Fig 1: BoNT/A induces Syt1-SV2 interactions and receptor nanoclustering A–C(A) Representative low-resolution image of SV2Awt-pH (green), and corresponding dual-color super-resolved uPAINT trajectories of SV2Awt-pH/At565nb (cyan) imaged simultaneously with BoNT/Aiwt-At647N (magenta). Regions (i–ii) are magnified below, and (iii) is magnified in (B) showing a representative time-lapse sequence of the single molecules of SV2Awt-pH/At565nb (cyan) and BoNT/Aiwt-At647N (magenta) detected coincidentally in a neuronal projection. The 10 × 10 pixel boxed region (iv) is magnified in (C), showing frame-by-frame coincidental detection of BoNT/Aiwt-At647N and SV2Awt-pH/At565nb obtained using a 50 Hz (20 ms exposure time).D, E(D) Representative NASTIC nanocluster analysis image of BoNT/Aiwt-At647N and SV2Awt-pH/At565nb showing outlined nanoclusters in magenta and cyan, respectively. BoNT/Aiwt-At647N and SV2Awt-pH/At565nb co-clusters are indicated by colored arrowheads and hotspots with repeated nanoclustering events by white outlines. Boxed area in the bottom is shown magnified in (E), which shows examples of (i) a BoNT/Aiwt-At647N and SV2Awt-pH/At565nb co-cluster, (ii) an SV2Awt-pH/At565nb cluster, (iii, iv) two BoNT/Aiwt-At647N clusters, and a hotspot (v) as indicated.F3D representation of the 2D image in (E), with corresponding i–v clusters.GMSD graph of unclustered and clustered BoNT/Aiwt-At647N (magenta) and SV2Awt-pH/At565nb (cyan).H, IRepresentative images of NASTIC nanocluster analysis of SV2Awt-pH/At565nb in (H) the control condition (high K+; Ctrl) and (I) following high K+ stimulation in the presence of 100 pM BoNT/Aiwt-At647N, with 2D kernel density estimation (KDE, high density in red), instantaneous Log10 diffusion coefficient map (low mobility in red), and nanoclusters with their respective trajectories (white) and centroids (orange) shown. Boxed areas are magnified on the right, with arrowheads indicating nanoclusters.J–NQuantification of the (J) SV2Awt-pH/At565nb, (K) SV2AT84A-pH/At565nb, (L) Syt1wt-pH/At565nb, (M) Syt1K326A,K328A-pH/At565nb and (N) Syt1K52A-pH/At565nb nanoclustering in control conditions (high K+; Ctrl) and following BoNT/Aiwt-At647N treatment (diluted in high K+).O In situ proximity ligation assay (PLA) of Syt1-SV2C interactions in naïve (ctrl), high K+ stimulated (vehicle) conditions, and following high K+ stimulation with the indicated toxins. Nucleus (DAPI; blue) and Syt1-SV2C PLA (red) signals from a representative confocal maximum intensity projection of the somatodendritic area.PNumber of PLA (Syt1-SV2C) dots per nucleus in the indicated conditions. Data information: Error bars are shown as standard error of the mean (± SEM). (J–N) N = 11–19 technical replicates from three to five independent biological replicates, and (P) n = 25 regions of interest (200 × 200 pixel ROI surrounding DAPI staining)/condition from two independent biological replicates. Unpaired t test in (J–N) and nonparametric Kruskal–Wallis multiple comparison test in (P). *P < 0.05, ***P < 0.001, ns—nonsignificant.