Journal: Nature Cell Biology

Article Title: VAMP2 regulates phase separation of α-synuclein

doi: 10.1038/s41556-024-01451-6

Figure Lengend Snippet: a , Co-expression of VAMP2 and wild-type αSYN–YFP in HeLa cells showing condensate formation. Cells co-expressing VAMP2 and αSYN(A30P)–YFP lack condensate formation. b , 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. c , Co-expression of αSYN–YFP, VAMP2 and mScarlet synaptotagmin showing partial co-localization of mScarlet synaptotagmin with αSYN–YFP condensates. d , Quantification of Pearson correlation coefficient for αSYN–YFP and mScarlet synaptotagmin co-localization. Three biological repeats were conducted; n indicates biological repeats. Data are mean ± s.d. Unpaired two-tailed t -test. e , Zoomed-in areas highlighting co-localization of αSYN condensates with co-expressed mScarlet synaptotagmin and mScarlet synaptotagmin outside αSYN condensates. Fluorescence intensity distribution for αSYN–YFP (yellow) and mScarlet synaptotagmin (magenta). f , Quantification of mScarlet synaptotagmin intensity outside and within αSYN condensates. n = 10 cells, pooled from four biological repeats. Data are mean ± s.d. Unpaired two-tailed t -test. g , HeLa cells with ectopic expression of αSYN–YFP, VAMP2 and 4xMTS-mScarlet, electron microscopy image overlaid with fluorescence microscopy, showing assemblies of vesicles colocalizing with αSYN–YFP condensates. Also see Extended Data Fig. for individual images. h , Electron microscopy images for individual vesicle clusters in Fig. 7g. Scale bar, 1 µm. i , Histogram showing size distribution of vesicles contained within αSYN condensates. Data are mean ± s.d. n = 14 vesicle clusters pooled from two cells from two biological repeats. j , Co-expression of αSYN–YFP, VAMP2 and complexin-1/2 mScarlet demonstrating enrichment of complexins within αSYN–YFP condensates. k , Quantification of complexin in αSYN condensates versus cytosolic complexin levels. Complexin-2 levels were significantly higher than complexin-1. n = 21 and 23 cells for complexin-1 and complexin-2, respectively, pooled from three biological repeats. Data are mean ± s.d. Unpaired two-tailed t -test.

Article Snippet: SYT1 , encoding synaptotagmin-1, was cloned from SH-SY5Y cDNA into the pCDNA3.1 vector with an N-terminal mScarlet tag, CPLX1 and CLPX2 , encoding complexin-1 and complexin-2 were cloned from SH-SY5Y cDNA into the pCDNA3.1 vector with a C-terminal mScarlet tag (Addgene 16015 and Addgene 85045).

Techniques: Expressing, Derivative Assay, Comparison, Two Tailed Test, Fluorescence, Electron Microscopy, Microscopy