Nanoscale domains govern local diffusion and ageing within fused-in-sarcoma 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, 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 labelled RNA and protein molecules within liquid-like condensates. Using RNA-binding protein fused-in-sarcoma as a model for condensates implicated in amyotrophic lateral sclerosis, we discover that RNA and protein diffusion is confined within distinct nanometre-scale domains, or nanodomains, which exhibit unique connectivity and chemical environments. The properties of these nanodomains are tunable by guest molecules. As condensates age, nanodomains reposition, facilitating fused-in-sarcoma fibrilization at the condensate surface, a process further enhanced by anti-amyotrophic lateral sclerosis drugs. Our findings demonstrate that nanodomain formation governs condensate function by modulating the residence time and spatial organization of constituent biomolecules, providing previously unattainable insights into condensate ageing and mechanisms underlying disease.