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.
Highlights
The Wnt signalling pathway is highly conserved between animals and humans (Logan and Nusse, 2004)
The formation of Dvl2 puncta correlates with Wnt signalling activity Confirming previous reports, we found that Dvl2 forms striking puncta when overexpressed in transfected simian COS-7 cells (Fig. 1A-D)
Our results strongly argue against the idea, widespread in the literature, that Dvl puncta reflect stable associations of Dvl with cytoplasmic vesicles
Summary
The Wnt signalling pathway is highly conserved between animals and humans (Logan and Nusse, 2004). It controls numerous steps in normal development and has been linked to disease, in particular to cancer. Their assembly into a signalling pathway, were discovered through Drosophila genetics (Perrimon, 1994). It is well established that there are different Wnt pathway branches that signal through distinct components to nuclear or cytoplasmic targets. The noncanonical Wnt pathways – including planar cell polarity (PCP) signalling – are less well defined in terms of their components and ultimate targets (Logan and Nusse, 2004)
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