Hsp90 Regulates Nongenetic Variation in Response to Environmental Stress

Molecular Cell

Article Hsp90 Regulates Nongenetic Variation in Response to Environmental Stress Yu-Ying Hsieh,1 Po-Hsiang Hung,1,2 and Jun-Yi Leu1,2,* 1Institute

of Molecular Biology, Academia Sinica, Taipei 115, Taiwan and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei 106, Taiwan *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.01.026 2Genome

SUMMARY

Nongenetic cell-to-cell variability often plays an important role for the survival of a clonal population in the face of fluctuating environments. However, the underlying mechanisms regulating such nongenetic heterogeneity remain elusive in most organisms. We report here that a clonal yeast population exhibits morphological heterogeneity when the level of Hsp90, a molecular chaperone, is reduced. The morphological heterogeneity is driven by the dosage of Cdc28 and Cla4, a key regulator of septin formation. Low Hsp90 levels reduce Cla4 protein stability and cause a subpopulation of cells to switch to a filamentous form that has been previously suggested to be beneficial under certain hostile environments. Moreover, Hsp90-dependent morphological heterogeneity can be induced by environmental stress and is conserved across diverse yeast species. Our results suggest that Hsp90 provides an evolutionarily conserved mechanism that links environmental stress to the induction of morphological diversity.

INTRODUCTION Since Delbruck’s observation of cell-to-cell differences in phage T1 production in the 1940s (Delbruck, 1945), scientists have gradually realized the importance of nongenetic variation in different aspects of life (reviewed by Eldar and Elowitz, 2010). At the population level, nongenetic variation has also been demonstrated to facilitate microbial survival in fluctuating laboratory environments (Bishop et al., 2007; Blake et al., 2006; Fraser and Kaern, 2009). Bacterial persistence against antibiotics and the constant presence of acid-resistant subpopulations in the spoilage yeast cells further support that phenotypic heterogeneity in a clonal population may be important for coping with environmental fluctuations that microbes often encounter (Balaban et al., 2004; Steels et al., 2000). Several different molecular mechanisms in which the participating molecules are limited in number have been shown to cause nongenetic variation, including transcription, translation, alternative splicing, and protein partitioning (Huh and Paulsson, 2011; Ozbudak et al., 2002; Waks et al., 2011). However, whether other cellular 82 Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc.

processes, such as protein folding, also modulate nongenetic variation remains unclear. Protein folding is a specific step of posttranslational processing susceptible to many environmental factors, like temperature fluctuations or the molecular crowding effect (reviewed by Hartl and Hayer-Hartl, 2009). In both prokaryotic and eukaryotic cells, molecular chaperones orchestrate a hierarchical network to decrease errors during protein folding, ensure maturation of functional proteins, and further maintain protein homeostasis (Gong et al., 2009). These functions probably allow molecular chaperones to reduce fluctuations in the number of functional protein molecules below the level expected from the stochastic variation in transcript and translation, helping cells control stochastic fluctuations in gene synthesis. Among these molecular chaperones, Hsp90 is of particular interest because its client proteins are broadly involved in signal transduction and gene regulation (reviewed by Taipale et al., 2010). Large-scale studies in yeast indicate that Hsp90 occupies a central position in both physical and genetic interaction networks (McClellan et al., 2007; Zhao et al., 2005). In addition, it has been shown that the activity or stability of some Hsp90 client proteins could become Hsp90 independent after the introduction of mutations (Citri et al., 2006; Xu et al., 2005). These results suggest that the dependency on Hsp90 may not be completely due to physical constraints of the protein structure and raise the possibility that the link between Hsp90 and some of its clients may be selected and maintained for specific regulation. Other than its critical role in cell physiology, Hsp90 has been proposed to function as an evolutionary ‘‘capacitor.’’ The model hypothesizes that Hsp90 helps a population accumulate cryptic genetic variation under normal conditions and further increases the population’s phenotypic variation by enhancing the effect of many mutations once Hsp90 is compromised under stress (Sangster et al., 2004). So far, Hsp90-mediated genetic capacitance has been observed in different organisms including yeast, flies, and plants, suggesting that it may be a common mechanism allowing organisms to reveal genetic variation in response to environmental cues (reviewed by Jarosz et al., 2010). However, other than a few cases in yeast, the genetic bases of this phenomenon remain enigmatic (Jarosz and Lindquist, 2010). It is still unclear what fraction of the mutations in Hsp90’s client proteins can be buffered by Hsp90 and how much of those cryptic variation will be adaptive under stress conditions. Whether such an evolutionary capacitor has a general effect on the evolutionary trajectory of organisms remains untested.

