Electrochemical cues regulate assembly of the

Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization

Aurin VosSabrina Berkamp

Supervisor: Madelon Maurice

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?