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. 2024 Oct 30;109:105416. doi: 10.1016/j.ebiom.2024.105416

Fig. 3.

Fig. 3

C2B and C2A domain SYT1 variants induce a dominant-negative slowing of exocytosis. Hippocampal neurons transfected with SYT1-pHluorin variants or wild-type SYT1-pHluorin were stimulated with 1200 AP at 10 Hz in the presence of bafilomycin A1, and then perfused with ammonia buffer to reveal total SYT1-pHluorin fluorescence. (a, b) Synaptic vesicle recycling pool sizes in the presence of C2B (a) or C2A (b) domain variants, expressed as a percentage of the total pool of vesicles (peak SYT1-pHluorin fluorescence with ammonia buffer). One-way ANOVA with Dunnett's multiple comparison test compared to WT; a: M303V p = 0.014, S309P p = 0.0077, Y365C p = 0.87, G369D p = 0.99, I368T p = 0.14. b: L159R p = 0.60, T196K p = 0.87, E209K p = 0.98, E219Q p = 0.41, I368T p = 0.91. (c, d) Time course of mean ± SEM ΔF/F0 of C2B (c) or C2A (d) domain SYT1-pHluorin variants normalised to peak amplitude of fluorescence change induced by stimulation. SEM indicated by shading. Coloured bars above graph represent time points of significant difference between the SYT1 variants and WT, analysed through repeated measures ANOVA with Dunnett's multiple comparison test. (e, f) One-phase curves were fit over the entire stimulation period, from which tau constant values were obtained; tau values shown for C2B (e) or C2A (f) domain variants. e) One-way ANOVA with Dunnett's multiple comparison test compared to WT for C2B variants; M303V p = 0.021, S309P p = <0.0001, Y365C p = 0.23, G369D p = 0.30, I368T p = 0.017. f) Kruskal–Wallis test with Dunn's multiple comparison test compared to WT for C2A variants; L159R p = 0.84, T196K p > 0.99, E209K p = 0.23, E219Q p = 0.074, I368T p = 0.0092. All data displayed as mean ± SEM, n = 7–11 (individual field of view from an independent coverslip, from at least 3 independent cultures each comprising at least 3 embryos; WT n = 9 for a, c, e, n = 10 for b, d, f; I368T n = 8 for a, c, e, n = 7 for b, d, f; M303V n = 8; S309P = 8; Y365C = 8; G369D = 9; L159R = 9; T196K = 11; E209K = 9; E219Q = 9). In a, b, e, f ∗ indicates p < 0.05, ∗∗ indicates p < 0.01, ∗∗∗∗ indicates p < 0.0001 compared to WT; also see Supplementary Table S2d–g.