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

Cholesterol Ozonolysis Products Atheronal A Cholesterol Aldolization Science 2003;302 (5647):1053-6. Atheronal B

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):7162-70) Cause aggregation of b-amyloid protein (Proc Natl Acad Sci USA.2006 ;103(10):3581-5.) 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):7162-70

Intracellular cholesterol transport Nature 2005,438:612-621

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

Quantum Dots Fluorescent semiconductor nanocrystals Resistance to photobleaching Broad excitation, narrow emission spectrum TRENDS in Cell Biology 2004;14(9):497-504

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):7162-70

Emission spectrum of QD and conjugated QD

PartII: Fluorescent microscopy Murine Macrophage Raw 264.7 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?

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

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

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

Intracellular cholesterol transport Nature 2005;438,612-621

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

Colocalization study with early endosome marker Rab5 QAtB Merge

Colocalization study with late endosome marker M6PR QAtB Merge

Colocalization with endoplasmic reticulum marker ERp19 QAtB Merge

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

Future Studies Live cell imaging Receptor determination

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)