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 (STORM, STED and DNA-PAINT) we show that integrins α ₅ β ₁ and α _v β ₃ and their main adaptor proteins exhibit similar functional nanoscale segregation across all adhesion proteins examined, independent of FA maturation state. Notably, both integrins never mix at the nanoscale indicating that their functions might be spatially regulated. We find a nearly 1:1 relationship 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 near 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 are arranged as modular nanoscale units that distinctively organize inside FAs to spatially regulate integrin activation and function.