β-catenin-Mediated Wnt Signal Transduction Proceeds Through an Endocytosis-Independent Mechanism

The Wnt pathway is a key intercellular signaling cascade that regulates development, tissue homeostasis, and regeneration. However, gaps remain in our understanding of the molecular events that take place between ligand-receptor binding and target gene transcription. Here we used a novel tool for quantitative, real-time assessment of endogenous pathway activation, measured in single cells, to answer an unresolved question in the field – whether receptor endocytosis is required for Wnt signal transduction. We combined knockdown or knockout of essential components of Clathrin-mediated endocytosis with quantitative assessment of Wnt signal transduction in mouse embryonic stem cells (mESCs). Disruption of Clathrin-mediated endocytosis did not affect accumulation and nuclear translocation of β-catenin, as measured by single-cell live imaging of endogenous β-catenin, and subsequent target gene transcription. Disruption of another receptor endocytosis pathway, Caveolin-mediated endocytosis, did not affect Wnt pathway activation either. These results, confirmed in multiple cell lines, suggest that endocytosis is not a general requirement for Wnt signal transduction. We show that off-target effects of a drug used to inhibit endocytosis may be one source of the discrepancy among reports on the role of endocytosis in Wnt signaling.