Proton-induced fusion of oleic acid-phosphatidylethanolamine liposomes.
Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become destabilized, and fuse below pH 6.5 in 150 mM NaCl. Fusion is monitored by (i) the intermixing of internal aqueous contents of liposomes, utilizing the quenching of aminonaphthalene-3,6,8-trisulfonic acid (ANTS) by N,N'-p-xylylenebis(pyridinium bromide) (DPX) encapsulated in two separate populations of vesicles, (ii) a resonance energy transfer assay for the dilution of fluorescent phospholipids from labeled to unlabeled liposomes, (iii) irreversible changes in turbidity, and (iv) quick-freezing freeze-fracture electron microscopy. Destabilization is followed by the fluorescence increase caused by the leakage of coencapsulated ANTS/DPX or of calcein. Ca2+ and Mg2+ also induce fusion of these vesicles at 3 and 4 mM, respectively. The threshold for fusion is at a higher pH in the presence of low (subfusogenic) concentrations of these divalent cations. Vesicles composed of phosphatidylserine/phosphatidylethanolamine or of oleic acid/phosphatidylcholine (3:7 mole ratio) do not aggregate, destabilize, or fuse in the pH range 7-4, indicating that phosphatidylserine and phosphatidylcholine cannot be substituted for oleic acid and phosphatidylethanolamine, respectively, for proton-induced membrane fusion. Freeze-fracture replicas of oleic acid/phosphatidylethanolamine liposomes frozen within 1 s of stimulation with pH 5.3 display larger vesicles and vesicles undergoing fusion, with membrane ridges and areas of bilayer continuity between them. The construction of pH-sensitive liposomes is useful as a model for studying the molecular requirements for proton-induced membrane fusion in biological systems and for the cytoplasmic delivery of macromolecules.
D002118:Calcium; D055598:Chemical Phenomena; D002621:Chemistry; D004735:Energy Transfer; D005452:Fluoresceins; D005614:Freeze Fracturing; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D008081:Liposomes; D008274:Magnesium; D008854:Microscopy, Electron; D009281:Naphthalenes; D019301:Oleic Acid; D009829:Oleic Acids; D010714:Phosphatidylethanolamines; D011726:Pyridinium Compounds; D013050:Spectrometry, Fluorescence
D005452:Fluoresceins; D007202:Indicators and Reagents; D008081:Liposomes; D009281:Naphthalenes; D009829:Oleic Acids; D010714:Phosphatidylethanolamines; D011726:Pyridinium Compounds; C046466:N,N'-4-xylylenebis(pyridinium); D019301:Oleic Acid; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D008274:Magnesium; D002118:Calcium; C007740:fluorexon