Formation of Subnuclear Foci Is a Unique Spatial Behavior of Mating MAPKs during Hyperosmotic Stress  Simon E. Vidal, David Pincus, Jacob Stewart-Ornstein, Hana El-Samad  Cell Reports  Volume 3, Issue 2, Pages 328-334 (February 2013) DOI: 10.1016/j.celrep.2013.01.022 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 3, 328-334DOI: (10.1016/j.celrep.2013.01.022) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Ste12, Fus3, and Kss1 Colocalize in Foci under High-Osmolarity Stress (A) The MAPKs Fus3 and Kss1, but not Slt2 and Hog1, form punctuate and transient foci under high-osmolarity stress. Endogenous Fus3, Kss1, Hog1, and Slt2 were fused on the C termini to YFP and spatial localization was followed by confocal microscopy after addition of 0.5 M sorbitol. (B) Fus3 and Kss1 colocalize under high-osmolarity stress. Endogenous Fus3 and Kss1 were fused on the C termini to YFP and mKate, respectively. Foci formation was determined by confocal microscopy 15 min after the addition of 0.5 M sorbitol. (C) Fus3 and Ste12 colocalize under high-osmolarity stress. Endogenous Fus3 and Ste12 were fused on the C termini to mKate and YFP, respectively. Foci formation was determined by confocal microscopy 15 min after adding 0.5 M sorbitol. (D) Fus3 and Kss1 fail to form foci following osmotic stress in cells lacking Ste12. However, Ste12 continued to form foci following osmotic stress even in a fus3Δkss1Δ strain. Endogenous Ste12 was fused on the C termini to YFP. Fus3, Kss1, and Ste12 were deleted by homologous insertion. Foci formation was determined by confocal microscopy 15 min after addition of 0.5 M sorbitol. (E) Fus3 colocalizes with the nuclear pore under high-osmolarity stress. Endogenous Fus3 and nup49 (nuclear pore marker) were tagged with YFP and mKate, respectively. Foci formation was followed by confocal microscopy in cells treated with 0.5 M sorbitol. Images were taken 10–15 min after adding sorbitol. See also Figure S1. Cell Reports 2013 3, 328-334DOI: (10.1016/j.celrep.2013.01.022) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Fus3/Kss1/Ste12 Foci Formation Requires Hog1 Kinase Activity (A) Hog1 residence in the nucleus correlates with Fus3/Kss1 foci formation. Endogenous Fus3 and Hog1 were fused on the C termini to YFP and mKate, respectively. Distribution of Fus3 and Hog1 was followed by confocal microscopy at different time points after the addition of 0.5 M sorbitol. (B) Quantification of Hog1 nuclear residence and Fus3 foci formation. The percentage of cells with foci and nuclear Hog1 was calculated using ImageJ. (C) The kinase activity of Hog1 is a prerequisite for Fus3 and Kss1 foci formation under high-osmolarity stress. Following hyperosmotic stress, Fus3-YFP failed to form foci in cells harboring a hog1-as allele pretreated with the cell-permeable adenine analog 1-NM-PP1 (1 μM). (D) The percentage of cells with foci following hyperosmotic stress was calculated using ImageJ for cells with wild-type HOG1, hog1-as allele untreated with 1-NM-PP1 and cells with a hog1-as allele after 1-NM-PP1 treatment. Compromised Hog1 kinase activity generates dramatic reduction in foci formation. Error bars represent the mean ± SD of three independent experiments. See also Figure S2. Cell Reports 2013 3, 328-334DOI: (10.1016/j.celrep.2013.01.022) Copyright © 2013 The Authors Terms and Conditions

Figure 3 Preactivation of the Pheromone Pathway Inhibits the Formation of Fus3 Foci under High-Osmolarity Stress (A) Cells harboring Fus3-YFP were treated with either 0.5 M sorbitol, 0.5 M sorbitol, and 1 μM pheromone at the same time or 1 μM pheromone for 10 min followed by 0.5 M sorbitol. While the simultaneous stimulation by pheromone and sorbitol had little qualitative effect on foci formation, foci were absent when pheromone preceded sorbitol addition. Foci formation resumed under this scenario in a ste7Δ strain. (B and C) Preactivation of cells harboring a pheromone transcriptional reporter (PAGA1-YFP) exhibits full induction under different concentrations of sorbitol. The time-resolved expression of a pheromone transcriptional reporter (PAGA1-YFP) was recorded for cells treated at the same time (B) with 1 μM pheromone and different concentrations of sorbitol (0.1–0.8 M), or (C) with 1 μM pheromone for 10 min followed by different concentrations of sorbitol (0.1–0.8 M). YFP expression was measured by flow cytometry. While increasing amounts of sorbitol attenuate signaling in the mating pathway if cells experience sorbitol and salt simultaneously, this effect disappears if cells are pretreated with pheromone. Error bars represent the mean ± SD of three independent experiments. See also Figure S3. Cell Reports 2013 3, 328-334DOI: (10.1016/j.celrep.2013.01.022) Copyright © 2013 The Authors Terms and Conditions

