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There is growing evidence that stochastic events play an important role in determining individual longevity. Studies in model organisms have demonstrated that genetically identical populations maintained under apparently equivalent environmental conditions display individual variation in lifespan that can be modeled by the Gompertz-Makeham law of mortality. Here we report that within genetically identical haploid and diploid wild type populations, shorter-lived cells tend to arrest in a budded state, while cells that arrest in an unbudded state are significantly longer-lived. This relationship is particularly notable in diploid BY4743 cells, where mother cells that arrest in a budded state have a shorter mean lifespan (25.6 vs. 35.6) and larger coefficient of variance with respect to individual lifespan (0.42 vs. 0.32) than cells that arrest in an unbudded state. Mutations that cause genomic instability tend to shorten lifespan and increase the proportion of the population that arrest in a budded state. These observations suggest that randomly occurring damage may contribute to stochasticity during replicative aging by causing a subset of the population to terminally arrest prematurely in the S or G2 phase of the cell cycle.
Aging is an inherently stochastic process. Even among genetically homogeneous populations under nearly identical environmental conditions, there is variation in individual longevity and healthspan (Herndon, et al., 2002, Kirkwood, et al., 2005, Rea, et al., 2005, Pincus, et al., 2011). The budding yeast Saccharomyces cerevisiae provides a useful model system for studying the genetic and environmental factors that underlie stochasticity of aging.
In the yeast replicative aging paradigm, replicative life span (RLS) is defined as the number of daughter cells produced prior to irreversible cell cycle arrest under nutrient replete conditions (Mortimer & Johnston, 1959, Kaeberlein, 2010). Similar to other model organisms, genetically identical yeast within a population do not senescence at the same age (Kaeberlein, et al., 2001). Replicative senescence in yeast typically occurs in the G1 phase of the cell cycle, where cells arrest in the unbudded state; however, even in genetically homogenous yeast populations a significant proportion (often 40% or higher) of yeast arrest with small or large buds, or in some cases with a cluster of interconnected cells(Pichova, et al., 1997, Nestelbacher, et al., 1999, McVey, et al., 2001, Merker & Klein, 2002, Falcon & Aris, 2003). In yeast, bud emergence occurs at the beginning of S phase (Hartwell, et al., 1974, Di Talia, et al., 2007), suggesting that cells that have a budded terminal morphology have failed to exit the cell cycle in G1 and have progressed into S phase.
Determining the terminal morphology of yeast mother cells is experimentally straightforward and can be easily accomplished by microscopic visualization of cells at the end of the replicative life span experiment. Despite the experimental simplicity of the assay, there is currently only limited data available on this subject, comprising terminal morphology data for a total of approximately 250 wild type mother cells across three strain backgrounds (Table 1). The relative lack of datain this area likely stems from the fact that tracking terminal morphology of individual yeast mother cells is a labor intensive process generally requiring full replicative life span analysis involving manual micromanipulation of daughter cells from mother cells(Steffen, et al., 2009). For this reason, prior studies have identified only a few specific mutations that alter the proportion of cells with unbudded or budded terminal morphologies. For example, deletion of the RecQ helicase gene, SGS1, increases the proportion of budded cells at senescence.(Gangloff, et al., 2000, Johnson, et al., 2001). Deletion of the gene encoding the RNA polymerase II component Hpr1 also results in a similar phenotype (Merker & Klein, 2002). These two genes share a function in promoting genomic stability at the ribosomal DNA cluster and elsewhere, and both mutants also have reduced replicative life span (Sinclair, et al., 1997, Defossez, et al., 1999, Merker & Klein, 2002).
We have recently begun collecting terminal morphology data for individual mother cells as part of an ongoing effort to quantify replicative life span for each of the viable haploid strains in the yeast ORF deletion collection(Kaeberlein & Kennedy, 2005, Kaeberlein, et al., 2005, Kaeberlein, et al., 2005). In the wild type background of the yeast ORF deletion collection, mother cells typically arrest in an unbudded state about two-thirds of the time; however, we have observed several mutations thatsignificantlyalter the percentage of budded yeast at the end of life. Here we present an initial report of these observations containing terminal morphology data for more than 5,000 individual S. cerevisiae mother cells. These data indicate that, within the wild type population, mother cells that terminally arrest in a budded state tend to have shorter replicative life spans than cells that arrest in the unbudded state. This is most notable in the diploid population where budded cell cycle arrest contributes substantially to individual variation with respect to replicative lifespan. We expand upon prior evidence that a budded terminal morphology is associated with genomic instability by showing that cells lacking DNA repair enzymes arrest with a significantly higher percentage of budded cells, as do cells lacking the sirtuin histone deacetylase enzyme Sir2.
