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. 2020 Aug 31;117(37):22671–22673. doi: 10.1073/pnas.2009702117

Fig. 2.

Fig. 2.

α-Syn fibrils modulate RPT aggregation. (A) TEM images of unseeded α-syn and RPT fibrils, formed at pH 6 (20 mM 2-(N-morpholino)ethanesulfonic acid, 100 mM NaCl) under shaking conditions. N-terminally acetylated α-syn was used in all experiments. Brackets and arrows represent twisted and rod-like filaments, respectively. (B and C) Aggregation of 30 µM RPT (black in B) and α-syn (black in C) alone, or with 3 µM preformed α-syn (green in B; purple in C) and RPT (blue in B; teal in C) seeds at pH 6, monitored by (B) Trp or (C) ThT fluorescence at 37 °C under quiescent conditions. In B, the blue and red curves overlay. First-generation cross-seeded fibrils (green in B) were propagated over two more generations, second (yellow in B) and third (red in B). Five replicates (dots) were averaged and fit to a sigmoidal curve (solid lines). (D) TEM images of fibrils from B. Brackets and arrows represent twisted and rod-like filaments, respectively, with the observed relative percentages (n ≥ 300). (E) Fibrils with a twisted morphology were analyzed to determine the half-pitch lengths, which averaged 150 ± 28, 158 ± 21, and 159 ± 25 nm for first-, second-, and third-generation cross-seeded fibrils, respectively (n ≥ 100). (F) Raman spectra of self-seeded RPT (blue), first-generation cross-seeded RPT (green), and unseeded α-syn (black) fibrils. Averaged spectra (thick lines) from three spatial locations (thin lines) were normalized to the amide-III (1,225 cm−1; Left) or amide-I (1,665 cm−1; Right) band. Data are offset for clarity. Arrow indicates a distinctive feature (1,250 cm−1) in self-seeded RPT fibrils, that is less prominent in cross-seeded and α-syn fibrils. Self-seeded RPT data are reproduced from previous work (12). (G) Schematic representation of cathepsin L limited digestion, where lines represent cleavage sites (Top) and peptide fragments (Bottom) observed in both self-seeded and cross-seeded RPT fibrils. Cleavage locations and fragments that were less abundant in cross-seeded relative to self-seeded fibrils are colored green.