| We studied the lipid mixing and content release in SNARE mediated single vesicle-membrane fusion with two channel fluorescence movies. The v-SNARE vesicles, labeled with lipid and content markers of different color, dock and fuse with a planar t-SNARE bilayer supported on glass. A narrow (<5 ms duration), intense spike of calcein fluorescence due to content release and dequenching coincides with inner-leaflet lipid mixing within 10 ms. The spike provides more sensitive detection of productive hemifusion events than do lipid labels alone. Consequently, many fast events previously thought to be prompt, full fusion events are now reclassified as productive hemifusion. Both full fusion and hemifusion occur with a time constant of 5-10 ms. However, quantitative analysis shows that content is released into the space above the bilayer (vesicle bursting), rather than the thin aqueous space between the bilayer and glass. Evidently, at the instant of inner-leaflet mixing, flattening of the vesicle increases the internal pressure beyond the bursting point. This may be related to in vivo observations suggesting that membrane lysis often competes with membrane fusion.;A novel model membrane system has been developed by rupturing giant unilamellar vesicles (GUVs) onto a glass supported biotin-PEG-DSPE/POPC lipid bilayer through pH-sensitive electrostatic interactions between NeutrAvidin and cationic lipids. The resulting "GUV pancakes" form a single lipid bilayer that exhibits good lateral mobility and surface integrity. Atomic force microscopy (AFM) shows that the distance between the height of large GUV pancakes (∼30 microm in diameter) and the top of the underlying lipid bilayer is about 7.9 +/- 0 6 nm, implying the polymer cushion has a height of ∼4 nm. |
