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~
Outline
• Background
• Cholesterol vs. Analogs
• Results/summary
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
Purpose:
In order to understand the role, distribution and
retention of cholesterol in the cell by using
cholesterol analogs.
Chemical Structures
Cholesterol
DHE
Analog requirements:
-Intact alicyclic methyl
groups
-Branched 7-carbon alkyl
chain at the 17-Beta
position
25-NBD-cholesterol
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
Filipin
• A drug that binds to cholesterol
• Used to locate cholesterol in cells.
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
Figure 3
12.5 uM DHE
Auto-fluorescence
unlabeled cells
-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
Distribution of DHE
• Although DHE has similar binding patterns
as cholesterol, may effect its distribution
throughout cells.
• Unsure how DHE is metabolized by cells.
Figure 4
Distribution of 25 NBD cholesterol and DHE
TRVb-1/TacTGH38 cells labeled with:
12 uM DHE
1.5 uM 25 NBDcholesterol
--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)
5 uM MitoTracker Red in ethl
• 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
Summary
• Cholesterol is in outer leaflet
• DHE localize at intracellular membranes
than PM
• 25-NSD cholesterol localize more at mito.
Thank you!
Any Questions!