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Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

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. Secretory Pathway. Lipid raft. Ordered, highly packed

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Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

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  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. Secretory Pathway

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

  4. Lipid raft • 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!

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

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

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

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

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

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

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

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

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

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

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

  19. 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

  20. 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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