A new Schizosaccharomyces pombe chronological lifespan assay reveals that caloric restriction promotes efficient cell cycle exit and extends longevity.
ABSTRACT: We describe a new chronological lifespan (CLS) assay for the yeast Schizosaccharomyces pombe. Yeast CLS assays monitor the loss of cell viability in a culture over time, and this new assay shows a continuous decline in viability without detectable regrowth until all cells in the culture are dead. Thus, the survival curve is not altered by the generation of mutants that can grow during the experiments, and one can monitor the entire lifespan of a strain until the number of viable cells has decreased over 10(6)-fold. This CLS assay recapitulates the evolutionarily conserved features of lifespan shortening by over nutrition, lifespan extension by caloric restriction, increased stress resistance of calorically restricted cells and lifespan control by the AKT kinases. Both S. pombe AKT kinase orthologs regulate CLS: loss of sck1(+) extended lifespan in over nutrition conditions, loss of sck2(+) extended lifespan under both normal and over nutrition conditions, and loss of both genes showed that sck1(+) and sck2(+) control different longevity pathways. The longest-lived S. pombe cells showed the most efficient cell cycle exit, demonstrating that caloric restriction links these two processes. This new S. pombe CLS assay will provide a valuable tool for aging research.
Project description:The fission yeast Schizosaccharomyces pombe carries a cytosine 5-methyltransferase homolog of the Dnmt2 family (termed pombe methyltransferase 1, Pmt1), but contains no detectable DNA methylation. Here, we found that Pmt1, like other Dnmt2 homologs, has in vitro methylation activity on cytosine 38 of tRNA(Asp) and, to a lesser extent, of tRNA(Glu), despite the fact that it contains a non-consensus residue in catalytic motif IV as compared with its homologs. In vivo tRNA methylation also required Pmt1. Unexpectedly, however, its in vivo activity showed a strong dependence on the nutritional status of the cell because Pmt1-dependent tRNA methylation was induced in cells grown in the presence of peptone or with glutamate as a nitrogen source. Furthermore, this induction required the serine/threonine kinase Sck2, but not the kinases Sck1, Pka1 or Tor1 and was independent of glucose signaling. Taken together, this work reveals a novel connection between nutrient signaling and tRNA methylation that thus may link tRNA methylation to processes downstream of nutrient signaling like ribosome biogenesis and translation initiation.
Project description:BACKGROUND:Ketosis in dairy cattle has been shown to cause a high morbidity in the farm and substantial financial losses to dairy farmers. Ketosis symptoms, however, are difficult to identify, therefore, the amount of ketone bodies (mainly ?-hydroxybutyric acid, BHB) is used as an indicator of subclinical ketosis in cows. It has also been shown that milk BHB concentrations have a strong correlation with ketosis in dairy cattle. Mid-infrared spectroscopy (MIR) has recently became a fast, cheap and high-throughput method for analyzing milk components. The aim of this study was to perform a genome-wide association study (GWAS) on the MIR-predicted milk BHB to identify genomic regions, genes and pathways potentially affecting subclinical ketosis in North American Holstein dairy cattle. RESULTS:Several significant regions were identified associated with MIR-predicted milk BHB concentrations (indicator of subclinical ketosis) in the first lactation (SCK1) and second and later lactations (SCK2) in Holstein dairy cows. The strongest association was located on BTA6 for SCK1 and BTA14 on SCK2. Several SNPs on BTA6 were identified in regions and variants reported previously to be associated with susceptibility to ketosis and clinical mastitis in Jersey and Holstein dairy cattle, respectively. One highly significant SNP on BTA14 was found within the DGAT1 gene with known functions on fat metabolism and inflammatory response in dairy cattle. A region on BTA6 and three SNPs on BTA20 were found to overlap between SCK1 and SCK2. However, a novel region on BTA20 (55-63?Mb) for SCK2 was also identified, which was not reported in previous association studies. Enrichment analysis of the list of candidate genes within the identified regions for MIR-predicted milk BHB concentrations yielded molecular functions and biological processes that may be involved in the inflammatory response and lipid metabolism in dairy cattle. CONCLUSIONS:The results of this study confirmed several SNPs and genes identified in previous studies as associated with ketosis susceptibility and immune response, and also found a novel region that can be used for further analysis to identify causal variations and key regulatory genes that affect clinical/ subclinical ketosis.
Project description:CTP synthase (CTPS) has been demonstrated to form evolutionarily-conserved filamentous structures termed cytoophidia whose exact cellular functions remain unclear, but they may play a role in intracellular compartmentalization. We have previously shown that the mammalian target of rapamycin complex 1 (mTORC1)-S6K1 pathway mediates cytoophidium assembly in mammalian cells. Here, using the fission yeast Schizosaccharomyces pombe as a model of a unicellular eukaryote, we demonstrate that the target of rapamycin (TOR)-signaling pathway regulates cytoophidium formation (from the S. pombe CTPS ortholog Cts1) also in S. pombe Conducting a systematic analysis of all viable single TOR subunit-knockout mutants and of several major downstream effector proteins, we found that Cts1 cytoophidia are significantly shortened and often dissociate when TOR is defective. We also found that the activities of the downstream effector kinases of the TORC1 pathway, Sck1, Sck2, and Psk1 S6, as well as of the S6K/AGC kinase Gad8, the major downstream effector kinase of the TORC2 pathway, are necessary for proper cytoophidium filament formation. Interestingly, we observed that the Crf1 transcriptional corepressor for ribosomal genes is a strong effector of Cts1 filamentation. Our findings connect TOR signaling, a major pathway required for cell growth, with the compartmentalization of the essential nucleotide synthesis enzyme CTPS, and we uncover differences in the regulation of its filamentation among higher multicellular and unicellular eukaryotic systems.
