PT  - JOURNAL ARTICLE
AU  - Michael M. Lacy
AU  - David Baddeley
AU  - Julien Berro
TI  - New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics
AID  - 10.1101/092536
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 092536
4099  - http://biorxiv.org/content/early/2016/12/08/092536.short
4100  - http://biorxiv.org/content/early/2016/12/08/092536.full
AB  - 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.BARBin/Amphiphysin/Rvs domainFRAPFluorescence Recovery After PhotobleachingmEGFPmonomeric Enhanced Green Fluorescent ProteinSiR647silicon-rhodamine 647TIRFTotal Internal Reflection Fluorescence