanical contexts tested, whereas targets like CTGF and AREG require permissive mechanical conditions. These results suggest a preliminary model in which the mechanical state of the cell tunes the activation barrier for YAP-dependent transcription, enabling context-dependent responses to universal biochemical triggers. We show that destabilizing the F-actin cytoskeleton by promoting depolymerization or sequestering actin monomers results in attenuation of the calcium-mediated YAP activity, while stabilizing F-actin results in amplification YAP activity in response to calcium stimulation. This work reveals a fundamental mechanism by which transient signals integrate with matrix mechanics to produce distinct YAP-dependent outcomes, suggesting new strategies for controlling cell fate in regenerative medicine and engineered tissues. STATEMENT OF SIGNIFICANCE: Although YAP regulation by steady-state mechanical cues is well established, how these cues interact with dynamic biochemical signals such as calcium transients to control gene-specific transcription remains unclear. We show that intracellular calcium transients act as rapid, tunable inputs that promote YAP nuclear localization and target gene expression, even on soft substrates that normally suppress YAP activity. Low-threshold gene targets are activated broadly, whereas high-threshold targets require stiffer environments, positioning substrate mechanics as a biophysical gate that shapes the magnitude, kinetics, and gene specificity of calcium-mediated YAP responses. These findings provide insights into how cells integrate transient calcium signals with steady-state matrix mechanics to regulate proliferation, differentiation, and tissue behavior, and provide a framework for guiding cell fate in engineered tissues, organoids, and regenerative medicine.