A Novel FRET-based Method for Monitoring Protein Phase Separation in Living Cells

Protein phase separation (biomolecular condensation) may be a universal phenomenon in various cellular processes. Protein aggregation resulting from biomolecular condensation is a potential therapeutic target for many diseases, particularly neurodegenerative diseases. In this thesis, we present a FRET-based (Förster Resonance Energy Transfer based) method for continuous and high-throughput monitoring of protein self-assemblies to reveal well-resolved transient intermediate states. Intermolecular FRET with both the donor and acceptor proteins at the same target protein provides high sensitivity while retaining the advantage of straightforward ratiometric imaging. We apply this method to monitor the self-assembly of three proteins. We show that the mHttex1 first forms less-ordered assemblies, which develop into fibril-like aggregates. We demonstrate that the chaperone protein DNAJB6b increases the critical saturation concentration of mHttex1 in a concentration and temperature-dependent manner. We also monitor the structural changes in FUS condensates and investigate the influence of osmotic stress. This novel FRET-based method adds to the toolbox for protein self-assembly structure and kinetics determination, and implementation with native or non-native proteins can inform studies involving protein condensation or aggregation.