Figure 1: Combinations of FRET/2pFLIM probes for SNAREs.

(a–f) Schematic drawing of combinations of probes whose constructs are depicted in Supplementary Fig. 1. mTurquoise was used as a donor and Venus as an acceptor. The N terminal end of synaptosomal-associated protein 25 (SNAP25b) was labelled with either mTurquoise (mtq, a) or in tandem with mtq and Venus (Ven, b) to measure the folding fraction of Ven. The binding fractions (or A1 fractions) of syntaxin1A (Syx) with vesicle-associated membrane protein 2 (VAMP2) in the cytosol were detected by mtq-Syx and Ven-VAMP2 (c). The cis-complex of Syx with VAMP2 was detected by Syx-mtq and VAMP2-Ven (d), because FRET occurs only when they are present in the same membrane. The assembly of SNAP25 with VAMP2 or Syx was detected by mtq-SNAP25 and Ven-VAMP2 (e) or Ven-Syx (f). (g) Images of synaptic boutons in rat cortical dissociated culture lipofected with mtq-SNAP25 (scale bar, 5 μm). The left image shows the fluorescence intensity of mtq-SNAP25, while the right image shows the fluorescence decay of the A1 fraction for each pixel in pseudocolour coding shown in j. (h) A lifetime decay curve in a semi-logarithmic plot, which was obtained from eight boutons. The decay fits well with a single exponential function with a time constant of 3.5 ns. (i) The residual data after subtracting the exponential function. (j) Images of synaptic boutons lipofected with the tandem mtq-Ven-SNAP25b probe (scale bar, 5 μm). (k) A lifetime decay curve in a semi-logarithmic plot obtained from 13 boutons, which fits well with a double-exponential function, with time constants of 0.7 ns (τ₁) and 3.5 ns (τ₂) and weights of 66% (A1) and 34% (A2), respectively. (l) The residual data after subtracting the A2 component (red) or both components (black). (m) The A1 fractions of mtq-SNAP25 (mean±s.e.m.=0.28±1.9%; 19 boutons) and mtq-Ven-SNAP25 probes (63.5±1.2%; 12 boutons). Wilcoxon signed-rank test versus 0%: P=0.71 and **P=0.002, respectively.