Role of endocytosis in Wnt signal transduction

The Wnt pathway is an intercellular signaling cascade that regulates development, tissue homeostasis, and regeneration. Wnt signaling is initiated upon ligand-receptor interaction at the membrane and results in activation of target genes by the central signal transducer B-catenin. However, we do not fully understand the molecular events that take place within the cell between ligand-receptor binding and target gene transcription. One open question in the field is whether receptor endocytosis upon ligand binding is required for Wnt signal transduction. Some studies have reported endocytosis as a positive regulator of signal transduction while some have reported it as a negative regulator, and the molecular mechanism behind each possibility is unclear. To determine the role of endocytosis in Wnt signal transduction, I used mouse embryonic stem cells (mESCs) in culture, a system well-suited for this investigation with its inherent Wnt-responsiveness. A B-catenin knock-in reporter was generated in mESCs to monitor the behavior of endogenous B-catenin. I combined knockdown or CRISPR-mediated knockout of essential components of Clathrin-mediated endocytosis with quantitative assessment of Wnt signal transduction upon pathway activation. Disruption of Clathrin-mediated endocytosis did not affect accumulation and nuclear translocation of B-catenin, as measured by single-cell live imaging of endogenous B-catenin, and subsequent target gene transcription. Clathrin-mediated endocytosis was not required for Wnt signaling in two additional cell lines. Lastly, disruption of another receptor endocytosis pathway, Caveolin-mediated endocytosis, did not affect Wnt pathway activation. These results led me to the conclusion that endocytosis is not a general requirement for Wnt signal transduction, a conclusion that undermines an existing model of signal transduction that requires receptor endocytosis. The discrepancy among reports may be attributed to a combination of factors: pleiotropic effects of endocytosis disruption such as impaired mitosis and protein degradation, use of overexpressed Wnt pathway proteins to activate Wnt signaling or to evaluate signal transduction, and off-target effects of drugs used to inhibit endocytosis. The second chapter describes an effort to apply proximity labeling to study molecular changes at the cell membrane upon Wnt pathway activation. To investigate changes in the interaction network of the Wnt receptor Frizzled7 following pathway activation, I tagged Frizzled7 with APEX2, an enzyme that rapidly biotinylates interactors near the protein of interest. Mass spectrometry analysis of biotinylated interactors identified known Wnt pathway proteins such as Dvl2, APC, and the kinase CK2. Other proteins enriched in the Wnt-treated Frizzled7 interactome, such as PKN2 and PI5K1, do not have well-established roles in Wnt signaling and are interesting candidates to follow up. However, some proteins known to come in contact with Frizzled were missing from the list. To better recapitulate the native behavior of Frizzled, future proximity labeling studies could be improved by knocking in the APEX2 tag in the endogenous locus. I conclude that with optimization, proximity labeling can be a powerful biochemical approach to identify changes in interaction networks of Wnt signal transducers. Cell biological, genetic, and biochemical tools at our disposal, such as live cell imaging and proximity labeling, are rapidly advancing, granting us greater specificity and spatiotemporal resolution. Application of these new tools will help us tackle the most challenging questions remaining in the field of Wnt signal transduction

Keywords: Developmental Biology.
Advisor: Nusse Roel
Axelrod Jeffrey (Jeffrey David)
Fuller Margaret T.
O'Brien Lucy Erin
Weis William I.