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Science 332 (6030): 732-735

Copyright © 2011 by the American Association for the Advancement of Science

Transient Activation of the HOG MAPK Pathway Regulates Bimodal Gene Expression

Serge Pelet1,*, Fabian Rudolf1,{dagger}, Mariona Nadal-Ribelles2, Eulàlia de Nadal2, Francesc Posas2, and Matthias Peter1,*

1 ETH-Zurich, Department of Biology, Institute of Biochemistry, Schafmattstrasse 18, CH-8093 Zurich, Switzerland.
2 Cell Signaling Unit, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain.


Figure 1
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Fig. 1. Bimodal expression of fluorescent reporters upon osmotic stress. (A) Dose response of the quadruple-Venus (qV) fluorescence reporter driven by the STL1 promoter (pSTL1) measured by flow cytometry. (B) Dose responses of wild-type or pbs2{Delta} cells harboring the indicated osmostress-inducible reporters driven by the STL1, ALD3, or HSP12 promoters, or α-factor–inducible reporter pFIG1-qV in a cdc28-as background with 1-NM-PP1 inhibitor (10 μM) or dimethyl sulfoxide (DMSO). The mean of the log-normal distribution fitted to the flow cytometry histograms is plotted. The best fit between single or double log-normal distributions was selected for each curve. The open and closed circles represent, respectively, the population of nonreacting and reacting cells. Circle size is proportional to the population under each distribution. (C and D) Intrinsic and extrinsic noise revealed by microscopy in a strain that contains both CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) expression reporters driven by the pSTL1 promoter stressed with 0.1 M (red) or no (black) NaCl (C), or in cdc28-as cells inhibited by 1-NM-PP1 with expression reporters driven by the pFIG1 promoter treated with 0.03 μM (red) or no (black) α-factor (D). (E) Percentage of intrinsic (gray) and extrinsic (white) noise over total noise quantified for osmotic stress or α-factor treatment in cells bearing two pSTL1 or pFIG1 expression reporters, respectively.

 

Figure 2
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Fig. 2. Comparison between Hog1 nuclear relocation and pSTL1-qV reporter expression in single cells. (A) Wild-type cells expressing the nuclear marker Hta2-CFP, Hog1-mCherry, and the pSTL1-qV expression reporter imaged before or after addition of 0.4 M NaCl. (B and C) Quantification of single-cell traces; the integral below the nuclear accumulation curve is used as a measure of the signaling output (B). The difference between final and initial average intensity allows quantification of the expression output (C). (D) The signaling and expression outputs were quantified in single cells exposed to 0.1 M (red) or no (black) NaCl. (E and F) Traces of three single cells marked in (D) displaying almost identical Hog1 relocation dynamics (E) but different expression outcomes (F).

 

Figure 3
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Fig. 3. Influence of chromatin remodeling on reporter expression. (A) Dynamics of histone H3 eviction at the STL1, ALD3, and HSP12 promoters was measured by quantitative ChIP experiments after addition of the indicated NaCl concentration. Histone H3 binding was normalized to a TEL2 sequence control. The error bars correspond to the standard deviation of three independent measurements. (B) Mean of the log-normal fit of flow cytometry histograms of pSTL1-qVenus expression quantified in wild-type (wt), arp8{triangleup}, and gcn5{triangleup} cells after addition of NaCl. (C) Histone H3 occupancy in wt and gcn5{triangleup} cells was quantified as in (A) 5 min after osmotic stress.

 

Figure 4
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Fig. 4. Duration and intensity of Hog1 nuclear accumulation controls bimodality. (A to C) Flow cytometric measurement of pSTL1-qV expression and mean of log-normal fits of the histograms (C) upon transient or sustained activation with a pulse of 0.2 M NaCl (A) or a ramp from 0.05 to 0.4 M NaCl (B) Cycloheximide (CHX) is added after 45 min to block protein synthesis. (D) Modulation of the Hog1 activation pattern in flow chambers studied by microscopy. Mean Hog1 nuclear accumulation upon transient (0.4 M NaCl for 3 min, green) and sustained (from 0.075 to 0.4 M NaCl in 20 min, red) activation of the pathway. Stepwise activation (dashed lines) for 0.075 M (red), 0.1 M (blue), 0.2 M (cyan), and 0.4 M NaCl (green) are shown for comparison. (E) Average signaling (red) and expression outputs (blue) for different activation patterns. The open and closed circles represent, respectively, the population of nonreacting and reacting cells. Circle size is proportional to the population under each distribution. The error bars show the standard deviation of 40 to 130 single cells.

 


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