Abstract
Extracellular matrix (ECM) stiffness is a critical physical cue that directs stem cell lineage specification and tissue homeostasis, yet the molecular mechanisms decoding mechanical signals into transcriptional programs remain incompletely understood. Here, we investigate the dynamics of YAP/TAZ nucleocytoplasmic shuttling as a central mechanotransduction module in stiffness-mediated cell fate decisions. Using a combination of live-cell imaging, fluorescence recovery after photobleaching (FRAP), and pharmacological perturbations in mesenchymal stem cells (MSCs) cultured on tunable polyacrylamide hydrogels (1–50 kPa), we demonstrate that substrate stiffness modulates the frequency, amplitude, and duration of YAP/TAZ nuclear translocation events. On stiff matrices (25–50 kPa), YAP/TAZ exhibited sustained nuclear localization with rapid shuttling kinetics (mean nuclear residence time 12.3 ± 2.1 min), whereas on soft matrices (1–5 kPa), shuttling was sporadic with prolonged cytoplasmic retention. Mathematical modeling revealed that nuclear shuttling frequency correlates with osteogenic differentiation, while sustained nuclear localization promotes proliferation. Pharmacological inhibition of actin polymerization (latrunculin A) and Rho-associated kinase (Y-27632) abolished stiffness-dependent shuttling dynamics and disrupted lineage commitment. Furthermore, we identified that YAP/TAZ shuttling cooperates with Notch signaling to fine-tune cell fate decisions. Our findings establish YAP/TAZ nuclear shuttling dynamics as a key determinant of mechanosensitive cell fate, providing a framework for understanding how physical microenvironmental cues are encoded into discrete transcriptional outputs.
Keywords
YAP/TAZ, nuclear shuttling, mechanotransduction, matrix stiffness, stem cell fate, osteogenesis, Notch signaling, live-cell imaging