Beta-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3beta. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of beta-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged beta-catenin, and found that a small mobile pool of beta-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t(0.5) of approximately 30 s. Thus, beta-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of beta-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t(0.5) approximately 5 s) is indicative of high turnover and transient association. In contrast, beta-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t(0.5) approximately 8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of beta-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than beta-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3beta increased beta-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of beta-catenin. In summary, beta-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.
Amino Acid Substitution Animals