Molecular Cell Hsp90 Regulates Nongenetic Variation in Yeast

Cell morphology is a crucial survival factor shaped by selective pressures such as growth competition, nutrient acquisition and predation. Microorganisms often change their morphology in response to different environmental conditions (reviewed by Young, 2007). In Saccharomyces cerevisiae, cells switch to filamentous growth when certain nutrients become limited. Filamentous growth in budding yeast is different from other fungal hyphae in which neighboring cells often have continuous cytoplasm. It is characterized by unipolar cell division and chains of elongated cells that remain physically connected after cytokinesis (Palecek et al., 2002). This morphological program is shared between haploid and diploid cells. However, haploid cells often penetrate into agar after a long time of growth even in rich medium, so it is also called haploid invasive growth (Roberts and Fink, 1994). Filamentous growth has been speculated to be adaptive for nonmotile yeast cells since it allows a colony to explore the environment at reduced biomass cost under stress (Kron, 1997; Palecek et al., 2002). In pathogenic isolates of S. cerevisiae, filamentous growth was also observed to be associated with virulence or host infection (Gognies et al., 2006; McCusker et al., 1994). Moreover, a dimorphic switch has also been observed in several pathogenic fungi during the infection process (Sa´nchez-Martı´nez and Pe´rez-Martı´n, 2001). The dimorphic switch is probably an ancient survival strategy widely used in the fungal kingdom (Lengeler et al., 2000). Hsp90 has been suggested to reduce stochastic variation in plant development (Samakovli et al., 2007; Sangster et al., 2007). However, it is unclear about the underlying mechanism and whether the regulation is a general phenomenon in other organisms. We tested whether Hsp90 can regulate nongenetic variation in the budding yeast Saccharomyces cerevisiae. Our results show that when Hsp90 is reduced, about 40%–60% of cells switch to the filamentous form by altering their septin organization. The observed morphological heterogeneity is mediated through reduced levels of a cyclin-dependent kinase Cdc28 and Cla4, whose stability is regulated by Hsp90. We found that the Hsp90-mediated morphological switch exists in diverse yeast species, including S. bayanus (a distant relative of S. cerevisiae in the S. sensu stricto complex), S. exiguus (a S. sensu lato species), and Z. rouxii (a pre-whole-genome duplication species) (Dujon, 2006). Finally, we showed that this Hsp90-mediated morphological switch allows a clonal population to have different distributions of phenotypes under different environmental conditions. RESULTS Reduction of Hsp90 Increases Morphological Heterogeneity in a Clonal Yeast Population The yeast genome contains two Hsp90-encoding genes, HSC82 and HSP82. To test how a reduced level of Hsp90 affects yeast cells, we generated a strain in which HSP82 was deleted and HSC82 was under the control of a Tet-regulated promoter repressed by doxycycline (dox) (see the Supplemental Experimental Procedures and Figure S1A available online) (Davierwala et al., 2005). Interestingly, individual cells of a clonal population revealed heterogeneous bud morphologies when Hsp90 expression was reduced. About 60% of low-Hsp90 cells (hsp82D tetHSC82 + dox) showed elongated buds, whereas almost 100%

