Long-duration 3D single-molecule tracking maps transport landscapes within tau condensates
Abstract
Biomolecular condensates are spatially heterogeneous, but most measurements either average over molecular populations or yield trajectories too short to reveal how individual molecules traverse this heterogeneity. Here, we use active-feedback three-dimensional single-molecule tracking to follow individual fluorescently labelled tau molecules continuously within reconstituted condensates for tens to more than 100 s. The resulting long trajectories reveal recurrent switching among three apparent mobility states, spatially revisited residence hotspots enriched in slow and subdiffusive motion, and restricted shell-like motion in representative boundary-associated trajectories. We further capture repeated single-molecule transfer between adjacent condensates. A representative continuous trajectory resolves inter-condensate passages lasting tens of milliseconds. The configurational free-energy difference predicted from the molecule-accessible volume ratio closely matches the occupancy-derived value, revealing a quantitative correspondence between molecular occupancy and accessible configurational space. Thus, long-duration 3D single-molecule tracking preserves sufficient molecular history to connect heterogeneous transport, local confinement, interfacial motion, exchange kinetics, and trajectory-derived thermodynamic partitioning within the same measurement, providing a framework for reconstructing the transport and effective free-energy landscapes experienced by individual molecules in biomolecular condensates.
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