Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions

Integrins in focal adhesions (FAs) mediate adhesion and force transmission to extracellular matrices crucial for cell motility, proliferation and differentiation1,2. Different αβ-integrins binding to fibronectin (FN) perform distinct functions3-5 and are simultaneously present in FAs. Although the static nanoscale organization of FAs was described6, explaining how individual dynamics of specific integrins control biochemical and biomechanical events in FAs is still elusive. Combining single protein tracking and super-resolution imaging we show that β3- and β1-integrins act as distinct adhesion units displaying specific dynamics and nano-organizations within FAs. Integrins reside in FAs through free-diffusion and immobilization cycles, thus integrins are not constantly active inside FAs. Integrin activation promotes immobilization, stabilized in FAs by simultaneous FN and actin binding proteins (ABPs) connections. The integrin activator talin7, is recruited in FAs from the cytosol without membrane free-diffusion, spatially restricting integrin immobilization to FAs. Talin immobilization zones are concentrated and mainly stationary in FA, consistent with the formation of stable β-integrin/talin connections. β3-integrin immobilizations are concentrated and stationary within FAs, whereas β1-integrins are sparse displaying eventually rearward movements. Thus, differential transmission of F-actin motion to FN occurs through specific integrins within FAs. This dynamic nano-partitioning of β-integrins within FAs could control local forces and signaling necessary for distinct cellular functions such as migration and extracellular matrices remodeling