1. Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization Aurin Vos Sabrina Berkamp Supervisor: Madelon Maurice

3. Introduction Canonical Wnt signalling –Amount of B-catenin reaching nucleus Non-canonical (PCP) Wnt signalling –Actin skeleton rearrangement

4. Introduction Frizzled (Fz) –G-protein coupled receptor (membrane protein) –Key role in development and cell polarity Dishevelled (Dsh) contains 3 domains: –DIX multimerization –PDZ weak binding to frizzled –DEP membrane targeting

5. Research goal To identify and characterize new components involved in forming the Dsh- Fz complex at the plasma membrane.

6. The Screening

7. Introducing Nhe2 Identification of Nhe2 –Homology with (human) NHE3 –NHE3 = plasma membrane exchanger (Na/H) –NHEs are involved in: Formation of alkaline PH and charge microenvironments Activation of Rho family GTPases, actin polymerization

8. Introducing Nhe2

9. Preliminary conclusions Knockdown of Nhe2 causes a defect in recruitment of Dsh Nhe2 has a homologue in human: Nhe3 –A Na/H exchanger causes local higher pH Changing cellular pH changes localization Proton translocation by Nhe2 is required for correct localization Dsh

10. Nhe2 – Fz interaction

11. Preliminary conclusions Nhe2 is essential protein Overexpression/knockdown of Nhe2 causes PCP phenotypes in Drosophila eyes Overexpression of Fz causes PCP phenotype –This phenotype is rescued by Nhe2 knockdown

12. Dsh lipid binding

13. Dsh lipid binding II

14. Preliminary conclusions (positively charged) DEP domain of Dsh binds negatively charged lipids R,K-to-E mutations abolish this affinity

15. Localization of Dsh mutants

16. Preliminary conclusions R,K-to-E mutation mislocalizes Dsh Positively charged lipid (sphingosine) mislocalizes Dsh, but rescues DshKR/E

17. Macroscopic effect of mutant

19. Preliminary conclusions Membrane association of positively charged stretch on DEP domain in Dsh is essential in vivo Mutation does not affect canonical pathway, only non-canonical pathway

20. Overview

21. Questions?