Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking |
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Authors: | Avery L McIntosh Barbara P Atshaves Huan Huang Adalberto M Gallegos Ann B Kier Friedhelm Schroeder |
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Affiliation: | (1) Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, USA;(2) Department of Pathobiology, Texas A&M University, TVMC, College Station, TX 77843-4467, USA |
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Abstract: | Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent
of dehydroergosterol ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar
to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular
sterol trafficking in living organisms. DHE is a naturally occurring, fluorescent sterol analog that faithfully mimics many
of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies
require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins,
is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is
easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock
solution to cell culture media is effective, but this process forms an aqueous dispersion of DHE crystals which can result
in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of
living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma
membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques
can deliver large quantities of monomeric DHE with significant distribution into the plasma membrane. The properties and behavior
of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging
in living cells indicate that this naturally occurring fluorescent sterol is a useful mimic for probing the properties of
cholesterol in these systems. |
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Keywords: | Dehydroergosterol Cholesterol Ergosterol Fluorescent sterols Microscopy |
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