of cells had round buds in the normal-Hsp90 population with or without doxycycline treatments (Figures 1A and 1B and Movie S1). The image analysis software Calmorph was employed to characterize morphological heterogeneity in detail (Ohya et al., 2005). This program analyzed a variety of quantifiable parameters according to cell size, bud axis, and the angle of bud (see the Experimental Procedures). As expected, the low-Hsp90 population exhibited higher coefficients of variance in those parameters related to bud formation such as axis ratios in mother and daughter cells, bud direction, and bud neck position (Figure S1B). To test whether morphological heterogeneity was due to fluctuations in HSC82 gene expression regulated by the Tet promoter, in the same hsp82D tet-HSC82 construct we tagged Hsc82 with GFP and compared the levels of Hsc82 between individual normal-budded cells and elongated-budded cells in the low-Hsp90 population. No significant difference in the Hsc82 level was observed between these two types of cells (Figure 1C). Moreover, when wild-type cells were treated with Hsp90specific inhibitors, macbecin II or geldanamycin (GdA), about 30% of the cells exhibited elongated buds (Figure S1C). Thus, reduction of Hsp90 activity is sufficient to trigger morphological heterogeneity. Some cell-cycle mutants generate elongated cell morphology, which represents a terminal phenotype, meaning that cells are completely arrested or dead (Hartwell et al., 1973). We performed a time-lapse experiment and observed that elongated cells could produce progeny with elongated buds for several generations, suggesting that elongated cells did not result from complete cell-cycle arrest (Figure S1D and Movie S1). We also tested whether morphological change in low-Hsp90 cells was caused by genetic mutations (Chen et al., 2012; Specchia et al., 2010). We selected individual elongated cells under doxycycline pressure and propagated them in normal medium without doxycycline for a few generations, allowing cells to reproduce in the round shape again. These cells were then treated with doxycycline to shut down Hsp90. If bud elongation in low-Hsp90 cells was caused by genetic mutations, we would expect to see that the progeny of an elongated cell should all become elongated when Hsp90 was reduced. However, we observed a distribution of morphological heterogeneity similar to that in wild-type cells (Figure S1E). Hsp90-Dependent Morphological Heterogeneity Is Conserved among Diverse Yeast Species Laboratory yeast strains have been reported to accumulate many mutations due to relaxed selection (Gu et al., 2005). To rule out the possibility that the observed Hsp90-dependent morphological heterogeneity is a phenotype specific to our laboratory strains (BY4741 and S1278b), we used geldanamycin to test the effect of reduced Hsp90 activity on a wild isolate, UWOPS05-217.3, which is genetically highly divergent from the BY and sigma strains (Liti et al., 2009). We observed similar morphological heterogeneity in the wild isolate (Figure S1F). To understand whether Hsp90-dependent morphological heterogeneity also occurs in other yeasts, we examined three other species, Saccharomyces bayanus, Saccharomyces exiguus, and Zygosaccharomyces rouxii. These species are estimated to diverge from the common ancestors to S. cerevisiae Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc. 83

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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 mg/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.

between 20 and 100 million years ago (Dujon, 2006). Z. rouxii is a species that separated from the lineage of S. cerevisiae before whole-genome duplication. The average sequence identity between orthologous proteins of Z. rouxii and S. cerevisiae is only about 50% (Souciet et al., 2009). Nonetheless, when cells were treated with geldanamycin, they all showed heterogeneous bud morphology (Figure 1D). These results suggest that the influence of Hsp90 on bud morphology is conserved across a wide range of the yeast species. Morphological Heterogeneity Is Caused by an Altered Septin Formation Pathway Yeast cell morphogenesis can be roughly divided into two stages during the cell cycle. The daughter bud grows apically during the 84 Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc.

G1/S phase and then switches to isotropic growth in G2/M (Lew, 2003). A protein kinase, Swe1, prevents the switch by inhibiting Cdc28/Clb2 activity, which is required for isotropic growth (Barral et al., 1999). We speculated that the elongated bud morphology observed in low-Hsp90 cells might result from a prolonged apical growth phase. To test this idea, we deleted SWE1 in low-Hsp90 cells and then examined their morphology. Indeed, deletion of SWE1 drastically reduced the proportion of cells with elongated bud morphology, suggesting a mechanism by which reduction of Hsp90 activity could cause a prolonged apical growth phase in a subpopulation of the cells (Figure 2A). The septin ring is a hetero-oligomeric complex formed at the mother bud neck. The organization of the septin complex is crucial for the morphology of daughter buds (Gladfelter et al., 2001).

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Previous studies have shown that perturbing the septin pathway results in Swe1-dependent bud elongation (Lew, 2003). We examined the septin structure by tagging the endogenous copy of a septin component, Cdc10, with GFP. In the low-Hsp90 population, more than 70% of cells (which were mostly elongated cells) exhibited mislocalized or misorganized septins (see the Experimental Procedures; Figures 2B and 2C). To investigate whether the altered septin structure is the cause or effect of elongated buds, we examined the swe1 mutant cells that displayed round bud morphology in the majority of the population when Hsp90 was reduced. We observed abnormal septin structures in a large proportion of the rounded cells (Figure 2D). These results suggest that when Hsp90 is reduced, septin formation is altered in a subpopulation of cells, resulting in the activation of Swe1 which in turn inhibits Cdc28/Clb2 kinase activity and causes elongated bud morphology. Cla4 Protein Stability Is Reduced in Low-Hsp90 Cells In yeast, bud emergence is initiated by an essential Rho GTPase, Cdc42 (Ziman et al., 1991). Cdc42 and its downstream effectors recruit actin, septin, and components of membrane trafficking to establish polarized bud growth. In a later step, Cdc42 activates Cla4, a kinase in the p21-activated kinase family, to promote the complete structure of the septin ring (Versele and Thorner, 2004). Previous large-scale studies have shown that Hsp90 genetically or physically interacts with several components involved in this pathway, including Cdc42, Cla4, Cdc10, and Cdc12 (McClellan et al., 2007; Zhao et al., 2005). To understand the effect of Hsp90 on the septin pathway, we directly examined

