Figures and data
AMPK suppresses senescence through mitochondrial Acetyl-CoA import pathways
A: Experimental scheme for analysis of replicatively aged yeast B: Impact of SAK1 overexpression on Tom70-GFP (marker of senescence onset) and WGA (marker of replicative age). p-values calculated by two way ANOVA, n=7 C: Separation of wild type and PGPD-SAK1 low senescence (high WGA low Tom70-GFP) and high senescence (low WGA high Tom70-GFP) populations by flow cytometry, and quantification of low senescence population at 48 h. p-value calculated by t test, n=7 D: Comparison of gene expression profiles of wild type and PGPD-SAK1 cells at log phase and after 48 h ageing by mRNA-seq. Data is an average of 3 biological replicates, significantly different genes (p<0.01 by DEseq2) are shown in purple, genes both substantially (>3x indicated by dotted lines) and significantly different are highlighted in green. Substantially and significantly upregulated genes are annotated. E: Simple schematic of the relationship between AMPK, mitochondria and glycolysis pathways. Central glucose processing by glycolysis leading to fermentation is shown by blue arrows, AMPK activated pathways as purple arrows. Key enzymes in this study are shown in red. F: Representative flow plots and quantification of the low senescence population at 48 h in wild type and PGPD-SAK1 mutants lacking the carnitine shuttle (cat2Δ) and mutants lacking both the glyoxylate cycle (mls1Δ) and cat2Δ. p-values calculated by one way ANOVA, n=3-4
Combining AMPK activity and fatty acid synthesis to avoid senescence
A: Schematic highlighting the regulation of fatty acid synthesis by AMPK. Key proteins for this figure are highlighted in red. B: Representative flow plots and quantification of the low senescence population in acc1S1157A, PGPD-SAK1 and A2A at 48 h. p values calculated by one way ANOVA, n=8-9 C: Population medians for Tom70-GFP and WGA in acc1S1157A, PGPD-SAK1 and A2A at log phase, 24 h and 48 h. p values calculated by two way ANOVA, n=7-9 D: Representative flow plots of wild type and A2A at matched average replicative age (48 h for wild type versus 40 h for A2A), with quantification of the low senescence population and of median WGA (bud scars). p values calculated by one way ANOVA, n=4-5 E: Percentage cell viability in cultures aged for 40 and 48 h. p value calculated by t test, n=4. F: Size of colonies formed in 24 h on a YPD plate by log phase and age-matched wild type and A2A. Only cells that eventually formed colonies within 3 days were included to ensure all tested cells were viable. p values calculated by two way ANOVA, n=14-39 G: Size of colonies formed in 24 h on a YPD plate by log phase and 48 h-aged wild type, acc1S1157A, PGPD-SAK1 and A2A. Only cells that eventually formed colonies within 3 days were included to ensure all tested cells were viable. p values calculated by two way ANOVA, n=15-61
Separate classes of A2A cells depend on respiration and fatty acid availability
A: Schematic of acetyl coenzyme A biosynthesis and the emergence of a negative feedback loop through AMPK regulation by Sip2. Key proteins for this figure are highlighted in red. B: Western blot analysis of acetyl lysine on H3 in log phase wild type, acc1S1157A, PGPD-SAK1 and A2A cells. Membranes were probed with mouse anti-H3 and rabbit anti-pan-acetyl lysine using a 2-colour detection system. p values calculated by one-way ANOVA, n=3-4. C: Representative flow plots and quantification of low senescence population in sip2Δ and sip2Δ PGPD-SAK1 at 48 h. p-values calculated by t-test, n=5 D: Representative flow plots and quantification of low senescence population in wild type and ald6Δ at 48 h. Population medians are also shown for Tom70-GFP and WGA in wild type and ald6Δ at 48 h. p values calculated by t-test, n=5-6 E: Size of colonies formed in 24 h on a YPD plate by log phase and 48 h-aged wild type and ald6Δ. Only cells that eventually formed colonies within 3 days were included to ensure all tested cells were viable. p values calculated by one-way ANOVA, n=36-57 F: Representative flow plots and quantification of low senescence population in A2A mutants lacking both carnitine acetyltransferase activity (cat2Δ) and the glyoxylate cycle (mls1Δ) at 48 h. Arrows indicate two populations, p-value calculated by t-test, n=4 G: Size of colonies formed in 24 h on a YPD plate by log phase and 40 h-aged A2A and A2A cat2Δ mls1Δ. Only cells that eventually formed colonies within 3 days were included to ensure all tested cells were viable. Arrows indicate two populations, p values calculated by two way ANOVA, n=29-55
Fatty acids are particularly important in ageing cells that do not respond to AMPK activation
A:Schematic of potential mechanisms for the beneficial effect of ACC1 activity: Inhibition of mTOR mediated by malonyl coenzyme A, the direct product of Acc1, or efficient synthesis of fatty acids, compared to a null hypothesis that Acc1 acts to remove cytosolic acetyl-CoA, for example if these cells are respiratory-deficient. B: Representative flow plots and quantification of low senescence population at 48 h in sip2Δ PGPD-SAK1 acc1S1157A and sip2Δ PGPD-SAK1 supplemented with 2.5 µM cerulenin (condition 3), 10 µM cerulenin + 0.04% oleic acid + 0.04% Tween 80 (condition 4) or 0.04% oleic acid + 0.04% Tween 80 (condition 5). p-values calculated by one-way ANOVA, n=2-3 C: Representative flow plots and quantification of low senescence population at 48 h in wild type, PGPD-SAK1 and A2A supplemented with 0.04% oleic acid + 0.04% Tween 80. p-values calculated by one-way ANOVA, n=4 D: Size of colonies formed in 24 h on a YPD plate by log phase and 48 h-aged wild type, acc1S1157A, PGPD-SAK1 and A2A supplemented with 0.04% oleic acid + 0.04% Tween 80. Only cells that eventually formed colonies within 3 days were included to ensure all tested cells were viable. p values calculated by one-way ANOVA, n=19-52
Model of different classes of ageing mediated by Acetyl-CoA and lipid metabolism
Schematic demonstrating the two wild type senescence fates. (i) Adaptation to future scarcity by synthesising excess cytosolic Acetyl-CoA to store as much energy as possible in lipids against future scarcity, and (ii) Adaptation to rapid growth by maximising growth from the current food supply by synthesising the bare minimum of cytosolic Acetyl-CoA.
