Directly Observed Membrane Fusion Between Oppositely Charged Phospholipid Bilayers |
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Authors: | DP Pantazatos RC MacDonald |
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Institution: | (1) Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208-3500, US |
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Abstract: | A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged
phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4–20 μm in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with
individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion
within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid
vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused
while the inner monolayers remained intact) was inferred from membrane-bound dye transfer 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 charged phospholipids
plus zwitterionic phospholipids. Hemifusion occurred in one 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 immediate hemifusion;
the resultant hemifused state was stable and seldom underwent 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 fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs
in cells, in the absence of proteins.
Received: 25 November 1998/Revised: 23 March 1999 |
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Keywords: | : Hemifusion — Fluorescence microscopy — Cationic lipid — EDOPC |
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