Author Archives: jb246

New paper from the Berro lab!

The preprint of our new single-molecule study of the eisosome Bar protein Pil1p is now available on bioRxiv

Lacy M, Baddeley D, Berro J
New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics.
bioRxiv
BIORXIV/2016/092536

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.

New paper from the Berro lab about fast CRISPR/Cas9 edition in fission yeast thanks to a new plasmid selection using fluoride

Congratulations to Ronan Fernandez for his new paper!

Fernandez R, Berro J
Use of a fluoride channel as a new selection marker for fission yeast plasmids and application to fast genome editing with CRISPR/Cas9.
Yeast. 2016 Oct;33(10):549-557.
PMID: 27327046

Abstract:
Fission yeast is a powerful model organism that has provided insights into important cellular processes thanks to the ease of its genome editing by homologous recombination. However, creation of strains with a large number of targeted mutations or containing plasmids has been challenging because only a very small number of selection markers is available in Schizosaccharomyces pombe. In this paper, we identify two fission yeast fluoride exporter channels (Fex1p and Fex2p) and describe the development of a new strategy using Fex1p as a selection marker for transformants in rich media supplemented with fluoride. To our knowledge this is the first positive selection marker identified in S. pombe that does not use auxotrophy or drug resistance and that can be used for plasmids transformation or genomic integration in rich media. We illustrate the application of our new marker by significantly accelerating the protocol for genome edition using CRISPR/Cas9 in S. pombe.

New release of the PatchTrackingTools

A new release of the PatchTrackingTools is now available!

This new release now integrates the functionalities of Trackmate to find and track patches. It also fixes many glitches and performance issues. If you have issues or find glitches please contact Julien Berro directly (email: julien dot berro at yale.edu)

Please cite: Berro J, Pollard TD. Mol Biol Cell. 2014 Nov 5;25(22):3515-27. Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin-mediated endocytosis and cell polarization in fission yeast. PMID: 25143407