Single-Molecule Dynamics Of Dishevelled At The Plasma Membrane And Wnt Activation

Dvl (Dishevelled) is one of several essential nonenzymatic compo-nents of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of beta-catenin degradation. Al-though this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus of dishevelled2 gene using CRISPR/Cas9-induced homologous recom-bination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes dur-ing Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is pre-dominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concen-tration of membrane-bound Dvl2 from 0.12/mu m2 to 0.54/mu m(2). Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The olig-omerized Dvl2 complexes have increased dwell time, 2 similar to 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of as-sembly and stability at the membrane in response to Wnt ligands.