Increased AMPK activity suppresses senescence in a subset of cells
A: mRNA expression level of constitutive over-expression of the upstream kinase Sak1 using a PGPD promoter fused to SAK1 at log phase, 24 h and 48 h. p-values calculated by 1 way ANOVA, n=3 B: Impact of SAK1 overexpression on Rpl13a-mCherry in young and aged cells. p-values calculated by two way ANOVA, n=7 C: Representative flow plots and quantification of the low senescence population at 48 h in wild type and PGPD-SAK1 mutants lacking the glyoxylate cycle (mls1Δ) or succinate import (sfc1Δ). p-values calculated by one way ANOVA, n=4-6 D: Representative flow plots and quantification of wild type and PGPD-SAK1 low senescence populations at 48 h in mitochondrial mutants lacking a functional electron transport chain (cox9Δ) or pyruvate import (mpc1Δ). p-values calculated by one way ANOVA, n=4-5
Combining AMPK activity and fatty acid synthesis to avoid senescence
A: Growth curve of wild type and A2A cells in YPD media starting from log-phase pre-cultures. B: Lifespan measured in YPD media for wild type and A2A cells based on %age of viable cells remaining at each time point; n = 5. C: Population medians for Rpl13a-mCherry in wild type, acc1S1157A, PGPD-SAK1 and A2A at log phase, 24 h and 48 h. p values calculated by two way ANOVA, n=7-10. D: Population medians for Tom70-GFP and WGA (bud scars) in wild type, PGPD-SAK1, and A2A at log phase, 24 h and 48 h made from a compilation of experiments performed during this study. p values calculated by two way ANOVA, n=27-38. E: Manual bud scar counts of log phase and 48 h-aged wild type and A2A cells. p values calculated by one way ANOVA, n=14-20
Genetic evidence that cytosolic Acetyl-CoA promotes senescence
A: Representative flow plots of the low senescence population at 48 h in sip23R, sip23R PGPD-SAK1 and sip23R A2A. B: Representative flow plots and quantification of low senescence population in wild type and sip2Δ PGPD-SAK1 ald6Δ at 48 h. Population medians are also shown for Tom70-GFP and WGA in wild type and ald6Δ at 48 h. p values calculated by t-test, n=5-6 C: Representative flow plots and quantification of low senescence population at 48 h in A2A mutants lacking the electron transport chain (cox9Δ), p-value calculated by t-test, n=3-4.
Fatty acids in senescence.
A: Population medians for Tom70-GFP and WGA at 48 h in sip2Δ PGPD-SAK1 acc1S1157A and sip2Δ PGPD-SAK1 supplemented with cerulenin and/or 0.04% oleic acid + 0.04% Tween 80. p-values calculated by one-way ANOVA, n=2-3 B: Manual bud scar counts of 48 h-aged sip2Δ PGPD-SAK1 cells with and without 0.04% oleic acid. p values calculated by one way ANOVA, n=20 C: Population medians for Tom70-GFP and WGA at 48 h in wild type, PGPD-SAK1 and A2A with and without 0.04% oleic acid + 0.04% Tween 80. p-values calculated by one-way ANOVA, n=4 D: Size of colonies formed in 24 h on a YPD plate by log phase and 48 h-aged ald6Δ cells with 0.04% oleic acid + 0.04% Tween 80. Only cells that eventually formed colonies within 3 days were included to ensure all tested cells were viable. p values calculated by one-way ANOVA, n=28-53
Strains used in this work.
All strains are diploid derivatives of the MEP system (47). TOM70-GFP and RPL13a-mCherry markers are heterozygous to avoid growth defect.
