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Science 339 (6118): 460-464

Copyright © 2013 by the American Association for the Advancement of Science

Tunable Signal Processing Through Modular Control of Transcription Factor Translocation

Nan Hao1,2, Bogdan A. Budnik1, Jeremy Gunawardena3, and Erin K. O'Shea1,2,*

1 Harvard University Faculty of Arts and Sciences Center for Systems Biology, Cambridge, MA 02138, USA.
2 Howard Hughes Medical Institute, Department of Molecular and Cellular Biology, and Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
3 Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.


Figure 1
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Fig. 1. Tunable signal-processing behaviors of Msn2. (A) Illustration of the distinct single-cell dynamic responses of Msn2 to various stresses. (B) Steady-state abundance of Msn2 in the nucleus in response to various concentrations of 1-NM-PP1. In response to each concentration of 1-NM-PP1, Msn2 exhibited uniform and stable nuclear localization in single cells and did not exhibit stochastic fluctuations as observed in natural stress responses. Open circles: responses to different concentrations of 1-NM-PP1; closed circles: responses to 3 μM and 0.2 μM 1-NM-PP1, which are used as high- and low-amplitude inputs, respectively, for the following analyses. AU, arbitrary unit. (C) Averaged single-cell time traces of Msn2 nuclear translocation (bottom: n {approx} 50 cells; error bar: single-cell variances) in response to oscillatory inputs with high and low amplitudes (top). (Left) High-amplitude input produced by 3 μM 1-NM-PP1; (right) low-amplitude input produced by 0.2 μM 1-NM-PP1. Pulse duration of 3 min; pulse interval of 2 min. To emphasize the fact that 3 μM 1-NM-PP1 elicits a steady-state response that is about twice the response elicited by 0.2 μM 1-NM-PP1, the top y axes are not presented on a linear scale.

 

Figure 2
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Fig. 2. A theoretical analysis of TF translocation. (A) Nomenclature used to define the status of phosphorylation and localization of the TF: for example, the first "P/U": the NES is phosphorylated (P) or unphosphorylated (U); "c/n": superscript shows location in the cytoplasm (c) or nucleus (n). (B) Phosphorylation states determine the rate constants of nucleocytoplasmic transport. Unphosphorylated or phosphorylated NES has slow (dashed line) or fast (solid line) nuclear export rates (kout, kout'), respectively; unphosphorylated or phosphorylated NLS has fast (solid line) or slow (dashed line) nuclear import rates (kin', kin), respectively. Thus, each phosphoform has a specific combination of nuclear import and export rates. (C) The translocation model. (Left) Schematic of WT and phosphosite mutants; (right) model structures and reaction flows (gray arrows) in response to strong or weak inputs and input removal. First row: WT; second row: NLS 4A at S582A, S620A, S625A, and S633A; third row: NLS 4E at S582E, S620E, S625E, and S633E; fourth row: NES 2A at S288A and S304A. We did not specifically study the case in which the NES sites are constitutively phosphorylated because Ser-to-Glu mutants of the NES sites behaved similarly to Ser-to-Ala mutants, which suggests that Glu cannot mimic phosphorylation on NES sites (fig. S2A). (D) Predicted responses to various dynamic inputs—first column: oscillatory high-amplitude input, second column: oscillatory input with varied amplitudes, third column: input fluctuating between high and low amplitudes. Color key—black: responses of WT, blue: NLS 4A, green: NLS 4E, red: NES 2A. The ranges of input time scales necessary to generate the predicted responses are determined by the fast and slow time scales of transport rates and are listed above each column. Model output was generated by a steady-state analysis of the translocation system (supplementary materials).

 

Figure 3
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Fig. 3. Distinct signal processing by WT, NLS, and NES phosphosite mutants of Msn2. (A) Averaged single-cell time traces of Msn2 nuclear translocation in response to sustained inputs with low (0.2 μM 1-NM-PP1, solid triangles) or high (3 μM 1-NM-PP1, solid circles) amplitudes. Inputs were applied at time point zero. (B) Averaged single-cell time traces of Msn2 nuclear translocation in response to removal of high-amplitude input (3 μM 1-NM-PP1). (C) Averaged single-cell time traces of Msn2 nuclear translocation in response to oscillatory high-amplitude (3 μM 1-NM-PP1) inputs. Pulse duration of 3 min; pulse interval of 4 min. (D) Time traces of Msn2 nuclear translocation in response to oscillatory inputs with a mixture of low- and high-amplitude pulses (0.2 μM 1-NM-PP1 and 3 μM 1-NM-PP1, respectively). (E) Time traces of Msn2 nuclear translocation in response to input fluctuating between high (3 μM 1-NM-PP1) and low (0.2 μM 1-NM-PP1) amplitudes. For (A) to (E), data points are averaged single-cell time traces (n {approx} 50 cells; error bar: single-cell variances). The simple model in Fig. 2 has been fitted to the time trace data in this figure, and the solid lines in (A) to (E) are model fitting results (see supplementary materials, "Model parameters are constrained by experimental data" for details). The dependence of the responses on the time scales of input and transport rates is presented in the supplementary materials, "The relation between time scales of input and time scales of transport rates."

 

Figure 4
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Fig. 4. Distinct responses of WT, NLS, and NES phosphosite mutants of Msn2 to natural stresses. Single-cell responses of WT, NLS 4A, NLS 4E, and NES 2A to glucose limitation (A), osmotic stress (B), and oxidative stress (C) (n {approx} 50 cells, each stress condition). Representative single-cell time traces of Msn2 nuclear translocation are shown. Asterisks emphasize the conditions under which the mutants fail to distinguish two different stresses. Quantification of the time traces is presented in fig. S4. (D) Time traces of WT Msn2-mCherry and mutant Msn2-YFP, monitored in the same cells, in response to glucose limitation (black, WT; blue, NLS 4A; green, NLS 4E; red, NES 2A). More single-cell traces are shown in fig. S5.

 


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