Fig. 3: Analysis of αS fibril disaggregation by the Hsc70 chaperone machinery using confocal microfluidic diffusional sizing.

a Design of the microfluidic device for confocal microfluidic diffusional sizing. The chip design is the same as for the epifluorescence microscopy experiments (see Fig. 1). b The confocal volume scans across the four innermost channels of the microfluidic chip made of polydimethylsiloxane (PDMS) (highlighted in panel a), thereby capturing the diffusive broadening of the reaction mixture with increased diffusion time. (Right) Typical diffusion profiles for pure fibrils and monomers. Pure fibrils show large fluorescence bursts due to the high number of fluorophores detected per fibril, as well as little broadening due to the large size of the fibrils. Profiles for pure monomer samples exhibit no bursts, due to the bulk concentrations employed and because each detected monomer only carries one label. Monomer profiles are broadened significantly in comparison to fibrils due to the small hydrodynamic radius of the monomeric protein. c Diffusion profiles for confocal scanning across the microfluidic channel for αS fibrils at different time points during the disaggregation reaction. Consistent with Fig. 2, the width of the profile base broadens significantly. Furthermore, the bursts, indicating the presence of numerous fluorophores as found in a fibrillar state, vanish at later time points, consistent with the disappearance of fibrils as a result of Hsc70-mediated disaggregation. d Fitting generated for profiles in panel (c). Normalised experimentally obtained diffusional profiles are shown in blue and the obtained fits in orange. e Histograms showing the size and fractional distribution of the two species from diffusion profile analysis. At 0 and 360 min, R_h values of single-component fits are reported. The individual data points are overlaid on the bar plot. Data in e are represented as mean ± standard deviation of n = 3 independent experiments.