Real-time imaging of beta-catenin dynamics in cells and living mice

beta-Catenin (beta-cat) is a key signaling component of the canonical Wnt pathway as well as an increasingly studied contributor to various pathways that regulate cell adhesion, proliferation, and differentiation. For its best known function, posttranslational stabilization of beta-cat is required for T cell factor-dependent transcription of numerous downstream targets of Wnt, and this process is aberrantly active in a wide array of cancers. To enable direct monitoring of posttranslational stabilization of beta-cat in cells and living animals, we constructed and characterized the bioluminescent fusion reporters beta-cat firefly luciferase (beta-cat-FLuc) and beta-cat click beetle green luciferase (beta-cat-CBG). These reporters provided real-time, noninvasive readout of modulators of beta-cat stability in cellulo and, furthermore, enabled monitoring of changes in total beta-cat levels in vivo in intact animals. In addition, using spectral unmixing, green beta-cat-CBG was deconvoluted from a red TCF-dependent FLuc reporter (TOPFLASH), enabling analysis of beta-cat processing and downstream transcriptional activation simultaneously. By using this system, the natural product epigallocatechin 3-gallate was found to block Wnt signaling, independent of beta-cat processing. These beta-cat reporters represent a powerful new strategy for identifying in cellulo and in vivo dynamic regulators and mechanism-based therapeutics of signaling pathways mediated by beta-cat stabilization.