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 mm. (B and C) Low Hsp90 induces altered septin structures in a subpopulation. hsp82D 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.

the steady-state protein levels of these potential Hsp90 clients and two other septin components, Cdc3 and Cdc11, in low-Hsp90 cells. Only Cla4 was significantly influenced by the changed Hsp90 level (Figures 3A and S2). However, the level of CLA4 messenger RNA (mRNA) was not decreased, suggesting that the effect of reduced Hsp90 is not at the transcriptional level (Figure 3B). Since Hsp90 is a molecular chaperone that acts in the later stage of protein folding, it is possible that the protein stability of Cla4 is influenced by Hsp90. We used a strain containing a hemagglutinin (HA)-tagged Cla4 protein under the control of a Tet-Off promoter to address this question. Expression of the HA-tagged Cla4 was shut off by adding doxycycline in the presence or absence of the Hsp90-inhibitor geldanamycin. Cells were collected at different time points after the addition of doxycycline, and the protein level of Cla4-HA was examined by western blot. As shown in Figure 3C, the half-life of the Cla4 protein fell from 3.05 ± 0.16 hr to 1.95 ± 0.27 hr (two-tailed t test, p = 0.02) when the activity of Hsp90 was inhibited, suggesting that the protein stability of Cla4 is influenced by Hsp90. If we corrected the protein half-life with the difference in the doubling time of cells (1.89 ± 0.04 hr with geldanamycin versus 1.67 ± 0.08 hr without geldanamycin), the stability of Cla4 was estimated to decrease to about 48% of the wild-type level. However, the steady-state level of Cla4 in low-Hsp90 cells is about 20%– 30% of that in wild-type cells. It is possible that other factors also contribute to this reduction. The CLA4 gene has been shown to genetically interact with HSC82 in a genome-wide screen (McClellan et al., 2007). To Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc. 85

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(A) The protein level of Cla4 decreases significantly in the low-Hsp90 population. Total cell protein was 0.8 extracted, and the western blot was hybridized Cla4-HA with antibodies against HA or glucose-6-phosphate dehydrogenase (G6PDH). G6PDH was used 0.4 G6PDH as an internal control. (B) Transcription of CLA4 is not affected when 0 Hsp90 is reduced (paired t test, wild-type, p = WT hsp82Δ tet-HSC82 0.371; tet-HSC82, p = 0.343). Total RNA was exC 0h 2h 4h 6h tracted from cells with normal or low levels of Hsp90, reverse transcribed, and subjected to GdA _ + _ + _ + _ + quantitative PCR with CLA4-specific and ACT1D Cla4-HA specific primers. The CLA4 mRNA levels were Input IP normalized to the ACT1 mRNA levels. CLA4UnCLA4Un(C) The protein half-life of Cla4 is reduced when G6PDH TAP-HA tagged TAP-HA tagged Hsp90 activity is inhibited. tet-CLA4-HA cells were treated with or without geldanamycin for 3 hr. Time (hour) Doxycycline was then added into these cultures Cla40 2 4 6 8 (0 hr) to shut off the expression of CLA4. Cells were 0 HA collected at different time points. Total cell protein -GdA -0.2 was extracted and examined by western blot. +GdA Protein half-life was calculated with the change of Hsp90 -0.4 relative protein intensity after the doxycycline treatment (see the Experimental Procedures). -0.6 (D) Coimmunoprecipitation (IP) of Cla4 and Hsp90. Cells carrying a plasmid with or without galactose-0.8 inducible Cla4 fused with a TAP-HA tag were grown in galactose, and Cla4-TAP-HA was im-1.0 munoprecipitated 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.

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determine whether Cla4 physically interacts with Hsp90, we performed a coimmunoprecipitation assay using a strain carrying a plasmid in which the Cla4 protein is fused with a tandem affinity purification (TAP) tag that includes an HA tag and its expression is under the control of the GAL1 promoter. Cla4 was first induced by growing cells in galactose-containing medium and then was immunoprecipitated with IgG sepharose beads after cells were lysed. Our result showed that Hsp90 was copurified with the tagged Cla4 protein, suggesting a physical interaction between these two proteins (Figure 3D). Cdc28 Is Involved in Morphological Change in Low-Hsp90 Cells If Cla4 is the only component affected by Hsp90 and influencing morphological changes in low-Hsp90 cells, we expect that overexpressing CLA4 should completely eliminate morphological heterogeneity. However, we only observed a partial rescue when single-copy or multiple-copy plasmids carrying CLA4 were introduced into low-Hsp90 cells to overexpress Cla4 (Figure 4A and data not shown). It suggests that Cla4 may not be the only factor that is affected by Hsp90 and contributes to bud elongation. Cdc28 mutant alleles have been shown to influence cell morphology in natural isolates of S. cerevisiae (Lee et al., 2011). A recent study in the pathogenic fungus Candida albicans indicates that Cdc28 is an Hsp90 client protein and reducing Hsp90 activity destabilizes Cdc28 and promotes fila86 Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc.

