Fig. 4: Single round disaggregation experiments.

a To measure the outcome of a single disaggregation round, αS fibrils (2 µM monomer equivalent) were incubated with Hsc70 and DnaJB1. Subsequently, a 200-fold molar excess of Hsc70-binding peptide GSGNRLLLTG was added, to block Hsc70 re-binding to fibrils. b Outcome of a single disaggregation round, as measured by epifluorescence detection. Two species were detectable, the larger of which had a similar size to the initial fibril, the second of which had the size of monomer. Red represents pure fibrils, blue the outcome after a single disaggregation round. c Fraction of monomer generated as a function of fibril length at constant αS concentration, corresponding to an increased number of fibril ends. Shorter fibrils lead to more monomer release per single disaggregation round than longer fibrils, whereby this effect plateaus for fibrils longer than 100 nm. d Fraction of monomer measured at different Hsc70 concentrations. No disaggregation took place for Hsc70 concentrations below 1 µM, suggesting that there is a critical Hsc70 concentration required for efficient disaggregation. Data in b–d are represented as mean ± standard deviation of n = 3 independent experiments. e Diffusion profile for confocal scanning across the microfluidic channel for pure αS fibrils. The profile does hardly broaden, as expected for a large amyloid fibril. f Diffusion profile for confocal scanning across the microfluidic channel after a single disaggregation round. This leads to significant broadening, whilst the burst height remains similar, indicating that fibrils of similar length and a small species are generated. g Comparison of the signal of pure fibrils (blue), single round disaggregation (red) and pure monomer (black). The red signal after a single disaggregation round is the overlay between the two sets.