aurin vos sabrina berkamp supervisor: madelon maurice
DESCRIPTION
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. Frizzled (Fz) G-protein coupled receptor (membrane protein) - PowerPoint PPT PresentationTRANSCRIPT
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Electrochemical cues regulate assembly of the
Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization
Aurin VosSabrina Berkamp
Supervisor: Madelon Maurice
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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
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Research goal
To identify and characterize new components involved in forming the Dsh-
Fz complex at the plasma membrane.
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The Screening
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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
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Introducing Nhe2
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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
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Nhe2 – Fz interaction
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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
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Dsh lipid binding
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Dsh lipid binding II
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Preliminary conclusions
• (positively charged) DEP domain of Dsh binds negatively charged lipids
• R,K-to-E mutations abolish this affinity
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Localization of Dsh mutants
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Preliminary conclusions
• R,K-to-E mutation mislocalizes Dsh• Positively charged lipid (sphingosine)
mislocalizes Dsh, but rescues DshKR/E
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Macroscopic effect of mutant
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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
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Overview
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Questions?
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