Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. Our current outline of Wnt signaling focuses around several key players. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. However, mechanisms underlying destruction complex function and those by which Wnt signaling inactivates it remain much less clear. Based on work in cultured cells, we hypothesize the destruction complex is a supermolecular entity that self-assembles by Axin and APC polymerization, and that regulating complex assembly and dynamics underlie function. We took these insights into the Drosophila embryonic epidermis, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins accumulate at roughly similar levels, countering the accepted dogma that Axin accumulates at much lower levels. By expressing Axin:GFP at near endogenous levels we found Axin assembles into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger complex recruitment to the membrane, while diffuse cytoplasmic Axin levels increase, suggesting slowed assembly. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.

Author Summary

Cell-cell communication is critical for cells to choose fates during embryonic development and often goes wrong in diseases like cancer. The Wnt cell signaling pathway provides a superb example. Mutations in negative regulators like the proteins APC and Axin take the brakes off cell proliferation and thus contribute to colon cancer. We study how APC, Axin and their protein partners keep cell signaling off, and how cell-to-cell Wnt signals reverse this. We use the fruit fly embryo, combining biochemical, and genetic tools with advanced microscopy. We found that APC2 and Axin proteins are present in cells in similar numbers, challenging the previous dogma. We further find that the ability of Wnt signaling to turn off this negative regulatory machine is influenced both by the levels of Axin and APC2 and by the ratio of their protein levels. We also visualize the active destruction complex in the animal, and count the number of Axin proteins in this complex. Finally, we find that Wnt signals have two effects on the destruction complex—recruiting it to the cell’s plasma membrane and reducing its ability to assemble. Based on this, we propose a new model for how this important signaling pathway is regulated.

Abbreviations

ßcat beta-catenin APC adenomatous polyposis coli GSK3 glycogen synthase kinase-3 CK1 casein kinase 1 Dsh Dishevelled DIX domain Dishevelled/Axin domain LRP5/6 low-density lipoprotein receptor-related proteins 5/6 GFP green fluorescent protein RFP red fluorescent protein ROI rectangular region of interest TCF T-cell factor Wg Wingless