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• W2016231318 abstract "Enzymes scavenging reactive oxygen species (ROS) are important for cell protection during stress and aging. A deficiency in these enzymes leads to ROS imbalance, causing various disorders in many organisms, including yeast. In contrast to liquid cultures, where fitness of the yeast population depends on its ROS scavenging capability, the present study suggests that Saccharomyces cerevisiae cells growing in colonies capable of ammonia signaling use a broader protective strategy. Instead of maintaining high levels of antioxidant enzymes for ROS detoxification, colonies activate an alternative metabolism that prevents ROS production. Colonies of the strain deficient in cytosolic superoxide dismutase Sod1p thus developed the same way as wild type colonies. They produced comparable levels of ammonia and underwent similar developmental changes (expression of genes of alternative metabolism and center margin differentiation in ROS production, cell death occurrence, and activities of stress defense enzymes) and did not accumulate stress-resistant suppressants. An absence of cytosolic catalase Ctt1p, however, brought colonies developmental problems, which were even more prominent in the absence of mitochondrial Sod2p. sod2Δ and ctt1Δ colonies failed in ammonia production and sufficient activation of the alternative metabolism and were incapable of center margin differentiation, but they did not increase ROS levels. These new data indicate that colony disorders are not accompanied by ROS burst but could be a consequence of metabolic defects, which, however, could be elicited by imbalance in ROS produced in early developmental phases. Sod2p and homeostasis of ROS may participate in regulatory events leading to ammonia signaling. Enzymes scavenging reactive oxygen species (ROS) are important for cell protection during stress and aging. A deficiency in these enzymes leads to ROS imbalance, causing various disorders in many organisms, including yeast. In contrast to liquid cultures, where fitness of the yeast population depends on its ROS scavenging capability, the present study suggests that Saccharomyces cerevisiae cells growing in colonies capable of ammonia signaling use a broader protective strategy. Instead of maintaining high levels of antioxidant enzymes for ROS detoxification, colonies activate an alternative metabolism that prevents ROS production. Colonies of the strain deficient in cytosolic superoxide dismutase Sod1p thus developed the same way as wild type colonies. They produced comparable levels of ammonia and underwent similar developmental changes (expression of genes of alternative metabolism and center margin differentiation in ROS production, cell death occurrence, and activities of stress defense enzymes) and did not accumulate stress-resistant suppressants. An absence of cytosolic catalase Ctt1p, however, brought colonies developmental problems, which were even more prominent in the absence of mitochondrial Sod2p. sod2Δ and ctt1Δ colonies failed in ammonia production and sufficient activation of the alternative metabolism and were incapable of center margin differentiation, but they did not increase ROS levels. These new data indicate that colony disorders are not accompanied by ROS burst but could be a consequence of metabolic defects, which, however, could be elicited by imbalance in ROS produced in early developmental phases. Sod2p and homeostasis of ROS may participate in regulatory events leading to ammonia signaling. When organisms, including yeast, grow under aerobic conditions, they produce ROS 2The abbreviations used are: ROSreactive oxygen speciesDHEdihydroethidiumGSglutamine synthetaseMet+methionine prototrophyPQrParaquat-resistantwtwild typeMES4-morpholineethanesulfonic acid. as a consequence of aerobic respiration. Their excess is scavenged by different stress defense enzymes. Various clinical human disorders such as porphyria, hypertension, atherosclerosis, and some neurodegenerative diseases are believed to be partly induced by unbalanced ROS levels or by a deficiency in one of the stress defense enzymes (1.Eberhardt M.K. Reactive Oxygen Metabolites: Chemistry and Medical