Mapping of the α-syn–VAMP2 binding domain and requirement of α-syn–VAMP2 interactions for α-syn–mediated SV attenuation. Mapping of the α-syn–VAMP2 binding domain and requirement of α-syn–VAMP2 interactions for α-syn–mediated SV attenuation. (A and B) Both α-syn 1–140 and 1–110 bind VAMP2 with equal affinity, and scrambling of amino acid sequences in α-syn 96–110 attenuates α-syn–VAMP2 binding (co-IP in neuro2a, repeated twice). (C and D) Sequential amino acids (from α-syn 96) were mutated to alanine, and association of these mutants (myc-tagged) with VAMP2 was evaluated (co-IP in neuro2a cells). Note that mutations in α-syn 96–104 show the greatest disruption (repeated twice). (E) Scrambled and KKD mutations in the α-syn 96–110 sequence abrogated α-syn–mediated synaptic attenuation, as determined by pHluorin assays in hippocampal neurons. (F) Quantification of data in E (mean ± SEM from at least 3 independent experiments); control, 0.423 ± 0.029, n = 6; α-syn 1–140, 0.178 ± 0.032, n = 6; KKD, 0.453 ± 0.070, n = 5; Scr-1, 0.464 ± 0.041, n = 6; **P = 0.0017 (one-way ANOVA followed by Dunnett’s post hoc test). (G–I) Optical single vesicle clustering experiments were carried out as described in ref. 10. Briefly, VAMP2-containing synaptic-like vesicles were first immobilized on a glass slide assembled in a microfluidic chamber, and then WT or mutant α-syn protein was added. After extensive washing (to remove unbound α-syn), DiI-labeled VAMP2-containing vesicles were added to the chamber, and clustering of the labeled vesicles was visualized by prism-type total internal reflection fluorescence microscopy (after extensive washing to remove unbound vesicles). As shown in representative images (G) and quantitative data (H), α-syn induced vesicle clustering, and deletions or subtle mutations in the VAMP2-binding site markedly abrogated the number of vesicle clusters. Mean ± SEM from 4 independent experiments where observer was blinded to the conditions; α-syn 1–140, 100%; α-syn 1–110, 83.15% ± 6.439%; no α-syn, 21.83% ± 7.437%; α-syn 1–95, 29.94% ± 8.332%; KKD, 33.89% ± 3.465%; Scr-1, 30.49% ± 8.138%; NS, nonsignificant; ****P < 0.001 (one-way ANOVA followed by Dunnett’s post hoc test). (I) Scatter plots showing number of vesicle clusters (on y axis) and fluorescence intensities (on x axis) of all Dil-labeled clusters, along with a smoothened curve through the data points. Jichao Sun et al. PNAS 2019;116:23:11113-11115 ©2019 by National Academy of Sciences