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Fig. 4. SNARE clusters are heterogeneous. Intensity images and FLIM maps showing mCerulean-Syx_1-288, in the presence of EYFP-SNAP-25_1-206 and munc18-1 in live N2a cells, imaged by FLIM and two-photon microscopy. (A) mCerulean-Syx_1-288 (donor) fluorescence exhibited a plasma membrane distribution. The colour scale [1900 (red) – 2400 pseconds (blue)] in the FLIM map represents the donor fluorescence lifetime in resting cells (top) and after depolarisation with 55 mM KCl (lower panels). These data were plotted as a frequency distribution histogram, showing the 99% confidence interval of the donor lifetime distribution (red dashed line; bars are mean±s.e.m., n=5 experiments). Bar, 5 µm. (B) Similar experiments performed in the presence of ionomycin, but a Ca²⁺-free environment also revealed clusters at the base of the cell. The colour scale in the FLIM map represents the donor fluorescence lifetime. Bar, 5 µm. [Colour scale: 1500 (red) – 2000 pseconds (blue).] The boxed region of interest is shown in the lower panels and illustrates clusters at the plasma membrane. Pixels containing fluorescence lifetimes below the 99% confidence interval of the non-FRET distribution in panel A are shown in red. Bar, 1 µm. SNARE clusters where no FRET could be detected are highlighted with a dashed circle. (C) In the presence of ionomycin and Ca²⁺, mCerulean-Syx_1-288 (donor) fluorescence in the presence of EYFP-SNAP-25_1-206 (acceptor) and munc18-1 showed clusters at the base of the cell. The colour scale [1500 (red) – 2000 pseconds (blue)] in the FLIM map represents the donor fluorescence lifetime in the presence of ionomycin and Ca²⁺. The donor fluorescence lifetime was significantly shortened, indicative of FRET between the t-SNAREs. Bar, 5 µm. The boxed region of interest is shown in the lower panels and illustrates clusters at the plasma membrane. Pixels containing fluorescence lifetimes below the 99% confidence interval of the non-FRET distribution in panel A are shown in red. SNARE clusters where no FRET could be detected are highlighted with a dashed circle. Bar, 1 µm. (D) Similar results were obtained using KCl depolarization. The boxed region of interest is shown in the right-hand panels as a zoomed image, showing that the proportion of FRET-positive t-SNARE clusters increased after KCl-induced depolarization. (E) The donor fluorescence lifetime data for each sample were plotted as a frequency distribution histogram. The fluorescence lifetimes in the KCl-treated samples (open circles, grey fit line) were significantly reduced compared with those from resting cells (filled circles, black fit line).

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