Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles

Abstract

Cell-derived GPMVs (giant plasma-membrane vesicles) enable investigation of lipid phase separation in a system with appropriate biological complexity under physiological conditions, and in the present study were used to investigate the cholesterol-dependence of domain formation and stability. The cholesterol level is directly related to the abundance of the liquid-ordered phase fraction, which is the majority phase in vesicles from untreated cells. Miscibility transition temperature depends on cholesterol and correlates strongly with the presence of detergent-insoluble membrane in cell lysates. Fluorescence correlation spectroscopy reveals two distinct diffusing populations in phase-separated cell membrane-derived vesicles whose diffusivities correspond well to diffusivities in both model systems and live cells. The results of the present study extend previous observations in purified lipid systems to the complex environment of the plasma membrane and provide insight into the effect of cholesterol on lipid phase separation and abundance.

Figure 1. Fluorescence images and quantification of the L_d (non-raft) phase fraction in GPMVs (stained with rhoPE to label the L_d phase) as a function of cholesterol modulation

The cholesterol level relates inversely to the abundance of the L_d phase in GPMVs and the L_o phase is the majority phase in vesicles from untreated cells. Ribbon-like gel-phase domains are observed when cholesterol is entirely depleted by direct treatment of GPMVs. Values are means + S.D. from > 35 vesicles per condition. Scale bars = 5 μm. chol, cholesterol; depl, depleted.

Figure 2. Temperature-dependence of phase separation in GPMVs isolated from untreated cells (black), cholesterol-depleted cells (red circles) and cholesterol-loaded cells (blue squares)

Pictures are superpositions of red and green images from epifluorescence micrographs of GPMVs prepared from untreated cells stained with rhoPE (L_d, red) and nap (L_o, green) at 10 °C (left-hand side, phase-separated) and 37 °C (right-hand side, homogeneous). cont, control; chl/chol, cholesterol; depl, depleted; PL, phospholipid.

Figure 3. Correlation between the temperature-dependence of phase separation of GPMVs (diamond points, line is a sigmoidal fit) and the abundance of detergent-resistant membranes (detergent solubilization performed at the indicated temperatures) as quantified by the percentage of cholesterol in the detergent-resistant fractions (striped bars)

Values are means ± S.D. Low temperatures, which induce GPMV phase separation, also induce Triton-resistant membrane fractions as quantified by the presence of cholesterol in low-density fractions (fractions 1–3 for all temperatures, except 10 °C where detergent-resistance membranes were in fractions 1–6; see Supplementary Figure S3 at http://www.BiochemJ.org/bj/424/bj4240163add.htm). DRM, detergent-resistant membrane.

Figure 4. Histograms of diffusion coefficients obtained by FCS of rhoPE diffusing in (A) phase-separated vesicles at 10 °C and (B) microscopically uniform vesicles at 37 °C

Diffusion coefficients were calculated by fitting autocorrelation data to a two-component two-dimensional diffusion equation (for experimental details see the Supplementary Experimental section at http://www.BiochemJ.org/bj/424/bj4240163add.htm). These results show a single diffusing population of tracers in uniform vesicles and two distinct populations in phase-separated vesicles [bold lines are Gaussian fits to all data and thin lines in (A) show the component Gaussians]. Histograms are from > 70 measurements on seven to nine vesicles per condition.