Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells
Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in beta-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of beta-catenin are not fully understood. Here, w...
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| Hauptverfasser: | , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
11 January 2022
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| In: |
eLife
Year: 2022, Jahrgang: 11, Pages: 1-27 |
| ISSN: | 2050-084X |
| DOI: | 10.7554/eLife.64498 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.7554/eLife.64498
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| Verfasserangaben: | Giulia Ambrosi, Oksana Voloshanenko, Antonia F. Eckert, Dominique Kranz, G. Ulrich Nienhaus, Michael Boutros |
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| 520 | |a Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in beta-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of beta-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of endogenous beta-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 beta-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 GSK3 beta inhibitor or a truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant beta-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants. | ||
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