Fig. 9: Synapsin 2a^WT-mEos3.1 fully rescues the SynTKO reserve SV pool mobility phenotype.

a Graphical representation of the timeline for dual-colour single-particle tracking of the reserve pool of synaptic vesicles (SVs; black line) and either wild-type (WT) Synapsin 2a (Syn2a^WT-mEos3.1; cyan line) or the Synapsin 2a tetramerization deficient mutant (Syn2a^K337Q-mEos3.1; magenta line) in Synapsin triple knockout (SynTKO) hippocampal neurons. b Representative epifluorescence image of a neuronal segment expressing Synaptotagmin1-pHluorin (Syt1pH). c–e Maximum intensity projections of (c) Syt1pH-bound anti-green fluorescent protein (GFP) Atto647N nanobodies (Syt1pH/At647Nb; reserve pool) and (d) Syn2a^WT-mEos3.1. e Merged maximum intensity projections of (c) and (d). f, h Average mean square displacement (MSD; µm²) of reserve SVs in SynTKO neurons (Synapsin TKO; black), reserve SVs when Syn2a^K337Q-mEos3.1 is expressed (+Syn2a^K337Q magenta), and reserve SVs when Syn2a^WT-mEos3.1 is expressed (+Syn2a^WT; cyan), within the (f) presynapses and (h) axons of SynTKO hippocampal neurons. g, i Area under the MSD curve (AUC; µm²s) for the (g) presynapses and (i) axons of SynTKO hippocampal neurons. j, k Average diffusion coefficient of reserve SVs in SynTKO neurons (Synapsin TKO; black), reserve SVs when Syn2a^K337Q-mEos3.1 is expressed (+Syn2a^K337Q; magenta), and reserve SVs when Syn2a^WT-mEos3.1 is expressed (+Syn2a^WT; cyan), within the (j) presynapses and (k) axons of SynTKO hippocampal neurons. l Ratio of reserve SVs detected in presynapses to reserve SVs detected in the axons in SynTKO neurons (black), SynTKO neurons when Syn2a^K337Q-mEos3.1 is expressed (magenta), and SynTKO neurons when Syn2a^WT-mEos3.1 is expressed (cyan). Data are displayed as mean ± SEM. Values were obtained from n  =  36 presynapses (Synapsin TKO), n  =  33 presynapses (+Syn2a^WT) and n  =  35 presynapses (+Syn2a^K337Q) in (f, g), from n  =  18 axons (Synapsin TKO), n  =  7 axons (+Syn2a^WT) and n  =  13 axons (+Syn2a^K337Q) in (h, i), from n  =  35 presynapses (Synapsin TKO), n  =  34 presynapses (+Syn2a^WT) and n  =  34 presynapses (+Syn2a^K337Q) in (j), from n  =  18 axons (Synapsin TKO), n  =  8 axons (+Syn2a^WT) and n  =  13 axons (+Syn2a^K337Q) in (k) and from n  =  17 presynapses (Synapsin TKO), n  =  11 presynapses (+Syn2a^WT) and n  =  7 presynapses (+Syn2a^K337Q) in (l). Data were obtained from three biological replicates. Statistical comparisons were performed using the one-way ANOVA and Dunnett’s or Tukey’s multiple comparisons test in (f–l). Scale bar 1 µm (b–e). Source data are provided as a Source Data file.