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

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

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

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

The Screening

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

Introducing Nhe2

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

Nhe2 – Fz interaction

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

Dsh lipid binding

Dsh lipid binding II

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

Localization of Dsh mutants

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

Macroscopic effect of mutant

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

Overview

Questions?