Directly observed membrane fusion between oppositely charged phospholipid bilayers

A novel method was developed for the direct examination of pairwise encount ers between positively and negatively charged phospholipid bilayer vesicles . Giant bilayer vesicles (unilamellar, 4-20 mu m in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with individu al anionic phospholipid vesicles. Fluorescence video microscopy revealed th at such vesicles commonly underwent fusion within milliseconds (1 video fie ld) after contact, without leakage. Fusion occurred at constant volume and, since flaccid vesicles were rare, the excess membrane was not available af ter fusion. Hemifusion (the outer monolayers of each vesicle fused while th e inner monolayers remained intact) was inferred from membrane-bound dye tr ansfer and a change in the contact area. Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of char ged phospholipids plus zwitterionic phospholipids. Hemifusion occurred in o ne of three ways following adhesion: either delayed with an abrupt increase in area of contact, immediately with a gradual increase in area of contact , or with retraction during which adherent vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted imm ediate hemifusion; the resultant hemifused state was stable and seldom unde rwent complete fusion. Although sometimes single contacts between vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has unequivocally demonstrated the fusio n of two, isolated bilayer-bounded bodies to yield a stable, non-leaky prod uct, as occurs in cells, in the absence of proteins.