Investigating the Factors Affecting the Aggregation of Alpha-Synuclein using Single Molecule Fluorescence and Fast Flow Microfluidics

The conversion of α-synuclein (αs) into oligomeric and fibrillar species and its deposition into Lewy bodies is the pathological hallmark of Parkinson's disease (PD). It is therefore of great importance to understand the mechanism of αs aggregation and its relationship to PD pathogenesis. We use single molecule fluorescence techniques with fast flow microfluidics to follow the early stages of this process in vitro. The methodology is based on the detection of fluorescent bursts from red or blue fluorophore-tagged αs species as they flow through a blue confocal laser volume. Forster Resonance Energy Transfer (FRET) occurs between blue and red fluorescently-tagged αs within the oligomers, giving rise to a signal in both the acceptor and donor channels. As only oligomers generate a coincident signal, they can be isolated from a solution which is >99% monomer, allowing their size and structure to be determined. By taking regular time-points, the kinetics of the aggregation can be deduced.This technique has allowed us to gain a unique insight into the effects of nanobodies and molecular chaperones on the aggregation of both the wild-type and pathological mutants of αs.View Large Image | View Hi-Res Image | Download PowerPoint Slide