Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

摘要: 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] fluorescent sterol most structurally and functionally similar to cholesterol date, proven be a major asset probing/elucidating environment intracellular trafficking organisms. DHE is naturally occurring, analog that faithfully mimics many properties cholesterol. Because these are sensitive structure degradation, such studies require use extremely pure (>98%) quantities sterol. readily bound by cholesterol-binding proteins, incorporated into lipoproteins (from diet animals or exchange vitro), easily cultured cells where it co-distributes with endogenous Incorporation from an ethanolic stock solution cell culture media effective, but this process forms aqueous dispersion crystals which can result endocytic cellular uptake distribution lysosomes problematic at plasma membrane In contrast, monomeric unilamellar vesicles exchange/fusion DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes membrane. Both latter techniques deliver large significant The behavior protein-binding, lipoproteins, model membranes, biological lipid rafts/caveolae, indicate occurring useful mimic probing systems.