mentous growth in C. albicans (Senn et al., 2012). It raises the possibility that Cdc28 is also affected by Hsp90 in S. cerevisiae. When the protein level of Cdc28 was examined, it showed that Cdc28 was reduced in low-Hsp90 cells (Figure 4B). If, in lowHsp90 cells, the reduced Cla4 and Cdc28 levels act together to induce morphological heterogeneity, we expect that overexpressing both CLA4 and CDC28 in low-Hsp90 cells should completely eliminate morphological heterogeneity. Indeed, most low-Hsp90 cells had round buds when both CLA4 and CDC28 were mildly overexpressed (Figures 4A and S3). To investigate whether Cdc28 works directly upstream of Cla4, we measured the Cla4 protein level in the low-Hsp90 cells overexpressing Cdc28. We observed a similar Cla4 level in the lowHsp90 cells with or without Cdc28 overexpression, indicating that the effect of Cdc28 is independent of Cla4 (Figure 4C). The Protein Level of Cla4 Is Critical for Hsp90Dependent Morphological Heterogeneity Cla4 has been shown to phosphorylate some septin components and promote septin organization (Versele and Thorner, 2004). This raised the possibility that the dosage of Cla4 protein might be a critical factor controlling Hsp90-dependent morphological heterogeneity. We examined the maximum level of Cla4 in individual daughter cells using a GFP-tagged CLA4 construct (Figure S2B). Single-cell analysis revealed an interesting pattern. First, the distribution of Cla4 intensity in normal- and low-Hsp90

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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. hsp82D 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 lowHsp90 cells. The Cla4 protein levels of wild-type or hsp82D tet-HSC82 cells carrying pRS425 or pRS425-CDC28 were determined by western blotting. See also Figure S3.

populations exhibited a significant overlap. Second, although Cla4 intensity in the low-Hsp90 cells had a lower mean value, its variance is similar to that of the normal-Hsp90 population (2905 ± 3120 pixels versus 6620 ± 3230 pixels; Figure 5A). To test whether Cla4 levels were correlated with subsequent development of cell shape in the low-Hsp90 population, we performed a time-lapse experiment using the same GFP-tagged CLA4 strain. The Cla4 intensity was first recorded when small buds emerged, and then cell morphology was measured after these small buds grew to the normal size. The result showed that elongated cells had a lower Cla4-GFP intensity than normal rounded cells (Mann-Whitney test, p < 0.01, n = 120; Figures 5B and S4). It is likely that in a low-Hsp90 population the Cla4 level is reduced to a value closer to a threshold, below which cells will form elongated buds and above which cells form normal buds. The combination of these two types of cells resulted in the observed morphological heterogeneity in a clonal population. When we deleted CLA4 and examined bud morphology in low-Hsp90 cells, almost all cells showed elongated buds, supporting the idea that Cla4 is important for maintaining round

