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. 2013 Jun 15;24(12):2045–2057. doi: 10.1091/mbc.E12-12-0870

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© 2013 Petrenko et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 7: — Nuclear localization of Msn2 in response to stress is stochastic. (A) Y3630 (MSN2-GFP msn4∆ prototroph) cells immobilized in a microfluidic chamber were imaged while perfused with SD for 30 min, then subjected to 1 M sorbitol stress for 100 min, allowed to recover in SD for 30 min, and again stressed by SD + 1 M sorbitol for the duration of the time course. Shown are traces of Msn2-GFP nuclear localization in four different cells. (B) Y3630 (MSN2-GFP msn4∆ prototroph) cells immobilized in a microfluidic chamber were imaged while perfused with SD for 40 min, shifted to glucose-limited medium for 140 min, and then returned to SD. The top graph plots the average Msn2 nuclear localization over all cells, and the shaded area in the bottom graph shows the total number of cells with nuclear localization above a threshold of background fluorescence as a function of time. The inset shows a kymograph of individual cell behavior for the last 80 min of the experiment, spanning the transition from limited glucose to SD. Data were collected at 1-min intervals. (C) Top, kymograph of nuclear localization of Msn2 in Y3630 cells subjected to 3-min cycle of glucose addition and withdrawal. Data were collected at 20-s intervals. Bottom, simulated kymograph of wild-type cells subjected to 3-min cycles of glucose addition and withdrawal, obtained by Gillespie simulations of the model depicted in Figure 5. (D) Msn2-GFP nuclear localization was monitored in WT and two of the pka-wimp strains subjected to 3-min cycles of glucose removal and addition. The green line in each plot shows the averaged nuclear occupancy for all cells over the experiment, and the blue shading is the average ± SD. Gillespie simulations were performed as described in the Supplemental Methods, based on the model in Figure 5. The red line is averaged nuclear localization from these simulations, and the yellow shading indicates ± SD. See also Supplemental Figure S5.