End-of-life terminal morphology wasrecorded and analyzed for wild type haploid MATa (BY4741, 1792 cells), wild type haploid MATα (BY4742, 3794 cells), and wild type diploid (BY4743, 529 cells) yeast subjected to replicative lifespan analysis under standard conditions(Steffen, et al., 2009). In all three backgrounds a majority of cells arrested in a budded state (Table 2). Likewise, in each case, the cells that arrested in a budded state lived shorter on average than those that arrested in an unbudded state (Figure 1). The difference in longevity of cells that arrest in an unbudded state versus a budded state is particularly apparent in the diploid cell population, where cells showing a budded terminal morphology had an average replicative life span of 26.6 compared to 35.6 for cells that arrest in the unbudded state. Interestingly, the coefficient of variance was larger for diploid cells that arrest in a budded state (0.42) relative to cells that arrest in an unbudded state (0.32), indicating that this premature cell cycle arrest contributes to individual variation in lifespan within this population.
A prior study found that plasmid accumulation can reduce replicative life span in the W303AR5 strain background and, at least in some cases, alter the terminal morphology distribution in this strain (Falcon & Aris, 2003). We have recently been using the pMoBY CEN marked plasmid collection (Ho, et al., 2009) to perform overexpression studies for replicative life span. Consistent with the results of Falcon and Aris(Falcon & Aris, 2003), we find that BY4742 wild type cells containing a CEN marked pMoBY vector are slightly short-lived relative to untransformed cells and also tend to arrest more frequently as budded cells (Table 3). As in the prior cases, the subset of plasmid-containing cells arresting in the budded state are shorter-lived than those that arrest in the unbudded state.
Sincethe longest-lived wild type cells arrest in the unbudded state, we hypothesized that mutations that extend wild type replicative life span might also cause more cells to arrest in the unbudded state.To test this hypothesis, we examined the terminal morphologies of long-lived mutants for which we had terminal morphology data for at least 60 mother cells. Gpr1, Tor1, and Sch9 are important nutrient sensors that have been previously implicated in mediating increased replicative life span in response to dietary restriction, and deletion of the genes coding for any one of these proteins is sufficient to increase life span(Lin, et al., 2000, Fabrizio, et al., 2004, Kaeberlein, et al., 2004, Kaeberlein, et al., 2005). In the case of gpr1Δand sch9Δcells, the percentage of cells arresting in the unbudded state was significantly increased relative to wild type BY4742 cells (Table 3). In the case of tor1Δcells, there was a trend toward a higher percentage of unbudded terminal cells that did not reach statistical significance.
Theassociation between increased unbudded terminal morphology and longevity was not apparent in all long-lived mutants, however. For example, deletion of the isocitrate dehydrogenase gene IDH2or the meiotic gene SPS1 also increases replicative life span(Kaeberlein, et al., 2005, Managbanag, et al., 2008). Yet, idh2Δand sps1Δcells showed a significant decrease in the percent of cells with unbudded terminal morphology (Table 3). Unlike the other long-lived mutants tested, both idh2Δand sps1Δcells are also respiratory deficient (Merz & Westermann, 2009), but whether this defect is related to the terminal morphology phenotype remains unclear. In every genotype examined, however, the cells that arrested in the unbudded state trended towardlonger RLS than those that arrested in the budded state (Table 3), and in most genotypes unbudded cells lived significantly longer (Figure 2).
The ribosomal DNA (rDNA) has been repeatedly shown to be an important locus determining replicative longevity of yeast cells (Sinclair & Guarente, 1997, Ganley, et al., 2009, Lindstrom, et al., 2011). The yeast rDNA exists as a tandem repeat of 50-200 copies of a 9.1 kb sequence (Petes & Botstein, 1977, Rustchenko & Sherman, 1994). This repeated structure leads to elevated rates of homologous recombination within the rDNA. The replication fork block protein Fob1 has been previously shown to limit replicative life span by promoting rDNA instability and the resulting formation of extrachromosomal rDNA circles (Defossez, et al., 1999). The sirtuin histone deacetylase Sir2 antagonizes Fob1 by promoting enhanced rDNA stability and reduced formation of extrachromosomal rDNA circles (Kaeberlein, et al., 1999). Deletion of FOB1 extends replicative life span while deletion of SIR2 shortens it. Correspondingly, deletion of FOB1 caused a greater proportion of cells to terminally arrest in the unbudded state, and deletion of SIR2 caused more cells to terminally arrest in the budded state (Table 3).
