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Oscillatory stress stimulation uncovers an Achilles’ heel of the yeast MAPK signaling network

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Science  11 Dec 2015:
Vol. 350, Issue 6266, pp. 1379-1383
DOI: 10.1126/science.aab0892
  • Fig. 1 Osmotic oscillations at an intermediate frequency cause slow proliferation.

    (A) Schematic of the flow chamber used in our experiment. (B) Cell growth under various frequencies of mild osmostress (0.4 M KCl). The graphs show the average number of progeny cells relative to the number of cells before stress is applied (n indicates the number of parental cells monitored). Growth without osmotic stress is depicted in gray. The insets at right show representative images of cells. (C) Systematic frequency scan of mild osmotic oscillations (0.4 M KCl). The graph shows the mean doubling time over a period of 8 hours. Each point marks the mean generation time calculated from at least 50 individual sets of progeny in two biological repeats. Error bars indicate SE.

  • Fig. 2 Mathematical modeling of adaptive signaling of the osmotic pathway predicts downstream pathway hyperactivation at resonance stress frequency.

    (A) Schematic of the osmotic pathway (3). Changes in turgor pressure activate Hog1-dependent and Hog1-independent response arms that act to reduce deviation from the optimal turgor pressure. (B) Pathway activation according to the perfect-adaptation model (3). (Top) Predicted amounts of Hog1 phosphorylation in response to a 0.4 M increase in osmolality with induction and adaptation phases. (Bottom) Integral under the Hog1-PP curve, taken as an approximation of the accumulated transcriptional output. (C) Pathway activation at three representative pulse durations (ON and OFF intervals are marked in red and gray, respectively). The area under the predicted signaling curve (Top) was normalized to the entire pulse period (ON + OFF) (Bottom). (D) Model-predicted signaling and transcriptional dynamics under representative oscillation periods. (E) Experimentally observed signaling dynamics under representative oscillation periods, as measured by tracking Hog1-GFP nuclear localization. The graphs show the mean intensity ratio of nuclear Hog1-GFP over total Hog1-GFP in 40 to 100 cells (relative to the basal ratio at t = 0 min). (F) Measured signaling integral (normalized per minute) in a frequency scan. The blue bars show the average integral in two biological repeats (error bars indicate SD). The dashed black curve marks the model predictions.

  • Fig. 3 Pathway hyperactivation and cross-talk underlie growth inhibition at the sensitive frequency.

    (A) Network diagram of the high-osmolality and invasive-growth pathways. YFP, yellow fluorescent protein. (B) Transcriptional output of the pathways in response to alternative inputs. The graphs show the mean fold induction in florescence per cell and the single-cell traces of cells within the interquartile range. The graph with the dotted purple line shows full pathway activation in response to butanol. Although pathway isolation is maintained under a step input profile, osmotic oscillations lead to hyperactivation of the osmotic response and full activation of the invasive-growth pathway. The microscopy images show representative cells 8 hours after their first exposure to stress. Arrowheads indicate cells that died during the stress period. (C) Transcriptional response at various frequencies of osmotic stress (0.4 M KCl). The activity of both reporters behaves as a band-pass filter, with peaked activity at intermediate frequencies (8 to 16 min). Error bars indicate SE of maximal fold fluorescence measured for 70 to 200 individual cells. (D) Frequency-dependent model of the MAPK network that explains growth inhibition at the resonance frequency. (E) Mutational analysis points to a contribution from both pathways in growth inhibition under osmotic oscillations (0.4 M, 8-min period). The color code marks the fold improvement of the deleted strain relative to the WT strain. Statistical significance was evaluated with the t test (comparing the mean growth rate of multiple progeny of the deleted strain and multiple progeny of the cocultured WT strain).

  • Fig. 4 Introducing a synthetic feedback loop resolves osmotic hyperactivation and relieves growth inhibition under osmotic oscillations but also reduces proliferation under more natural input dynamics.

    (A) Diagram of the genetic circuit that underlies the conditional negative feedback. The bacterial effector OspF (fused to an osmotic stress–responsive promoter) deactivates phosphorylated Hog1 by removing a hydroxyl group (14), leading to a longer delay in retriggering of the pathway. (B) Transcriptional response of the osmotic pathway in the WT strain and engineered strain. Both strains show a transient response after an osmotic shock but respond differently to an oscillating input. The graphs show the mean fold induction in florescence per cell and the single-cell traces of cells within the interquartile range. (C) Comparative growth assays of the WT and engineered strains under alternative inputs. (D) Growth inhibition under oscillatory input originates from the adaptive nature of the osmotic response. Although the signaling cascade effectively filters oscillatory inputs at a high frequency (15), oscillations at a lower frequency lead to repeated stimulation of the osmotic pathway. In this frequency range, the cascade circuitry perceives an oscillatory input as gradually increasing osmolarity and hence keeps the osmotic pathway continuously active to counteract the seemingly increasing high osmolality. Growth inhibition is maximized at an intermediate frequency because it is interpreted as the steepest stepwise (i.e., staircase) increase in osmolality, which leads to peaked levels of downstream hyperactivation.

Supplementary Materials

  • Oscillatory stress stimulation uncovers an Achilles' heel of the yeast MAPK signaling network

    Amir Mitchell, Ping Wei, Wendell A. Lim

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S5
    • Tables S1 and S2
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    Images, Video, and Other Other Media

    Movie S1
    Cell growth and osmoreponse under time-variant osmostress in a wild-type strain. A representative time-lapse microscopy movie depicting the cell growth and the osmotic response under different osmostress profiles (no stress, single step osmo-stress, and osmotic oscillations). The movie shows an overlay of images acquired from the DIC and yECitrine channels (images acquired every 15min). Stress starts one hour after the experiment started.
    Movie S2
    Cell growth and osmoreponse of wild-type and OspF strains. A representative time-lapse microscopy movie depicting the cell growth and the osmotic response under osmotic oscillations (period 16min) in the wild-type and feedback engineered strains. The movie shows an overlay of images acquired from the DIC and yECitrine channels (images acquired every 15min). Stress starts one hour after the experiment started.

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