Figure 3: FRET/2pFLIM of cis-SNARE complexes between Syx-mtq and VAMP2-Ven in neurons. | Nature Communications

Figure 3: FRET/2pFLIM of cis-SNARE complexes between Syx-mtq and VAMP2-Ven in neurons.

From: Two-photon fluorescence lifetime imaging of primed SNARE complexes in presynaptic terminals and β cells

Figure 3

(a) Schematic drawing of the conformation change of the cis-probes via exocytosis. (b) Fluorescence and A1 fraction images of Syx-mtq/VAMP2-Ven-expressing boutons (scale bar, 5 μm). (c) The average A1 fractions of Syx-mtq/VAMP2-Ven-stained boutons (black) and axons (open) in the absence and presence of the light chain of botulinum toxin (BoNTE). The A1 fractions in boutons (24.7±2.1%; 24 boutons) and axons (21.5±2.0%; 19 axons) were significantly different from 0% (**P=0.0001 and 0.00001 using the Wilcoxon signed-rank test) in the absence of BoNTE at the mean expression levels of VAMP2-Ven of 524±45 a.u. and 508±26 a.u., respectively (Supplementary Fig. 3d). They were not significantly different from 0% in the boutons (3.5±2.1%; 22 boutons) and axons (0.9±2%; 12 axons) in the presence of BoNTE using the Wilcoxon signed-rank test (P=0.12 and 0.53). The Mann–Whitney U-test yielded ††P=0.00001 for boutons without and with toxin and ††P=0.00001 for axons. (d) The correlation between the A1 fractions in the boutons (A1b) and those of axons surrounding the boutons (A1a; P=0.0001 using Spearman’s rank correlation coefficient; r=0.80; n=27). (e) Schematic drawing of the cis-configuration and the site of BoNTE action. (f) Fluorescence and A1 fraction images of boutons expressing Syx-mtq, VAMP2-Ven and BoNTE (scale bar, 5 μm). (g) Fluorescence lifetime images of Syx-mtq/VAMP2-Ven-stained boutons during 300 mM sucrose stimulation (scale bar, 1 μm). (h) The time courses of the A1 fractions estimated either by decay curve fitting (filled circles, 21 boutons) or average lifetimes (open symbols, 21 boutons) in the boutons (TA1b, open circles, n=19) and axons surrounding the boutons (TA1a, open triangles). All values after sucrose stimulation were significantly (P<0.01) different from those before stimulation. (i) The time courses of A1b estimated by average lifetimes during 300 mM sucrose stimulation in the preparation treated with BoNTE (n=19).

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