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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 [ergosta5,7,9(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.
Keywords Dehydroergosterol * Cholesterol * Ergosterol * Fluorescent sterols * Microscopy
Abbreviations
DHE Dehydroergosterol.
DHE-MβCD DHE-methyl-β-cyclodextrin
SP Spin labeled
BODIPY 4, 4-Difluoro-4-bora-3a, 4a-diaza-s-indacene
NBD N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl)amino
Dansyl 5-Dimethylaminonapthalene-1-sulfonyl
CTE Cholestatrienol
DiO 3, 3'-Dioctadecyloxacarbocyanine Perchlorate
ECFP Enhanced cyan fluorescent protein
BHT Butylatedhydroxytoluene
APCI (Atmospheric pressure chemical ionization) mass spectroscopy
HPLC High-performance liquid chromatography
LUV Large unilamellar vesicle
SCP-2 Sterol carrier protein-2
L-FABP Liver fatty acid binding protein
MDCK Madin-Darby canine kidney
EPR Electron spin resonance
Historical Perspective
In order to resolve the dynamics and factors governing cholesterol trafficking within cells, it is important to recognize that the cholesterol molecule has very few polar constituents (Fig. Ia). Consequently,...