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

Michael M. Lacy, David Baddeley, View ORCID ProfileJulien Berro
doi: https://doi.org/10.1101/092536
Michael M. Lacy
aDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
bNanobiology Institute, Yale University, West Haven, CT 06516, USA
cIntegrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06520, USA
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David Baddeley
bNanobiology Institute, Yale University, West Haven, CT 06516, USA
cIntegrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06520, USA
dDepartment of Cell Biology, Yale University, New Haven, CT 06520, USA
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Julien Berro
aDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA
bNanobiology Institute, Yale University, West Haven, CT 06516, USA
cIntegrated Graduate Program in Physical and Engineering Biology, Yale University, New Haven, CT 06520, USA
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  • ORCID record for Julien Berro
  • For correspondence: julien.berro@yale.edu
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Abstract

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.

BAR
Bin/Amphiphysin/Rvs domain
FRAP
Fluorescence Recovery After Photobleaching
mEGFP
monomeric Enhanced Green Fluorescent Protein
SiR647
silicon-rhodamine 647
TIRF
Total Internal Reflection Fluorescence
Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted December 08, 2016.
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New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics
Michael M. Lacy, David Baddeley, Julien Berro
bioRxiv 092536; doi: https://doi.org/10.1101/092536
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New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics
Michael M. Lacy, David Baddeley, Julien Berro
bioRxiv 092536; doi: https://doi.org/10.1101/092536

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