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Fluorescence Imaging of Cholesterol Distribution in Living Cells

Explore the role of cholesterol and its analogs in cellular function and distribution within living cells using fluorescence imaging techniques. Chemical structures and behaviors of cholesterol analogs Dehydroergosterol and 25-NBD Cholesterol are detailed, along with their impact on cell activities.

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Fluorescence Imaging of Cholesterol Distribution in Living Cells

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  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/PurposeCholesterol • 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 Analog requirements: -Intact alicyclic methyl groups -Branched 7-carbon alkyl chain at the 17-Beta position 25-NBD-cholesterol

  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 -CH3 • 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 Auto-fluorescence unlabeled cells 12.5 uM DHE -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

  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 4Distribution of 25 NBD cholesterol and DHE TRVb-1/TacTGH38 cells labeled with: • --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) 12 uM DHE 1.5 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!

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