Fig. 1

aSyn fibrils with distinct polymorphs have in vitro seeding and toxicity in cells. a, b Negative-stain EM (a) of full-length aSyn fibrils showing two distinct polymorphs—rod (non-twisted filaments, white arrow) and twister (twisted filaments, black arrow)—and a fibril width around 10 nm (b). c, d Direct visualization (c) and FRET-based quantification (d) of seeded intracellular aSyn aggregates. Fluorescent images obtained using the FITC channel (ex. 488 nm, em. 525 nm) showed aSyn aggregates as indicated by bright fluorescent puncta (white arrows in c). The diffuse background fluorescence came from endogenously expressed soluble, non-aggregated YFP-aSyn in the cells. Transduction of sonicated aSyn fibril seeds into the cells induced intracellular aSyn aggregation, which was also quantified using FRET analysis (d and Supplementary Fig. 3). e Cytotoxicity of aSyn fibrils evaluated by MTT-based cell viability assay of differentiated PC12, neuron-like cells. The aSyn fibrils used in the seeding experiment (c, d) have significant cytotoxicity (p < 0.0001) at 500 nM. Data are presented as mean ± standard error. Results are from multiple independent biological experiments with n = 3–5 per experiment. *p ≤ 0.01, ***p ≤ 0.0001 vs. control (buffer used to produce the aSyn fibrils). Scale bar: (a) 100 nm (c) 50 μm