A variety of Wnt receptors bind a range of specific ligands to regulate the Wnt pathway. Accurate quantification of these interactions in living cells should help us understand better signaling specificity in complex cellular environments where multiple interactions occur. The overall aim of our research is to provide a comprehensive, quantitative analysis of ligand-receptor interactions for the Wnt pathway under physiological conditions. We are establishing new methods that are based on advanced fluorescence microscopy techniques, such as dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) for the measurement of ligand-receptor interactions on live specimens. We will also utilize alternative fluorescence imaging-based methods to measure Wnt ligand affinities toward various receptors.
We will also employ high resolution imaging techniques to study Wnt signaling events. Advanced fluorescence microscopy techniques including super-resolution imaging (STED and PALM/STORM) will be used to visualize the dynamics of activated Wnt receptors, with an initial focus on LRP6. CRISPR/Cas gene editing is being used to generate endogenously tagged Wnt pathway components in order to perform our analysis under physiologically relevant expression conditions.