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Presented by: Marjolein Haagsman Natasja Schoonen Supervised by: Dr. P. van Bergen en Henegouwen

Traffic 2009; 10: 590-599. Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie Porat-Shliom, Roberto Weigert, Wells W. Wu, Rong-Fong Shen and Julie G. Donaldson. Presented by: Marjolein Haagsman

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Presented by: Marjolein Haagsman Natasja Schoonen Supervised by: Dr. P. van Bergen en Henegouwen

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  1. Traffic 2009; 10: 590-599 Discovery of New Cargo Proteins that Enter Cellsthrough Clathrin-Independent EndocytosisCraig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie Porat-Shliom, Roberto Weigert, Wells W. Wu, Rong-Fong Shen and Julie G. Donaldson Presented by: Marjolein Haagsman Natasja Schoonen Supervised by: Dr. P. van Bergen en Henegouwen

  2. Arf6 regulated CIE • Independently of clathrin & dynanim • Requires free cholesterol • Regulated by GTP-binding proteins (Arf6) • Alters cortical actin cytoskeleton • Alters lipid composition PM • Arf6Q67L: • Stimulation CIE • Accumulation cargo in vacuolar-type structure

  3. CIE vs CDE Route depends on specific targeting sequences at cargo proteins

  4. Research aim • Understanding the machinery responsible for ARF6 mediated endocytosis • By identifying PM proteins that enter the cells through the Arf6 route How?

  5. Isolating early endosomes originating from A6-CIE • Use of a receptor for this route: MHC1 and intracellular marker: GFP-syn3 • Inhibit trafficking: Arf6-Q67L • Preparation cell lysates • Affinity purification with anti-GFP-coupled magnetic beads

  6. Isolating early endosomes originating from A6-CIE

  7. Identifying new proteinsby LC-MS/MS

  8. Co-localization of identified proteins within Arf6 route? Transfected with: MEM-GFP: marker Arf6 route Arf6Q67L

  9. Co-localization of identified proteins within Arf6 route? Transfected with: MEM-GFP: marker Arf6 route Arf6Q67L

  10. MHC1 and Tfn colocalize with CD55 and Glut1 60 min.

  11. CD44, CD98 and CD147 colocalizes partly with MHC1 and not with Tfn 60 min.

  12. Recap • Identification of new cargo proteins in CIE • New cargo proteins differ in their intracellular trafficking

  13. CD98 MHC1 colocalization varies during internalization

  14. CD55, MHC1, Glut1 traffic via EEand CD98, CD147 and CD44 not MHC1 HA-Glut1 CD55 EEA1 EEA1 CD98 CD147 CD44

  15. Recap • CD98 internalizes faster than MHCI • Different route of internalization between CD55/Glut1 and CD44/98/147

  16. MHC1 and CD98 internalize dynamin independent K44 = dynamin2 mutant

  17. MHC1 and CD98 internalize cholesterol dependent Filipin binds PM cholesterol

  18. Conclusion

  19. Future research • How are CIE vesicles formed? • Cellular function of CIE pathways?

  20. Discussion • Why not EM pictures? • Why not inhibiting clathrin? • Why cell lysates?

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