Depletion with Cyclodextrin Reveals Two Populations of Cholesterol in Model Lipid Membranes

Abstract

Recent results provide evidence that cholesterol is highly accessible for removal from both cell and model membranes above a threshold concentration that varies with membrane composition. Here we measured the rate at which methyl-β-cyclodextrin depletes cholesterol from a supported lipid bilayer as a function of cholesterol mole fraction. We formed supported bilayers from two-component mixtures of cholesterol and a PC (phosphatidylcholine) lipid, and we directly visualized the rate of decrease in area of the bilayers with fluorescence microscopy. Our technique yields the accessibility of cholesterol over a wide range of concentrations (30-66 mol %) for many individual bilayers, enabling fast acquisition of replicate data. We found that the bilayers contain two populations of cholesterol, one with low surface accessibility and the other with high accessibility. A larger fraction of the total membrane cholesterol appears in the more accessible population when the acyl chains of the PC-lipid tails are more unsaturated. Our findings are most consistent with the predictions of the condensed-complex and cholesterol bilayer domain models of cholesterol-phospholipid interactions in lipid membranes.

Substrate-led cholesterol extraction from supported lipid membranes

“…Changes in lipid membranes supported on PDMS substrates Supported lipid membrane patches offer a convenient system for studying membrane-substrate interactions because they allow for simultaneous microscopic observation of the inplane membrane changes and for a quantitative analysis of the membrane surface area. 31,32 The patches can be routinely formed by fusing giant unilamellar lipid vesicles to a substrate (see Methods for further details). The substrate must be hydrophilic enough to interact with the lipid headgroups, but the interaction is usually non-specific and mediated by the thin interstitial water layer.…”

“…1), it is possible to directly correlate the relative changes in the membrane surface area to the changes in the membrane cholesterol mole fraction. To do this, we adapted the model proposed by Litz et al 32 for cyclodextrin-mediated cholesterol extraction: the relative patch area A r (t ) = A(t )/A(t 0 ) is related to the time-dependent cholesterol mole fraction, χ chol (t ), according to:…”

“…The well-known effects of cholesterol condensing [39][40][41] are accounted for in the expression for a avg , which depends exponentially on the cholesterol mole fraction. 32 To determine how χ chol (t ) varies with time, we must make two assumptions: (1) the PDMS substrate acts as an infinitely large cholesterol sink and the kinetics of cholesterol extraction can hence be modelled as a first order rate equation.…”

“…(2) There are two populations of membrane cholesterol, one more accessible for removal than the other, in line with pre- vious studies on cholesterol extraction. 32,42 Based on these assumptions, we can write:…”

Shape Transformations of Lipid Bilayers Following Rapid Cholesterol Uptake

“…S1). To account for the cholesterol condensing effect, we use the experimental data and method given in (20) for DOPC SLB patches (Eq. S3 in the Supporting Material).…”