Extended Data Fig. 1: α-Syn, VAMP2, and SVs form a multi-component condensed phase.
From: VAMP2 chaperones α-synuclein in synaptic vesicle co-condensates
(a) Dynamic light scattering measurement of the size distribution of SVM in solution. (b) Molecular weight and concentration of PEG pairs, (c) SVM lipid, and (d) NaCl concentrations scoring positive (blue dots) or negative (red dots) for the appearance of droplets. 100 μM VAMP21-96, and 100 μM α-Syn were used for all the titrations. Additionally, 1 mM SVMs, 5% (w/v) PEG 3350, or 1 mM SVMs and 5% (w/v) PEG 3350 were used for the titration in (b), (c), or (d), respectively. Fluorescence images of (e, top) 100 μM α-Syn (containing 1% Alexa555-α-Syn), (e, middle) 100 μM VAMP21-96 (containing 1% Alexa647-V2), (e, bottom) 100 μM α-Syn (containing 1% Alexa555-α-Syn) and 100 μM VAMP21-96 (containing 1% Alexa647-V2), (f) 100 μM α-Syn (containing 1% α-Syn-Alexa488) and 1 mM SVM with 100 μM other synaptic proteins (90 μM SNAP-25, 5 μM Munc18, and 5 μM Munc13), or (g) 100 μM α-Syn (containing 1% Alexa555-α-Syn) and 1 mM carboxyfluorescein-green-labeled SVM with 100 μM VAMP21-96 (containing 1% Alexa647-V2) in the buffer A (10 mM HEPES-Na pH 7.4, 5 mM Zn2+, 5% PEG 3350), respectively. DIC: differential interference contrast. (h) The final SV product was fractionated into three fractions by gradient centrifugation. The SVs in the bottom layer were used for experiments. (i) Western blot of fractions collected from each step of SV purification. The fraction containing SV is highlighted in red boxes. Other fractions are labeled in abbreviations (see details in Methods). The fractions were immunoblotted for tubulin (cytosol marker), synaptophysin (SV marker), Rpt4 (proteasome marker), VDAC (mitochondria marker), and Sec61B (endoplasmic reticulum marker), respectively. (j) Representative negative staining TEM image of endogenous SVs (bottom layer) purified from mouse brains. Uncropped blots are available in the Source Data Extended Data Fig. 1.
