The Wnt signalling effector Dishevelled forms dynamic protein assemblies rather than stable associations with cytoplasmic vesicles

Abstract

Dishevelled is a crucial effector upstream in the Wnt signalling pathway, but the molecular mechanism by which it transduces the Wnt signal remains elusive. Dishevelled is a cytoplasmic protein with a strong tendency to form puncta, which correlates with its potent activity in stimulating Wnt signal transduction when overexpressed. These puncta are thought to reflect cytoplasmic vesicles. However, we show here that the mammalian Dishevelled protein Dvl2 does not colocalise with known vesicle markers for clathrin-mediated or clathrin-independent endocytic pathways. Furthermore, Dvl2 puncta do not stain with lipid dyes, indicating that these puncta do not contain membranes. Instead, our evidence from live imaging by TIRF microscopy of Dvl2 tagged with green fluorescent protein (GFP-Dvl2) revealed that these puncta move in and out of the evanescent field near the plasma membrane in an undirected fashion, and that they can grow by collision and fusion. Furthermore, high-resolution confocal microscopy and photobleaching experiments indicate that the GFP-Dvl2 puncta are protein assemblies; there is a constant exchange of GFP-Dvl2 between puncta and a diffuse cytoplasmic pool, which, therefore, are in a dynamic equilibrium with each other. The same is true for the DIX domain of Dvl2 itself and also for Axin-GFP, which equilibrates between the punctate and cytosolic pools. Our evidence indicates that Dvl2 and Axin puncta are dynamic protein assemblies rather than cytoplasmic vesicles.