Hsp90 Regulates Nongenetic Variation in Response to Environmental Stress  Yu-Ying Hsieh, Po-Hsiang Hung, Jun-Yi Leu  Molecular Cell  Volume 50, Issue 1, Pages 82-92 (April 2013) DOI: 10.1016/j.molcel.2013.01.026 Copyright © 2013 Elsevier Inc. Terms and Conditions

Molecular Cell 2013 50, 82-92DOI: (10.1016/j.molcel.2013.01.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 1 Low Hsp90 Results in Morphological Heterogeneity in a Clonal Yeast Population (A) A subpopulation of low-Hsp90 cells shows elongated buds or multibuds. Log-phase cells were treated with or without 5 μg/ml doxycycline for 15 hr, and then bud morphology was scored (see the Experimental Procedures). Cells with multiple buds were probably due to failures in cytokinesis. (B) A population of low-Hsp90 cells with different bud morphologies. A normal bud is indicated by an arrowhead, and an elongated bud is indicated by an arrow. (C) The morphological change is not correlated with the protein level of Hsp90 (two-tailed t test, p = 0.509, n = 300). Yeast cells in which the Tet-regulated Hsp90 was tagged with GFP were treated with doxycycline and then examined under a fluorescence microscope. The mean intensity of Hsp90-GFP in individual cells was calculated as a ratio of the total background-subtracted fluorescence intensity of a single cell divided by the cell size. (D) Inhibition of Hsp90 causes morphological heterogeneity in diverse yeast species. The phylogenetic relationship among S. cerevisiae, S. bayanus, S. exiguus, and Z. rouxii is shown in the upper panel with arbitrary branch lengths. Cells were treated with optimal concentrations of an Hsp90-specific inhibitor geldanamycin for 15 hr and then bud morphology was scored. In all morphological scores, at least 300 cells were counted for each sample and the data represent the mean ± SEM of three biological replicates. See also Figure S1 and Movie S1. Molecular Cell 2013 50, 82-92DOI: (10.1016/j.molcel.2013.01.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 2 Altered Septin Structures Induce Elongated Bud Morphology in Low-Hsp90 Cells (A) Deletion of SWE1 drastically reduces the elongated bud morphology. The top panels show representative images of each genotype with the addition of doxycycline. The bottom panel shows the quantitative data. The scale bar represents 7 μm. (B and C) Low Hsp90 induces altered septin structures in a subpopulation. hsp82Δ tet-HSC82 cells carrying a GFP-tagged Cdc10 were treated with doxycycline to reduce Hsp90 and then the septin structure was examined. Cells with normal or low levels of Hsp90 are shown in (B), and the quantitative data are shown in (C). The septin structures were classified as mislocalized if the majority of Cdc10 localized to the bud tip instead of the bud neck. They were classified as misorganized if Cdc10 forms irregular shapes at or near the bud neck (Gladfelter et al., 2005). Typical images of mislocalized and misorganized septin are shown. (D) Deletion of SWE1 suppresses the morphological change but not the altered septin structure. Only the round cells were examined and the septin structure was regarded as abnormal if Cdc10-GFP was mislocalized or misorganized. At least 100 cells were counted for each sample and the data represent the mean ± SEM of three biological replicates. Molecular Cell 2013 50, 82-92DOI: (10.1016/j.molcel.2013.01.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 3 The Protein Stability of Cla4 Is Reduced in Low-Hsp90 Cells (A) The protein level of Cla4 decreases significantly in the low-Hsp90 population. Total cell protein was extracted, and the western blot was hybridized with antibodies against HA or glucose-6-phosphate dehydrogenase (G6PDH). G6PDH was used as an internal control. (B) Transcription of CLA4 is not affected when Hsp90 is reduced (paired t test, wild-type, p = 0.371; tet-HSC82, p = 0.343). Total RNA was extracted from cells with normal or low levels of Hsp90, reverse transcribed, and subjected to quantitative PCR with CLA4-specific and ACT1-specific primers. The CLA4 mRNA levels were normalized to the ACT1 mRNA levels. (C) The protein half-life of Cla4 is reduced when Hsp90 activity is inhibited. tet-CLA4-HA cells were treated with or without geldanamycin for 3 hr. Doxycycline was then added into these cultures (0 hr) to shut off the expression of CLA4. Cells were collected at different time points. Total cell protein was extracted and examined by western blot. Protein half-life was calculated with the change of relative protein intensity after the doxycycline treatment (see the Experimental Procedures). (D) Coimmunoprecipitation (IP) of Cla4 and Hsp90. Cells carrying a plasmid with or without galactose-inducible Cla4 fused with a TAP-HA tag were grown in galactose, and Cla4-TAP-HA was immunoprecipitated with IgG sepharose beads. Cla4-TAP-HA and Hsp90 were detected with the anti-HA and anti-Hsp90 antibodies, respectively. A higher-molecular-weight band appearing in both tagged and untagged inputs is due to nonspecific hybridization of the anti-HA antibody. The quantitative data shown in (B) and (C) represent the mean ± SEM of three biological replicates. See also Figure S2. Molecular Cell 2013 50, 82-92DOI: (10.1016/j.molcel.2013.01.