Data and code from Durrieu et al. (IScience, 2022). Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources
Description
Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources Several assays exist to measure nuclear transport rates, but not at the single-cell level. Here, we developed a fluorescent recovery after photobleaching (FRAP)- based method to determine nuclear import and export rates independently, in individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential FRAPs on the same cell and optimization to an elaborate model that accounts for both the biological and the experimental aspects. We found large cell-to-cell variation in transport rates within isogenic yeast populations. Our data suggest that a main determinant of this heterogeneity may be variability in the number of nuclear pore complexes (NPCs). For passive transport, this component explained most of the variability. Actively transported proteins were influenced by variability in additional components, including general factors such as the Ran-GTP gradient as well as specific regulators of the export rate. By considering mother-daughter pairs, we showed that mitotic segregation of the transport machinery is too noisy to control cellular inheritance. Finally, we studied mother-daughter cell asymmetry in the localization of the transcription factor Ace2, which is specifically concentrated in daughter cell nuclei. We found that this asymmetry is the outcome of a higher ratio of import rate to export rate in daughters. Uploaded here: Code - the NuclearFRAP R package, for the fitting of FRAP experiments in single cells. - Matlab scripts for simulation of data from FRAP experiments - R scripts for analysis of the data (internal scripts used to create the plots from the paper, comments are mostly in spanish) Data - Raw data of train of FRAP experiments in yeast. Performed on the proteins YFP, Fus3-YFP, YFP-Ace2 and YFP-Ace2 G128E. - Results of fitting the experimental data of train of FRAP experiments in yeast. Output from the NuclearFRAP package. - Simulated train of FRAP experiments raw data (for different signal/noise levels) - Results of fitting the simulated train of FRAP data - Raw measurements of the abundance of nuclear pore complex proteins. Result of the quantification of confocal images of the proteins Mpl1-GFP, Mpl2-GFP, Prm3-GFP and Nup49-GFP. - Raw data from the train of FRAPs experiments on Ace2, in cells Crm1* treated with LMB. Methods and protocols - Protocols from the lab for performing and analyzing the train of FRAPs and NPC quantification. In spanish. The microscopy images from the train of FRAPs experiments are available upon request. For more data or any questions please write to: luciadurrieu@gmail.com