Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization

Abstract

Oxidation of unsaturated lipids is a fundamental process involved in cell bioenergetics as well as in cell death. Using giant unilamellar vesicles and a chlorin photosensitizer, we asymmetrically oxidized the outer or inner monolayers of lipid membranes. We observed different shape transitions such as oblate to prolate and budding, which are typical of membrane curvature modifications. The asymmetry of the shape transitions is in accordance with a lowered effective spontaneous curvature of the leaflet being targeted. We interpret this effect as a decrease in the preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation (cleaved-lipids). Permeabilization of giant vesicles by light-induced oxidation is observed after a lag and is characterized in relation with the photosensitizer concentration. We interpret permeabilization as the opening of a pore above a critical membrane tension, resulting from the budding of vesicles. The evolution of photosensitized giant vesicle lysis tension was measured and yields an estimation of the effective spontaneous curvature at lysis. Additionally photo-oxidation was shown to be fusogenic.

Document S2: Figure

Figure 1

Time sequences of giant vesicles with photosensitizers targeted to the outer leaflet (upper row) or to the inner leaflet (lower row). The bar is 10 μm; the time lapse between two images is 1 s. In the first sequence of the upper row (phase contrast microscopy), a GUV photosensitized in its outer leaflet endures a short-time deformation (second image) followed by a budding toward the inside (open arrow). In the second sequence of the upper row (fluorescence of chlorin), a GUV photosensitized in its outer leaflet endures a deformation (second image) followed by multiple budding toward the inside (open arrows). In the sequence of the lower row (phase contrast microscopy), three GUVs photosensitized in their inner leaflet show deformations: the upper vesicle exhibits a transition from oblate to prolate (due to the projection, oblate appears as circle and prolate as ellipse), whereas the two other vesicles are deformed into pear shapes. These shape transitions are followed by a budding to the outside on each GUV (solid arrows). The morphology transitions observed when the outer leaflet is targeted denote a decrease of the membrane's equivalent curvature, whereas those observed when the inner leaflet is targeted denote an increase of the membrane's equivalent curvature.

Figure 2

Proportion of each type of photoinduced morphology transition as a function of photosensitizer localization. (Top to bottom) Outward budding (open dots on solid), oblate to prolate transition (close dots), long-time fluctuations (>20 s) (spaced dots), short-time fluctuations (<2 s) (wide hatching), and inward budding (close hatching). The midline between long- and short-time fluctuations materializes the separation between events associated with an increase of the effective spontaneous curvature (above the line) and those associated with a decrease of the effective spontaneous curvature (below the line).

Figure 3

Permeabilization of vesicles. (Left) Typical evolution of the contrast between the inside and outside of a photosensitized vesicle at 25 μM Ce6 in the outer medium. The decay of the contrast shows equilibrium between the inside solute (sucrose) and the outside solute (glucose). The experimental points are fitted by a decreasing exponential, from which are extracted a starting and a characteristic time of permeabilization. (Center and right) Starting time (t₀) and characteristic time (τ) of permeabilization for 50–120 vesicles for each concentration of Ce6. (Upward triangles) Vesicles with Ce6 in the outer medium; (downward triangles) vesicles with Ce6 in the inner medium; and (diamonds) in both media. The dependence on the vesicle radius is corrected (for τ at all concentrations and for t₀ at 50- and 125-μM outer medium).

Figure 4

Evolution of lysis tension over time during photodamage. The time after the start of the illumination at which lysis occurs was recorded for different membrane tensions (113 vesicles total). The boundaries of the boxes are the upper and lower quartiles (median 50% inside the box). The whiskers boundaries are the upper and lower deciles (median 80% inside the whisker).

Figure 5

Diagram of the lipid photo-oxidation processes in the vesicle membrane. The lipid oxidation, initiated via singlet oxygen, is a chain reaction.