Figure 1.
Radiating amyloid fibril (RAF) formation on the surface of lipid membranes.
(A) Transmission electron microscopy (TEM) image of radiating fibrils that were developed on the surface of liposomes. (B) With congo red staining, birefringency of the radiating fibrils on PC-liposomes has been monitored with fluorescence microscope under polarized light. The corresponding image revealed with light microscope of differential interference contrast (DIC) is also shown in the inset. (C) Thioflavin-T (ThT) binding fluorescence of the PC-liposomes (0.57 mg/ml) treated with either the oligomers (open dots) or the monomers (closed dots) of α-synuclein at their protein concentrations indicated was monitored at 482 nm with an excitation at 450 nm. (D) Circular dichroism (CD) spectra obtained for the PC-liposomes (1.25 mg/ml) incubated with either the oligomers (red dots) or the monomers (black dots) of α-synuclein at 0.5 mg/ml after an extended period of quiescent incubation for 18 hours at 37°C.
Figure 2.
Characterization of the RAF-forming oligomeric species of α-synuclein.
(A) The fibrillation kinetics of α-synuclein was monitored with ThT binding fluorescence following the incubation of α-synuclein at 1 mg/ml in 20 mM Mes, pH 6.5, at 37°C with shaking at 200 rpm. (B) TEM images showing the RAF formation on the surface of PC-liposomes. The liposomes (1.25 mg/ml) were treated with various states of α-synuclein (0.5 mg/ml) collected during the fibrillation kinetics at the times indicated with arrow heads in panel (A). (C) CD spectra of the α-synuclein collected at various time points during the fibrillation process (0 hr, red; 4 hr, orange; 8 hr, yellow; 12 hr, green; 16 hr, cyan; 18.5 hr, light blue; 24 hr, blue; 28 hr, violet; 37.5 hr, black; 44 hr, dark gray; 50 hr, gray; 60 hr, white color). (D) Plot of negative ratios of the differences in molar ellipticities obtained at 193 nm and 220 nm (-Δθ193 nm/Δθ220 nm) for the various states of α-synuclein in comparison with those of the monomeric form. The inset shows the oligomeric state of α-synuclein visualized with TEM giving rise to the maximum value of - Δθ193 nm/Δθ220 nm.
Figure 3.
Maturation of the RAFs with either oligomers or monomers of α-synuclein.
(A) The PC-liposomes with burgeoning RAFs prepared by incubating PC-liposomes (0.4 mg) with the oligomers (50 µg) in 20 mM Mes, pH 6.5, (0.25 ml) at room temperature for 5 min were sequentially fed with either the oligomers (upper panels) or the monomers (lower panels) of α-synuclein (50 µg per each addition) at every 30 min for total incubation of 2 hr under a quiescent incubation at 37°C. The fibrillar extension of RAFs was monitored with TEM. (B) Average lengths of RAFs were estimated by analyzing 200 separate PC-liposomes with RAFs at each time point. Gray and black bars indicate the average fibrillar lengths obtained with either oligomeric or monomeric form of α-synuclein, respectively.
Figure 4.
Disruption of lipid membranes upon the surface-dependent amyloid fibril formation.
(A) PC-liposomes (1.25 mg/ml, 0.2 ml) revealed with TEM following a brief incubation in 20 mM Mes, pH 6.5, for 5 min at 25°C under a quiescent condition with the oligomeric α-synuclein at 220 µg (left panel) and 330 µg (right panel). (B) Thermodynamic assessment with isothermal titration calorimetry (ITC) for the molecular assembly-disassembly process of the PC-liposomes as treated with either the oligomers (red line) or the monomers (black line) of α-synuclein. The PC-liposomes (2.5 mg/ml, 1.7 ml) in 62.5 mM Mes, pH 6.5 were sequentially combined with α-synuclein in either form at 54.7 µg per addition for 20 sec at 5 min-interval. Negative value of y-axis represents exothermic reaction. (C) Disruption of the liposome containing magnetic nanoparticles (MNPs). The PC-liposomes enclosing the Fe3O4 magnetic nanoparticles were examined with TEM before (left) and after (right) the treatment of oligomeric α-synuclein at 0.5 mg/ml in 20 mM Mes, pH 6.5, for 5 min at 25°C. Arrows in the right panel indicate MNPs localized on the fibrils. (D) Calcium release from the Ca2+-entrapped PC-liposomes in the presence of monomers, oligomers, and amyloid fibrils of α-synuclein. The PC-liposomes containing 1 mM CaCl2 were incubated with monomers, oligomers, and fibrils of α-synuclein at 0.11 mg/ml in 20 mM Mes, pH 6.5, for 2 hr at 25°C. The release of Ca2+ ions was monitored with the Ca2+ ion-indicative fluorescent dye of BTC by detecting light emitting at 529 nm with an excitation at 401 nm. (E and F) Disruption of mitochondria (E) and lysosomes (F) with the oligomer treatment of α-synuclein. Mitochondria and lysosomes were visualized with TEM before (inset in a) and after (a) the oligomer treatment (0.5 mg/ml) in 20 mM Mes at pH 6.5 for 2 hr at 37°C. Presence of α-synuclein in the fibrils was confirmed with immunogold labeling with rabbit anti-α-synuclein antibody as indicated with arrows. Releases of lactate dehydrogenase (LDH) and cathepsin D were separately monitored upon the addition of oligomers with chromogenic substrates as a measure of disruption of mitochondria and lysosomes, respectively (b). Values are shown in means±s.d. (n = 2).