Project description:Genetic factors underlying aging are remarkably conserved from yeast to human. The fission yeast Schizosaccharomyces pombe is an emerging genetic model to analyze cellular aging. Chronological lifespan (CLS) has been studied in stationary-phase yeast cells depleted for glucose, which only survive for a few days. Here, we analyzed CLS in quiescent S. pombe cells deprived of nitrogen, which arrest in a differentiated, G0-like state and survive for more than 2 months. We applied parallel mutant phenotyping by barcode sequencing (Bar-seq) to assay pooled haploid deletion mutants as they aged together during long-term quiescence. As expected, mutants with defects in autophagy or quiescence were under-represented or not detected. Lifespan scores could be calculated for 1199 mutants. We focus the discussion on the 48 most long-lived mutants, including both known aging genes in other model systems and genes not previously implicated in aging. Genes encoding membrane proteins were particularly prominent as pro-aging factors. We independently verified the extended CLS in individual assays for 30 selected mutants, showing the efficacy of the screen. We also applied Bar-seq to profile all pooled deletion mutants for proliferation under a standard growth condition. Unlike for stationary-phase cells, no inverse correlation between growth and CLS of quiescent cells was evident. These screens provide a rich resource for further studies, and they suggest that the quiescence model can provide unique, complementary insights into cellular aging.
Project description:Schizosaccharomyces pombe detects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 G? binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2(K270E)-activated G? relative to Gpa2(+) G?. Deletion of sck1(+) or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2(+) or Gpa2(K270E), suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2(K270E) GDP-GTP exchange rate mutant, GTPase-defective Gpa2(R176H) weakly binds Sck1 in the two-hybrid screen and a deletion of sck1(+) in a Gpa2(R176H) strain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deleting sck1(+) in a gpa2? strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allow S. pombe to produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.
Project description:Target of rapamycin (TOR), an evolutionarily conserved serine/threonine protein kinase, plays pivotal roles in several important cellular processes in eukaryotes. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1), which includes Tor2 as a catalytic subunit, manages the switch between cell proliferation and differentiation by sensing nutrient availability. However, little is known about the direct target of TORC1 that plays key roles in nutrient-dependent TORC1 signaling in fission yeast. Here we report that in fission yeast, three AGC kinase family members, named Psk1, Sck1 and Sck2, which exhibit high homology with human S6K1, are phosphorylated under nutrient-rich conditions and are dephosphorylated by starvation conditions. Among these, Psk1 is necessary for phosphorylation of ribosomal protein S6. Furthermore, Psk1 phosphorylation is regulated by TORC1 in nutrient-dependent and rapamycin-sensitive manners in vivo. Three conserved regulatory motifs (the activation loop, the hydrophobic and the turn motifs) in Psk1 are phosphorylated and these modifications are required for Psk1 activity. In particular, phosphorylation of the hydrophobic motif is catalyzed by TORC1 in vivo and in vitro. Ksg1, a homolog of PDK1, is also important for Psk1 phosphorylation in the activation loop and for its activity. The TORC1 components Pop3, Toc1 and Tco89, are dispensable for Psk1 regulation, but disruption of pop3(+) causes an increase in the sensitivity of TORC1 to rapamycin. Taken together, these results provide convincing evidence that TORC1/Psk1/Rps6 constitutes a nutrient-dependent signaling pathway in fission yeast.
Project description:Yeast chronological lifespan (CLS) is extended by multiple genetic and environmental manipulations, including caloric restriction (CR). Understanding the common changes in molecular pathways induced by such manipulations could potentially reveal conserved longevity mechanisms. We therefore performed gene expression profiling on several long-lived yeast populations, including anade4?mutant defective in de novo purine (AMP) biosynthesis, and a calorie restricted WT strain. CLS was also extended by isonicotinamide (INAM) or expired media derived from CR cultures. Comparisons between these diverse long-lived conditions revealed a common set of differentially regulated genes, several of which were potential longevity biomarkers. There was also enrichment for genes that function in CLS regulation, including a long-lived adenosine kinase mutant (ado1?) that links CLS regulation to the methyl cycle and AMP. Genes co-regulated between the CR and ade4? conditions were dominated by GO terms related to metabolism of alternative carbon sources, consistent with chronological longevity requiring efficient acetate/acetic acid utilization. Alternatively, treating cells with isonicotinamide (INAM) or the expired CR media resulted in GO terms predominantly related to cell wall remodeling, consistent with improved stress resistance and protection against external insults like acetic acid. Acetic acid therefore has both beneficial and detrimental effects on CLS.
