Fig. 1: The recycling pool of SVs exhibit higher mobility than the total pool.

a SdTIM of VAMP2-pHluorin-bound At647N-GBP nanobodies in SVs, indicative of recycling pool SV mobility. (i) Epifluorescence image of a neuronal segment expressing VAMP2-pHluorin acquired before incubation with At647N-GBP. Inset (red outline) highlights a presynaptic compartment, shown at higher magnification in (ii). (iii) Fluorescence intensity, (iv) diffusion coefficient (the color bar represents log₁₀[μm²s⁻¹]) and (v) trajectory maps of recycling SVs. b SptPALM of vGLUT1-mEos2-containing vesicles, indicative of the total pool SV mobility. (i) Epifluorescence image of a neuronal segment expressing vGLUT1-mEos2. Inset (red outline) highlights a presynaptic compartment, shown at higher magnification in (ii). (iii) Fluorescence intensity, (iv) diffusion coefficient (the color bar represents log₁₀[μm²s^-1]) and (v) trajectory maps of total SVs. c, Average MSD of VAMP2-pHluorin/At647N-GBP trajectories (Recycling pool; black), and vGLUT1-mEos2 (Total pool; red) as a function of time. d Area under the MSD curve (AUC; µm² s). e Frequency distribution of the diffusion coefficients [D] shown in a semi-log plot. Grey dashed line indicates the threshold used to distinguish the immobile (Log₁₀[D] ≤ −1.6) from the mobile (Log₁₀[D] > −1.6) fraction of molecules. f Plot of the mobile fraction of molecules. g Three-state model of diffusive states inferred by vbSPT analysis of VAMP2-pHluorin/At647N-GBP trajectories. h, Three-state model of diffusive states inferred by vbSPT analysis of vGLUT1-mEos2 trajectories. Circles in (g) and (h) represent diffusive states, where (D) is the diffusion coefficient. Immobile state (State 1), confined state (State 2) and highly mobile state (State 3). The areas of the circles represent the average state occupancy (%) of SVs in their respective states. The arrows indicate the transition probability of an SV moving from one state to the other. i Example of a trajectory from a recycling SV undergoing stochastic switching between the three diffusive states inferred by vbSPT analysis. Data in (c–f) are displayed as mean ± SEM. Values were obtained from n = 15 neurons (Recycling pool), and n = 16 neurons (Total pool), from over 3 independent neuronal cultures. 131 presynapses were analyzed in (g) and 35 presynapses were analyzed in (h). Statistical comparisons in (d, f) were performed using unpaired two-tailed Student’s t-test with Welch’s correction. Source data are provided as a Source Data file.