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

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Abstract
β-Catenin (β-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 β-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 β-cat in cells and living animals, we constructed and characterized the bioluminescent fusion reporters β-cat firefly luciferase (β-cat-FLuc) and β-cat click beetle green luciferase (β-cat-CBG). These reporters provided real-time, noninvasive readout of modulators of β-cat stability in cellulo and, furthermore, enabled monitoring of changes in total β-cat levels in vivo in intact animals. In addition, using spectral unmixing, green β-cat-CBG was deconvoluted from a red TCF-dependent FLuc reporter (TOPFLASH), enabling analysis of β-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 β-cat processing. These β-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 β-cat stabilization.