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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

Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

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Page 1: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

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 HaagsmanNatasja Schoonen

Supervised by: Dr. P. van Bergen en Henegouwen

Traffic 2009; 10: 590-599

Page 2: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

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

Page 3: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

CIE vs CDE

Route depends

on specific

targeting

sequences at

cargo proteins

Page 4: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Research aim

• Understanding the machinery responsible for ARF6 mediated endocytosis

• By identifying PM proteins that enter the cells through the Arf6 route

How?

Page 5: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

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

Page 6: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Isolating early endosomes originating from A6-CIE

Page 7: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Identifying new proteinsby LC-MS/MS

Page 8: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Co-localization of identified proteins within Arf6 route?

Transfected with:

MEM-GFP: marker Arf6 route

Arf6Q67L

Page 9: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Transfected with:

MEM-GFP: marker Arf6 route

Arf6Q67L

Co-localization of identified proteins within Arf6 route?

Page 10: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

MHC1 and Tfn colocalize with CD55 and Glut1

60 min.

Page 11: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

60 min.

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

Page 12: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Recap

• Identification of new cargo proteins in CIE

• New cargo proteins differ in their intracellular trafficking

Page 13: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

CD98 MHC1 colocalization varies during internalization

Page 14: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

CD55, MHC1, Glut1 traffic via EEand CD98, CD147 and CD44 not

CD55 HA-Glut1MHC1

CD98 CD147 CD44

EEA1

EEA1

Page 15: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Recap

• CD98 internalizes faster than MHCI

• Different route of internalization between CD55/Glut1 and CD44/98/147

Page 16: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

MHC1 and CD98 internalize dynamin independent

K44 = dynamin2 mutant

Page 17: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Filipin binds PM cholesterol

MHC1 and CD98 internalize cholesterol dependent

Page 18: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Conclusion

Page 19: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Future research

• How are CIE vesicles formed?

• Cellular function of CIE pathways?

Page 20: Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis Craig A. Eyster, Jason D. Higginson, Robert Huebner, Natalie

Discussion

• Why not EM pictures?

• Why not inhibiting clathrin?

• Why cell lysates?