Consequences. CRC Press, Boca Raton, FL2001: 303-365Google Scholar). reactive oxygen species dihydroethidium glutamine synthetase methionine prototrophy Paraquat-resistant wild type 4-morpholineethanesulfonic acid. The yeast Saccharomyces cerevisiae can protect itself against various stresses (including oxidative stress) with a variety of ROS-scavenging enzymes, including superoxide dismutases, catalase, glutathione peroxidases, peroxiredoxins, and others. As do other eukaryotes, S. cerevisiae contains cytosolic CuZn-Sod1p and mitochondrial Mn-Sod2p superoxide dismutases responsible for the removal of superoxide radicals. Another S. cerevisiae enzyme that plays a role in ROS detoxification and yeast resistance to various stresses (2.Izawa S. Inoue Y. Kimura A. Biochem. J. 1996; 320: 61-67Crossref PubMed Scopus (203) Google Scholar, 3.Schüller C. Brewster J.L. Alexander M.R. Gustin M.C. Ruis H. EMBO J. 1994; 13: 4382-4389Crossref PubMed Scopus (442) Google Scholar) and that appears to be important for replicative life span (4.Van Zandycke S.M. Sohier P.J. Smart K.A. Mech. Ageing Dev. 2002; 123: 365-373Crossref PubMed Scopus (25) Google Scholar) and programmed cell death (5.Guaragnella N. Antonacci L. Giannattasio S. Marra E. Passarella S. FEBS Lett. 2008; 582: 210-214Crossref PubMed Scopus (42) Google Scholar) is the cytosolic catalase Ctt1p, which decomposes hydrogen peroxide. Mutants deficient in mitochondrial Sod2p are sensitive to hyperoxia and exhibit growth inhibition on respiratory carbon sources, but during aerobic growth on glucose the absence of this enzyme has little effect (6.Westerbeek-Marres C.A. Moore M.M. Autor A.P. Eur. J. Biochem. 1988; 174: 611-620Crossref PubMed Scopus (48) Google Scholar). The loss of Sod1p leads to more pleiotropic defects, including sensitivity to various exogenous stresses and reduced growth under all aerobic conditions because of oxidative injury by internal oxygen species (7.Davidson J.F. Whyte B. Bissinger P.H. Schiestl R.H. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5116-5121Crossref PubMed Scopus (372) Google Scholar, 8.Gralla E.B. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar, 9.Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). A consequence of endogenous oxidative stress is damage of particular biosynthetic enzymes, resulting in lysine and methionine auxotrophy (10.Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Defective phenotypes associated with sod1Δ strains are usually suppressed by the accumulation of stress-resistant mutants of the two main groups. The first group includes mutations in the BSD1 and BSD2 genes involved in Mn2+ homeostasis (11.Lapinskas P.J. Cunningham K.W. Liu X.F. Fink G.R. Culotta V.C. Mol. Cell. Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). The sod1Δbsd mutants increase the cytosolic concentration of Mn2+, which possesses some intrinsic superoxide dismutase activity and partially compensates for the absence of Sod1p. The second group, designated seo (for suppressors of endogenous oxygen toxicity), includes various mutations that restore Met and Lys prototrophy, although sensitivity to environmental oxidants (e.g. Paraquat) is not reverted (10.Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In contrast to liquid microbial cultures commonly studied in laboratories, microorganisms in their natural environments often organize as multicellular communities (e.g. colonies and biofilms) with unique properties (12.Nadell C.D. Xavier J.B. Foster K.R. FEMS Microbiol. Rev. 2009; 33: 206-224Crossref PubMed Scopus (457) Google Scholar, 13.Palková Z. Váchová L. FEMS Microbiol. Rev. 2006; 30: 806-824Crossref PubMed Scopus (92) Google Scholar). During long term development, colonies of different yeasts can undergo developmental changes characterized by extracellular alkalization and production of volatile ammonia, which functions as a signaling molecule (14.Palková Z. Janderová B. Gabriel J. Zikánová B. Pospísek M. Forstová J. Nature. 