title: Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells.
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.
authors.affiliation: From the ‡Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany;
authors.forename: Bjoern
authors.initials: B
authors.lastname: Traenkle
authors.affiliation: From the ‡Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany;
authors.forename: Felix
authors.initials: F
authors.lastname: Emele
authors.affiliation: From the ‡Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany;
authors.forename: Roman
authors.initials: R
authors.lastname: Anton
authors.affiliation: §Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany;
authors.forename: Oliver
authors.initials: O
authors.lastname: Poetz
authors.affiliation: §Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany;
authors.forename: Ragna S
authors.initials: RS
authors.lastname: Haeussler
authors.affiliation: From the ‡Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany;
authors.forename: Julia
authors.initials: J
authors.lastname: Maier
authors.affiliation: From the ‡Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany; §Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany;
authors.forename: Philipp D
authors.initials: PD
authors.lastname: Kaiser
authors.affiliation: ¶Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University Munich, Oberschleissheim, Germany;
authors.forename: Armin M
authors.initials: AM
authors.lastname: Scholz
authors.affiliation: ¶Livestock Center of the Faculty of Veterinary Medicine, Ludwig Maximilians University Munich, Oberschleissheim, Germany;
authors.forename: Stefan
authors.initials: S
authors.lastname: Nueske
authors.affiliation: ‖ChromoTek GmbH, Planegg-Martinsried, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Buchfellner
authors.affiliation: ‖ChromoTek GmbH, Planegg-Martinsried, Germany.
authors.forename: Tina
authors.initials: T
authors.lastname: Romer
authors.affiliation: From the ‡Pharmaceutical Biotechnology, Eberhard-Karls University Tuebingen, Germany; §Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany; ulrich.rothbauer@uni-tuebingen.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
mesh_terms: D000818:Animals; D001665:Binding Sites; D002161:Camelids, New World / Q000276:immunology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods; D045325:HCT116 Cells; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000378:metabolism*; D051176:beta Catenin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D061905:Single-Domain Antibodies; D051176:beta Catenin