Post on 05-Feb-2016
description
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?