Figure 1.

Properties of early-fibrils and late-fibrils from αS condensates. (A) Confocal images of spherical condensates of αS that readily form at 37 °C (see Experimental Section). The droplets show a round shape (see also Figure S1) and liquid-like properties in FRAP analyses (Figure S2). Samples included 1% of AF488-αS_N122C to fluorescently label the condensates. Scale bars are 1 μm (white) and 5 μm (red). (B) After 1 day of incubation, TEM images (top, scale bar 1 μm) showed the presence of curly early-fibrils that possess the typical X-ray diffraction pattern of amyloid fibrils (down), with reflections at 4.8 Å (red arrow) and 10 Å (cyan arrow). The diffraction patterns of the early-fibrils are typical of unoriented fibrillar samples, likely owing to the curly nature of these amyloids.⁶⁷ (C) After 3 weeks of incubation, TEM images (top, scale bar 1 μm) detected straight late-fibrils, with a conserved amyloid pattern of X-ray diffraction (down). (D) early-fibrils imaged at the confocal microscope (scale bar 20 μm) after 1 day of incubation at 37 °C. The aggregates disassemble after overnight incubation at 4 °C and can be observed again after 1 day of incubation at 37 °C. The sample included 2% of AF488-αS_N122C to fluorescently label the aggregates. (E) Cell dysfunction monitored by the reduction of MTT in human neuroblastoma SH-SY5Y cells upon incubation with increasing concentrations (0.03, 0.3, and 3 μM, monomer equivalents) of early-fibrils and late-fibrils from αS spherical condensates formed after 1 day and 3 weeks, respectively. * and *** indicate P values <0.05 and <0.001 with respect to untreated cells. Cells exposed to monomeric αS (M) and buffer of the early-fibrils (buffer SIF) were also shown. (F) Cell dysfunction monitored by intracellular ROS production in human neuroblastoma cells [details as in panel (E)]. Error bars in panels E and F are S.E.M.