1. 1 Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network Kai Simons group, 2009, J. Cell Biology Deniz Ugurlar Robbert Kim Lecturer: Gerrit van Meer

2. 2 Secretory Pathway

3. 3 Lipid raft Ordered, highly packed Sterols, sphingolipids, glycerophospholipids, proteins

4. 4 Signal transduction, virus assembly, membrane trafficking Proposed to be involved in the generation of lipid gradients Lipid sorting at TGN Low conc in ER and accumulate toward PM ( Simons and van Meer,1988) Raft involvement in TGN sorting – no direct evidence! Lipid raft

5. 5 Hypothesis Raft plays a functional role in the sorting machinery  The secretory vesicles should be selectively enriched in sterols and sphingolipids Previous Research Shortcomings of the methodology insufficient purity of isolated TGN-derived vesicles not be able to characterize lipid composition

6. 6 Experimental Outline S.cerevisiae Ergosterol  cholesterol in mammals LDSV transporting FusMidGFP (TGN to PM) Immunoisolation procedure Purify secretory vesicles Late Golgi compartments Western Blot & EM Quantitative shotgun lipidomics

7. 7 The Bait for Immunoisolation FusMidGFPLTLM9  FusMidGFP  High affinity 9xmyc(M9) tag  T TEV Protease site  L linker  FusMidGFPLTLM9  FusMidp

8. 8 Expression Exocyst mutant sec6-4, temperature sensitive 24°C, permissive 37°C, restrictive  no PM translocation

9. 9 EM & Tomography intracellular accumulation of vesicles at 37 ° C http://jcb.rupress.org/cgi/ content /full/jcb.200901145/DC1/ 1

10. 10 Immunoisolation Vesicle isolation cell lysis differential fractionation Isopycnic sucrose gradient Immunoisolation Mouse anti-myc antibody Sheep anti-mouse immunoadsorbent Vesicle recovery TEV protease

11. 11 Purification assessment Distinct vesicles Raft protein Late endosome ER Late Golgi

12. 12 Morphology Purified vesicles with EM Homogenous ~100 nm (comparable with the ones in living cells)

13. 13 Recap Isolated FusMidp vesicles Good purification Spherical vesicles Homogenous ~100nm Comparable with living cells Ready for analysis Composition Comparison with donor organelle

14. 14 Immunoisolation TGN/E Distinct vesicles Raft protein Late endosome ER Late Golgi

15. 15 Comparison Morphology TGN/E Heterogenous population ~100-300 nm and ~40-50 nm Different from yeast PM Morphology FusMidp vesicles Distinct from the donor organelle Immunoisolation protocol Efficient tool Suitable different vesicles

16. 16 Lipidomics study Quantitative shotgun lipidomics Mass Spectrometry 83 lipids of 12 classes

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18. 18 Lipidomics results Most abundant ones FusMidp Ergosterol (22.8 mol%) Sphingolipids TGN/E Ergosterol (9.8 mol%) Sphingolipids FusMidp Less PS, PE, PC

19. 19 Membrane order assay C-Laurdan spectrophotometry Calculate general polarization (GP)

20. 20 Conclusions Nice method for purifying vesicles Analyzed LDSV compared with TGN/E High in ergosterol and sphingolipids Low PS, PE, PC Higher membrane order Raft involvement Article conclusion Membrane architecture modulated

21. 21 Discussion  Trustworthy results  Many methods  Complementary results  COPI coated vesicles low in sphingomyelin and cholesterol  Mammal system  Brügger et al, 2000. J. Cell Biol.  Endosomes and lysosomes  No known factors raft clustering  Candidates have been proposed (Prozynski et al. 2005)

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