Single molecule tracking reveals nanodomains in biomolecular condensates

Biomolecular condensates regulate cellular physiology by sequestering and processing RNAs and proteins, yet how these processes are locally tuned within condensates remains unclear. Moreover, in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), condensates undergo liquid-to-solid phase transitions, but capturing early intermediates in this process has been challenging. Here, we present a surface multi-tethering approach to achieve intra-condensate single-molecule tracking of fluorescently labeled RNA and protein molecules within liquid-like condensates. Using RNA-binding protein Fused in Sarcoma (FUS) as a model for condensates implicated in ALS, we discover that RNA and protein diffusion is confined within distinct nanometer-scale domains, or nanodomains, which exhibit unique connectivity and chemical environments. During condensate aging, these nanodomains reposition, facilitating FUS fibrilization at the condensate surface, a transition enhanced by FDA-approved ALS drugs. Our findings demonstrate that nanodomain formation governs condensate function by modulating biomolecule sequestration and percolation, offering insights into condensate aging and disease-related transitions.