Project description:Methionine restriction (MR) is one of only a few dietary manipulations known to robustly extend healthspan in mammals. For example, rodents fed a methionine-restricted diet are up to 45% longer-lived than control-fed animals. Tantalizingly, ongoing studies suggest that humans could enjoy similar benefits from this intervention. While the benefits of MR are likely due, at least in part, to improved cellular stress tolerance, it remains to be determined exactly how MR extends organismal healthspan. In previous work, we made use of the yeast chronological lifespan (CLS) assay to model the extension of cellular lifespan conferred by MR and explore the genetic requirements for this extension. In these studies, we demonstrated that both dietary MR (D-MR) and genetic MR (G-MR) (i.e., impairment of the cell's methionine biosynthetic machinery) significantly extend the CLS of yeast. This extension was found to require the mitochondria-to-nucleus retrograde (RTG) stress signaling pathway, and was associated with a multitude of gene expression changes, a significant proportion of which was also dependent on RTG signaling. Here, we show work aimed at understanding how a subset of the observed expression changes are causally related to MR-dependent CLS extension. Specifically, we find that multiple autophagy-related genes are upregulated by MR, likely resulting in an increased autophagic capacity. Consistent with activated autophagy being important for the benefits of MR, we also find that loss of any of several core autophagy factors abrogates the extended CLS observed for methionine-restricted cells. In addition, epistasis analyses provide further evidence that autophagy activation underlies the benefits of MR to yeast. Strikingly, of the many types of selective autophagy known, our data clearly demonstrate that MR-mediated CLS extension requires only the autophagic recycling of mitochondria (i.e., mitophagy). Indeed, we find that functional mitochondria are required for the full benefit of MR to CLS. Finally, we observe substantial alterations in carbon metabolism for cells undergoing MR, and provide evidence that such changes are directly responsible for the extended lifespan of methionine-restricted yeast. In total, our data indicate that MR produces changes in carbon metabolism that, together with the oxidative metabolism of mitochondria, result in extended cellular lifespan.
Project description:Schizosaccharomyces pombe regulates intracellular cAMP levels, and thus cAMP-dependent protein kinase (PKA) activity, in response to changes in nutrient conditions. Mutations in any of eight git genes inhibit glucose repression of fbp1 transcription, alter the cell morphology, and cause a reduction in the growth rate. The eight git genes encode components of an adenylate cyclase activation pathway, adenylate cyclase itself, and the catalytic subunit of PKA. Three of these genes have been identified in other studies as regulators of meiosis. Here we show that the sck1 gene, cloned as a high copy number suppressor of a mutation in git3, is able to suppress the defects conferred by a mutation in any of these git genes. Sequence analysis suggests that sck1 encodes a protein most closely related to the Saccharomyces cerevisiae SCH9 protein kinase that had previously been identified as a high copy number suppressor of mutations in S. cerevisiae that reduce or eliminate PKA activity. Disruption of the sck1 gene causes a significant delay in exit from stationary phase when combined with a disruption of the pka1 (git6) gene encoding the catalytic subunit of PKA. However, the sck1 disruption by itself has little or no effect upon fbp1 transcription, meiosis, or exit from stationary phase, and does not enhance the constitutive fbp1 transcription observed in a pka1 mutant. Therefore, sck1 appears to function in a redundant fashion to pka1, but to varying degrees, in the pathways regulated by pka1.
Project description:Chronologically aging yeast cells are prone to adaptive regrowth, whereby mutants with a survival advantage spontaneously appear and re-enter the cell cycle in stationary phase cultures. Adaptive regrowth is especially noticeable with short-lived strains, including those defective for SNF1, the homolog of mammalian AMP-activated protein kinase (AMPK). SNF1 becomes active in response to multiple environmental stresses that occur in chronologically aging cells, including glucose depletion and oxidative stress. SNF1 is also required for the extension of chronological lifespan (CLS) by caloric restriction (CR) as defined as limiting glucose at the time of culture inoculation. To identify specific downstream SNF1 targets responsible for CLS extension during CR, we screened for adaptive regrowth mutants that restore chronological longevity to a short-lived snf1? parental strain. Whole genome sequencing of the adapted mutants revealed missense mutations in TPR motifs 9 and 10 of the transcriptional co-repressor Cyc8 that specifically mediate repression through the transcriptional repressor Mig1. Another mutation occurred in MIG1 itself, thus implicating the activation of Mig1-repressed genes as a key function of SNF1 in maintaining CLS. Consistent with this conclusion, the cyc8 TPR mutations partially restored growth on alternative carbon sources and significantly extended CLS compared to the snf1? parent. Furthermore, cyc8 TPR mutations reactivated multiple Mig1-repressed genes, including the transcription factor gene CAT8, which is responsible for activating genes of the glyoxylate and gluconeogenesis pathways. Deleting CAT8 completely blocked CLS extension by the cyc8 TPR mutations on CLS, identifying these pathways as key Snf1-regulated CLS determinants.