1997; 390: 532-536Crossref PubMed Scopus (172) Google Scholar). In S. cerevisiae colonies, ammonia elicits expression changes leading to metabolic reprogramming (e.g. activation of peroxisomes and fatty acid β-oxidation, the methyl glyoxylate cycle, amino acid metabolism, and the production of different plasma membrane transporters) and a parallel decrease in the expression of stress-related genes (including CTT1 and SOD1) and genes of mitochondrial oxidative phosphorylation (15.Palková Z. Devaux F. Ricicova M. Mináriková L. Le Crom S. Jacq C. Mol. Biol. Cell. 2002; 13: 3901-3914Crossref PubMed Scopus (117) Google Scholar). Ammonia signaling is also important for constraining apoptotic-like cells to the colony center (16.Váchová L. Palková Z. J. Cell Biol. 2005; 169: 711-717Crossref PubMed Scopus (153) Google Scholar). This study assesses the importance of the individual stress defense enzymes (a deficiency of which poses a serious problem for liquid yeast cultures) during the development of S. cerevisiae colonies. New data suggest that in colonies activation of the ammonia-induced adaptive metabolism is more important for proper development and survival than the presence of ROS scavenging enzymatic activities. Nevertheless, ROS homeostasis appears to be important for the induction of ammonia signaling and activation of metabolic changes. S. cerevisiae strain BY4742 (MATα, his3Δ, leu2Δ, lys2Δ, ura3Δ) and all isogenic mutants (sod1Δ, sod2Δ, ctt1Δ) were obtained from the EUROSCARF collection. Yeast giant colonies (14.Palková Z. Janderová B. Gabriel J. Zikánová B. Pospísek M. Forstová J. Nature. 1997; 390: 532-536Crossref PubMed Scopus (172) Google Scholar) were grown 6/plate at 28 °C on GMA (1% yeast extract, 3% glycerol, 2% agar, 30 mm CaCl2) or on GMA-bromcresol purple (GMA, 0.01% bromcresol purple). Liquid yeast cultures were grown at 28 °C in a shaker in either YPD (2% glucose, 1% yeast extract, 1% pepton), GM (GMA without agar), or SD (2% glucose, 100 mm KH2PO4, 15 mm (NH4)2SO4, 0.8 mm MgSO4, 0.15% Wickerham's yeast nitrogen base supplemented with 150 mg/liter of histidine, leucine, lysine, and methionine and 60 mg/liter of uracil), buffered with 50 mm potassium Pi (KH2PO4/K2HPO4), pH 6.5, when appropriate. Agar media (YPDA and SDA) were solidified with 2% agar. Gene, protein, and mutant strain symbols follow standard yeast terminology. Ammonia released by growing colonies was absorbed into acidic traps (14.Palková Z. Janderová B. Gabriel J. Zikánová B. Pospísek M. Forstová J. Nature. 1997; 390: 532-536Crossref PubMed Scopus (172) Google Scholar). The amount of ammonia was determined using Nessler's reagent. The methods used were described previously (17.Váchová L. Devaux F. Kucerová H. Ricicová M. Jacq C. Palková Z. J. Biol. Chem. 2004; 279: 37973-37981Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In brief, cells from the central and margin regions of 6–12 colonies were broken in 10 mm MES buffer, pH 6, with Complete, EDTA-free protease inhibitor mixture (Roche Applied Science) and 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride (Sigma) with glass beads in a FastPrep instrument (QBiogene). After cell debris removal, the supernatant (5–15 μg of proteins/slot determined using a Bio-Rad protein detection kit) was subjected to PAGE under nondenaturing conditions. Superoxide dismutase and catalase activity in the gels was determined (18.Beauchamp C. Fridovich I. Anal. Biochem. 1971; 44: 276-287Crossref PubMed Scopus (9904) Google Scholar, 19.Clare D.A. Duong M.N. Darr D. Archibald F. Fridovich I. Anal. Biochem. 1984; 140: 532-537Crossref PubMed Scopus (216) Google Scholar). Enzyme activities were quantified by image analyses using UltraQuant 6.0 software (Media Cybernetics). Total RNA from ∼100 mg of wet colony biomass was extracted by a hot phenol method (15.Palková Z. Devaux F. Ricicova M. Mináriková L. Le Crom S. Jacq C. Mol. Biol. Cell. 2002; 13: 3901-3914Crossref PubMed Scopus (117) Google Scholar). For northern blots, 15 μg of total RNA was separated in 1.5% agarose gel and transferred to a positively charged nylon membrane (Amersham Biosciences). The Northern blots were densitometrically quantified using UltraQuant 6.0 software. The values presented are relative densities calculated for the maximal value of each film (set as 100%). RDN18 was the nonregulated control gene. Survival of cells from colonies was analyzed in liquid SD, YPD, or GM (inoculated to A560 = 0.2). The number of