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New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics

By Michael M. Lacy, David Baddeley, Julien Berro

Posted 08 Dec 2016
bioRxiv DOI: 10.1101/092536 (published DOI: 10.1091/mbc.E17-04-0238)

Molecular assemblies can have highly heterogeneous dynamics within the cell, but the limitations of conventional fluorescence microscopy can mask nanometer-scale features. We have developed a novel, broadly applicable, fluorescent labeling and imaging protocol, called Single-molecule Recovery After Photobleaching (SRAP), which allowed us to reveal the heterogeneous dynamics of the eisosome, a multi-protein structure on the cytoplasmic face of the plasma membrane in fungi. By fluorescently labeling only a small fraction of cellular Pil1p, the core eisosome BAR domain protein in fission yeast, we visualized whole eisosomes and, after photobleaching, recorded the binding of individual Pil1p molecules with ~20 nm precision. Further analysis of these dynamic structures and comparison to computer simulations allowed us to show that Pil1p exchange is spatially heterogeneous, supporting a new model of the eisosome as a dynamic filament.

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