Extended Data Fig. 3: Amyloid aggregation pathways in a phase-separated α-Syn solution.
From: Mass photometric detection and quantification of nanoscale α-synuclein phase separation
a. (Left) Representative fluorescence microscopy images of 200 μM α-Syn + 20% (w/v) PEG samples labeled with 100 nM Alexa488-α-Syn (upper panel) and 50 μM ThT (lower panel) at 0, 48 and 72 h are shown. The inset is an enlarged image of the droplets at 0 h. (Right) Fluorescence microscopy images (thermal LUT) of a 48 h old α-Syn droplet at different times (0–40 s) post-bleaching showing no recovery of fluorescence. b. (Left) Fluorescence microscopy image of a 72 h old microemulsion droplet containing 200 μM α-Syn + 20% (w/v) PEG (250 mM NaCl) is shown. The sample contains 50 μM ThT as a reporter for aggregation. The red dashed circle indicates the boundary of the microemulsion droplet (Right) Fluorescence microscopy images of 240 h old microemulsion droplets containing 200 μM α-Syn + 20% (w/v) PEG (0 mM NaCl) are shown. The samples contain 100 nM Alexa488-α-Syn (upper panel) and 50 μM ThT (lower panel) as reporters. c. (Upper panel) Bulk ThT aggregation kinetics of 200 μM α-Syn + 20% (w/v) PEG (250 mM NaCl) under quiescent conditions at 25 °C. (Lower panel) The first phase (marked with a rectangle) is magnified for better visualization. d. ThT fluorescence intensity (background corrected) from microscopic measurements of individual phase-separated droplets. Values represent mean±SD calculated from 45 individual droplets (n = 2 independent experiments). The slight decrease of the intensity over the first 5 h arises from passive bleaching. Representative snapshots of a droplet at different time-points are shown (upper panel). e. Representative fluorescence microscopy images after addition of 200 nM Alexa488-α-Syn monomer to an aged (~48 h) LLPS sample is shown. Subsequent incubation (~72–96 h) of this sample leads to aggregation of the labeled monomer on the surface of unlabeled droplets (indicated with white triangular pointers). A fluorescence signal intensity plot across a droplet showing no partitioning of the Alexa488-α-Syn inside the solidified droplet at ~48 h. f. Bulk ThT aggregation kinetics of freshly made 200 μM α-Syn + 20% (w/v) PEG (250 mM NaCl) under quiescent conditions, at 25 °C and in presence of 10% (v/v) isolated droplets (aged for ~48 h) and 10% (v/v) 50 μM α-Syn amyloid fibrils (pre-made in solution). The inset shows a schematic of the experimental approach. g. ThT aggregation kinetics of 200 μM α-Syn + 20% (w/v) PEG in absence of NaCl. h. Schematic depicting the three amyloid aggregation processes that can concurrently occur in an α-Syn LLPS sample. i. Representative TEM images of 200 μM α-Syn + 20% (w/v) PEG (250 mM NaCl) at 0 h, 72 h, 96 h and 168 h. Fibrillar aggregates and typical amyloid fibrils are indicated with yellow triangular pointers. The inset shows presence of amyloid fibrils even in the dilute phase after ageing for 168 h. The experiments (a-i) are performed n = 2 independent times with similar results. PEG: PEG-8000 unless mentioned otherwise. Detailed results are provided in the supplementary information.
