Nanopillar force measurements reveal actin-cap-mediated YAP mechanotransduction

The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms.

How different integrins that bind to the same type of extracellular matrix protein mediate specific functions is unclear. We report the functional analysis of β1- and αv-class integrins expressed in pan-integrin-null fibroblasts seeded on fibronectin. Reconstitution with β1-class integrins promotes myosin-II-independent formation of small peripheral adhesions and cell protrusions, whereas expression of αv-class integrins induces the formation of large focal adhesions. Co-expression of both integrin classes leads to full myosin activation and traction-force development on stiff fibronectin-coated substrates, with αv-class integrins accumulating in adhesion areas exposed to high traction forces. Quantitative proteomics linked αv-class integrins to a GEF-H1-RhoA pathway coupled to the formin mDia1 but not myosin II, and α5β1 integrins to a RhoA-Rock-myosin II pathway. Our study assigns specific functions to distinct fibronectin-binding integrins, demonstrating that α5β1integrins accomplish force generation, whereas αv-class integrins mediate the structural adaptations to forces, which cooperatively enable cells to sense the rigidity of fibronectin-based microenvironments.

Integrins in focal adhesions (FAs) mediate adhesion and force transmission to extracellular matrices essential for cell motility, proliferation and differentiation. Different fibronectin-binding integrins, simultaneously present in FAs, perform distinct functions. Yet, how integrin dynamics control biochemical and biomechanical processes in FAs is still elusive. Using single-protein tracking and super-resolution imaging we revealed the dynamic nano-organizations of integrins and talin inside FAs. Integrins reside in FAs through free-diffusion and immobilization cycles. Integrin activation promotes immobilization, stabilized in FAs by simultaneous connection to fibronectin and actin-binding proteins. Talin is recruited in FAs directly from the cytosol without membrane free-diffusion, restricting integrin immobilization to FAs. Immobilized β3-integrins are enriched and stationary within FAs, whereas immobilized β1-integrins are less enriched and exhibit rearward movements. Talin is enriched and mainly stationary, but also exhibited rearward movements in FAs, consistent with stable connections with both β-integrins. Thus, differential transmission of actin motion to fibronectin occurs through specific integrins within FAs.