Overexpression of Ssd1 and calorie restriction extend yeast replicative lifespan by preventing deleterious age-dependent iron uptake
- Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, United States
- BCMB Graduate Program, Weill Cornell Graduate School of Biomedical Sciences, United States
Figures
Figure 1 with 8 supplements
Overexpression of Ssd1 extends lifespan and forms transient cytoplasmic foci during replicative aging.
(A) Levels of GFP tagged Ssd1 in the indicated strains, for 10,000 cells per strain. (B) RLS of strains shown in panel A for 90 ssd1∆ cells, 63 SSD1-GFP cells and 63 PGPD1-SSD1-GFP cells, p-values are as follows: for control strain versus ssd1Δ strain is 0, for control versus PGPD1-SSD1-GFP is 8x10–8. (C) Transient formation of Ssd1-GFP foci in PGPD1-SSD1-GFP strain during aging at the indicated cell division number of the RLS for one cell. Arrows indicate Ssd1-GFP foci. (D) Single cell trajectories showing total cell Ssd1-GFP intensity and number of Ssd1-GFP foci formation for the indicated strains throughout the RLS. Each rectangle represents one cell cycle.
-
Figure 1—source data 1
Replicative lifespan data from the microfluidic imaging used to generate the graphs.
- https://cdn.elifesciences.org/articles/108892/elife-108892-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
SSD1 foci formation in a single cell during replicative aging.
Images were taken for every cell cycle in its lifespan in the bright field and GFP channel for SSD1-GFP and in the bright field for PGPD1-SSD1-GFP and correspond to images taken in the GFP channel shown in Figure 1C.
Figure 1—figure supplement 2
Relative Ssd1-GFP protein amount per cell throughout the lifespan.
Total cellular fluorescence intensity of strains SSD1-GFP and PGPD1-SSD1-GFP during the cell division in which 100% of the population (mothers) are actively dividing for the data shown in Figure 1D. Average and standard deviation for 63 cells per strain is shown. The box indicates interquartile range (IQR), whiskers extend to the smallest and largest non-outlier values and outliers are plotted as individual points outside Quartile(Q)1–1.5×IQR or Q3+1.5 × IQR, where IQR = Q3−Q1. Data are shown for cell divisions in which 100% of the mothers are still dividing.
Figure 1—figure supplement 3
Properties of the Ssd1 foci.
(A) Ssd1-GFP foci formed during aging are cytoplasmic. mCherry-NLS indicates the nucleus. (B) 1-6-hexanediol dissolves Ssd1-GFP foci, images taken before, or at 10 or 20 min after, addition of 1-6-hexanediol.
Figure 1—video 1
Bright field and corresponding fluorescence microscopy.
Figure 1—video 2
Bright field of Ssd1-GFP for an SSD1-GFP cell during the RLS.
Bright field.
Figure 1—video 3
Bright field and corresponding fluorescence microscopy.
Figure 1—video 4
Bright field of Ssd1-GFP for a PGPD1-SSD1-GFP cell during the RLS.
Numbers indicate the number of cell divisions into the lifespan.
Figure 1—video 5
Fluorescence microscopy of Ssd1-GFP condensates undergoing fusion in an aging PGPD1-SSD1-GFP cell.
Figure 2 with 1 supplement
Ssd1 foci formed during aging are cell cycle regulated and distinct from Hsp104 foci, P-bodies, and stress granules.
(A) Example showing Ssd1-GFP foci in G2 phase during aging. The images are taken of the same cell every 20 min and the cartoons indicate the bud size. Below are shown single cell trajectories of cell cycle stage at which Ssd1 foci are present during aging (left) and whether the foci dissolved at the following cytokinesis (shown in blue) or were persistent through cytokinesis (shown in red; right). (B) Colocalization of Ssd1 foci with P-bodies and Hsp104 foci in cells treated with 10 mM Sodium azide. Quantitation is shown below for over 100 cells per condition. (C) Colocalization of Ssd1 foci with P-bodies (marked by Edc3) and stress granules (marked by Pab1) under nutrient deprivation and quantitation. (D) Failure of Ssd1 foci to colocalize with age-induced Hsp104 foci or P-bodies.
-
Figure 2—source data 1
Replicative lifespan data from the microfluidic imaging used to generate the graphs.
