Structural and functional analysis of LIM domain-dependent recruitment of paxillin to αvß3 integrin-positive focal adhesions.

ABSTRACT

The LIM domain-dependent localization of the adapter protein paxillin to ß3 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 ß3 integrin at a putative paxillin binding site (ß3VE/YA) leads to rapidly inward-sliding FAs, correlating with actin retrograde flow and enhanced paxillin dissociation kinetics. Induced mechanical coupling of paxillin to ß3VE/YA integrin arrests the FA-sliding, thereby disclosing an essential structural function of paxillin for the maturation of ß3 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 ß3 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 ß3 integrin-FAs.