| Popis: |
Wnt signaling plays important roles in embryonic development and during tissue homeostasis, and its deregulation contributes to the onset and progression of tumors. beta-catenin is a key component of the canonical Wnt pathway. Upon activation of the pathway, beta-catenin is stabilized and enters the nucleus to bind transcription factors and activate Wnt target genes. Mutations that activate beta-catenin have been found in many gastrointestinal tumors, including colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of beta-catenin are not fully understood. Here, we engineered fluorescently tagged alleles of the endogenous beta-catenin by CRISPR/Cas9 in a colorectal cancer cell line. We generated a cell model which carries wild-type and the oncogenic delta45 mutant allele tagged with different fluorescent proteins, enabling the functional and biochemical analysis of both variants in the same cell. Endogenously tagged beta-catenin remains functional, responds to Wnt pathway activation and does not interfere with the role of beta-catenin in cell-cell adhesion. We then analyzed their biochemical and biophysical properties using immunoprecipitation, immunofluorescence and fluorescence correlation spectroscopy approaches. Our results show that wild-type and mutant beta-catenin have distinct biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3beta; inhibitor or truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant beta-catenin allele found in colorectal cancer. The one-step tagging strategy also demonstrates how CRISPR/Cas9-mediated genome engineering can be employed for the concurrent functional analysis of wild-type and mutant alleles. |
