Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation

Abstract

Raft formation and enlargement was investigated in liposomes and supported bilayers prepared from sphingomyelin (SM), cholesterol, and unsaturated phospholipids; NBD-DPPE and rhodamine-(DOPE) were employed as fluorescent probes. Rafts were created by lowering temperature. Maintaining 20 mol % SM, fluorescence microscopy showed that, in the absence of photooxidation, large rafts did not form in giant unilamellar vesicles (GUVs) containing 20 or more mol % cholesterol. But if photooxidation was allowed to proceed, large rafts were readily observed. In population, cuvette experiments, small rafts formed without photooxidation at high cholesterol concentrations. Thus, photooxidation was the cause of raft enlargement during microscopy experiments. Because photooxidation results in peroxidation at lipid double bonds, photosensitization experiments were performed to explicitly produce peroxides of SM and an unsaturated phospholipid. GUVs of high cholesterol content containing the breakdown products of SM-peroxide, but not phospholipid-peroxide, resulted in large rafts after lowering temperature. In addition, GUV production by electroswelling can result in peroxides that cause large raft formation. The use of titanium electrodes eliminates this problem. In conclusion, lipid peroxides and their breakdown products are the cause of large raft formation in GUVs containing biological levels of cholesterol. It is critical that experiments investigating rafts in bilayer membranes avoid the production of peroxides.

FIGURE 1

Large rafts do not form for a high cholesterol-content membrane in the absence of photooxidation, unless temperature is extremely low. Rafts do not appear at 25°C (A) or 10°C (B), but some rafts do appear at 1°C (C). GUVs were formed from a 20:40:40% eSM/cholesterol/DOPC mixture. NBD-DPPE was used as probe for each of these panels.

FIGURE 2

Photooxidation promotes large rafts for high cholesterol content bilayers. In the absence of antioxidants, large rafts formed within 15 s after illumination. GUVs prepared as in Fig. 1. Rho-DOPE was used as probe, so here nonraft circular domains are surrounded by a raft region. The times after starting illumination are shown (in units of seconds) in the upper left corners.

FIGURE 3

In the absence of photooxidation, the appearance of large rafts depended on the ratios of SM and cholesterol rather than on a simple increase in the amounts of the two components. (A) Large rafts formed for a 20:10% SM/cholesterol mixture. (B) Large rafts were scarce for a 20:20% SM/cholesterol mixture. Small, barely visible rafts are marked by arrows. (C) Large rafts were absent for a 10:20% mixture. NBD-DPPE was used as probe for this figure. All images are for GUVs at 25°C.

FIGURE 4

Using fluorimetry to measure the formation of small rafts upon lowering temperature. Liposomes of 200-nm diameter were prepared from a 20%/40%/40% eSM/cholesterol/DOPC mixture. For liposomes containing NBD-DPPE (0.1%) as a sole probe (open circles), fluorescence varies with the same slope for temperatures above and below 40°C. In contrast, partially quenching the NBD-DPPE fluorescence by Rho-DOPE (1%) caused NBD fluorescence to increase more steeply below 30°C–35°C than above this temperature range (solid triangles). The unquenched (open circles) and quenched (solid triangles) fluorescence was normalized to their values at 50°C, plotted as F/F₀. Records are shown for a typical experiment. Inset: NBD-DPPE fluorescence in liposomes composed of eSM/DOPC 20:80%, illustrated for a typical experiment.

FIGURE 5

Breakdown products of SM-peroxide, but not those of DOPC-peroxide, induce raft enlargement. (A) GUVs that included 5 mol % of SM-peroxide breakdown products in a 15:40:36% eSM/cholesterol/DOPC (plus 1% NBD-DPPE and 3% Rho-DOPE) mixture yielded large rafts at 25°C. (B) A greater percentage of the GUVs exhibited rafts at 10°C (C). GUVs that included 10 mol % of DOPC-peroxide products in a 20:40:26% eSM/cholesterol/DOPC (plus 1% NBD-DPPE and 3% Rho-DOPE) mixture did not yield visible rafts, even when temperature was lowered to 1°C. Note that the lipid mixtures were the same in all cases, except that in panels A and B the 5 mol % of SM-peroxide breakdown products replaced an equivalent amount of SM, whereas in (C) the 10 mol % DOPC-peroxide breakdown products substituted for this amount of DOPC. All three images display the fluorescence of NBD-DPPE.

FIGURE 6

Forming GUVs with ITO-glass electrodes, but not with titanium electrodes, generates peroxides. Bar graph: ITO-glass and titanium electrodes were used to prepare GUVs composed of 20:20:60% eSM/cholesterol/DOPC, and peroxide content was measured by a FOX assay and normalized to the amount of lipid. (This amount was calculated from the measured phospholipid content.) The ordinate thus displays the percentage of peroxide relative to the total lipid content in mol %. Error bar is mean ± SE, n = 4. The use of ITO-glass led to peroxides (column 1), but titanium electrodes (column 2) did not generate peroxides. GUVs were prepared in 200 mM sucrose using a 3 V p-p, 10-Hz sine wave across the electrodes. Images: When ITO-glass electrodes were used to prepare GUVs composed of 20:40:40% eSM/cholesterol/DOPC, some large rafts formed at 25°C (A); more formed at 4°C (B). Only a fraction of the GUVs could exhibit rafts because only those vesicles that came within proximity of the electrodes could have their lipids electrochemically peroxidized. Preparing GUVs under precisely the same conditions, but using titanium electrodes, did not lead to rafts, shown here at 10°C (C; see also Fig. 1). NBD-DPPE was used as probe for all images.

FIGURE 7

Adhering a bilayer directly onto a substrate promotes large rafts. (A) For a 20:20:40:20% eSM/cholesterol/DOPC/DOPE bilayer adhered to a mica substrate, large rafts form at 25°C in the presence of antioxidants. (This contrasts to GUVs of the same composition, for which rafts did not form.) (B) Photooxidation produced by illumination in the absence of antioxidants led to larger rafts. The arrows mark the boundary of the illuminated area. (C) Providing a cushion (here, made with 65 kD PEI) between the bilayer and substrate reduces large raft formation. In the presence of antioxidants, large rafts do not appear at 25°C for a cushioned bilayer. (D) Rafts appear in the cushioned bilayer when photooxidation is not prevented. Rho-DOPE was used as probe.

FIGURE 8

Photoexcitation of a fluorescent probe results in a cascade of reactions. One such cascade is illustrated. A photoexcited fluorescent probe raises molecular oxygen to its singlet excited state. The excited oxygen reacts with a double bond on an acyl chain to create a peroxide. The peroxide is unstable in the presence of any trace ferrous (or other transition metals) and decays to a lipid-free radical which removes (i.e., abstracts) a hydrogen from an unoxidized, unsaturated lipid. This creates a hydroxylated lipid (i.e., a peroxide product) and a new free radical. Molecular oxygen combines with the new radical, abstracting a hydrogen from another unsaturated lipid, and again producing a free radical and a lipid peroxide. This free radical and lipid peroxide initiates an additional round of reactions. The two feedback loops result in substantial lipid oxidation. These reactions occur without any destruction of the excited fluorescent lipid probe that decays back to its ground state, from which it can again be photoexcited.