This directory contains scripts and files supporting the publication: Schubert et. al.: Super-resolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates, PNAS, 2022. Specifically, to reproduce Figures 4F, 4G, and S8C and the example image analysis used to quantify condensates.
During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and -independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, how it mechanistically functions remains relatively unknown. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl’s role in Wnt signaling, we genome-engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for super-resolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and -independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supra-molecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large puncta, supra-molecular condensate formation at physiological protein levels is only observed in a subset of cells with approx. one per cell. We show that in these condensates, Dvl2 co-localizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt-dependent. Single-molecule localization microscopy using PALM of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supra-molecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.
Should you encounter any issues or have any questions please contact Antonia Schubert a.schubert@dkfz.de or Christian Scheeder c.scheeder@dkfz.de.