Matrix Stiffness-driven FAK Splicing Tunes Cell Mechanosensing

The extracellular matrix (ECM) stiffness influences many physiological and pathological processes. Cells can sense mechanical changes within the ECM through mechanosensors, most notably through the focal adhesion kinase (FAK). Using human-derived data and 2D/3D engineered models, we identified a ubiquitous and uncharacterized FAK isoform lacking exon 4 (FAKΔe4). We demonstrated that FAKΔe4 splicing is regulated by substrate stiffness in a biphasic manner, in which an optimal stiffness is required for a maximal FAKΔe4 expression. We further showed that FAKΔe4 dictates at which stiffness optimal migration speed and invasion occur, impairs focal adhesion dynamics and maturation, and shifts its autophosphorylation and downstream YAP nuclear translocation toward lower stiffness compared to the canonical FAK isoform. Moreover, the FAKΔe4 expression levels to canonical FAK determine at which stiffness cells will converge during durotaxis. Our results reveal how FAKΔe4 acts as a fine-tuning mechanosensing switch and reframe our understanding of mechanotransduction.