living cells was determined after 1–7 days of cultivation by drop assay. Five-microliter drops of serial 10-fold dilutions of the cell cultures were spotted on an YPDA, and the number of colony-forming cells was determined after 3 days of cultivation. The appearance of suppressor mutants in sod1Δ cultures was determined by drop assay on SDA without methionine and on YPDA supplemented with 1.5 mm Paraquat (methyl viologen; MP Biomedicals) or 5 mm MnSO4. Harvested colonies were suspended in distilled water to a concentration of 7.5 mg/ml. 10-fold serial dilutions of these suspensions were incubated at 52 °C for 150 min in microtitre plates, and then 5 μl drops were spotted on YPDA. The number of colony-forming cells was determined after 3 days of cultivation. Control cells were spotted at the same dilutions before the heat treatment. For 4′,6′-diamidino-2-phenylindole staining of the DNA, the cells were permeabilized with 60% ethanol. 4′,6′-Diamidino-2-phenylindole was added to a final concentration of 2.5 μg/ml, and the cells were observed under a fluorescence microscope using an A filter (Leica). Nuclear morphology was determined from a minimum of 500 cells/sample. Cell morphology and the presence of shrunken cells were visualized with Nomarski contrast (supplemental Fig. S3A). For DHE staining, 50 μl of 15 mg/ml cell suspension in 25 mm MES, pH 6, was incubated with 5 μl of 25 μg/ml DHE (Sigma; 1 mg/ml stock solution in Me2SO) for 25 min. The suspension was then transferred to a cuvette with 1.95 ml of distilled water. Mitochondrial superoxide production was monitored by MitoSOX Red staining. Twenty-five μl of 0.5 mm MitoSOX (Molecular Probes; 5 mm stock solution in Me2SO) was added to a cuvette with 2 ml of cell suspension (containing 0.75 mg of wet biomass) and incubated for 5 min. The release of hydrogen peroxide from cells was measured with an Amplex Red horseradish peroxidase kit (Molecular Probes). Fifty μl of cell suspension (15 mg/ml in 25 mm MES buffer, pH 6.0) incubated for 30 min in the presence of 50 μm Amplex Red, and 5 units/ml of horseradish peroxidase was diluted with 1.95 ml of distilled water. Fluorescence was measured using a FluoroMax 3 spectrofluorometer (Jobin Yvon) with the following excitation/emission wavelengths: 480/604 nm for DHE, 510/580 nm for MitoSOX Red, and 525/585 nm for Amplex Red. Readings from unstained cells were used as an autofluorescence control and subtracted from the stained cell fluorescence. The enzymatic activities of glutathione peroxidase and peroxiredoxins were measured in cell extracts prepared as described above, with the exception of using 50 mm potassium Pi, pH 7.8, for the extraction. The samples of 100 μg of protein were assayed with a glutathione peroxidase cellular activity assay kit (Sigma) detecting changes in NADPH absorbance after the addition of tert-butyl hydroperoxide. Peroxiredoxin activity was assayed by glutamine synthetase (GS) protection assay (20.Kim K. Kim I.H. Lee K.Y. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1988; 263: 4704-4711Abstract Full Text PDF PubMed Google Scholar, 21.Netto L.E. Chae H.Z. Kang S.W. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1996; 271: 15315-15321Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), which is based on the sensitivity of GS to oxidative damage caused by a thiol/Fe3+/O2 mixed function oxidase system. The ability of a cell extract to protect GS from oxidative damage is mostly ascribed to the activity of peroxiredoxins (21.Netto L.E. Chae H.Z. Kang S.W. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1996; 271: 15315-15321Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In the assay, 1 unit of glutamine synthetase (Sigma) in 50 μl was incubated with a mixture of 3 μm FeCl3 and 10 mm dithiothreitol in the presence of the cell extract (containing 50 μg of protein). After 10 min, the remaining GS activity was determined by adding 950 μl of a GS activity assay mixture (0.4 mm ADP, 150 mm glutamine, 10 mm KH2AsO4, 20 mm NH2OH, 0.4 mm MnCl2, 100 mm HEPES, pH 7.4). After 15 min, the reaction was stopped with 200 μl of a mixture of 5.5% FeCl3, 2% trichloroacetic acid, and 2% HCl. The γ-glutamylhydroxamate-Fe3+ complex was determined at 420 