Directly Observed Membrane Fusion Between Oppositely Charged Phospholipid Bilayers

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 micron 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.

The fusion of early endosomes induces molecular motor-driven tubule formation and fission.

“…Subsequent to the depolymerization of the microtubules no tubules were observed after fusion. On the contrary, we observed both tubular and vesicular intralumenal structures, reminiscent of observations made during in vitro experiments (Pantazatos and MacDonald, 1999). We hypothesize, that by depolymerizing the microtubules the excess membrane presumably invaginates into the luminal space in order to maintain the surface to volume equilibrium.…”

“…The stimulus for the extensive tubule formation may be that when two vesicles with identical volume fuse, the newly formed vesicle has 20% excess membrane, regardless of vesicle size (Pantazatos and MacDonald, 1999). This additional membrane has to be removed in order to balance the surface to volume ratio after fusion, which could be achieved by tubule formation.…”

Flat and sigmoidally curved contact zones in vesicle–vesicle adhesion

“…We show that with increasing surface-to-volume ratio, the range of adhesion strengths corresponding to the flat-contact doublet should diminish and eventually vanish, leaving the sigmoid-contact doublet as the only stable bound morphology. These findings also provide an insight into the mechanics of aggregates of more than two vesicles, which may be relevant for many biological systems and processes involving attraction of flexible membranes, such as cell adhesion (17), cell fusion (18), and tissue formation (19). Theory Doublet Energy.…”

Coupling of the fusion and budding of giant phospholipid vesicles containing macromolecules

“…Thus, spontaneous incorporation of lipids is unlikely. Before a protocell obtained an ability to synthesize lipids on its own, the vesicle size increase could be achieved by vesicle-to-vesicle fusion initiated by various external stimuli (19)(20)(21)(22) or surface functionalization (23)(24)(25)(26). Thus, it is conceivable that membrane fusion could be one of the primitive growth pathways for protocells composed of phospholipids.…”