Endocytosis and wingless signalling

Wingless (Drosophila Wnt-1) is secreted glycoprotein that triggers an evolutionary conserved signal transduction pathway. The role of endocytosis in Wnt/Wingless signaling is not clearly understood and highly debated. In my thesis I explore the role that endocytosis/endocytic trafficking has on Wingless signalling activation and termination. In the canonical pathway Wingless binds to a member of the Frizzled family of seven-pass transmembrane receptor (Frizzled1 or Frizzled2) and to Arrow. Formation of this trimeric complex leads to the inactivation of the Armadillo degradation complex and translocation of Armadillo into the nucleus where it contributes to the activation of target genes. I show that internalization of the ligand-receptor complex is not required for signalling activation. I also show that Wingless has different effects on the trafficking route of its receptors: it induces the degradation of Frizzled2 and the recycling of Arrow. To identify post translational modifications that regulate Arrow trafficking I conducted an RNAi screen in Drosophila S2R+ cells for de-ubiquitylating enzymes (DUBs) and ubiquitin conjugating enzymes (E2) that modulate signalling. To carry out this screen, improvements on the current TOPFlash Wnt/Wingless signalling reporter were made. I also directly assessed the role of endocytic trafficking on signalling using a chemical inhibitor of endocytosis, Dynasore. I find that Dynasore inhibits signalling by causing a strong decrease in Armadillo levels. Future experiments will determine whether it is the stability or the rate of production of Armadillo that is affected