nm. The intrinsic GS activity of the cell extract was subtracted. The activity of GS prior to treatment with FeCl3/dithiothreitol was taken as 100%. Without the protection by cell extract, the GS completely lost its activity. The data points representing the means ± S.D. were calculated from at least four independent experiments. Where appropriate, data significance was determined using the two-tailed t test. p values of 0.05 or less were considered statistically significant: *, p < 0.05; **, p < 0.01 or commented in the figure legends. The growth of colonies of strains deficient in Sod1p, Sod2p, or cytosolic Ctt1p on GMA was comparable with the growth of wild type (wt) BY4742 colonies (supplemental Fig. S1), exhibiting a linear increase in cell biomass that is typical for yeast colonies growing on solid media (16.Váchová L. Palková Z. J. Cell Biol. 2005; 169: 711-717Crossref PubMed Scopus (153) Google Scholar, 22.Palková Z. Váchová L. Gásková D. Kucerová H. Mol. Membr. Biol. 2009; 26: 228-235Crossref PubMed Scopus (13) Google Scholar). The colony development, however, substantially differed among strains, some being impaired in ammonia signaling. Whereas sod1Δ colonies exhibited a morphology, alkalization, and ammonia production comparable with the wt phenotype, sod2Δ colonies displayed altered morphology and failed to produce ammonia and accomplish transition to the alkali phase (Fig. 1, A and B). ctt1Δ colonies produced a reduced level of ammonia. To find out whether sod1Δ, sod2Δ, and ctt1Δ colonies are able to activate adaptive metabolic changes ascribed to ammonia signaling (15.Palková Z. Devaux F. Ricicova M. Mináriková L. Le Crom S. Jacq C. Mol. Biol. Cell. 2002; 13: 3901-3914Crossref PubMed Scopus (117) Google Scholar), we monitored expression of the most relevant genes involved in these changes. Expression of carbon metabolism and transporter genes (CIT3, POX1, ATO1, ATO3, and JEN1) was activated in ammonia-producing sod1Δ colonies as well as in wt colonies. The expression of these genes reached significantly lower levels in sod2Δ colonies and was slightly diminished in ctt1Δ colonies (Fig. 1C and supplemental Fig. S2A). On the other hand, sod2Δ colonies maintained a partially higher level of stress-related gene expression (MSN4, HSP30, and CTT1), contrary to sod1Δ and wt colonies (Fig. 1D and supplemental Fig. S2B). These data showed that colonies of all three mutants initiated adaptive metabolic changes in approximately the same developmental period as did wt strain colonies, but in sod2Δ (and to some extent also in ctt1Δ) colonies the changes did not reach a level comparable with that of the wt and sod1Δ colonies. Because sok2Δ colonies lacking Sok2p transcription factor and defective in ammonia signaling also exhibit an altered distribution of dying apoptotic-like cells (16.Váchová L. Palková Z. J. Cell Biol. 2005; 169: 711-717Crossref PubMed Scopus (153) Google Scholar), we examined center margin colony differentiation in sod1Δ, sod2Δ, and ctt1Δ colonies. First, we analyzed the occurrence of cells exhibiting late dying features (chromatin condensation and fragmentation as well as the presence of “shrunken,” i.e. partially digested, cells) (Fig. 2A and supplemental Fig. S3A) in outer margin and central colony areas. As in wt colonies, the cells exhibiting modified nuclei and shrunken cells were preferentially localized to central regions in ammonia-producing sod1Δ colonies, whereas cells located at the margin were mostly normal and healthy. In contrast, the cells with both dying features were spread throughout the whole of sod2Δ and ctt1Δ colonies (impaired in ammonia production) (Fig. 2A) similarly as in sok2Δ colonies (16.Váchová L. Palková Z. J. Cell Biol. 2005; 169: 711-717Crossref PubMed Scopus (153) Google Scholar). Furthermore, we determined stress-related characteristics of cells harvested from the central and margin regions of colonies from each strain. We determined ROS levels, ROS-scavenging enzyme activity, and cell tolerance to heat shock (Fig. 2, B–F). Cellular ROS production is characteristic of oxidative stress and has also been linked to ongoing apoptosis (23.Perrone G.G. Tan S.X. Dawes I.W. Biochim. Biophys. Acta. 2008; 1783: 1354-1368Crossref