Figure 1.
Preparation of asymmetric LUVs.
(A) Flow chart of method for preparation of asymmetric POPCo/POPE:POPSi/cholesterol LUVs. Preparation of vesicles with SM or SM/POPC outside is identical to POPC except that the initial dried lipids are different. The concentration of donor lipids and HPαCD after overnight incubation at 55°C is slightly higher (by ∼10–20%) than theoretical because of evaporation. (B) HP-TLC analysis of final pellet from asymmetric LUVs protocol. POPC MLVs (lane 1 to 4) or PBS (lane 5 and 6) were incubated with (lane 1, 3 and 5) or without (lane 2, 4, and 6) HPαCD overnight and 3∶3∶4 POPE/POPS/cholesterol LUVs (lanes 1, 2, 5 and 6) or PBS (lanes 3 and 4) were then added, and incubated for 30 min at 55°C, followed by two centrifugation steps.
Figure 2.
Sucrose density gradient centrifugation of POPCo/POPE:POPSi/cholesterol vesicles.
(A) Sucrose gradient fractions of exchange LUVs containing ∼400 µl of 1 mM (total lipid) POPCo/POPE:POPSi/40 mol% cholesterol. (B) Sucrose gradient fractions of a mixture of 200 µl of 1 mM POPC MLVs with HPαCD and 200 µl of 1 mM LUVs composed of 3∶3∶4 POPE/POPS/cholesterol with 25% (w/v) entrapped sucrose. Top: stained TLC plate; Bottom: Band intensity for each lipid. Fractions from left to right are from low to high density. Gradients were prepared by freeze-thawing 3.4 ml of 20% (w/v) sucrose. 400 µl of vesicle samples were loaded on top of the gradients and the gradients were centrifuged for 17 h at 19,000 g.
Figure 3.
Exchange efficiency of donor lipid into exchange vesicles containing varies amount of cholesterol.
Exchange vesicles were prepared using pure POPC or pure SM (A), 1∶1 SM/POPC (B), or 4∶1 SM/POPC (C) in the donor vesicles. Acceptor vesicles were composed of 1∶1 POPE/POPS with different amounts of cholesterol. % POPC or % SM of total phospholipid in exchange vesicles was determined by HP-TLC versus standard curves in which different amounts of each lipid was loaded on the HP-TLC plate. Average values (mean) and S.D., or range if n = 2, are shown. Sample numbers were n = 6 for POPCo/POPE:POPSi/cholesterol; n = 4 for SMo/POPE:POPSi/cholesterol and 1∶1 SM:POPCo/POPE:POPSi/cholesterol; and n = 2 for 4∶1 SM:POPC/POPE:POPSi/cholesterol.
Figure 4.
Assay of lipid asymmetry detected by binding of hydrophobic helix pL4A18.
(A–B) Dependence of fluorescence emission λmax of pL4A18 peptide upon the fraction of 1∶1 POPE/POPS in symmetric vesicles composed of 1∶1 POPE/POPS mixed with POPC (A), or 1∶1 SM/POPC (B), plus different amounts of cholesterol. % POPE:POPS equals sum of % POPE plus % POPS. Average values (mean) and S.D. from three samples are shown. (C–D) Fluorescence emission λmax (black bar) of pL4A18 peptide binding to exchange (asymmetric) vesicles and calculated outer leaflet lipids that were POPE:POPS (striped bars) are shown for POPCo/POPE:POPSi/cholesterol (C), or 1∶1 SM:POPCo/POPE:POPSi/cholesterol (D). The % of outer leaflet lipids that were POPE:POPS was calculated from the standard curves (A–B) fitted to Boltzmann Sigmoid curves (GraphPad Prism software, La Jolla, CA). Average values (mean) and S.D. are shown with n = 4 for POPCo/POPE:POPSi/cholesterol and n = 3 for 1∶1 SM:POPCo/POPE:POPSi/cholesterol vesicles.
Figure 5.
TNBS labeling of LUVs with or without Perfringolysin O (PFO).
Samples contained 100 µl of (A) 1∶1 POPC/POPE, (B) 3∶3∶4 POPC/POPE/cholesterol or (C–D) 2∶2∶2∶4 POPC/POPE/POPS/cholesterol LUVs with or without ∼20 µg PFO and 900 µl, 1mM TNBS in NaHCO3 pH 9. Final lipid concentration was 200 µM. After TNBS addition, samples were incubated at room temperature for various times. % POPE labeled = [1−(POPE/POPC)before TNBS labeling/(POPE/POPC)after TNBS labeling] ×100% (A–C) or % POPS labeled = [1−(POPS/POPC)before TNBS labeling/(POPS/POPC)after TNBS labeling] ×100% (D). POPE/POPC or POPS/POPC is the ratio of the amount of POPE or POPS to that of POPC calculated from HP-TLC of lipid extracts from vesicles. Average values and range from duplicates are shown.
Figure 6.
TNBS labeling of POPE in exchange (asymmetric) vesicles outer leaflet.
(A) POPCo/POPE:POPSi/cholesterol and (B) 1∶1 SM:POPCo/POPE:POPSi/cholesterol. Labeling was for 60 min using the protocol in Fig. 5. Black bar shows the % of POPE unlabeled, which equals (POPE/POPC)before TNBS labeling/(POPE/POPC)after TNBS labeling×100%. The % of outer leaflet lipids that was POPE after exchange (striped bar) = (100% – % POPE unlabeled) × the fraction of vesicle lipids that was POPE (see Table S2 in File S1)/53%. This assumes ∼53% of LUV lipid is in the outer leaflet. Average values (mean) and S.D. are shown and n = 4 for POPCo/POPE:POPSi/cholesterol and n = 3 for 1∶1 SM:POPCo/POPE:POPSi/cholesterol vesicles.