- https://cdn.elifesciences.org/articles/108892/elife-108892-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Frequency of Ssd1 foci formation.
Data are from exponentially growing cells, treated with sodium azide and under glucose starvation in strain PGPD1-SSD1-GFP. Average and standard deviation are shown for analysis of biological triplicate experiments for >60 cells per condition per experiment. p-values determined by Student’s t-test are indicated by ** for 0.01 and ***** for 0.0001.
Figure 3
CR and overexpression of Ssd1 are epistatic for lifespan extension, both influencing iron metabolism.
(A) Calorie restriction and overexpression of Ssd1 are epistatic for extension of RLS, for 54 control cells, 48 PGPD-SSD1 cells, 48 control cells undergoing CR, and 44 PGPD-SSD1 cells + CR. p-values determined by Student’s T-test for control versus PGPD-SSD1, CR, and PGPD-SSD1+CR are 1x10–6, 3x10–4 and 5x10–4 respectively. There is no significant difference between PGPD-SSD1, CR, and PGPD-SSD1+CR. (B) CR and overexpression of Ssd1 protect cells from lifespan reduction due to iron supplementation with 10 µM Fe+3, for 55 control cells, 53 control cells + Fe+3, 47 PGPD-SSD1 cells, 41 PGPD-SSD1 + Fe+3 cells, 37 CR cells, and 50 CR + Fe+3 cells. p-value for control versus Fe+3 is 6x10–7. There is no significant difference between PGPD-SSD1 with and without Fe+3 or for CR with and without Fe+3. (C) CR and Ssd1 overexpression protect cells from the lifespan reduction caused by iron depletion with 200 µM BPS, for 52 control cells, 40 control cells + BPS, 60 PGPD-SSD1 cells, 34 PGPD-SSD1 cells + BPS, 44 control cells under CR, and 53 cells under CR + BPS. p-values for the control + BPS, versus PGPD-SSD1+BPS and CR + BPS are 0, while there is no significant difference between the control with and without BPS treated and untreated control are not significantly different; control compared to PGPD-SSD1 and CR is 0.
-
Figure 3—source data 1
Replicative lifespan data from the microfluidic imaging used to generate the graphs.
- https://cdn.elifesciences.org/articles/108892/elife-108892-fig3-data1-v1.xlsx
Figure 4 with 4 supplements
Age-dependent induction of the iron regulon predicts the end of lifespan and is blocked by Ssd1 overexpression and CR.
(A) Schematic of iron regulon activation. (B) Single cell trajectories of nuclear localization of GFP-Aft1 during the RLS of control or CR yeast. (C) Earlier nuclear localization of Aft1 in the lifespan correlates with a shorter total lifespan. Analysis of the data shown in panel B. (D) Single cell trajectories of FIT2-mCherry expression during aging and Peak log fold change (FC) in mCherry intensity throughout the lifespan. (E) Single cell trajectories of ARN1-mCherry expression during aging and fold change intensity throughout the lifespan. Statistical difference is indicated where n.s. is not significant change with p>0.05, * p≤0.05, ** p≤0.01, *** p≤0.001 and **** p≤0.0001.
-
Figure 4—source data 1
Replicative lifespan data from the microfluidic imaging used to generate the graphs.
- https://cdn.elifesciences.org/articles/108892/elife-108892-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Aft1 localization and expression during aging.
(A) Example of old cell with (top) and without (bottom) nuclear localization of GFP-Aft1 on the left, profile after threshold (see Methods) on the right. (B) Localization of Aft1 to the nucleus occurs on average 5 divisions before the end of lifespan. The analysis is of the data shown in Figure 4B. (C) CR does not affect GFP-Aft1 expression as determined by fluorescence intensity. 50 cells were analyzed per conditions in each plot. n.s. indicates a not significant change of p>0.05 and **** is p≤0.0001.
Figure 4—figure supplement 2
Replicative lifespans of strains expressing FIT2-mCherry and ARN1-mCherry.
Replicative lifespan for experiments shown in Figure 4D and E. p-values for both graphs of control versus PGPD1-SSD1 and CR are 0, while for PGPD1-SSD1 versus CR is not significant, 50 cells were analyzed for each condition.
