Temporal profiling of redox-dependent heterogeneity in single cells
- The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram, The Hebrew University of Jerusalem, Israel
- Weizmann Institute of Science, Israel
- The Hebrew University of Jerusalem, Israel
- Benin School of Computer Science and Engineering, The Hebrew University of Jerusalem, Israel
Figures
Figure 1 with 2 supplements
Monitoring subcellular redox levels in yeast cells using the Grx1-roGFP2 variants and flow cytometry.
(A) Schematic of the Grx1-roGFP2 variant used in this study to monitor oxidation in the cytosol. Cellular GSSG reacts with the catalytic residues of the fused Grx1 which leads to oxidation of the …
Figure 1—figure supplement 1
Representation of FACS subpopulation gates.
(A) Representative data of an unsorted sample after 24 hr growth, plotted by the ratio between excitation at 405 and 488 nm (y and x axis, respectively). ‘GFP’ corresponds with the ‘reduced’ gate, …
Figure 1—figure supplement 2
Linear characterization of (A) peroxisomal and (B) mitochondrial ‘oxidation gate’.
Quantification of the redox status of fully reduced (blue) and fully oxidized (red) cells using FACS, using the peroxisomal and mitochondrial sensors.
Figure 2 with 1 supplement
Both cytosolic and mitochondrial oxidation levels increase with age and indicate the emergence of oxidized and reduced cellular subpopulations.
Cells expressing cytosolic Grx1-roGFP2 (A) or mitochondrial Grx1-roGFP2-Su9 (B) probes in wild type cells were grown for four days and their oxidation level monitored. In each experiment 10,000 …
Figure 2—figure supplement 1
Representative examples of OxD distribution curves at 24, 48, and 72 hr, in contrast to 405/488 nm ratios shown in Figure 2.
Normalization was calculated using median fluorescence values of 10,000 DTT- and diamide-treated cells. This normalization masks the actual fluorescence of the average population and natural …
Figure 3
Growth and division of reduced and oxidized subpopulations of yeast cells after sorting by FACS.
(A) Differences in minimal doubling time between the sorted oxidized and reduced subpopulations at different time points (48, 72, and 96 hr) measured by OD600 in a plate reader. (B) Corresponding …
Figure 4
Differential respiration profile of reduced and oxidized subpopulations after sorting by FACS.
(A) Normalized oxygen consumption rate (OCR) of reduced and oxidized yeast populations. (B) Normalized extracellular acidification rate (ECAR) of reduced and oxidized yeast populations. Three …
Figure 5
Correlation between redox status and replicative aging.
(A) Schematic of mother enrichment program in yeast, using estradiol induction for daughter cell death, growth, and flow cytometry analysis of both bud scar counts and oxidation level. (B) …
Figure 6 with 3 supplements
Proteomic analysis and functional enrichment analysis of the reduced and oxidized subpopulations.
(A) Hierarchical clustering of proteins identified in cells sorted from cultures at different time points (48 and 72 hr); downregulated proteins are in green, up-regulated are in red. (B) Functional …
Figure 6—figure supplement 1
Cellular localization annotation of all identified proteins (in at least two repeats within all four subpopulations).
Annotation of all identified proteins, corresponding with the localization as agreed upon in the literature.
Figure 6—figure supplement 2
Hierarchical clustering of the protein abundances.
log2 values of the log2 (LFQ) values of proteins identified in each post-sorting samples harvested at 48 and 72 hr (red corresponding to higher expression, green to lower). Median intensities were …
Figure 6—figure supplement 3
Functional enrichment of differentially expressed proteins (FDR < 0.05) in the oxidized (red) and reduced (blue) subpopulations (corresponding to the volcano plot in Figure 6).
https://doi.org/10.7554/eLife.37623.014
Figure 7 with 4 supplements
Analysis of differentially expressed proteins between the reduced and oxidized subpopulations.
(A) Oxidation levels in deletion strains of significantly changed proteins (∆tsa2, ∆dhh1, ∆pnc1, ∆hsp30, and wild type control) at different ages (24, 48, and 72 hr). (B) Differences in cell growth …
Figure 7—figure supplement 1
(A–C) Association maps of proteins associated with Dhh1, Pnc1, and Hsp30 (respectively), as identified in the mass spectrometry screen of differentially expressed proteins between the reduced (blue) and oxidized (red) subpopulations.
Protein associations are based on the STRING database (as of January 2017), under the highest confidence interaction and default active interaction sources. Visualization was done by using …
Figure 7—figure supplement 2
Dhh1 deletion decreases cell growth.
Minimal doubling times of wild type and deletion strains (Δdhh1, Δhsp30, Δpnc1) grown for 16 hr in casein supplemented medium, OD600 was measured in plate reader as described in Materials and methods…
Figure 7—figure supplement 3
Oxidation in mitochondria detected by mitochondrial sensor Su9 expressed in wild type, Hsp30 and Pnc1 knockout strains.