Deletion of FOB1 in sir2Δ cells restores replicative life span to the level of wild type mother cells (Kaeberlein, et al., 1999) and enhances the ability of dietary restriction to increase replicative life span (Kaeberlein, et al., 2004, Delaney, et al., 2011). Unexpectedly, sir2Δ fob1Δcells under both control and dietary restricted conditions arrested in a budded state more frequently than wild type cells (Table 3). Thus, deletion of FOB1 only partially suppresses the terminal morphology defect of sir2Δ cells and DR has no significant effect on terminal morphology in sir2Δ fob1Δcells, despite the large increase in replicative life span. In the BY4742 wild-type background, DR does not yield as great a lifespan increase and no significant change in terminal morphology is observed.
Since two prior studies had shownan increased proportion of senescent buddedcells in mutants with reduced genomic stability(McVey, et al., 2001, Merker & Klein, 2002), we tested whether DNA repair deficient mutants would also show altered terminal morphologies.For this experiment, we examined four highly conserved DNA repair genes: RAD50, RAD51, RAD52, and RAD53. Rad53 acts downstream of the Mec1 kinase to signal for G1 arrest and does not directly contribute to DNA damage repair (Sidorova & Breeden, 1997, Ma, et al., 2006). Rad50 is part of the Mre11 complex which acts in multiple DNA repair and G1 arrest signaling pathways, including homologous recombination, sensing of double stranded breaks, and suppression of chromosomal rearrangements (reviewed in (Symington, 2002, Krogh & Symington, 2004)). Rad51 and Rad52 are both directly involved in the repair of DNA double stranded breaks (New, et al., 1998, Shinohara & Ogawa, 1998, Krogh & Symington, 2004). In each case, the gene deletion reduced replicative life span (Figure 3), and cells arrested with a budded morphology greater than 70% of the time (Table 3). Even in these cases of decreased lifespan, the trend toward increased RLS among those mother cells that arrested in an unbudded state was preserved, although it did not achieve statistical significance on an individual strain basis.
Cells that arrest in a budded state can be further sub-classified based on whether their final arrest occurs with a small bud, a large (or symmetric) bud, or multiple daughter and/or granddaughter cells (clustered) that cannot be separated by micromanipulation (Figure 3B). The subtypes of budded arrest also differ in radΔ cells compared to wild type (Figure 3C). The percentage of cells arresting in the budded clustered and large-budded morphological categories was significantly increased in all radΔ mutants examined.
The stochastic nature of aging is evident in yeast from the variation in both longevity and terminal morphology of genetically identical mother cells maintained under essentially identical conditions. Here we have demonstrated a novel interaction between longevity and terminal morphology in wild type cells, by showing that, within the same population, mother cells arresting in the budded state tend to be shorter lived than mother cells arresting in the unbudded state. This was true in both haploid backgrounds, as well as the diploid background of the yeast deletion collection.This relationship also held true across severalmutantbackgrounds showing altered distributions of terminal morphologies. In some cases the instability which gives rise to higher terminal budding rates may be limiting for lifespan, since we observed an inverse correlation between strain end-of-life budding rates and overall mean lifespan (Figure 4).Taken together, these observations suggest that within a population of yeast cells, some individuals will succumb to a life span-limiting event that corresponds with budded cell cycle arrest, while others will escape this fate and achieve their full replicative potential.
The relationship between stochastic aging events and terminal morphology is most evident in the diploid BY4743 population. The coefficient of variation for individual cell lifespan was dramatically reduced in the unbudded cell population, relative to cells that arrested in the budded state. A similar relationship was not detected in either haploid background, perhaps due to the smaller difference in absolute lifespan between the budded and unbudded cell sets; however, when the results from all different genotypes were examined, those cells that arrested in a budded state tended to have a higher coefficient of variance in their lifespan compared to unbudded cells (Figure 5). This higher variation is consistent with the interpretation that terminal buds result from stochastically arising damage in the mother cell that contributes to individual variation in longevity.
What might be the nature of the event(s) resulting in premature arrest in the budded state? Prior data have suggested that genomic instability at the rDNA is associated with a higher percentage of mother cells arresting in a budded state(McVey, et al., 2001, Merker & Klein, 2002), and our data are consistent with this observation. Genomic instability or defects in DNA repair outside of the rDNA also appear to be associated with a higher proportion of cells terminally arresting in the budded state, as evidenced from the terminal morphologies of rad50Δ, rad51Δ, rad52Δ, and rad53Δ cells. It is interesting to note that rad52Δ cells, in particular, have very low levels of rDNA recombination (Park, et al., 1999), suggesting that the aging-related damage that results in a non-G1 arrest need not be restricted to the rDNA, although the rDNA may be the primary site of such damage in otherwise wild type cells. During chronological aging, DRor deletion of the gene encoding the S6 kinase ortholog SCH9 decrease the percent of cells that arrest with buds, and this is associated with less replication stress(Weinberger, et al., 2010). We have similarly found a reduction in the percent of budded under conditions that extendchronological lifespan as well as subsequent replicative lifespan(Murakami, et al., 2012).
