Master class Biomolecular Sciences Part 1: Molecular Cell Biology. October 9, 2008

1. Master class Biomolecular Sciences Part 1: Molecular Cell Biology. October 9, 2008 Madelon Maurice Department of Cell Biology, UMC, Utrecht (M.M.Maurice@umcutrecht.nl) “Cell Biology of Wnt signaling” Article: Franch-Marro X, Wendler F, Guidato S, Griffith J, Baena-Lopez A, Itasaki N, Maurice MM, Vincent JP (2008) Wingless secretion requires endosome-to-Golgi retrieval of Wntless-Evi-Sprinter by the retromer complex. Nat Cell Biol 10(2):170-77

3. Evolutionary conservation of the Wnt gene family.

4. Evolutionary conservation of the Wnt gene family. (Sea urchins) (flat worms) (jelly fish) Wnt homepage, R.Nusse

5. Destruction complex The Wnt signalling cascade Wnt OFF ON Lrp5/6 Fz ? Destruction complex Proteasomal degradation b-cat P b-catenin Ub TCF TCF No transcription Transcription • Wnt signalling: • Development (body axis, tissue patterning) and adult tissue homeostasis • Cell growth, lineage decisions, stem cell maintenance • Frequent target for mutations in cancer

6. Adult intestinal homeostasis KI67 A. Gregorieff, Gen Dev 2005

7. Destruction complex TCF TCF The Wnt signalling cascade OFF CANCER Destruction complex b-cat P Proteasomal degradation b-cat Ub No transcription Transcription

8. b-catenin dependent and independent Wnt signalling pathways Wnt/b-catenin Small GTPases, JNK signaling Strutt, D. Development 2003;130:4501-4513

9. Planar polarity and Fz phenotypes in the Drosophila wing Pupal stage Strutt, D. Development 2003;130:4501-4513

10. Wnts - Introduction Wnt family: - 19 members in man and mice - ~ 40kD - Secreted Cys-rich glycoproteins - Lipid-modified Wg Ser Palmitoleic acid Palmitate Wnt functions: - Embryogenesis and homeostasis of self- renewing tissues - Mitogenic stimulation - Cell fate specification / lineage decisions - Stem cell maintenance

11. Wnts act as morphogens • Morphogen: • - Secreted, diffusable signalling molecules • Generate discrete genetic programs in a concentration-dependent manner • (positional information) Spread: 20-25 cell diameters Wnt secretion Short-range vs. long-range signaling: The ‘hydrophobicity problem’

12. Extracellular components of Wnt (Wg) gradient formation • Wnt (Wg) gradient is stable and has defined steepness. • How is this accomplished? • Three factors proposed to contribute: • 1. Rate of production/secretion • 2. Rate of spreading • 3. Rate of degradation • Models of gradient formation • Cellular inheritance • Diffusion and lysosomal degradation • Planar transcytosis wt mut mut mut

13. Models for Wnt gradient formation • Cellular inheritance • (gradient established = 1 hour, • avg doubling time cells = 11 hours) * * * apical B. Diffusion and lysosomal degradation (Secretion apical, gradient forms basolaterally) * basal C. PLanar transcytosis (gradient forms extracellularly, Endocytosis does not affect Wg transport) *

14. What is the nature of secreted Wnt (Wg) particles? • Models proposed: • Multimeric complexes - Wnt ‘micelles’ • Vesicles (exosomes) - MVB derived • 3. Membrane protrusions - imaginal disc cells construct apical protrusions: cytonemes • 4. Lipoprotein particles - phospholipid monolayers surrounding a core of esterified cholesterol and triglyceride, scaffolded by apolipoproteins • - 10% Wg bound, required for long- range Wg signaling

15. Two-gradient model: explaining short- and long-range Wnt signaling (membrane association) - Lipoprotein particles (Transcytosis needed)

16. Questions Where in the cell is Wnt lipid-modified? How is Wnt inserted into lipoprotein particles? In what subcellular compartment does this take place? Does Wnt trafficking and secretion require specialized machinery? How do short-range signals differ from long-range signals?

17. Intracellular molecular machinery involved in Wg secretion • 1. Porcupine(EMBO 1993) - Acyl transferase, palmitoylation of Wnt • - ER resident protein, 8-span TM, highly conserved • - porc mutant (D.m.): retention of Wg in • producing cells • - Required for proper N-glycosylation and secretion of Wnt • 2. Retromer complex(Science 2006) - Recycling of proteins/receptors from • endosome to TGN • - Mutant (C.e/D.m.): No secretion • 3. Wntless/Evi(Cell 2006) - 7-span transmembrane molecule • - Localizes to Golgi and PM • - Mutant (C.e./D.m.): No secretion, accum. Wnt • in producing cells

18. Wnt gradient formation requires retromer function in Wnt-producing cells anterior posterior Retromer mutant b-cat mutant Coudreuse et al. (2006) Science 312:p921

19. Cellular function of the retromer complex Plasma membrane Endocytosis Early Endosome TEN/TSE Late Endosome Retromer Secretory pathway Degradative pathway Trans-Golgi Network Golgi Lysosome Nucleus / ER

20. The retromer complex: organization and function Seaman, Trends in Cell biology, 2005 SNX1 and 2: PH- and BAR-domain proteins Vps26/29/35: cargo selection Vps35: binds cargo

21. What cargo is transported by the retromer complex? N-ter SNX ? ? No structural information working model for the assembly and function of mammalian retromer

22. Example retromer-cargo: Mannose-6-phosphate receptor (CD-MPR) Binds to clathrin, concentrates MPRs ? Budding? ? Tethering? Fusion? Retromer = Lysosomal hydrolases

23. What is the relationship between Evi/Wls and retromer?

24. Drosophila as a model system for studying the Wnt (Wg) pathway Wing margin Common stages to look for effects of Wg signaling: Patterning epidermis Larvae encapsulate in puparium; metamorphosis (400/female) Wing disc - Larvae molt twice, ecdysis - Eat microorg on decaying fruit and fruit-derived sugar

27. Expression of wingless (Wg) and vestigial in the Drosophila wing disc Wg = green Vestigial = red D/V boundary

28. Wing disc expressing C93A HA-Wg (Dpp Gal4 –driven) 300x Fixation 2% PFA, 0.2%GA

29. Disc cells strongly expressing C93A HA-Wg are grouped together neg pos Peripodial membrane HA-Wg 15 nm

30. Disc cells expressing C93A HA-Wg are grouped together Peripodial lumen Peripodial membrane C93A HA-Wg15

31. Drosophila as a model system for studying the Wnt (Wg) pathway Wing margin Common stages to look for effects of Wg signaling: Patterning epidermis Wing disc

32. Epidermal patterning of Wg in fly embryo

33. Drosophila as a model system for studying the Wnt (Wg) pathway Wing margin Common stages to look for effects of Wg signaling: Patterning epidermis Wing disc

34. The wing disc and its adult derivatives wing margin notum wing blade wing hinge D V