Résumé
The LIM domain-dependent localization of the adapter protein paxillin to β3 integrin-positive focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology, photoactivation and FA-isolation experiments, we demonstrate specific contributions of each LIM domain of paxillin and reveal multiple paxillin interactions in adhesion-complexes. Mutation of β3 integrin at a putative paxillin binding site (β3 VE/YA ) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to β3 VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of β3 integrin/talin clusters. Moreover, bimolecular fluorescence complementation unveils the spatial orientation of the paxillin LIM-array, juxtaposing the positive LIM4 to the plasma membrane and the β3 integrin-tail, while in vitro binding assays point to LIM1 and/or LIM2 interaction with talin-head domain. These data provide structural insights into the molecular organization of β3 integrin-FAs.