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Journal of Cell Science
Phosphorylated paxillin and phosphorylated FAK constitute subregions within focal adhesions
Bachmann, M., Skripka, A., Weißenbruch, K., Wehrle-Haller, B., & Bastmeyer, M.
FAK, Focal adhesions, Paxillin, Paxillin phosphorylation
Integrin-mediated adhesions are convergence points of multiple signaling pathways. Their inner structure and their diverse functions can be studied with super-resolution microscopy. Here, we examined the spatial organization within focal adhesion by analyzing several adhesion proteins with structured illumination microscopy (SIM). We found that phosphorylated paxillin (pPax) and phosphorylated focal adhesion kinase (pFAK) form spot-like, spatially defined clusters within adhesions in several cell lines and confirmed these findings with additional super-resolution techniques. These clusters showed a more regular separation from each other compared to more randomly distributed labels of general FAK or paxillin. Mutational analysis indicated that the active (open) FAK conformation is a prerequisite for the pattern formation of pFAK. Live-cell super-resolution imaging revealed that organization in clusters is preserved over time for FAK constructs; however, distance between clusters is dynamic for FAK, while paxillin is more stable. Combined, these data introduce spatial clusters of pPax and pFAK as substructures in adhesions and highlight the relevance of paxillin-FAK binding for establishing a regular substructure in focal adhesions.