PubMed Scopus (315) Google Scholar). The occurrence of the superoxide was detected as superoxide-mediated oxidation of either DHE (detecting total cellular superoxide) or MitoSOX Red, an indicator of mitochondrial superoxide. After entering the alkali phase (at day 11), central and outer cells in colonies of both ammonia-producing strains (wt and sod1Δ) exhibited divergent trends in both mitochondrial and cellular superoxide levels. Whereas superoxide concentration gradually increased in the colony center, we observed a simultaneous decrease in the colony margin. A significantly smaller center margin difference was observed in ammonia production-impaired sod2Δ and ctt1Δ colonies. Interestingly, however, despite the similar profiles in central and outer cells, relative levels of mitochondrial (Fig. 2C) and cellular (Fig. 2B) superoxide differed. Total cellular superoxide levels in central cells of sod2Δ and ctt1Δ colonies did not increase as in wt central cells, whereas outer cell levels were comparable with wt (Fig. 2B). Conversely, mitochondrial superoxide levels in outer cells of sod2Δ and ctt1Δ did not decrease as in their wt counterparts, whereas central cell levels remained equivalent (Fig. 2C). These data showed that the overall superoxide level did not increase in sod1Δ, sod2Δ, and ctt1Δ colonies in general, although homeostasis of mitochondrial and cellular superoxide was differentially misbalanced. Cell production of hydrogen peroxide was determined through the Amplex Red assay (supplemental Fig. S4). The results confirmed that center margin H2O2 differences were nearly 40% more pronounced in wt and sod1Δ colonies as compared with sod2Δ and ctt1Δ colonies. However, the center margin difference in ammonia-producing colonies as compared with colonies defective in ammonia production was less prominent than in the case of the superoxide. This could be caused by more efficient diffusion of H2O2 across a colony. Overall amounts of H2O2 released by colonial cells were just 6–17% of the amounts released by 24-h-old wt cells cultivated in YPD liquid medium (data not shown). Because the cellular ROS homeostasis is determined by both ROS production and ROS-scavenging enzymes, we examined catalase and superoxide dismutase activity in colonies. Within wt and sod1Δ colonies, Ctt1p activity was significantly higher in central than in outer cells, whereas it remained uniform within sod2Δ colonies (Fig. 2D). Similarly, Sod1p activity was significantly higher in central than outer wt cells but homogeneous among both sod2Δ colony regions (Fig. 2E). Sod1p activity transiently increased in the central region of ctt1Δ colonies during their transition to the abortive alkali phase (Fig. 2E). Interestingly, both enzyme activities of central sod2Δ cells were lower than those of central wt cells. Variations in scavenging enzymatic activities can influence cell sensitivity to different stresses inducing ROS production. It was shown, for example, that sod1Δ is more sensitive to heat shock, presumably because of increased ROS production at elevated temperature (7.Davidson J.F. Whyte B. Bissinger P.H. Schiestl R.H. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5116-5121Crossref PubMed Scopus (372) Google Scholar). Heat shock tolerance of central and outer cells from both wt and sod1Δ colonies differed significantly (Fig. 2F). The survival rate of central cells was 10 times higher than that of outer cells from alkali phase and second acidic phase colonies after exposure to 52 °C for 150 min. Despite the similar trend, both central and outer cells from sod1Δ colonies were ∼5–10 times more sensitive to the heat shock than wt cells. In contrast, there was no difference in heat shock sensitivity between central and outer cells from sod2Δ and ctt1Δ colonies (Fig. 2F). Surprisingly, cells from sod2Δ and ctt1Δ colonies were in general relatively resistant. The above data show that the distribution of cells harboring dying and stress-related characteristics differed in the central and margin regions of ammonia-producing sod1Δ and wt colonies. In contrast, there was little or even no difference in their distribution in sod2Δ and ctt1Δ colonies, which are incapable of ammonia signaling. In