Presented by : Lecturer: Lidija Berke Dr. Madelon Maurice Renske Bolder Liliana Joach í n

1. Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex Presented by: Lecturer: Lidija Berke Dr. Madelon Maurice Renske Bolder Liliana Joachín October 9, 2008

2. Overview • Introduction • Experimental work • Results • Conclusions • General conclusions • Final model for Wnt secretion

3. IntroductionWnt proteins • Family of secreted glycoproteins (highly conserved). • Act as signaling molecules, local mediators and morphogens. • Regulate cell-to-cell interaction during embryogenesis. • Examples: Wingless, Hedgehog.

4. IntroductionDrosophila imaginal disc Group of cells that are set aside, apparently undifferenciated in the Drosophila embryo and which will develop into an adult structure, e.g eye, leg, wing.

5. IntroductionSignal proteins in the wing imaginal disc • Wingless • Dorso-ventral action • Long-range signal • Hedgehog • Posterior expression • Short-range signal Ventral Anterior Posterior Hedgehog Wingless Dorsal

6. IntroductionRetromer complex • Multiprotein complex that assembles on endosomes and form vesicles that return cargos to the Golgi apparatus. It is formed only when: • It can bind to the cytoplasmic tails of the cargo (Vps35). • It can interact directly with a curved phospholipid bilayer. • It can bind to a specific phosphorilated PI lipid.

7. IntroductionPrevious findings about Wnt • Wnts undergo up to two lipid modifications • Affect trafficking and spreading along epithelia. • Hypothesis: They are transported by lipoprotein particles which would shield lipid moieties, reducing membrane anchoring. • EGL-20 (a C. elegans Wnt) • Its long-range action is impaired in retromer-deficient worms. • Hypothesis: A retromer-dependent process might be required for Wnt-producing cells to package Wnts into lipoprotein particles.

8. Research question Is the retromer involved in Wingless secretion?

9. Important note Wingless ---- Wingless (Wg) Wntless ---- Wls/Evi (Wls)

10. Experimental workLoss of Vps35 affects the spread of Wingless • Generation of Drosophila mutants (deficient in Vps35) • Results: • Melanotic tumors • Wingless accumulation • Incomplete wings • Conclusion: Wingless trafficking might be affected in absence of Vps35 activity

11. Experimental workLoss of Vps35 affects the spread of Wingless • Mutation: posterior compartment deficient in Vps35 • Results: • Concentration of Wingless • Wingless spreading reduced • Reduction in extracellullar Wingless • Conclusion: In the absence of retromer function, progression of Wingless along the secretory pathway becomes blocked or impaired.

12. Experimental workEffects on short or long range wingless signaling • Senseless • Distalless • Conclusion: Vps35 has an important role in short range Wingless signalling

13. Experimental workIs vps35 also involved in Hedgehog secretion? • Hedgehog • Wingless • Patched • Conclusion: Vps35 is required for a specific step in the secretion of Wingless

14. Experimental workOverexpression of Wls/Evi • Conclusion: Overexpression of Wls/Evi rescues the wingless secretion phenotype of Vps35

15. Experimental workStability of Wls/Evi • HEK293 cells • Conclusion: Stability of Wls/Evi is reduced in the absence of Vps35 activity

16. Experimental workWhat happens after internalization of Wls/Evi in the absence of retromer? • Conclusion: In the absence of retromer function, Wls/Evi is diverted to MVBs.

17. Experimental workPhysical interactions between retromer components and Wls/Evi • Conclusion: hWls/Evi forms a a complex with retromer components.

18. Experimental workTracking internalized Wls/Evi • Conclusions: Wls/Evi undergoes retrograde transport to the Golgi in a retromer dependent manner.

19. General conclusions Schematic representation of the role of Wls/Evi in Wingless secretion

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