Figure 2
TI-VAMP2 rescues SV fusion upon TeNT treatment. (A) Schematic representation of TI-VAMP2. Q76V, F77W substitutions interfere with TeNT cleavage. N-terminal mCerulean (mCer) allows detection of the construct during live-cell experiments. (B) Western blot of neuronal lysate (DIV 16) infected with TI-VAMP2 (DIV 2) and a control construct or TeNT (DIV 14), incubated with VAMP2 antibody (original blots are shown in Figure S4). (C) Schematic representation of the method to measure synaptic vesicle exocytosis in neurons infected with synaptophysin-pHluorin (sypHy). Electrical stimulation (16 trains of 50 AP at 50 Hz (blue bars) interspaced by 0.5 s) elicits SV fusion with the plasma membrane, de-quenching sypHy through the increase in pH from 5.5 (in the SV lumen) to 7.4. Before stimulation, sypHy is quenched (F0). During stimulation, SVs fuse with the plasma membrane which is visualized by gradual increase in fluorescence (Fusion). Upon NH4+ perfusion, the total sypHy labeled SV pool is visualized (NH4+). Scale bar 5 µm. (D) ΔF/Fmax sypHy (infected at DIV 3–4) signal before, during and after electrical stimulation (blue bars) in control, TeNT (infected at DIV 9), and TeNT (infected at DIV 9) + TI-VAMP2 (infected at DIV 2 and 9) neurons imaged at DIV 11. Yellow bar represents NH4+ superfusion, which de-quenches all labeled SVs and reveals the total pool (used as max in the ΔF/Fmax plot). Shaded area represents SEM. (E) Average Fstimmax (peak in the ΔF/Fmax graph, see D) for control (n = 6, N = 2), TeNT (n = 4, N = 2) and TeNT + TI-VAMP2 (n = 17, N = 2) neurons. Kruskal–Wallis with Dunn's correction: *p = 0.0116, **p = 0.0067, non-significant (ns) p > 0.99. Bars represent mean + SEM. Detailed statistics are shown in Supplementary Table S1.