bud morphology (Figure 5C). On the other hand, in normalHsp90 cells, cla4 deletion only had a partial effect. This result, together with the CLA4/CDC28 overexpression data, suggests that the functions of Cla4 and Cdc28 in regulating morphological heterogeneity are overlapping. To further test the threshold hypothesis, we constructed a Tet-regulated CLA4 gene in a strain carrying a hypomorphic allele of cdc28 (cdc28-4) (Reed and Wittenberg, 1990). By adding doxycycline to the medium, we were able to adjust CLA4 expression to the level observed in low-Hsp90 cells (Figure 5D). When this strain was grown at a permissive temperature, most cells grew as round buds. However, if the Cla4 level was reduced to a level similar to that in low-Hsp90 cells, about 40% of cells became elongated (Figure 5E). In the same background, almost all cells became elongated if CLA4 was deleted. Together, these results suggest that reduced levels of Cla4 and Cdc28 are sufficient to induce morphological heterogeneity even in a normal-Hsp90 population. Hsp90 Regulates Morphological Heterogeneity in Response to Environmental Stress In yeast, it has been suggested that filamentous growth enables cells to escape from locally stressful environments or to forage for nutrients under conditions of starvation (Kron, 1997). The elongated bud morphology observed in low-Hsp90 cells is reminiscent of the cells undergoing filamentous growth. We therefore tested whether a reduced level of Hsp90 could induce cells to invade agar plates, a standard assay for filamentous growth in haploid cells. When Hsp90 was reduced in the S1278b strain, cells readily invaded YPD agar plates within 2 days (Figure S5A). It has been suggested that when cells encounter environmental stress, the amount of Hsp90 available for its regular clients may become limited even though the total cytoplasmic level of Hsp90 increases or remains constant (the titration model) (Sangster et al., 2004). This fall occurs because most Hsp90 molecules are used to contend with the increased quantity of misfolded proteins caused by stress. In such a scenario, morphological heterogeneity may be strongly induced. We tested this hypothesis by growing wild-type S1278b cells at 37 C. As shown in Figures 6A and 6B, the population became morphologically heterogeneous and was able to quickly invade agar plates when growing at high temperatures. To verify that morphological heterogeneity was triggered by limited availability of Hsp90, we introduced a multicopy plasmid carrying Hsp90 into the strain that would double the amount of Hsp90 in cells growing at high temperatures (Figures 6C and S6A). Overexpression of Hsp90 reduced morphological heterogeneity and also prevented agar invasion (Figures 6A and 6B). Since the amounts of Cla4 and Cdc28 are crucial for Hsp90-dependent morphological heterogeneity, we examined the protein levels of Cla4 and Cdc28 in wild-type cells growing at high temperatures. The levels of both proteins decreased significantly at 37 C (Figure 6D). To rule out the possibility that the observed phenotypes are sigma strain specific, we repeated similar experiments in the natural yeast strain UWOPS05-217.3. Similar results were observed in the wild isolate (Figures S5B–S5D). A previous study has also reported that high temperatures could induce Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc. 87

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0

0

(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.

normal

development, immune response, cancer biology, and adaptation strategies of organisms (Eldar and Elowitz, 2010; Snijder and Pelkmans, 2011). The identification of quantitative trait loci involved in the noise level of gene expression implies that individual organisms in a population may exhibit different noise levels (of a certain phenotype) depending on their genetic background (Ansel et al., 2008). Previous studies in yeast also indicate that the distribution of whole-genome gene expression noise is uneven, meaning that essential and complex-forming genes exhibit lower noise while stress-related genes produce higher noise (Bar-Even et al., 2006; Fraser et al., 2004; Newman et al., 2006). Noise is an inevitable consequence of the stochastic nature of chemical reactions that involve small numbers of molecules. The observation that genetic variation can affect noise levels suggests that the level of noise in biochemical pathways and its effect on organismal phenotypes is under natural selection, but we still lack direct evidence showing that increased phenotypic variation is beneficial outside the laboratory. In laboratory populations of microorganisms, phenotypic variation caused by increased transcriptional fluctuations has been shown to facilitate population survival upon acute environmental stress (Fraser and Kaern, 2009). Nonetheless, our knowledge about the underlying molecular mechanisms is still limited in most cases. Many types of phenotypic variation in an isogenic

tet-CLA4-HA cdc28-4 tet-CLA4-HA cdc28-4 +Dox

filamentous growth in a diploid strain (Zaragoza and Gancedo, 2000). Together, these results suggest that environmental stress-induced morphological heterogeneity is quite common in yeast. Finally, we tested whether high temperatures could also induce morphological heterogeneity in other yeast species. About 50%–60% of cells became elongated when S. exiguus and Z. rouxii cells were grown at higher temperatures, indicating that stress-induced morphological heterogeneity occurs in diverse yeast species (Figure S6B). Our results suggest that an Hsp90-mediated pathway can respond to environmental stresses and generate population heterogeneity, which may contribute to the ability of a population to cope with a fluctuating environment. DISCUSSION Nongenetic variation can be exploited to increase phenotypic complexity. Although not genetically heritable, nongenetic phenotypic variation impacts diverse aspects of life, including virus infection, pluripotency of embryonic stem cells, organ 88 Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc.

