Molecular & Cellular Proteomics 2015-03-01

Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.

Bjoern Traenkle, Felix Emele, Roman Anton, Oliver Poetz, Ragna S Haeussler, Julia Maier, Philipp D Kaiser, Armin M Scholz, Stefan Nueske, Andrea Buchfellner, Tina Romer, Ulrich Rothbauer

Index: Mol. Cell. Proteomics 14(3) , 707-23, (2015)

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Abstract

β-catenin is the key component of the canonical Wnt pathway and plays a crucial role in a multitude of developmental and homeostatic processes. The different tasks of β-catenin are orchestrated by its subcellular localization and participation in multiprotein complexes. To gain a better understanding of β-catenin's role in living cells we have generated a new set of single domain antibodies, referred to as nanobodies, derived from heavy chain antibodies of camelids. We selected nanobodies recognizing the N-terminal, core or C-terminal domain of β-catenin and applied these new high-affinity binders as capture molecules in sandwich immunoassays and co-immunoprecipitations of endogenous β-catenin complexes. In addition, we engineered intracellularly functional anti-β-catenin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. For the first time, we were able to visualize the subcellular localization and nuclear translocation of endogenous β-catenin in living cells using these chromobodies. Moreover, the chromobody signal allowed us to trace the accumulation of diffusible, hypo-phosphorylated β-catenin in response to compound treatment in real time using High Content Imaging. The anti-β-catenin nanobodies and chromobodies characterized in this study are versatile tools that enable a novel and unique approach to monitor the dynamics of subcellular β-catenin in biochemical and cell biological assays. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.