In vivo analysis of formation and endocytosis of the Wnt/β-Catenin signaling complex in zebrafish embryos

After activation by Wnt/beta-Catenin ligands, a multi-protein complex assembles at the clustering membrane-bound receptors and intracellular signal transducers into the so-called Lrp6-signalosome. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt-Fz-Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The mu 2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2 mu 2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2 mu 2-mediated endocytosis is important to maintain Wnt/beta-catenin signaling in vertebrates.