Molecular Cell Hsp90 Regulates Nongenetic Variation in Yeast

A

+pRS426 37°C

B

Wild-type Hsp90

+pRS426-HSC82 37°C

100

Normal temperature

% of total cells

+pRS426 28°C

Hsp90 Overexpressed High temperature

High temperature

60

Before wash

20 0

elongated multibud normal

C

+pRS426

After wash

+pRS426HSC82

28°C 37°C 37°C Individual cells

Hsp90

Normalized Hsp90/G6PDH

D

28°C 37°C

+pRS426-HSC82 37°C 3

+pRS426 37°C

Cla4-HA

+pRS426 28°C 2 1

G6PDH Cdc28-HA G6PDH

Normalized protein level

G6PDH

28°C 37°C 1.0 0.6 0.2 0

0

population could be attributed to the physiological states of cells (Sumner et al., 2003). In recent years, it has been recognized that stochasticity in gene transcription or translation plays a crucial role in nongenetic variation (Kaern et al., 2005). Since stochasticity strongly influences cellular reactions involving small numbers of molecules, it is unlikely to be restricted to transcription and translation alone. Other steps in gene synthesis or signal transduction probably also suffer from this inevitable fluctuation (Eldar and Elowitz, 2010; Raser and O’Shea, 2005). Our current study provides evidence that the stability of a critical regulatory protein could provide another mechanism for regulated phenotypic variation. Cla4 and Cdc28 function as two partially overlapping pathways in regulating morphological heterogeneity and the abundance of both proteins is under the control of Hsp90. Because Hsp90 is a chaperone that aids protein folding (therefore the protein stability) and Hsp90 physically interacts with Cla4, it is tempting to speculate that Cla4 may be a client protein of Hsp90. Nonetheless it is still possible that Hsp90 indirectly affects the stability of Cla4 through other pathways. The environmentally responsive molecular chaperone Hsp90 has been viewed as a promising candidate to explain a phenomenon termed ‘‘canalization’’ by C.H. Waddington: phenotypic invariance in spite of genetic or environmental perturbations during organismal development (Cowen and Lindquist, 2005; Jarosz et al., 2010; Waddington, 1942). However, since most of the mutations buffered by Hsp90 are likely to be deleterious

Cla4-HA

Cdc28-HA

Figure 6. High Temperatures Induce Hsp90Dependent Morphological Heterogeneity and Invasive Growth in the S1278b 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 HAtagged 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.

for the population even in stress conditions, it remains controversial whether genetic buffering makes an important contribution to evolution. Here, we showed that the level of functional Hsp90 regulates nongenetic variation in a clonal yeast population (Figure 7). Filamentous growth can help yeast cells forage nutrients or escape from local harsh environments (Kron, 1997). On the other hand, the yeast form has a faster growth rate and can be dispersed easily (Kron et al., 1994). A morphologically heterogeneous population probably has a higher chance to survive under a fluctuating environment. However, it is also possible that the observed variation is simply the consequence of evolutionary constraints on cellular function, or is neutral due to lack of selection. It will be interesting to determine whether such heterogeneity is really adaptive under conditions similar to natural environments. Unlike the stochastic state switching observed in some previous studies (Eldar and Elowitz, 2010; Snijder and Pelkmans, 2011), Hsp90 adds one more layer of regulation in population heterogeneity. Diminishment of Hsp90 function can reduce the level of Hsp90-dependent regulators toward a threshold where stochastic variations in gene expression produce phenotypic variation in the population. It allows cells to minimize stochastic noise under normal conditions and increase variation when encountering environmental stresses. Compared with genetic variation that takes a long time to establish in a population, nongenetic variation provides a fast means to increase population diversity, which is especially critical when facing the stress Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc. 89

Molecular Cell Hsp90 Regulates Nongenetic Variation in Yeast

Normal Condition

Stress Condition

Hsp90

Hsp90

misfolded protein

Cla4 Cdc28

Cla4

Cdc28

threshold Cla4 and Cdc28 levels in a clonal population

Cell morphology

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.

conditions that threaten the survival of the population (Zhuravel et al., 2010). We have shown that the Hsp90-dependent morphological heterogeneity is conserved in a wide range of yeast species covering 100 million years of evolution. It raises the possibility that this may be a common mechanism allowing cells to cope with fluctuating environments. Given that Hsp90 interacts with many key proteins involved in different functional networks and the function of Hsp90 is conserved from yeast to mammals, it is possible that Hsp90 acts as a common regulator of nongenetic variations in various cellular processes. EXPERIMENTAL PROCEDURES Image Analysis Unless otherwise specified, images were analyzed by ImageJ (http://rsbweb. nih.gov/ij/). At least 100 cells from three independent experiments were analyzed. Cell Counter (http://rsbweb.nih.gov/ij/plugins/cell-counter.html) was used as a plug in for ImageJ to count and categorize cells. Cells were defined as elongated when the ratio of long- to short-axis diameter of the cell was larger than 1.3 since the ratios range from 0.8 to 1.2 in the majority of wild-type cells (0.96 ± 0.11). Yeast cell morphological parameters were calculated by Calmorph (http://scmd.gi.k.u-tokyo.ac.jp/datamine/calmorph/) following its recommended procedure. In the Calmorph program, the bud angle was defined as the angle between the long axis of the bud and the long axis of the mother cell, the axis ratio was defined as the ratio of long- to short-axis of cells, the bud direction was defined as the angle between the line from the midpoint of the bud neck to the tip of the daughter bud and the long axis of the mother cell, and the bud neck position was defined as the angle between the line from the midpoint of the bud neck to the center of the mother cell and the long axis of the mother cell. To classify the septin structures, we followed the standard used in a previous study by Gladfelter and colleagues (Gladfelter et al., 2005). In normal wild-type cells, septin appears as an hourglass shape at the mother bud neck region when the daughter bud emerges. ‘‘Mislocalization’’ indicates that a majority of septin was detected as a cap at the bud tip instead of the

