Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells
Giulia Ambrosi,
Oksana Voloshanenko,
Antonia F Eckert,
Dominique Kranz,
G Ulrich Nienhaus,
Michael Boutros
Affiliations
Giulia Ambrosi
German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics and Heidelberg University, BioQuant and Medical Faculty Mannheim, Heidelberg, Germany
German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics and Heidelberg University, BioQuant and Medical Faculty Mannheim, Heidelberg, Germany
Antonia F Eckert
Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany
German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics and Heidelberg University, BioQuant and Medical Faculty Mannheim, Heidelberg, Germany
Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany; Institute of Nanotechnology, Karlsruhe Institute of Technology, Karlsruhe, Germany; Institute of Biological and Chemical Systems, Karlsruhe Institute of Technology, Karlsruhe, Germany; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States
German Cancer Research Center (DKFZ), Division of Signaling and Functional Genomics and Heidelberg University, BioQuant and Medical Faculty Mannheim, Heidelberg, Germany
Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in β-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of endogenous β-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both β-catenin alleles using immunoprecipitation, immunofluorescence, and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3β inhibitor or a truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant β-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants.
