1. 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 Natasja Schoonen Supervised by: Dr. P. van Bergen en Henegouwen Traffic 2009; 10: 590-599

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 proteins by LC-MS/MS

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

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

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

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

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 EE and CD98, CD147 and CD44 not CD55 HA-Glut1MHC1 CD98CD147CD44 EEA1

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. Filipin binds PM cholesterol MHC1 and CD98 internalize cholesterol dependent

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?