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 4 Both Cdc28 and Cla4 Are Involved in Morphological Change in Low-Hsp90 Cells (A) Overexpression of Cla4 or Cdc28 alone diminishes the number of elongated cells, while overexpression of both Cla4 and Cdc28 completely abolishes morphological heterogeneity in the low-Hsp90 population. hsp82Δ tet-HSC82 cells carrying different plasmids were treated with doxycycline and cell morphology was scored. Data represent the mean ± SEM of three biological replicates. (B) The protein level of Cdc28 decreases significantly in the low-Hsp90 population. Total cell protein was extracted, and the western blot was hybridized with antibodies against HA or glucose-6-phosphate dehydrogenase (G6PDH). G6PDH was used as an internal control. (C) Reduced Cla4 levels are not rescued by overexpression of Cdc28 in low-Hsp90 cells. The Cla4 protein levels of wild-type or hsp82Δ tet-HSC82 cells carrying pRS425 or pRS425-CDC28 were determined by western blotting. See also Figure S3. Molecular Cell 2013 50, 82-92DOI: (10.1016/j.molcel.2013.01.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 5 The Protein Level of Cla4 Is Critical for Hsp90-Dependent Morphological Heterogeneity (A) The Cla4 intensity in low-Hsp90 cells has a lower mean value, but its variance is similar to that of normal-Hsp90 cells. Log-phase cells carrying GFP-tagged Cla4 were treated with doxycycline, and the maximum Cla4-GFP intensity in the small bud was measured (see the Experimental Procedures). The Cla4-GFP intensities from individual cells were used to generate the histograms. At least 300 cells with small buds were analyzed for each sample. (B) Box plot of the Cla4-GFP intensity showing that elongated cells have a significantly lower level of Cla4 than normal rounded cells in the low Hsp90 population (Mann-Whitney test, p < 0.01, n = 120). (C) Deletion of CLA4 in cells with diminished Hsp90 expression results in elongated cell morphology in almost all cells examined but only has a partial effect if the Hsp90 level is normal. (D and E) Reduced levels of Cla4 and Cdc28 are sufficient to induce morphological heterogeneity even in a normal-Hsp90 population. (D) shows that the Cla4 protein can be adjusted to the level observed in low-Hsp90 cells with a Tet-regulated promoter. In the cdc28-4 (a hypomorphic allele of cdc28) background, reduced Cla4 levels induce elongated cell morphology at a permissive temperature. Data shown in (C) and (E) represent the mean ± SEM of three biological replicates. See also Figure S4. Molecular Cell 2013 50, 82-92DOI: (10.1016/j.molcel.2013.01.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 6 High Temperatures Induce Hsp90-Dependent Morphological Heterogeneity and Invasive Growth in the Σ1278b Strain (A) Cells growing at high temperatures induce morphological heterogeneity and the heterogeneity can be suppressed by overexpression of Hsp90. Cells carrying a multicopy plasmid with or without the HSC82 gene were grown at normal or high temperatures for 12 hr before cell morphology was scored. (B) High temperatures induce invasive growth that can be suppressed by overexpression of Hsp90. Cells with or without pRS426-HSC82 were spotted and grown on the YPD + 4% agar plate at normal or high temperatures for 2 days. After cells on the surface of plates were washed, many cells growing at high temperatures were embedded into the agar, indicating that cells had undergone invasive growth. (C) A 2-fold increase in Hsp90 is sufficient to suppress the effect caused by high temperatures. Cells carrying pRS426 or pRS426-HSC82 were grown at normal or high temperatures for 12 hr, and the protein levels were examined by western blotting. (D) The protein levels of Cla4 and Cdc28 are reduced at high temperatures. Cells carrying HA-tagged Cla4 or Cdc28 were grown at normal or high temperatures for 12 hr. Total cell protein was extracted and examined by western blotting. G6PDH was used as an internal control. The quantitative data shown in (A), (C), and (D) represent the mean ± SEM of three biological replicates. See also Figure S5. Molecular Cell 2013 50, 82-92DOI: (10.1016/j.molcel.2013.01.026) Copyright © 2013 Elsevier Inc. Terms and Conditions

Figure 7 A Model Showing how Hsp90 Modulates Morphological Heterogeneity in Response to Environmental Stress Under normal conditions, cells maintain a constant and above-threshold level of Cla4 and Cdc28 directly or indirectly by Hsp90. Thus, most cells produce homogeneous round buds. When the population encounters environmental stress, most Hsp90 proteins are recruited to contend with misfolded proteins caused by stress. Cla4 and Cdc28 become less stable and the protein abundance drops toward the threshold level, making the pathway more sensitive to fluctuations in the number of Cla4 and Cdc28 molecules. Individual cells with various regulator protein levels result in morphological heterogeneity in a clonal population. See also Figure S6. Molecular Cell 2013 50, 82-92DOI: (10.1016/j.molcel.2013.01.026) Copyright © 2013 Elsevier Inc. Terms and Conditions