Fig. 2: VAMP2 enables αSYN condensate formation in cells.
a, Screening of disease-relevant synaptic proteins on αSYN–YFP distribution upon co-expression in HeLa cells. Scale bar, 20 µm. SPH1, synphilin-1; Rab-3A, Ras-related protein Rab-3A; RPH3A, rabphilin-3A; VPS35, vacuolar protein sorting-associated protein 35; Endo-A1, endophilin-A1; HSC70, heat shock cognate 71 kDa protein; auxilin, putative tyrosine-protein phosphatase auxilin; SJ145 and SJ170, synaptojanin-1 isoforms 1-145 and 1-170. b, Cytosolic–nuclear distribution of αSYN–YFP upon ectopic expression in HeLa cells, condensate formation upon co-expression of αSYN–YFP and VAMP2, co-expression of YFP and VAMP2 shows no condensate formation. c, Zoomed-in regions and fluorescence intensity distribution for cells with αSYN–YFP only, αSYN with VAMP2 and YFP with VAMP2. FL, full-length. d, Quantification of cells forming condensates. Data are derived from Incucyte screening, with 16 images per well, three wells per biological repeat and four biological repeats. n indicates biological repeats. Data are mean ± s.d. One-way ANOVA with Dunnett’s multiple comparison test. e, αSYN–YFP condensates show fluid-like behaviour. Zoomed-in region showing individual fusion event and fission event. Scale bar, 1 µm. See also Supplementary Videos 1–4 and Extended Data Fig. 2. f, Photobleaching and recovery of αSYN condensate in cells. Quantification of FRAP experiments. Data are mean ± s.d. with four biological repeats, n = 22, n represents individual FRAP experiments. g, αSYN–YFP condensates show dispersal upon incubation with 3% 1,6-hexanediol. See also Extended Data Fig. 3 for recovery of αSYN condensates after 1,6-hexanediol washout. h, Quantification of staining in g before and after incubation with 3% 1,6-hexanediol. n = 11 cells, pooled from three biological repeats. The same cells were followed over 30 s. Paired two-tailed t-test. i, αSYN–YFP condensates are still present after incubation with 3% 1,3-propanediol. j, Quantification of staining in i before and after incubation with 3% 1,3-propanediol. n = 9 cells, pooled from three biological repeats. The same cells were followed over 30 s, 2 min and 5 min. Repeated measures one-way ANOVA with Dunnett’s multiple comparison test.
