Fig. 2. Syntaxin and SNAP-25 colocalise and can interact in the Golgi complex in live cells. Syx_1-288 and SNAP-25_1-206 coexpressed in live N2a cells and imaged by CLSM. (A) Residual map corresponds to weighted residuals indicating fluorescent channel covariance. The hue is from –1 to 1, with cyan corresponding to a zero residual. Bar, 10 µm. (B) The Golgi complex marker GRASP65-mCherry was coexpressed in live N2a cells and imaged by CLSM (left panel). Residual map of Syx_1-288 and SNAP-25_1-206 covariance in the Golgi complex, as defined by a GRASP65-mCherry mask (middle panel). The weighted residuals of Syx_1-288 and SNAP-25_1-206 contained within the Golgi complex, as defined by GRASP65-mCherry (black circles), alongside residual values from elsewhere in the cell (grey circles), were plotted as a frequency distribution histogram (right panel). (C) mCerulean-Syx_1-288 (donor) fluorescence in the absence of EYFP-SNAP-25_1-206 (acceptor) exhibited an intracellular distribution. The colour scale in the FLIM map represents the fluorescence lifetime [1900 pseconds (red) – 2400 pseconds (blue)]. The fluorescence lifetime values were plotted as a frequency distribution histogram, showing the 99% confidence interval of the Syx_1-288 fluorescence lifetime distribution (red line). Syx_1-288 alone has a single fluorescence lifetime of 2388±47 pseconds. The excited-state fluorescence decay of Syx_1-288 in the absence of an energy acceptor followed a mono-exponential decay (light grey circles) (right panel). (D) mCerulean-Syx_1-288 (donor) fluorescence in the presence of SNAP-25_1-206 (acceptor) exhibited an intracellular distribution. The colour scale in the FLIM map represents the fluorescence lifetime. The fluorescence lifetime values were plotted as a frequency distribution histogram, showing the 99% confidence interval of the Syx_1-288 fluorescence lifetime distribution (non-FRET; red line). The fluorescence lifetime of mCerulean-Syx_1-288 was shortened significantly to 2176 ±134 pseconds (Mann-Whitney, n=5, P<0.01) when coexpressed with EYFP-SNAP-25_1-206. In the presence of an energy acceptor, the fluorescence decay of Syx_1-288 was fit by a bio-exponential decay function (dark grey circles) (C, right panel). Syx_1-288 (donor) fluorescence in the presence of SNAP-25_1-206 (acceptor) showing in red the pixels containing donor fluorescence lifetimes below than the 99% confidence interval (right panel). Data are expressed as the mean±s.e.m.