Differential spatial regulation and activation of integrin nanoclusters inside focal adhesions

Abstract

Abstractα5β1and αvβ3integrins are key components of focal adhesions (FAs) that play important roles in cell adhesion, migration and mechanobiology-dependent processes. Whether both integrins work in concert or their activities are differentially regulated inside FAs is not fully understood. Here, we exploited dual-color super-resolution stochastic optical reconstruction microscopy (STORM) to quantitatively assess the lateral nanoscale organization and activation state of both integrins and main adaptors inside FAs. Our results reveal that key FA proteins α5β1, αvβ3, paxillin, talin and vinculin organize into segregated nanoclusters of similar size (∼50 nm) and number of molecules, regardless of their partitioning in the membrane, confirming that integrin nanoclustering is force-independent. Both integrins further segregate as active or inactive nanoclusters that do not intermix at the nanoscale and with a higher fraction of active α5β1nanoclusters as compared to αvβ3. We find a nearly 1:1 stoichiometry between active integrin and adaptor nanoclusters indicating that coordinated integrin nanocluster activation occurs via the concurrent engagement of talin and vinculin nanoclusters. Interestingly, active α5β1nanoclusters preferentially localize at the FA periphery in proximity to paxillin, talin and vinculin nanoclusters, establishing multiprotein nano-hubs, whereas αvβ3nanoclusters uniformly distribute throughout FAs. Overall, we reveal a highly complex lateral organization of integrins and main adaptors within FAs, where adhesion proteins arrange as modular nanoscale units that distinctively organize inside FAs to spatially regulate integrin activation. Such spatial control of integrin activity provides a physical mechanism to tune their functions having implications for their key roles in physiology and disease.Significance StatementFocal adhesions (FAs) are places for cell attachment to the extracellular matrix that influence multiple cellular processes as well as being sites for sensing and transducing mechanical stimuli. Integrins, core components of FAs, have different activation states and execute diversity of functions, but how their activity and roles are regulated remain mysterious. Using quantitative dual-color super-resolution microscopy we reveal that integrin activation is spatially regulated inside FAs. We show that main adhesion proteins organize as universal modular nanoscale units, spatially segregating from each other inside FAs. Remarkably, these modular units distinctively organize inside FAs selectively activating one type of integrin or another at specific locations within FAs, providing a physical mechanism to regulate integrin activation and possibly function.