1. Study of Cholesterol Ozonolysis Product Trafficking in Cells by Using Quantum Dots
Kanlaya Prapainop
6th June 2007
Scripps-Oxford Laboratory
Department of Biochemistry
Professor Paul Wentworth

2. Cholesterol Ozonolysis Products
Atheronal A
Cholesterol
Aldolization
Science 2003;302 (5647):
Atheronal B

3. Cholesterol Ozonolysis products
Cause misfolding of LDL (Science 2003;302 (5647):1053-6)
Trigger foam cell formation in atherosclerosis
(Science 2003;302 (5647):1053-6, Biochemistry 2006;45(23): )
Cause aggregation of b-amyloid protein
(Proc Natl Acad Sci USA.2006 ;103(10): )
Cause aggregation of a-synuclein (Nat Chem Biol. 2006;2(5):249-53)
May be associated with various protein misfolding diseases
Macrophages treated with a dansyl atheronal B analog
Biochemistry 2006;45(23):

4. Intracellular cholesterol transport
Nature 2005,438:

5. Organic Fluorophores Susceptible to photodamage
Have narrow excitation and wide emission spectrum

6. Quantum Dots Fluorescent semiconductor nanocrystals
Resistance to photobleaching
Broad excitation, narrow emission spectrum
TRENDS in Cell Biology 2004;14(9):

7. PartI: Synthesis of Atheronal B-Qdot conjugates
2. AcOH/H2O/Zinc dust
Atheronal A
cholenic acid
L-Proline
1. EEDQ in DMF
Atheronal B 2.
QAtB
Biochemistry 2006;45(23):

8. Emission spectrum of QD and conjugated QD

9. PartII: Fluorescent microscopy
Murine Macrophage Raw cells
130 nM Qdot
in 1%FCS RPMI
130 nM QChol
in 1%FCS RPMI
130 nM QAtB
In 1%FCS RPMI
Can Qdots be transported into the cells?

10. Signal of Qdots are from conjugated Qdots only
(negative control)
QChol
QAtB
Signal of Qdots are from conjugated Qdots only

11. Time course Study
30 mins
40 mins
45 mins
130 nM QAtB in 1% FCS RPMI

12. Murine macrophage cell
Green: Phalloidin (Actin staining)
Blue:DAPI (Nucleus staining)
Red:QAtB

14. Intracellular cholesterol transport
Nature 2005;438,

15. Is lipoprotein necessary for transporting
conjugated Qdots into the cells?
QChol
QAtB
W/O FCS
Qdot(neg.control)
1%FCS
Lipoprotein may help to transport conjugated Qdots

16. Colocalization study with early endosome marker
Rab5
QAtB
Merge

17. Colocalization study with late endosome marker
M6PR
QAtB
Merge

18. Colocalization with endoplasmic reticulum marker
ERp19
QAtB
Merge

19. Summary QChol and QAtB do not change spectral property of Qdots
Unconjugated Qdots cannot transport into the cells
QChol and QAtB may require lipoprotein to transport
into the cells
There is some degree of colocalization of QAtB and ER
marker

20. Future Studies
Live cell imaging
Receptor determination

21. Acknowledgements Professor Paul Wentworth Daniel Witter Dr. John Offer
Dr. Sarah Tully
Adria Lombardo
Alicia Izharuddin
Jason Chang
Johanna Scheinost
Kavitha Baruah
Michael Cole
Natalie Cygan
Royal Thai government
(Higher Educational Strategic Scholarships
for Frontier Research Network)