(A) Degree of mitochondrial oxidation (OxD) in yeast samples of different ages (24 and 48 hr), (B-D) Distribution of fluorescence intensity ratios obtained at 405 and 488 nm of wild type (B), Δhsp30 …
Figure 7—figure supplement 4
Oxidation in peroxisome detected by peroxisomal sensor SKL-roGFP expressed in wild type, dhh1, hsp30 and pnc1 knockout strains.
(A) Degree of peroxisomal oxidation (OxD) in yeast samples after growth in standard medium for 24 hr. The analysis was done using 5–6 replicates. (B-E) Distribution of fluorescence intensity ratios …
Figure 8 with 2 supplements
Differentially expressed transcripts between the reduced and oxidized subpopulations.
(A) Hierarchical clustering of the median expression values of all differentially expressed genes (FDR < 0.05) identified in the post-sorting cells harvested at different time points (48 and 72 hr); …
Figure 8—figure supplement 1
Hierarchical clustering of expression values of all differentially expressed genes (FDR < 0.05) identified in the biological replicates of the post-sorting cells harvested at 48 and 72 hr.
Transcripts above the median value of the row are in green, below in red. Each row was normalized by its median and log was taken for visualization purposes. The data was clustered using centered …
Figure 8—figure supplement 2
Correlation plot between mRNA and protein expression at 72 hr.
Significantly differentially regulated proteins are emphasized in bold and color according to their coupling status (bright red – coupled upregulation in oxidized subpopulation, dark red – uncoupled …
Figure 9
Changes in oxidative status of individual cells over time under confocal microscopy.
(A) Oxidation over time of a single cell, from reduced to highly oxidized, imaged using confocal microscopy. (B) Samples treated with 40 mM DTT and 8 mM Diamide for 15 min and imaged using confocal …
Figure 10
Changes in oxidative status between mother and daughter cells over time under confocal microscopy.
(A–C) Oxidation levels over time in mother and respective daughter cells during and after budding, showing the shared oxidative status until separation.
Videos
Video 1
Growth and replication of the wild type strain.
The wild type cells were grown at 30°C on synthetic media for six hours and images were taken every five minutes using time-lapse confocal microscope as described in the Materials and methods part.
Video 2
Growth and replication of the pnc1 knockout strain.
The wild type cells were grown at 30°C on synthetic media for six hours and imageswere taken every five minutes using time-lapse confocal microscope as described in the Materials and methods part.
Video 3
Growth and replication of the hsp30 knockout strain.
The wild type cells were grown at 30°C on synthetic media for six hours and imageswere taken every five minutes using time-lapse confocal microscope as described in the Materials and methods part.
Video 4
Growth and replication of the dhh1 knockout strain.
The wild type cells were grown at 30°C on synthetic media for six hours and imageswere taken every five minutes using time-lapse confocal microscope as described in the Materials and methods part.
Tables
Table 1
Number of budding events
https://doi.org/10.7554/eLife.37623.008| Growth (h) | Oxidized (%) | Reduced (%) |
|---|---|---|
| 48 | 10 | 30 |
| 72 | 9 | 21 |
| 96 | 6 | 15 |
Additional files
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Supplementary file 1
Comparison of OxD values of wild type and knockout strains (related to Figure 2F).
- https://doi.org/10.7554/eLife.37623.029
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Supplementary file 2
Protein list.
- https://doi.org/10.7554/eLife.37623.030
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Supplementary file 3
Significant proteins (48 hr).
- https://doi.org/10.7554/eLife.37623.031
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Supplementary file 4
Significant proteins (72 hr).
- https://doi.org/10.7554/eLife.37623.032
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Supplementary file 5
Comparison of wild type and knockout strains OxD values (related to Figure 6E).
- https://doi.org/10.7554/eLife.37623.033
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Supplementary file 6
Transcript list (48 hr).
- https://doi.org/10.7554/eLife.37623.034
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Supplementary file 7
Transcript list (72 hr).
- https://doi.org/10.7554/eLife.37623.035
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Supplementary file 8
Transcripts upregulated in red (48 hr).
- https://doi.org/10.7554/eLife.37623.036
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Supplementary file 9
Transcripts upregulated in ox (48 hr).
- https://doi.org/10.7554/eLife.37623.037
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Supplementary file 10
Transcripts upregulated in red (72 hr).
- https://doi.org/10.7554/eLife.37623.038
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Supplementary file 11
Transcripts upregulated in ox (72 hr).
- https://doi.org/10.7554/eLife.37623.039
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Supplementary file 12
Comparison of wild type and knockout strains OxD values (related to Figure 7).
- https://doi.org/10.7554/eLife.37623.040
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Transparent reporting form
- https://doi.org/10.7554/eLife.37623.041
