Limits...
Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells.

Smith MC, Sumner ER, Avery SV - Mol. Microbiol. (2007)

Bottom Line: Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p.Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance.The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Institute of Genetics, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Phenotypic heterogeneity among individual cells within isogenic populations is widely documented, but its consequences are not well understood. Here, cell-to-cell variation in the stress resistance of Saccharomyces cerevisiae, particularly to cadmium, was revealed to depend on the antioxidant glutathione. Heterogeneity was decreased strikingly in gsh1 mutants. Furthermore, cells sorted according to differing reduced-glutathione (GSH) contents exhibited differing stress resistances. The vacuolar GSH-conjugate pathway of detoxification was implicated in heterogeneous Cd resistance. Metabolic oscillations (ultradian rhythms) in yeast are known to modulate single-cell redox and GSH status. Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p. Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance. This enabled manipulation of the degree of gene expression noise in cultures. It was shown that cells expressing Gts1p heterogeneously had a competitive advantage over more-homogeneous cell populations (with the same mean Gts1p expression), under continuous and fluctuating stress conditions. The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

Show MeSH

Related in: MedlinePlus

Homogeneous GTS1 expression causes homogeneous stress resistance. A. GTS1 expression (culture-averaged) in exponential-phase PTETGTS1 cells was determined at varying doxcycline concentrations and plotted relative to GTS1 expression measured in wild-type (BY4743) cells. GTS1 mRNA in extracts was determined quantitatively by quantitative PCR in triplicate, with reference to ACT1 mRNA. B. Western blotting was used to determine Gts1p levels in exponential-phase wild-type and PTETGTS1 cells, incubated at 0.8 μg ml−1 doxycycline in either the absence or presence of 12.5 or 37.5 μM Cd(NO3)2. Each lane was loaded with protein extracted from 2 × 106 cells [the decrease in Gts1p levels in Cd-treated cells is consistent with Cd-dependent inhibition of protein synthesis, as reported elsewhere (Lafaye et al., 2005); Cd has no effect on Gts1p as a proportion of total cellular protein (Vido et al., 2001)]. The lower panel shows densitometry analyses of Gts1p in each lane. The first lane contains protein markers. C. Exponential-phase cultures of wild-type (○) or PTETGTS1 strains (•) were plated onto YPD supplemented with Cd(NO3)2 or H2O2 and 0.8 μg ml−1 doxycycline (to give equivalent culture-averaged GTS1 expression; see A). Viability (colony formation) was determined after up to 8 days incubation at 30°C, and converted to percentages by reference to growth on unsupplemented control medium. The points represent means from three replicate determinations ± SEM. Typical results from one of at least three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2167119&req=5

fig06: Homogeneous GTS1 expression causes homogeneous stress resistance. A. GTS1 expression (culture-averaged) in exponential-phase PTETGTS1 cells was determined at varying doxcycline concentrations and plotted relative to GTS1 expression measured in wild-type (BY4743) cells. GTS1 mRNA in extracts was determined quantitatively by quantitative PCR in triplicate, with reference to ACT1 mRNA. B. Western blotting was used to determine Gts1p levels in exponential-phase wild-type and PTETGTS1 cells, incubated at 0.8 μg ml−1 doxycycline in either the absence or presence of 12.5 or 37.5 μM Cd(NO3)2. Each lane was loaded with protein extracted from 2 × 106 cells [the decrease in Gts1p levels in Cd-treated cells is consistent with Cd-dependent inhibition of protein synthesis, as reported elsewhere (Lafaye et al., 2005); Cd has no effect on Gts1p as a proportion of total cellular protein (Vido et al., 2001)]. The lower panel shows densitometry analyses of Gts1p in each lane. The first lane contains protein markers. C. Exponential-phase cultures of wild-type (○) or PTETGTS1 strains (•) were plated onto YPD supplemented with Cd(NO3)2 or H2O2 and 0.8 μg ml−1 doxycycline (to give equivalent culture-averaged GTS1 expression; see A). Viability (colony formation) was determined after up to 8 days incubation at 30°C, and converted to percentages by reference to growth on unsupplemented control medium. The points represent means from three replicate determinations ± SEM. Typical results from one of at least three independent experiments are shown.

Mentions: Oscillations and heterogeneity in cellular GSH content are suppressed in cells expressing GTS1 under PTET regulation. Exponential-phase cells were stained with mBCl and analysed by flow cytometry. A. The dot plots show fluorescence versus forward scatter properties of cells in cultures of wild-type cells (BY4743) and of cells in which the genomic GTS1 promoter is replaced with PTET. The doxycycline concentration (1 μg ml−1) used to regulate PTET expression corresponded to culture-averaged expression of GTS1 at ∼0.9× the level in wild-type cultures (see Fig. 6A). Similar results to those shown were obtained also at other doxycycline concentrations (data not shown). Each dot plot shows data accumulated from 20 000 cells. B. Subpopulations comprising cells with the highest ∼20% mBCl fluorescences from wild-type (○) or PTETGTS1 (•) cultures were sorted and then incubated with shaking in doxycycline-supplemented YPD broth at 30°C. The mean mBCl fluorescences of the subpopulations were determined at intervals with flow cytometry. The doxycycline concentration (0.8 μg ml−1) used to regulate PTET expression corresponded to culture-averaged expression of GTS1 at ∼1.0× the level in wild-type cultures (see Fig. 6A). Each point represents data accumulated from 50 000 cells. AU, arbitrary units of fluorescence.


Glutathione and Gts1p drive beneficial variability in the cadmium resistances of individual yeast cells.

