A few plasmids that were created in the Berro lab and can be useful for the S. pombe community are now available on Addgene. These include plasmids to perform CRISPR/Cas9 edition using fluoride selection (Fernandez and Berro, 2016), and plasmids to C-terminally tag proteins with SNAP, CLIP, Halo, mEOS3.2, PAmKate, PATagRFP or PAmCherry (unpublished).
New single-molecule imaging of the eisosome BAR domain protein Pil1p reveals filament-like dynamics.
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.
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
We recently published two new studies on quantitative analysis of clathrin-mediated endocytosis in fission yeast:
Molecular Biology of the Cell. 2014 Nov 5;25(22):3501-14. PMID: 25143395
Berro J and Pollard TD. Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin mediated endocytosis and cell polarization in fission yeast. Molecular Biology of the Cell. 2014 Nov 5;25(22):3515-27. PMID: 25143407
The first paper presents new methods to collect and analyze quantitative microscopy data. We describe a new “temporal super-resolution” method to realign datasets obtained with low temporal resolution and reconstruct a signal with higher temporal resolution. This method allowed us to demonstrate the high reproducibility of endocytic events in fission yeast, and to show that endocytic vesicles move with a diffusive motion that is modulated by actin disassembly. In this paper, we also present a new method to automatically estimate the number of endocytic events in a cell at a given time point.
Application of these new methods allowed us to demonstrate in a second paper that Aip1p caps the barbed ends of actin filaments in a specific context and can be replaced by the canonical capping protein heterodimer Acp1p/Acp2p. However, Aip1p cannot replace Acp1p/Acp2p. Our data also point out new independent functions for Acp1p and Acp2p in cell polarity. Finally, our quantitative analysis allowed us to infer geometric properties of the endocytic structures and to show that the actin meshwork compresses before scission of the endocytic vesicle.