A Molecular Rotor-Based Platform for Dissecting Protein Phase Separation and Aggregation

Biomolecular condensates formed via phase separation play critical roles in regulating cellular processes, and their dysregulation is increasingly associated with neurodegeneration. A key challenge in understanding condensate pathology lies in distinguishing dynamic condensates from neurotoxic solid aggregates, the formation of which can, in some systems, occur within existing condensates. Yet current detection techniques often lack the sensitivity and resolution to directly monitor these assemblies and their transitions in dynamic and complex environments. Here, we combined fluorescent probes, based on molecular rotors, with fluorescence lifetime imaging microscopy to quantify changes in protein dynamics during phase separation-associated aggregate formation. Molecular rotors respond to local viscosity changes, which in our study allowed discrimination between fluid condensates and rigid amyloids based on rotors' fluorescence lifetime. Using -synuclein, a hallmark protein in Parkinson's disease, as a model system, we demonstrate real-time visualization of phase separation and condensate maturation within a unified experimental framework. Our method preserves protein integrity and provides quantitative, spatially resolved insights into structural transitions. This technology bridges a critical gap in the study of pathological protein aggregation and offers a powerful platform for mechanistic studies, biomarker discovery, and drug discovery in neurodegenerative disease research.