90 Molecular Cell 50, 82–92, April 11, 2013 ª2013 Elsevier Inc.

mother bud neck. ‘‘Misorganization’’ refers to the septin structures localizing at or near the bud neck but with an aberrant appearance (usually irregular, asymmetric, or extended structures in contrast to the wild-type hourglass shape). For quantification of the Cla4-GFP intensity in individual cells, the intensity of Cla4-GFP around the edge of small daughter buds (defined as the ratio of daughter cell size to mother cell size is smaller than 0.2) was measured and only the maximum intensity was used for further analysis. The background intensity was always subtracted. For cell wall labeling, cells were washed with PBS and then labeled with NHS-Rhodamine (Pierce, Rockford, IL) for 8 min. Protein Stability Assay A promoter shut-off assay was performed to determine the protein half-life of Cla4 (Zhou, 2004). Log-phase cells carrying the tet-CLA4-HA construct were grown in fresh complete supplement mixture (CSM) medium with or without geldanamycin (50 mM) for 3 hr, and then doxycycline was added to the culture (5 mg/ml) to turn off the transcription of CLA4 (defined as the 0 hr time point). Cells were collected every 2 hr (t = 0, 2, 4, and 6 hr) and frozen at 80 C. After all samples were collected, total protein was extracted and the Cla4 protein level was examined by immunoblotting. The relative intensity of Cla4 was calculated as the ratio of Cla4/G6PDH at each time point. All the data points were fit to the function of ln(Pt / P0) = [ln(1/2) / T1/2]t, in which P represents the relative intensity of Cla4 and T1/2 represents the half-life. The degradation constant [k = ln(1/2) / T1/2] was estimated as the slope of this linear regression line on the log(Pt / P0)–t plot. Cell Invasion Assay About 5 3 104 cells were inoculated on the YPD + 4% agar plate for 2 days at the indicated temperature. Colonies on top of the agar were washed out by ddH2O, and those cells able to invade into agar appeared as a clear white mark after washing. SUPPLEMENTAL INFORMATION Supplemental Information includes six figures, one table, Supplemental Experimental Procedures, and one movie and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2013.01.026. ACKNOWLEDGMENTS We thank D. Jarosz, A. Murray, and M. Piel for comments on the manuscript. We also thank A. Pen˜a for manuscript editing and the Imaging Core of IMB for technique help. We thank S. Lindquist, W.-S. Lo, J. Thorner, and C. Wang for providing reagents. This work was supported by Academia Sinica of Taiwan (grant number 100-CDA-L04) and the National Science Council of Taiwan (grant number NSC100-2321-B-001-021). Received: July 10, 2012 Revised: November 28, 2012 Accepted: January 15, 2013 Published: February 21, 2013 REFERENCES Ansel, J., Bottin, H., Rodriguez-Beltran, C., Damon, C., Nagarajan, M., Fehrmann, S., Franc¸ois, J., and Yvert, G. (2008). Cell-to-cell stochastic variation in gene expression is a complex genetic trait. PLoS Genet. 4, e1000049. Balaban, N.Q., Merrin, J., Chait, R., Kowalik, L., and Leibler, S. (2004). Bacterial persistence as a phenotypic switch. Science 305, 1622–1625. Bar-Even, A., Paulsson, J., Maheshri, N., Carmi, M., O’Shea, E., Pilpel, Y., and Barkai, N. (2006). Noise in protein expression scales with natural protein abundance. Nat. Genet. 38, 636–643. Barral, Y., Parra, M., Bidlingmaier, S., and Snyder, M. (1999). Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast. Genes Dev. 13, 176–187.

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