Organization of fluorescent cholesterol analogs in lipid bilayers — Lessons from cyclodextrin extraction

Abstract

To characterize the structure and dynamics of cholesterol in membranes, fluorescent analogs of the native molecule have widely been employed. The cholesterol content in membranes is in general manipulated by using water-soluble cyclodextrins. Since the interactions between cyclodextrins and fluorescent-labeled cholesterol have not been investigated in detail so far, we have compared the cyclodextrin-mediated membrane extraction of three different fluorescent cholesterol analogs (one bearing a NBD and two bearing BODIPY moieties). Extraction of these analogs was followed by measuring the Förster resonance energy transfer between a rhodamine moiety linked to phosphatidylethanolamine and the labeled cholesterol. The extraction kinetics revealed that the analogs are differently extracted from membranes. We examined the orientation of the analogs within the membrane and their influence on lipid condensation using NMR and EPR spectroscopies. Our data indicate that the extraction of fluorescent sterols from membranes is determined by several parameters, including their impact on lipid order, their hydrophobicity, their intermolecular interactions with surrounding lipids, their orientation within the bilayer, and their affinity with the exogenous acceptor.

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

“…We suggest that the large-sized PEG-linker in KK114-PEG-Chol and Star512-PEG-Chol prevents sterol fl ip-fl op and, therefore, only allows for vesicular uptake in these cells. Accordingly, their enrichment in Chol-containing organelles, such as the ERC in healthy cells or the lysosomal storage organelles in NPC cells, is lower than that of analogs with fast trans-bilayer movement ( 17,70 ).…”

Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol

“…However, exogenously added fluorophore-labeled cholesterol is also located in both leaflets of membrane bilayers (Milles et al, 2013). Furthermore, the addition of the fluorophore alters the properties and dynamics of cholesterol (Milles et al, 2013;Solanko et al, 2013). Although these probes remain useful for certain types of experimentation, the development of alternative probes is warranted.…”

“…An alternative to filipin that has been developed more recently is to use cholesterol molecules directly conjugated to a fluorophore, such as BODIPY-cholesterol (Börnig and Geyer, 1974;Li et al, 2006;Hölttä-Vuori et al, 2008). However, exogenously added fluorophore-labeled cholesterol is also located in both leaflets of membrane bilayers (Milles et al, 2013). Furthermore, the addition of the fluorophore alters the properties and dynamics of cholesterol (Milles et al, 2013;Solanko et al, 2013).…”

Interaction of fluorescent phospholipids with cyclodextrins

“…The assay for measuring the extraction of phospholipid analogs from membranes is similar to an approach described recently (Milles et al, 2013), in which the Förster resonance energy transfer (FRET) between fluorescent sterol analogs and Rh-PE was measured. Here, we measured the FRET between the NBD or BODIPY moiety linked to different phospholipid species and the rhodamine moiety linked to PE (Rh-PE).…”

“…Modifications of CDs at the hydroxyl groups of glucose such as methylations or hydroxypropylations modulate the affinity of lipids for CDs (Szente and Szejtli, 1999;Uekama, 2004). The high specificity of ßCD and its derivatives for sterols has been applied to characterize the physiological role of this lipid e.g., to manipulate the sterol content of membranes by extracting or incorporating cholesterol from/into membranes, to measure the cellular sterol dynamics and, to incorporate labeled sterol analogs into membranes (Müller and Herrmann, 2002;Wüstner, 2007;Zidovetzki and Levitan, 2007;Müller et al, 2011;Milles et al, 2013).…”