Single-molecule imaging of the BAR-domain protein Pil1p reveals filament-end dynamics

Molecular assemblies can have highly heterogeneous dynamics within the cell, but the limitations of conventional fluorescence microscopy can mask nanometer-scale features. Here we adapt a single-molecule strategy to perform single-molecule recovery after photobleaching (SRAP) within dense macromolecular assemblies to reveal and characterize binding and unbinding dynamics within such assemblies. We applied this method to study the eisosome, a stable assembly of BAR-domain proteins on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the main eisosome BAR-domain protein in fission yeast, we visualized whole eisosomes and, after photobleaching, localized recruitment of new Pil1p molecules with ∼30-nm precision. Comparing our data to computer simulations, we show that Pil1p exchange occurs specifically at eisosome ends and not along their core, supporting a new model of the eisosome as a dynamic filament. This result is the first direct observation of any BAR-domain protein dynamics in vivo under physiological conditions consistent with the oligomeric filaments reported from in vitro experiments.