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.