Differential spatial regulation and activation of integrin nanoclusters inside focal adhesions

α ₅ β ₁ and α _v β ₃ integrins are core components of focal adhesions (FAs) involved in cell attachment, migration and mechanobiology-dependent processes. Recent works indicate that both integrins organize in nanoclusters inside FAs, with sub-populations of active and inactive β ₁ nanoclusters. However, whether both integrins work in concert or their activities are spatially regulated is not fully understood. Using dual-color super-resolution microscopy we show that integrins α ₅ β ₁ and α _v β ₃ exhibit similar functional nanoscale segregation. Notably, both integrins never mix at the nanoscale indicating that their functions might be spatially regulated. We find a nearly 1:1 stoichiometry between active integrin and adaptor nanoclusters suggesting that coordinated integrin activation occurs via the concurrent engagement of adaptor nanoclusters. Interestingly, α ₅ β ₁ nanoclusters preferentially localize at the FA periphery in close proximity to adaptor nanoclusters, establishing regions of multi-nanocluster enrichment, whereas α _v β ₃ nanoclusters uniformly distribute throughout FAs. Overall, our results show that adhesion proteins arrange as modular nanoscale units that distinctively organize inside FAs to spatially regulate integrin activation and function.