Smith MC, Sumner ER, Avery SV - Mol. Microbiol. (2007)

Homogeneous GTS1 expression causes homogeneous stress resistance. A. GTS1 expression (culture-averaged) in exponential-phase PTETGTS1 cells was determined at varying doxcycline concentrations and plotted relative to GTS1 expression measured in wild-type (BY4743) cells. GTS1 mRNA in extracts was determined quantitatively by quantitative PCR in triplicate, with reference to ACT1 mRNA. B. Western blotting was used to determine Gts1p levels in exponential-phase wild-type and PTETGTS1 cells, incubated at 0.8 μg ml−1 doxycycline in either the absence or presence of 12.5 or 37.5 μM Cd(NO3)2. Each lane was loaded with protein extracted from 2 × 106 cells [the decrease in Gts1p levels in Cd-treated cells is consistent with Cd-dependent inhibition of protein synthesis, as reported elsewhere (Lafaye et al., 2005); Cd has no effect on Gts1p as a proportion of total cellular protein (Vido et al., 2001)]. The lower panel shows densitometry analyses of Gts1p in each lane. The first lane contains protein markers. C. Exponential-phase cultures of wild-type (○) or PTETGTS1 strains (•) were plated onto YPD supplemented with Cd(NO3)2 or H2O2 and 0.8 μg ml−1 doxycycline (to give equivalent culture-averaged GTS1 expression; see A). Viability (colony formation) was determined after up to 8 days incubation at 30°C, and converted to percentages by reference to growth on unsupplemented control medium. The points represent means from three replicate determinations ± SEM. Typical results from one of at least three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2167119&req=5

fig06: Homogeneous GTS1 expression causes homogeneous stress resistance. A. GTS1 expression (culture-averaged) in exponential-phase PTETGTS1 cells was determined at varying doxcycline concentrations and plotted relative to GTS1 expression measured in wild-type (BY4743) cells. GTS1 mRNA in extracts was determined quantitatively by quantitative PCR in triplicate, with reference to ACT1 mRNA. B. Western blotting was used to determine Gts1p levels in exponential-phase wild-type and PTETGTS1 cells, incubated at 0.8 μg ml−1 doxycycline in either the absence or presence of 12.5 or 37.5 μM Cd(NO3)2. Each lane was loaded with protein extracted from 2 × 106 cells [the decrease in Gts1p levels in Cd-treated cells is consistent with Cd-dependent inhibition of protein synthesis, as reported elsewhere (Lafaye et al., 2005); Cd has no effect on Gts1p as a proportion of total cellular protein (Vido et al., 2001)]. The lower panel shows densitometry analyses of Gts1p in each lane. The first lane contains protein markers. C. Exponential-phase cultures of wild-type (○) or PTETGTS1 strains (•) were plated onto YPD supplemented with Cd(NO3)2 or H2O2 and 0.8 μg ml−1 doxycycline (to give equivalent culture-averaged GTS1 expression; see A). Viability (colony formation) was determined after up to 8 days incubation at 30°C, and converted to percentages by reference to growth on unsupplemented control medium. The points represent means from three replicate determinations ± SEM. Typical results from one of at least three independent experiments are shown.
Mentions: Oscillations and heterogeneity in cellular GSH content are suppressed in cells expressing GTS1 under PTET regulation. Exponential-phase cells were stained with mBCl and analysed by flow cytometry. A. The dot plots show fluorescence versus forward scatter properties of cells in cultures of wild-type cells (BY4743) and of cells in which the genomic GTS1 promoter is replaced with PTET. The doxycycline concentration (1 μg ml−1) used to regulate PTET expression corresponded to culture-averaged expression of GTS1 at ∼0.9× the level in wild-type cultures (see Fig. 6A). Similar results to those shown were obtained also at other doxycycline concentrations (data not shown). Each dot plot shows data accumulated from 20 000 cells. B. Subpopulations comprising cells with the highest ∼20% mBCl fluorescences from wild-type (○) or PTETGTS1 (•) cultures were sorted and then incubated with shaking in doxycycline-supplemented YPD broth at 30°C. The mean mBCl fluorescences of the subpopulations were determined at intervals with flow cytometry. The doxycycline concentration (0.8 μg ml−1) used to regulate PTET expression corresponded to culture-averaged expression of GTS1 at ∼1.0× the level in wild-type cultures (see Fig. 6A). Each point represents data accumulated from 50 000 cells. AU, arbitrary units of fluorescence.

Bottom Line: Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p.Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance.The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Institute of Genetics, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
Phenotypic heterogeneity among individual cells within isogenic populations is widely documented, but its consequences are not well understood. Here, cell-to-cell variation in the stress resistance of Saccharomyces cerevisiae, particularly to cadmium, was revealed to depend on the antioxidant glutathione. Heterogeneity was decreased strikingly in gsh1 mutants. Furthermore, cells sorted according to differing reduced-glutathione (GSH) contents exhibited differing stress resistances. The vacuolar GSH-conjugate pathway of detoxification was implicated in heterogeneous Cd resistance. Metabolic oscillations (ultradian rhythms) in yeast are known to modulate single-cell redox and GSH status. Gts1p stabilizes these oscillations and was found to be required for heterogeneous Cd and hydrogen-peroxide resistance, through the same pathway as Gsh1p. Expression of GTS1 from a constitutive tet-regulated promoter suppressed oscillations and heterogeneity in GSH content, and resulted in decreased variation in stress resistance. This enabled manipulation of the degree of gene expression noise in cultures. It was shown that cells expressing Gts1p heterogeneously had a competitive advantage over more-homogeneous cell populations (with the same mean Gts1p expression), under continuous and fluctuating stress conditions. The results establish a novel molecular mechanism for single-cell heterogeneity, and demonstrate experimentally fitness advantages that depend on deterministic variation in gene expression within cell populations.

Show MeSH
Related in: MedlinePlus