DOI: 10.5061/dryad.80gb5mkzb
title: Data from: Live-cell analysis of IMPDH protein levels during yeast colony growth provides insights into the regulation of GTP synthesis

description: Here we present real-time, live-cell analysis of accumulation of the Imd2
 isoform of IMPDH in Saccharomyces cervisiae yeast cells forming a
 monolayer colony in a microfluidic device over a 50-hour time course. We
 observe two distinct phases of increased Imd2 accumulation: a
 guanine-insensitive phase early in outgrowth and a guanine-sensitive phase
 later, when cells become crowded. We show that the IMPDH inhibitor
 mycophenolic acid enhances both phases of increase. Deletion of a
 transcription attenuator upstream of the mRNA start site that decreases
 Imd2 mRNA synthesis in the presence of high GTP increases the baseline
 level of Imd2 protein ten-fold and abolishes guanine-sensitive but not
 guanine-insensitive induction. Our results suggest that at least two
 mechanisms of yeast Imd2 regulation exist, the known GTP-dependent
 attenuation of RNA polymerase II elongation and a GTP
 concentration-independent pathway that may be controlled by cell growth
 state.
description: Refer associated mBio paper.
description: # Data from: Live-cell analysis of IMPDH protein levels during yeast
 colony growth provides insights into the regulation of GTP synthesis
 [https://doi.org/10.5061/dryad.80gb5mkzb](https://doi.org/10.5061/dryad.80gb5mkzb) The microscopy data underlying each data figure in the manuscript is contained in a single folder, corresponding to a single time course experiment on a 4-channel CellASIC plate. Each folder includes a metadata spreadsheet, a subfolder containing 3-4 raw microscopy (nd2) files, a subfolder containing ~100 merged tiff files (an exposure every 30 minutes over 50 hours) corresponding to each of the four experimental channels (xy1-xy4) and three different light sources (DIC, GFP, or mCherry, either individually or merged), and a "cellMeasurements" spreadsheet containing the results of analysis of the GFP fluorescence from each identified cell in the frame. ## Description of the data and file structure Cells were identified and their GFP fluorescence measured from multichannel timelapse microscopy images using a custom MATLAB pipeline ([https://github.com/mccleanlab/cell_finder](https://github.com/mccleanlab/cell_finder)). Bio-Formats was used to load ND2 images directly into MATLAB and cells were identified from either the DIC or GFP channels based on a manual evaluation of image quality. In either case, the images were processed with a Laplacian of Gaussian filter to enhance cell contrast and circular regions of interest (ROIs) likely containing individual yeast cells were identified using MATLAB’s circle finder. The mean GFP fluorescence of each ROI was measured from the original (non-enhanced) images. ROIs with fluorescence values below a manually selected threshold were excluded as false positives. Percent confluency was calculated from the area occupied by the retained ROIs. The number of identified cells in the first timepoint ranged from 12-71 and in the last timepoint ranged from 2405-2900. Figure 2. Single-cell measurement of Imd2-GFP protein accumulation over 50 hours with and without guanine supplementation. Figure 3. Dose-dependent induction of Imd2-GFP by mycophenolic acid (MPA). Figure 4. Guanine-specific and dose-dependent suppression of Phase 2 induction of Imd2-GFP in the presence of MPA. Figure 6. Overexpression of catalytically active *IMD2* from a plasmid decreases endogenous Imd2-GFP levels in confluent cells. Figure 7. Addition of MPA shortly before or after confluence results in rapid, guanine-sensitive Imd2-GFP induction Figure 8. Deletion of nonproductive “G” transcription start sites from *IMD2* increases basal Imd2-GFP levels 10-fold and abolishes guanine sensitivity. Figure S6. Reproducibility of Imd2-GFP induction during 1.5 mg/mL MPA treatment in four biological replicates. Figure S8. The absence of the G start sites eliminates MPA induction of Imd2-GFP levels. ## Code/Software [https://github.com/mccleanlab/cell_finder](https://github.com/mccleanlab/cell_finder)