Ultrasensitive Biophysical Techniques to Investigate Detergent-Membrane and Protein-Membrane Interactions at the Single Vesicle Level

The utilisation of physicochemical indicators and Förster Resonance Energy Transfer (FRET) probes within model membrane vesicles has provided significant insights into complex membrane interactions, including pore formation, solution exchange, alterations to membrane curvature, and fusion dynamics. This research investigates a range of membrane-disrupting agents, including detergents and protein aggregates linked to neurodegenerative diseases such as Alzheimer’s Disease (AD), using high-throughput fluorescence assays. The methodology involves immobilizing highly-curved vesicles containing lipophilic membrane stains or encapsulated fluorescent Ca2+ indicators onto surfaces. Changes in fluorescence and FRET, resulting from membrane perturbations, were monitored with Total Internal Reflection Fluorescence (TIRF) microscopy. Unlike prior studies relying on ensemble measurements, our single-vesicle imaging techniques reveal a more complex and dynamic process for detergent-induced membrane solubilisation. Specifically, this work demonstrates that commonly-used non-ionic detergents Triton X-100 (TX-100) and Tween-20 induce structural changes in individual surface-tethered vesicles by inducing pore formation, solution exchange, vesicle expansion, and in the case of TX-100, vesicle fusion. Multiple intermediate stages during vesicle fusion by TX-100 at sub-Critical Micelle Concentration (CMC) concentrations were identified, enhancing our understanding of detergent-membrane interactions. Inital investigations with Amyloid-β (1-42) (Aβ(1-42)) (a major hallmark of AD) followed its aggregation with two fluorescent dyes: Thioflavin T (ThT) and a novel derivative of ThT, Thioflavin X (ThX). Results showed ThX exhibited enhanced brightness and binding in comparison to ThT. Incubation of Aβ(1-42) monomers and aggregate species with model membrane vesicles revealed that Aβ(1-42) species cause significant detergent-like perturbations in vesicles, including permeabilisation, swelling, and fusion, suggesting the disruption of essential transport pathways during AD. In conclusion, these innovative fluorescence-based methods offer high sensitivity, providing valuable insights into complex interactions involving proteins, detergents, and lipid membranes.