Figure 5: FRET/2pFLIM of mtq-SNAP25-based probes for SNARE complexes. | Nature Communications

Figure 5: FRET/2pFLIM of mtq-SNAP25-based probes for SNARE complexes.

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

Figure 5

(a,d) Schematic drawing of mtq-SNAP25/Venus-vesicle-associated membrane protein 2 (Ven-VAMP2) probes and the action of BoNTC1α-51. (b,e) Fluorescence and A1 fraction images of mtq-SNAP25/Ven-VAMP2-expressing boutons in the absence (b) and presence of BoNTC1α-51 (e) (scale bars, 5 μm). (c) The A1 fractions of mtq-SNAP25/Ven-VAMP2-expressing boutons (filled; 36.7±2.2%; 20 boutons at the endogenous expression level of VAMP2; Supplementary Fig. 3f) and axons (open; 22.7±1.3%; 22 axons) and those in the presence of BoNTC1α-51 in boutons (14.5±2.2%; 24 boutons) and in axons (−0.4±1.1%; 17 axons). **P<0.001 for control boutons and axons and **P=0.0001 for BoNTC1α-51-treated boutons using the Wilcoxon signed-rank test versus 0%. Using the Kruskal–Wallis test on the four groups, we obtained a P value of <0.00001, and the effects of BoNTC1α-51 on boutons and axons were †P=0.03 and †P=0.04, respectively, using Scheffé’s test. ††P=0.004 between control boutons and axons and ††P=0.002 between BoNTC1α-51-treated boutons and axons. (f,i) Schematic drawing of mtq-SNAP25/Ven-Syx probes and the action of TeNT. (g,j) Fluorescence and A1 fraction images of mtq-SNAP25/Ven-Syx-expressing boutons in the absence (g) and presence of tetanus toxin (TeNT; j) (scale bars, 5 μm). (h) The average A1 fractions of mtq-SNAP25/Ven-Syx-stained boutons (filled; mean±s.e.=29.5±3.1%; 21 boutons with the mean expression of Ven-Syx of 139 a.u.; Supplementary Fig. 3h) and axons (open; 16.2±1.3%; 28 axons), those of boutons in the presence of TeNT (12.0±1.1%; 45 boutons with the mean expression level Ven-Syx of 138 a.u.) and of axons (2.4±1.7%; 15 axons). **P<0.01 for control boutons, axons and TeNT-treated boutons using the Wilcoxon signed-rank test versus 0%. Using the Kruskal–Wallis test on the four groups, we obtained a P value of <0.00001. The A1 values of boutons and axons were different (††P=0.004) in the absence of the toxin, and the toxin’s effects were significant: ††P=0.0007 and †P=0.04 for boutons and axons using Scheffé’s test.

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