Fig 1.
Amyloid fibril characterization.
AFM height images of fibril samples adhered to mica surface (scale bar equals 1 μm) (A) α-syn, (B) Lysozyme and (C) Aβ40 with dot blot analysis of fibril samples (inset) by anti-fibril LOC antibody specific to generic epitopes common in amyloid fibrils.
Fig 2.
The effect of fibril fragmentation on length and its capacity to cause membrane disruption.
Liposome dye release assay using a model lipid membrane formed from 80% (w/w) phosphatidylcholine and 20% (w/w) phosphatidylserine encapsulating the fluorescent probe carboxyfluorescein. Dye-encapsulated vesicles were incubated with amyloid fibril samples (30 min): (A) α-Syn 100 μg/mL (B) Lysozyme 20 μg/mL and (C) Aβ40 20 μg/mL. Fluorescence was then recorded and the fold increase in dye release was normalised against unfragmented fibril fluorescence. Light grey bars denote unfragmented fibril samples and dark grey bars denote fragmented fibril samples (n = 3, error bars show SE). Statistical significance between the unfragmented and fragmented fibril dye release response was determined by a Mann Whitney U test P ≤ 0.05 (*) or P ≤ 0.005 (**).
Fig 3.
The effect of fragmented fibril on cell viability by cell membrane integrity assay.
SH-SY5Y cells (2x104/well) were plated with the addition of the CellTox dye and allowed to adhere. The unfragmented and fragmented fibril samples were then added (A) α-syn 7 μM and (B) Aβ40 10 μM. Fluorescence was recorded over a period of 72 hours (520Em/ 485Ex). Light grey bars denote unfragmented fibril samples and dark grey bars denote fragmented fibril samples (n = 3, error bars show SE). Unfragmented (light grey) and fragmented fibril samples (dark grey) (n = 6, SE). Statistical significance between toxicity of unfragmented and fragmented fibrils at each time point was determined by a Mann Whitney U test P ≤ 0.05 (*) or P ≤ 0.005 (**).
Fig 4.
TIRE measurements of membrane disruption at native lipid membrane by the incremental addition of amyloid fibrils.
Natively derived cells from the neuronal cell line; SH-SY5Y was deposited by Langmuir-Schaefer deposition and protein: lipid interactions were probed between amyloid fibril samples and plasma membranes. Left plots (A, C and E) show experiments with unfragmented samples and right plots (B, D and F) fragmented samples. Top row plots (A+D) show experiments with α-Syn, middle row (B+E) Lysozyme, and bottom row (C+F) Aβ40. Membrane thickness were measured by change in Δd (nm), which is ploted versus fibril concentration (n = 3, error bars show SE). Statistical significance for the differences in thickness within an experiment on exposure to fragmented fibril samples was determined by Kruskal Wallis test with an Conover-Inman post hoc-test. (*) or (**) denote P ≤ 0.05 and P ≤ 0.005, respectively. Statistical significance between separate unfragmented and fragmented experiments in the case of α-syn was also determined by Kruskal Wallis with an Inman post hoc-test with P ≤ 0.05 (#).