Figure 4—figure supplement 3
Single-cell examples of experiments shown in Figure 4D and E, to show variability between different cells.
The number indicates the cell division number in the RLS. Top is imaged under bright light and the bottom under mCherry fluorescence detection.
Figure 4—figure supplement 4
Replicative lifespans with and without cth2∆.
The number of cells counted per condition is control: 91, cth2∆: 150, PGPD1-SSD1: 39, PGPD1-SSD1/cth2∆: 91, CR: 101, CR/cth2∆: 98, p-values: control compared to cth2Δ is 6x10–3, cth2Δ compared to CR, PGPD1-SSD1/cth2Δ, cth2Δ under CR and PGPD1-SSD1 are 5x10–6, 1x10–5, 3x10–8 and 2x10–4, respectively.
Figure 5
CR and Ssd1 overexpression extend lifespan by preventing the accumulation of iron resulting from age-dependent activation of the iron regulon.
(A) Pre-induction of iron regulon by BPS treatment measured by mCherry-tagged expression of Fit2 and Arn1, for 10,000 cells per condition. (B) RLS of cells that had the iron regulon induced or uninduced from panel A measured by Fit2-mCherry expression, for 63 (a), 41 (b), 63 (c), 50 (d), 63 (e), and 31 (f) cells, p-values of control vs induced: 0.002, PGPD1-SSD1 vs induced: 0 and CR vs induced: 0. (C) RLS of yeast overexpressing Ssd1 as well as under CR are protected from sensitivity to Artemisinin. Left plot: for 62 control, 64 20 µM Art, 37 50 µM Art, 86 PGPD1-SSD1, 80 PGPD1-SSD1+20 µM Art and 107 PGPD1-SSD1+50 µM Art cells. p-values are control vs 20 µM Art: 4x10–6, control vs 50 µM Art: 0, PGPD1-SSD1 vs + PGPD1-SSD1 20 µM Art: 0.98 and PGPD1-SSD1 vs + PGPD1-SSD1+50 µM Art: 0.01. Right plot: 38 control, 34 20 µM Art, 30 50 µM Art, 61 CR, 61 CR + 20 µM Art, and 50 CR + 50 µM Art cells. p-values are control vs 20 µM Art: 0.01, control vs 50 µM Art: 1x10–7, CR vs CR 20 µM Art: 0.95 and CR vs+CR + 50 µM Art: 0.005. (D) Relative levels of Fe2+ at indicated cell division of the RLS measured by Phen Green. (E) Relative amount of Fe2+ in all cells of the indicated strains from panel D during the RLS, regardless of age. (F) RLS of the indicated strains / conditions with and without aft1∆, for 39 control, 71 aft1∆, 37 PGPD1-SSD1, 72 PGPD1-SSD1/aft1∆, 42 CR, and 74 CR/aft1∆ cells. p-values of control compared to all other strains are ≤1 x 10–4 while there is no significant difference between the strains / conditions other than the control.
-
Figure 5—source data 1
Replicative lifespan data from the microfluidic imaging used to generate the graphs.
- https://cdn.elifesciences.org/articles/108892/elife-108892-fig5-data1-v1.xlsx
Figure 6
Model for molecular mechanism of RLS extension by Ssd1 overexpression and CR.
