Single molecule tracking reveals nanodomains in biomolecular condensates

Abstract

AbstractProtein-rich biomolecular condensates formed by phase separation1play a crucial role in cellular RNA regulation by the selective recruitment and processing of RNA molecules2. The functional impact of condensates on RNA biology relies on the residence time of individual RNA molecules within a condensate3, which is governed by intra-condensate diffusion4– the slower or more confined the diffusion, the longer the residence time. However, the spatiotemporal organization of RNA and protein diffusion within a single condensate remains largely unknown due to the challenge of accurately profiling intra-condensate diffusion behaviors down to the single-molecule level. Here we introduced a general condensate-tethering approach that allows single molecule tracking (SMT) of fluorescently labeled proteins and RNAs within non-wetted spherical 3D condensates without the interference of condensate motions. We found that a significant fraction of RNA and protein molecules are locally confined, rather than freely diffusive, within a model condensate formed by full-length, tag-free, RNA-binding protein Fused-in-Sarcoma (FUS), known for its critical roles in RNA biology under both physiological and pathological conditions5. Dynamic Point Accumulation for Imaging in Nanoscale Topography (PAINT)6reconstruction further revealed that RNA and proteins are confined to distinct slow-moving nanometer-scale regions, termed nanodomains, within a single condensate. Remarkably, nanodomains affect the diffusion but not the density of the confined biomolecules, supporting local percolation1,7rather than a secondary phase separation within the condensate as their origin. Beyond their regulatory roles on RNA residence time, nanodomains engender both elevated local connectivity and altered chemical environment, a prerequisite for pathological liquid-to-solid transition of FUS during aging. In summary, our study uncovers a patterned spatial organization of both protein and RNA molecules within a single condensate, revealing distinct diffusion dynamics that affect molecular retention time and interactions underlying cellular function and pathology.