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Aurin Vos Sabrina Berkamp Supervisor: Madelon Maurice

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)

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Aurin Vos Sabrina Berkamp Supervisor: Madelon Maurice

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  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

  2. 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

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

  4. The Screening

  5. 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

  6. Introducing Nhe2

  7. 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

  8. Nhe2 – Fz interaction

  9. 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

  10. Dsh lipid binding

  11. Dsh lipid binding II

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

  13. Localization of Dsh mutants

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

  15. Macroscopic effect of mutant

  16. 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

  17. Overview

  18. Questions?

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