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Juan Wu BMMB 12/04/09

Juan Wu BMMB 12/04/09. Golgi Apparatus. http://micro.magnet.fsu.edu/cells/golgi/golgiapparatus.html. PtdIns (4)P/GOLPH3/MYO18A/F- actin apparatus. Apply tensile force that pulls on trans-Golgi membrane to produce the flattened-shaped cisternae.

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Juan Wu BMMB 12/04/09

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  1. Juan Wu BMMB 12/04/09

  2. Golgi Apparatus http://micro.magnet.fsu.edu/cells/golgi/golgiapparatus.html

  3. PtdIns(4)P/GOLPH3/MYO18A/F-actinapparatus • Apply tensile force that pulls on trans-Golgi membrane to produce the flattened-shaped cisternae. • Efficient vesicle budding and transporting from Golgi TGN to PM Dippold et al., 2009

  4. PtdIns(4)P (also named PI(4)P): most abundant phospholipid at Golgi membrane Phosphatidylinositol Metabolic cycle of PtdIns(4)P in mammals Matteis et al.,

  5. Lipid-binding Screen Identifies GOLPH3 as a Pl(4)P-binding protein IVT system Bacterial purified Dippold et al., 2009

  6. GOLPH3 A B PI(4)P reporter EYFP-FAPP1-PH (Godi, et al 2004) colocalize with mCherry-GOLPH3 over space and time. MS1 Dippold et al., 2009

  7. PI(4)P is Required for GOLPH3 Localization to the Golgi Hela yeast also see movieS2 Dippold et al., 2009

  8. GOLPH3 Binds PI(4)P via a Positively Charged Pocket Dippold et al., 2009

  9. GOLPH3 Binds PI(4)P via a Positively Charged Pocket Phosphatidylethanolamine Binding pocket mutants Dippold et al., 2009

  10. Summary I • GOLPH3 binds tightly, specifically and directly to PI(4)P in two assays. (Lipid blot and vesicle binding) • Binding is evolutionarily conserved from yeast to humans • Depletion or blocking PI(4)P results in dissociation of GOLPH3 from Golgi TGN • GOLPH3 requires the ability to bind PI(4)P to localize to the Golgi.

  11. GOLPH3 is Required for Anterograde Trafficking from Golgi to PM Ts045- Dippold et al., 2009

  12. GOLPH3 is Required for the Normal Extended Golgi Ribbon Examinnation of several Golgi markers in GOLPH3 knockdown cells: GM130 (cis-Golgi), glycosyltransferases( cis/medial-Golgi), P130 &TGN46( trans-Golgi) in Hela, HEK293, NIH 3T3 cells are revealed condensation of Golgi. Dippold et al., 2009

  13. Rescue of GOLPH3 Knockdown Requires the Ability to Bind PI(4)P Asterisks denotes transfected cells Dippold et al., 2009

  14. Why does Golgi Change Morphology upon GOLPH3 Depletion??? Actin? Microtubule? GOLPH3 PI(4)P GOLPH3 X G O L G I LatB treatment led to rapid Golgi condensation MOVIES3-Live Image

  15. GOLPH3 Links the Golgi to the Actin Cytoskeleton • Knockdown of GOLPH3 results in condensation of Golgi without altering F-actin • Depolymerization of the actin by LatB leads to compaction of Golgi without affecting • the Golgi association of GOLPH3 Dippold et al., 2009

  16. GOLPH3 Binds MYO18A Directly Hela whole cell lysate Dippold et al., 2009

  17. MYO18A depends on GOLPH3 for Golgi Localization Dippold et al., 2009

  18. Rescue of MYO18A Knockdown Requires an intact MYO18A ATPase Pocket Dippold et al., 2009

  19. Summary II PI(4)P/GOLPH3/MYO18A/F-Actin GOLPH3 p230 Depletion of PI(4)P P230 Knockdown of GOLPH3 Knockdown of MYO18A Depolymerization of action Dippold et al., 2009

  20. Trans-Golgi Cisternae Are Dilated in Cells Depleted of GOLPH3 or MYO18A Dippold et al., 2009

  21. PI(4)P/GOLPH3/MYO18A/F-Actin Are Required for Efficient Vesicle Budding Live Time-lapse images of EYFP-FAPP1-PH tubules or vesicles leaving Golgi. movie4 Dippold et al., 2009

  22. PI(4)P/GOLPH3/MYO18A/F-Actin Are Required for Efficient Vesicle Budding The initial trajectory of vesicles exit follows the orientation of the tensile force. Dippold et al., 2009

  23. Summary • GOLPH3 binds to PI(4)P and depends on PI(4)P for its Golgi localization • PI(4)P/GOLPH3/MYO18A/F-actin apparatus is required for maintain Golgi flattened cisternae morphology at trans-face. • PI(4)P/GOLPH3/MYO18A/F-actin apparatus is also required for efficient vesicle budding and trafficking from Golgi to PM

  24. Future Perspectives… ? ? ? • PI(4)P GOLPH3 MYO18A F-actin • How does this molecular overcomes the problem of actin being constantly polymerizing and depolymerizing? • How does this molecular apparatus balance between maintaining Golgi morphology and extracting vesicles? • How are the flattened cis-and medial-cisternae morphology maintained? • Can the similar molecular apparatus be extended to other organelle, like ER? • Scott et al., identified GOLPH3 as an oncogene.

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