Figure 4. Synaptic vesicles undergo multiple rounds of fusion.

(A) Synaptic vesicles were first fused with syntaxinH3/SNAP-25 proteoliposomes containing Oregon Green labelled lipid, followed by proteoliposomes containing Texas Red labelled lipid. Because fusion is strictly dependent on one of the fusion partners containing synaptobrevin, FRET only develops if a Texas Red liposome fuses with a synaptic vesicle that has acquired Oregon Green labelled lipids in a preceding fusion step. FRET was monitored fluorometrically using excitation at 490 nm and emission at both 520 nm (Oregon Green donor, green trace on graph) and 615 nm (Texas Red acceptor, red trace on graph).

SyntaxinH3/SNAP-25 proteoliposomes, labelled with Oregon Green or Texas Red, were added to synaptic vesicles at the indicated time points (circles OG and TR). The increase in OG fluorescence at the beginning of the experiment is due to dequenching of the dye on fusion. Both OG and TR fluorescence signals were normalised to an arbitrary value of 1 after the TR liposomes were added, to account for the dilution effect (dashed lines; F0). The inlay shows a portion of the graph expanded after addition of TR liposomes, to show the decrease in OG (donor) and increase in TR (acceptor) signals typical of FRET.

(B) Donor and acceptor emission signals at the end of the reaction (F) normalised to the point at which the TR proteoliposomes were added (F0). Addition of TR proteoliposomes to the reaction mixture caused a decrease in the OG signal and a concomitant increase in the TR signal (Fusion; n=12, average ± SEMs), when compared to protein free liposomes (No Fusion; n=11, average ± SEMs) (*p<0.001).