Cross-Correlated Fluctuation Analysis Reveals Phosphorylation-Regulated Paxillin-FAK Complexes in Nascent Adhesions

Abstract

We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions.

Focal adhesion stabilization by enhanced integrin-cRGD binding affinity

“…REF52 WT cells were plated for 4 h on nanopatterned surfaces with different inter-ligand spacings, then fixed and stained for paxillin, zyxin and actin. Paxillin resides in both initial adhesion contacts [58]; namely, focal complexes (FXs), and mature adhesions, while zyxin is recruited during the maturation of these FXs into FAs, and constitutes a distinctive protein of mature FAs [59]. Examination of the labeled cells revealed extensive cell adhesion and spreading on the 62 nm cRGD-nanopatterns, indicating successful integrin-ligand interactions with all the different compounds ( Figure 4, first column).…”

Signaling Networks that Regulate Cell Migration

“…FAK binds to paxillin following tyrosine phosphorylation of paxillin Y31 and Y118 (Choi et al 2011). The phosphorylation occurs after both molecules are in nascent adhesions and appears to result from a conformational activation of paxillin, because FAK binds to the carboxyl end of paxillin.…”

Dynamic Phosphorylation of Tyrosine 665 in Pseudopodium-enriched Atypical Kinase 1 (PEAK1) Is Essential for the Regulation of Cell Migration and Focal Adhesion Turnover

“…Src family kinases (SFKs) play a critical role in the regulation of FA dynamics and cell migration (10,11). SFKs that are recruited to FAs phosphorylate multiple FA-associated proteins, including focal adhesion kinase (FAK), paxillin, and p130CAS (12)(13)(14). These phosphorylation events lead to activation of the GTPase Rac1, which promotes lamellipodium protrusion, FA formation, and cell migration (15,16).…”