During normal aging, cells have limited intracellular iron, potentially due to mitochondrial dysfunction reducing the assembly of iron-sulfur clusters, which leads to nuclear translocation of Aft1 and activation of the iron regulon. This subsequent deleterious increase in intracellular iron limits lifespan. CR also extends lifespan in a manner dependent on failure to activate the iron regulon; mechanistically, CR indirectly prevents nuclear localization of Aft1, preventing production of the mRNAs from the iron regulon and the subsequent age-dependent increase in deleterious iron accumulation. In aging cells overexpressing Ssd1, transient cytoplasmic Ssd1 condensates appear that correlate with lifespan extension, where the lifespan extension is due to failure to activate the iron regulon preventing deleterious iron accumulation. We speculate that Ssd1 dephosphorylation by Sit4 after G1 phase likely leads to formation of Ssd1 condensates in aging cells overexpressing Ssd1 including mRNAs of the iron regulon genes FIT2 and ARN1, while dissolution of Ssd1 condensates occurs prior to cytokinesis upon Cbk1-mediated phosphorylation of Ssd1.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Saccharomyces cerevisiae) | BY4741 background, parental ssd1::NATMX | Kurischko et al., 2011 | FLY2184 | Parental strain for all others in study |
| Strain, strain background (Saccharomyces cerevisiae) | SSD1-GFP (GPD1 promoter) | This paper; pAG415-GPD1-SSD1-GFP (FLE1019 in Kurischko et al., 2011) | See Supplementary file 1 | Constructed by homologous recombination |
| Gene (Saccharomyces cerevisiae) | SSD1 | SGD | SGD:S000002701 | Systematic Name: YDR293C |
| Sequence-based reagent | GPD1 promoter | SGD | SGD:S000003709 (GPD1 gene) | Used as constitutive promoter |
| Strain, strain background (Saccharomyces cerevisiae) | PGPD1-GFP-AFT1 | This paper; pRS plasmid Baker Brachmann et al., 1998 | See Supplementary file 1 | Homologous recombination |
| Strain, strain background (Saccharomyces cerevisiae) | Gene deletions, mCherry and GFP tagging strains | Longtine et al., 1998 | See Supplementary file 1 | Homologous recombination |
| Chemical compound, drug | Yeast Nitrogen Base (no amino acids, no ammonium sulfate) | Difco / BD Biosciences | 233520 | Component of SCD medium |
| Chemical compound, drug | Complete drop-out mix without YNB | US Biological | D9515 | Component of SCD medium |
| Chemical compound, drug | Ammonium sulfate | Fisher Chemical | A702-3 | Component of SCD medium |
| Chemical compound, drug | D-glucose | Sigma-Aldrich | G8270 | Component of SCD and YPD media |
| Chemical compound, drug | Dextrose (for YPD) | Grainger | 31GE61, 31GC58 | YPD component |
| Chemical compound, drug | Artemisinin | Thermo Scientific | J65406.03 | Used in supplementation experiments |
| Other | Automated dissection/screening chip | iBio chips | Not specified | Used for RLS analysis |
| Chemical compound, drug | Iron(III) chloride | Sigma-Aldrich | 157740–100 G | Iron supplementation |
| Chemical compound, drug | Bathophenanthrolinedisulfonic acid disodium salt hydrate (BPS) | Sigma-Aldrich | 146617–1 G | Iron chelator in media |
| Other | Filter bottle, 0.22 um PES, 54.5 cm² | Corning | 431098 | Media sterilization |
| Other | EVOS II microscope | Thermo Fisher | Not specified | Used for all imaging |
| Other | EVOS Light Cube GFP 2.0 | Invitrogen Thermo Fisher | AMEP4951 | FITC/GFP filter, fluorescence imaging |
| Other | EVOS Light Cube TxRed 2.0 | Invitrogen Thermo Fisher | AMEP4955 | TxRed/mCherry filter, fluorescence imaging |
| Chemical compound, drug | Ascorbic acid | LabChem | LC115309 | 20 mM in fluorescence media to reduce photobleaching |
| Software, algorithm | ImageJ | NIH | https://imagej.nih.gov | Manual image analysis |
| Software, algorithm | GraphPad Prism | GraphPad | https://graphpad.com | Plotting and statistics |
| Software, algorithm | Excel | Microsoft | https://office.com | Data management & plotting |
| Software, algorithm | OASIS 2 | Han et al., 2016 | https://sbi.postech.ac.kr/oasis2/ | RLS survival curves |
| Chemical compound, drug | Phen Green FL, Diacetate | Thermo Fisher | P6763 | Iron measurement, 5 µg/mL |
| Chemical compound, drug | 1,10-Phenanthroline | Sigma-Aldrich | 131377–5 G | Iron chelation, 1 mM |
| Other | LSR II | BD Biosciences | Not specified | For FITC/TxRed flow cytometry |
Additional files
-
Supplementary file 1
Table of Yeast strains used in this study.
- https://cdn.elifesciences.org/articles/108892/elife-108892-supp1-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/108892/elife-108892-mdarchecklist1-v1.pdf
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Overexpression of Ssd1 and calorie restriction extend yeast replicative lifespan by preventing deleterious age-dependent iron uptake
eLife 14:RP108892.
https://doi.org/10.7554/eLife.108892.3
