1. Cholesterol Distribution in Living Cells: Fluorescence Imaging Using DHE as a Fluorescent Cholesterol Analog. Sushmita Mukherjee, et al. Oct. 1998. Danielle “Dede” English Fluorescence Chapter 5 Adv. Analytical Chemistry ~Spring 2007~

2. Outline Background Cholesterol vs. Analogs Results/summary

3. Background/Purpose Cholesterol Majority in outer leaflet of plasma membrane (PM) –33 mole % in PM of cells –Interaction with membrane proteins and lipids –Role in cellular function (signaling, adhesion and motility) Distributed throughout intracellular membranes

4. Purpose: In order to understand the role, distribution and retention of cholesterol in the cell by using cholesterol analogs.

5. Chemical Structures Cholesterol DHE 25-NBD-cholesterol Analog requirements: -Intact alicyclic methyl groups -Branched 7-carbon alkyl chain at the 17-Beta position

6. Analogs of Cholesterol: Cholesterol: Planar steroid ring system with 3-Beta-hydroxyl and hydrophobic alkyl tail ( anchors onto membrane) Dehydroergosterol (DHE): Similar to cholesterol (Fluorescent) 3 additional double bonds and extra -CH 3 Mimics behavior of cholesterol Does not effect growth, membrane or activities of cell 25-NBD Cholesterol: Cuts seven member alkyl chain short with a bulky reporter group

7. Filipin A drug that binds to cholesterol Used to locate cholesterol in cells.

8. Figure 2: -Excitation and emission of DHE -Absorbs in the UV region of spectrum and emits in UV and blue region - Absorption peaks at 310, 324, and 338nm -Emission peaks at 354, 357, 394, and 414nm

9. Figure 3 - Chinese Hamster Ovary cell line (CHO cell line) -Imaged at focal plane near middle of cell: a ring of fluorescence -Results similar-in PM and intracellular -DHE 2-3 fold brighter in intercellular spot than PM -Filipin fluorescence more at the PM -Below self-quenching levels: maintained linear intensity patterns with increased concentration * Auto-fluorescence-the fluorescence of other molecules other than fluorophore. 50 ug/ml Filipin 12.5 uM DHE Auto-fluorescence unlabeled cells

10. Distribution of DHE Although DHE has similar binding patterns as cholesterol, may effect its distribution throughout cells. Unsure how DHE is metabolized by cells.

11. Figure 4 Distribution of 25 NBD cholesterol and DHE --Labeled and plated for 16-20h -DHE Localized at PM and perinuclear location -25 NBD Chol. Localized in mito. -Co-localized with MitoTracker red (small arrow) -2-5% co-localized with DHE in intracellular compartments ( large arrow a/b) TRVb-1/TacTGH38 cells labeled with: 12 uM DHE1.5 uM 25 NBD- cholesterol 5 uM MitoTracker Red in ethl

12. Separate experiment –22 and 25 NBD chol. Incubated for 2 min. –Similar results –Due to 7 member alkyl chain cut short (NBD polar group) –Less similar to cholesterol

13. Summary Cholesterol is in outer leaflet DHE localize at intracellular membranes than PM 25-NSD cholesterol localize more at mito.

14. Thank you! Any Questions!