Figure 2.

Lipid-leaflet lamellarity and content asymmetry of GUVs and LUVs. a) GUVs loaded with carboxyfluorescein were monitored over time by fluorescence microscopy. GUVs exposed to alpha-hemolysin (blue, +aH) were compared with control liposomes (red, −aH) and the flux of fluorescent molecules from within the lumen monitored over time. Inset: electron microscopic image of a liposome studded with aH pores (n = 5) (error bars are standard error of the mean (s.e.m.)). Data sets were min–max normalized, with +aH compared to that of −aH. b) Images of GUVs exposed (lower panels) or not exposed (upper panels) to alpha-hemolysin over time (n = 5). Scale bar, 10 μm. c) Fluorescence intensity and quenching of NBD-PC (both leaflets: POPC/POPS/cholesterol/NBD-PC) (n = 3, error bars are s.e.m.). Arrow number indicates as follows: 1) addition of quencher, 2) fluorescence quenching, 3) addition of Triton X-100 followed by bilayer solubilization and complete quenching. Data sets were min–max normalized and averaged. d) Liposome lamellarity visualized by cryoEM (lipid composition: POPC/POPS/cholesterol; n = 199). e) Fluorescence intensity of liposomes synthesized with NBD-PC present either in both leaflets or a single leaflet (n = 3). Unpaired t-test, ***p ˂ 0.001. f) Fluorescence intensity quenching of NBD-PC deposited exclusively in the inner leaflet. Inner leaflet, POPC/NBD-PC. Outer leaflet, POPS (n = 3, error bars are s.e.m.). Comparing solvents for generating asymmetric liposomes: mineral oil versus squalene. Data sets were min–max normalized.