Figure S1 Foci Formation Is Triggered Specifically under High-Osmolarity Stress, Related to Figure 1 (A) MAPKs do not form foci under pheromone treatment. Fluorescently-tagged MAPKs were treated with 1 uM pheromone for 1 hr and then imaged. Cells adopted an elongated morphology and Fus3 accumulated on the tip of the cells (arrow) as previously described (Slaughter et al., 2007). (B) Foci formation is a general response to high-osmolarity stress. Fus3-YFP cells were treated with NaCl (0.5 M) and glucose (0.8 M). Foci formation was followed by confocal microscopy after 10-15 min after the addition of the stressor. (C) Foci formation is specific to hyper-osmotic stress. Fus3-YFP cells were exposed to hypo-osmotic shock (50% H2O) (A), oxidative stress (0.5% H202), heat shock (40°C), cell wall stress (100μg/ml zymolyase), ER stress (5mM DTT), Tor inhibition (20mM caffeine) and images were taken every 10 min for 30 min. No foci were observed in all cases. (D) Fus3 appears in foci in the absence of sorbitol in dig1Δ cells. A saturated culture of Fus3-YFP cells was diluted in fresh media to an OD600 ∼ 0.05, grown to an OD600∼ 0.2 and images taken. (E) Dig1 and Dig2 are part of the sorbitol-induced foci in wild-type cells. Endogenous Dig1 and Dig2 were fused on the C-termini to GFP and their spatial localization was followed by confocal microscopy after addition of 0.5 M sorbitol. (F) Pheromone responsive promoters localize in the hyper-osmotic stress induced foci. ChIP-PCR experiments were done using Ste12-YFP as bait in either untreated, pheromone treated, or sorbitol treated cells. PCR for pheromone target gene promoters (AGA1 and FUS1), osmo-stress target gene promoters (HOR2 and GPD1) and metabolic gene promoters (PGK1 and TDH3) was done using whole cell extract and IP. A 100bp fragment is expected, however, a smaller band sometimes is visible, which corresponds to unincorporated primers. (G) Quantification of ChIP-PCR. Bands from the gel were assigned values in ImageJ, and using the standard curve from the dilution series of WCE, IP bands were quantified as a percent of the input DNA. Cell Reports 2013 3, 328-334DOI: (10.1016/j.celrep.2013.01.022) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Fus3 Foci Formation and Hog1 Nuclear Residency Correlate for a Range of Osmotic Stress Magnitudes, Related to Figure 2 (A) Fus3 and Ste12 form foci with similar kinetics. Distribution of Fus3-YFP and Ste12-mKate2 was followed by confocal microscopy at different time points after the addition of 0.5 M sorbitol. (B) Quantification of percentage of cells with Fus3 and Ste12 foci. Using ImageJ to quantify the Ste12 and Fus3 foci containing cells, we determined that cells that have Ste12 foci are also highly likely to have Fus3 foci. (C) Different osmotic stress magnitudes produce different Hog1 nuclear enrichment and Fus3 foci formation dynamics. Hog1 residence in the nucleus correlates with Fus3/Kss1 foci formation. Endogenous Fus3 and Hog1 were fused to YFP and mKate, respectively. Distribution of Fus3-YFP and Hog1-mKate2 was followed by confocal microscopy at different time points after the addition of 0.2M Sorbitol (A) and 1 M sorbitol (B). The percentage of cells with foci and nuclear Hog1 was calculated using ImageJ. Results represent the mean ± SD of three independent experiments. (D) Fus3, Kss1 and Hog1 become phosphorylated/dephosphorylated with similar kinetics under osmotic stress. Wild-type S. cerevisiae (W303a) cells were grown at 30°C to an OD of ∼ 0.2-0.3 and 0.5M Sorbitol was added to the culture. Protein extracted at different time points were subjected to Western blot analysis using anti-Phospho p38 MAPK and anti Phospho-p44/42 MAPK (Erk1/2) (Cell signaling). Cell Reports 2013 3, 328-334DOI: (10.1016/j.celrep.2013.01.022) Copyright © 2013 The Authors Terms and Conditions

Figure S3 Inhibition of Pheromone Outputs by Hyperosmotic Stress Signaling is Specific, Related to Figure 3 A constitutive PTDH3-mCherry reporter was built and mCherry expression levels were measured by flow cytometry using increasing concentrations of sorbitol administered either simultaneously with pheromone, or 10 min after pheromone stimulation. Neither regime elicited a response in this non-pheromone target gene. Error bars represent the mean ± SD of three independent experiments. Cell Reports 2013 3, 328-334DOI: (10.1016/j.celrep.2013.01.022) Copyright © 2013 The Authors Terms and Conditions