Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells

eLife

Dominique Kranz¹  Michael Boutros¹  Giulia Ambrosi¹  Oksana Voloshanenko¹  Antonia F Eckert²  G Ulrich Nienhaus³ 

[1] 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 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;

关键词: Wnt signaling;    β-catenin;    endogenous tagging;    fluorescence correlation spectroscopy;    FCS;    Human;    Cancer;    CTNNB1;    CRISPR;    Oncogenic signaling;   

DOI  :  10.7554/eLife.64498

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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.

【 授权许可】

CC BY