Actin dynamics orchestrates assembly, turnover, and maturation of nascent adhesions in migrating cells

Using 2-color imaging and high resolution total internal reflection fluorescence (TIRF) microscopy, we investigated the assembly and maturation of nascent adhesions in migrating cells. We show that nascent adhesions assemble and are stable within the lamellipodium. The assembly is independent of myosin II but its rate is proportional to the protrusion rate and requires actin polymerization. At the lamellipodium back, the adhesions either disassemble or mature through growth and elongation. Maturation occurs along an alpha-actinin-actin template that elongates centripetally from nascent adhesions. alpha-Actinin mediates the formation of the template and organization of adhesions associated with actin filaments, suggesting that actin cross-linking has a major role in this process. Adhesion maturation also requires myosin II. Rescue of a myosin IIA knockdown with an actin bound but motor-inhibited mutant of myosin IIA shows that the actin cross-linking function of myosin II mediates initial adhesion maturation. From these studies, a model emerges for adhesion assembly that clarifies the relative contributions of myosin II and actin polymerization and organization. Also, to detect adhesion-associated protein-protein interactions in living cells, we developed a method for detecting molecular complexes using the TIRF imaging modality with an electron-multiplied charge-coupled device (EMCCD) camera. The method measures simultaneous fluctuations of fluorescence intensity in dual-channel time-lapse images, where each channel detects one type of protein exchanging at adhesions. From the fluctuation data, the ratio of cross-variance and mean intensity is analyzed to show the presence of molecular complexes and obtain their brightness. We applied this quantitative technique to determine whether paxillin and FAK are aggregated in nascent adhesions and show that these two proteins interact with one another as multimeric complexes. Overexpression of a tyrosine phosphomimetic mutant of paxillin, which promotes enhanced lamellipodium protrusion and adhesion turnover, significantly increases the complex size. In contrast, a non-phosphorylatable paxillin mutant that suppresses turnover dynamics reduces the paxillin aggregation nearly 2-fold. Taken together, these novel measurements demonstrate the feasibility of identifying complexes and quantifying their aggregation in adhesions of living cells. Moreover, the analysis clarifies the tyrosine phosphorylation-regulated interaction between paxillin and FAK, which has pertinent ramifications on nascent adhesion assembly and turnover.