Methyl-β-cyclodextrins preferentially remove cholesterol from the liquid disordered phase in giant unilamellar vesicles

Abstract

Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into lo and ld phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent on the MβCD concentration. At high concentrations, MβCD also removes phospholipids.

Fig. 1

Changes in Laurdan GP value for GUVs of POPC and POPC plus 33% molar Chol after incubation for 2 h with 800 μM MβCD. GP value of 10 GUVs before and after the addition was averaged. Working temperature was 25°C

Fig. 2

GP image analysis. a Laurdan GP image (equatorial z-section) of a GUV made of DOPC/DPPC/FC 1:1:1 at 25°C showing phase separation, l _o (orange) and l _d (green). The SimFCS program was used to analyze the GP image. Pixels with different GP values can be separated and the individual l _o (b) and l _d (c) phases followed in time. The number of pixels is related to the area of each phase (Color figure online)

Fig. 3

GP changes of coexisting l _o/l _d during interaction with 0.25 mM (a) and 1 mM (b) MβCD. Continuous lines at GP_i correspond to the GP value of the l _o and l _d domains in the DOPC/DPPC/FC (1:1:1 mol %) mixture and are the initial values for the kinetics. Dotted lines at GP_f are the GP values for the coexisting l _o and l _d phases in DOPC/DPPC (1:1) and correspond to the expected final GP values if all Chol is removed (Sánchez et al. 2007). Arrows represent the expected magnitude and direction for the GP changes when Chol is removed from the domains. Vertical dashed line at 45 min in b indicates the beginning of GUV shrinkage. The GP changes of the l _o (closed circles) and l _d (open circles) domains and volume reduction of the GUV (open squares) were followed for 2 h at 25°C. Images on top of the plots are GP images at the beginning (0 min) and end (110 and 120 min) of the incubation time, and the circular dotted line around the GUVs indicates the original size and shows the decrease in volume experimented by the target GUV (Color figure online)

Fig. 4

GP changes of coexisting l _o/l _d during interaction with 2 mM MβCD. GP_i, GP_f, arrows and symbols have the same meaning as in Fig. 3. Images on top of the plots are GP images at different points during the incubation time, and the circular dotted line around the GUVs indicates the original size. Numbers at the center of the GUVs in the GP images correspond to the experimental points shown in the plot (Color figure online)

Fig. 5

Kinetics for the separated l _o and l _d phases after addition of 1 mM MβCD. a Changes in GP value (open circles) and volume (closed squares) for the separated l _d phase (DOPC/DPPC/FC, 60:18:22 mol%). Comparison of GUV images at t = 0 and t = 100 min shows the decrease in size at the end of kinetics (dotted circular white line around the GUV on the image at t = 100, plot (a). b GUV of the l _d mixture without Chol (DOPC/DPPC/FC 77:23:0 mol%) with the corresponding average GP value for the homogeneous phase calculated from five independent GUVs. c Changes in GP value (closed circles) and volume (closed squares) for the separated l _o phase (DOPC/DPPC/FC, 5:53:42 mol%). Images of the GUV at t = 0 and t = 100 min show no changes in size during the kinetics. d Center and top views of a GUV of the l _o mixture without Chol (DOPC/DPPC/FC 9:91:0 mol%) showing phase separation. GP values for the different phases (green and orange) are given as an average of four GUVs. The same color scale was used for all images. Temperature 25°C (Color figure online)

Fig. 6

Model for the interaction of MβCD and membranes presenting phase separation. Empty circles symbolize Chol removed by MβCD