Supplementary Information Index: Supplementary Information 1: Characterization of monomeric and aggregated forms of α-syn Supplementary Information 2. Nanomotion control experiments Figures S1-S8 Tables S1-S2 Movies S1-S3 Additional References Supplementary Information 1. Characterization of monomeric and aggregated of The weak characterization of the protein aggregates is one of the major factors of the discrepancies in the studies involving the cell cytotoxicity of amyloids. Thus, as a preliminary step, we have performed a combination of bulk and single molecule characterization of the α-syn species, which is of fundamental importance for the investigation of the role of amyloids in the disease onset. We carried out full characterization of monomeric and its aggregated forms α-syn in vitro using an array of biophysical methods (Figure S1). At first, we performed a complete characterization of the biophysical and structural properties of the recombinant preparations. For this analysis of paramount importance in the α-syn understanding of their interaction with the cells, we used several bulk and single molecule techniques: Thioflavin T (ThT), Circular Dichroism (CD), Electron Microscopy (EM) and Atomic Force Microscopy (AFM) imaging. The AFM analyses showed that the monomeric solution was highly homogeneous contained only a residual amount of small spheroidal early oligomeric species (<5%) with height of ~1 nm (Figure S1a). After 4 weeks’ incubation at 37°C, the resulting aggregated solution presented the typical fingerprint of amyloids: aggregated oligomers, with larger height (1-9 nm) than the early spheroidal forms, and mature fibrillar species with height of approximately 6-9 nm, which we named crude mixture (Figure S1b). EM confirmed the presence of fibrillar aggregates in the incubated species (Figure S1c). Consistently, the samples before incubation did not have ThT fluorescence while, after 4 weeks
Supplementary Information Index: Supplementary Information 1: Characterization of monomeric and aggregated forms of α-syn Supplementary Information 2. Nanomotion control experiments Figures S1-S8 Tables S1-S2 Movies S1-S3 Additional References Supplementary Information 1. Characterization of monomeric and aggregated of The weak characterization of the protein aggregates is one of the major factors of the discrepancies in the studies involving the cell cytotoxicity of amyloids. Thus, as a preliminary step, we have performed a combination of bulk and single molecule characterization of the α-syn species, which is of fundamental importance for the investigation of the role of amyloids in the disease onset. We carried out full characterization of monomeric α-syn and its aggregated forms in vitro using an array of biophysical methods (Figure S1). At first, we performed a complete characterization of the biophysical and structural properties of the recombinant α-syn preparations. For this analysis of paramount importance in the understanding of their interaction with the cells, we used several bulk and single molecule techniques: Thioflavin T (ThT), Circular Dichroism (CD), Electron Microscopy (EM) and Atomic Force Microscopy (AFM) imaging. The AFM analyses showed that the monomeric solution was highly homogeneous contained only a residual amount of small spheroidal early oligomeric species (<5%) with height of ~1 nm (Figure S1a). After 4 weeks’ incubation at 37°C, the resulting aggregated solution presented the typical fingerprint of amyloids: aggregated oligomers, with larger height (1-9 nm) than the early spheroidal forms, and mature fibrillar species with height of approximately 6-9 nm, which we named crude mixture (Figure S1b). EM confirmed the presence of fibrillar aggregates in the incubated species (Figure S1c). Consistently, the samples before incubation did not have ThT fluorescence while, after 4 weeks