addition, contrary to liquid cultures, there was no increase in ROS and/or ROS-scavenging enzymes in colonies deficient in these enzymes. In fact, levels of ROS and of scavenging enzymes were even decreased in some cases. To determine whether the activation of some other stress defense mechanisms could be responsible for compensating the lack of superoxide dismutases or catalase, we measured glutathione peroxidase and peroxiredoxin activities. As shown in supplemental Fig. S5, these activities in cells harvested from 23- and 30-day-old colonies were approximately the same among all strains. Surprisingly, in contrast to the absence of mitochondrial Sod2p, the absence of cytosolic Sod1p was accompanied by no visible defect in colony development and long term survival, despite the fact that sod1Δ colonies were grown aerobically on respiratory glycerol medium GMA. In sharp contrast, it has been shown previously that the absence of Sod1p leads to pleiotropic defects in liquid yeast cultures (e.g. increased sensitivity to oxygen and various stresses, slow growth, and rapid cell dying) (7.Davidson J.F. Whyte B. Bissinger P.H. Schiestl R.H. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5116-5121Crossref PubMed Scopus (372) Google Scholar, 8.Gralla E.B. Valentine J.S. J. Bacteriol. 1991; 173: 5918-5920Crossref PubMed Google Scholar, 9.Longo V.D. Gralla E.B. Valentine J.S. J. Biol. Chem. 1996; 271: 12275-12280Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar) (supplemental Fig. S1A) and to high rate appearance of suppressor mutants resistant to oxidants and exhibiting reversal of methionine auxotrophy (10.Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11.Lapinskas P.J. Cunningham K.W. Liu X.F. Fink G.R. Culotta V.C. Mol. Cell. Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). To determine whether the wt-like behavior of GMA-grown sod1Δ colonies could be caused by the high accumulation rate of stress-resistant suppressor mutants, we tested the sensitivity of cells harvested from sod1Δ colonies to the superoxide-producing agent Paraquat (an extracellular stressor). We also monitored whether these cells still exhibit Met auxotrophy (a result of intracellular oxygen stress (10.Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar)) under aerobic conditions. As shown in Fig. 3A, even sod1Δ cells from 30-day-old GMA-grown colonies (GMA-sod1Δ) still exhibited high sensitivity to Paraquat and Met auxotrophy at normoxic O2. They were not hypersensitive to extracellular Mn2+. In contrast, a 10-day-old sod1Δ colony grown on YPDA glucose agar (YPDA-sod1Δ) contained ∼1% Paraquat-resistant cells (PQr) and 5% methionine prototrophic (Met+) cells (0.1% of Met+ cells was observed even in a fresh 2-day-old YPDA-sod1Δ colony). These data demonstrate that sod1Δ cells from aged GMA-grown colonies exhibited neither of the characteristics typical of either of the two previously described groups of suppressor mutations (bsd and seo) (10.Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11.Lapinskas P.J. Cunningham K.W. Liu X.F. Fink G.R. Culotta V.C. Mol. Cell. Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar) nor any other suppressor mutation that would increase their resistance to either extracellular or intracellular oxidative stress. The data described above excluded the accumulation of oxidative stress-resistant mutants in GMA-grown sod1Δ colonies. An interesting possibility remained, however, that cells within these colonies either transiently adapt or acquire mutation(s), resulting in properties optimal for their existence within this multicellular structure. To investigate this possibility, we compared sod1Δ cells forming colonies capable of ammonia signaling with sod1Δ cells forming colonies under" @default.
• W2016231318 created "2016-06-24" @default.
• W2016231318 creator A5018175143 @default.
• W2016231318 creator A5058570149 @default.
• W2016231318 creator A5070832697 @default.
• W2016231318 date "2009-11-01" @default.
• W2016231318 modified "2023-10-14" @default.
• W2016231318 title "Yeast Colony Survival Depends on Metabolic Adaptation and Cell Differentiation Rather Than on Stress Defense" @default.
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