It is also of interest that sir2Δ mutant cells phenocopy the DNA damage sensitive radΔ cells with respect to terminal morphology, yet deletion of FOB1 only partially alleviates this phenotype, despite restoring life span and rDNA recombination rates to wild type levels. In addition to destabilizing the rDNA, deletion of SIR2 also results in loss of silencing near telomeres, and recent studies have suggested that this function of Sir2 may be particularly important for ensuring replicative longevity (Dang, et al., 2009, Chan, et al., 2011). Thus, it may be that the increase in terminal budding in cells lacking Sir2 occurs in response to a DNA damage signal originating from destabilized telomeres.
The observation that some long-lived mutants have a higher percentage of cells arrest in the unbudded state suggests that one path to longer life span may be achieved by reducing the proportion of cells dying from premature cell cycle arrest outside of G1. This is clearly not the only way to achieve life span extension, however, since both idh2Δand sps1Δcells are long-lived while also having a greater proportion of cells terminally arrest with buds. An even more striking example of this is the case of sir2Δ fob1Δcells subjected to dietary restriction. These mother cells live, on average, about 50% longer than wild type cells, but have a greater proportion of cells terminally arrest in the budded state. Although many strains in the yeast ORF deletion collection are known to carry secondary mutations(Cheng, et al., 2008, Steffen, et al., 2012), we do not believe that secondary mutations account for the observed terminal morphology distributions in the mutants described here, because we have independently re-derived a majority of the strains examined in this study.
At least some aspects of the genetic control of longevity in yeast are conserved in multicellular eukaryotes (Smith, et al., 2008, Fontana, et al., 2010, Longo, et al., 2012). We speculate that themechanisms underlying control of terminal morphology in yeast, as well as their stochastic nature, may also be relevant in mammalian cells.TheATR tumor suppressor pathway acts to prevent ectopic entry into S phase. Mec1 is the ATR homologue in yeast(Cimprich, et al., 1996) and it activates Rad53 to induce cell cycle arrest(Sweeney, et al., 2005, Ma, et al., 2006), in particular due to MMS induced DNA damage(Sidorova & Breeden, 1997). Rad53 has been implicated in modulating appropriate entry into S phase in unstressed conditions as well (Sidorova & Breeden, 2002). The finding that rad53Δ mutant cells have a high proportion of cells that arrest in the budded state supports the idea thatthis regulation is importantduring aging. Thus, yeast replicative lifespanmay provide a useful method for researching the mechanisms of cell cycle arrest in a model system without the confounding effects of background mutations found in immortal cell lines.
There is little doubt that apparently stochastic events occurring during aging play an important role in determining individual life span(Kirkwood, et al., 2005). In yeast, one form of this stochasticity arises from damage that causes some cells to arrest prematurely, an event that can be detected by a budded terminal morphology. This stochasticity can be modulated genetically, but in all cases examined was never completely removed. Although the precise molecular nature of the damage leading to non-G1 arrest during yeast aging is unclear, one potential contributing cause is DNA damage or genomic instability. There is abundant evidence that elevated levels of DNA damage and genomic instability induceearly cell death and progeroid phenotypes in mammals(Burtner & Kennedy, 2010, Kennedy, et al., 2012). It seems plausible that stochastic events occurring during normal aging cause some cells in mammals to die prematurely, as well, perhaps akin to the sub-population of wild type yeast that arrest in a budded state. Thus, it may be that by understanding the genetic and environmental factors that modulate terminal morphology of yeast mother cells, we will gain insight into the mechanisms that govern stochastic aspects of aging both in yeast and in higher eukaryotes.
All mutant strains examined were derived from the MATα ORF deletion collection and are isogenic to the parental BY4742 strain(Brachmann, et al., 1998, Winzeler, et al., 1999) or were derived from direct transformation of BY4742 with a PCR generated disruption cassette. Strain genotypes were verified by PCR genotyping. The pMoBY strain contains the vector CEN marked plasmid and a dubious gene that does not code for a functional protein, YGR045C, and acts as a vector control. Yeast replicative lifespan assays were performed as previously described(Kaeberlein, et al., 2004, Steffen, et al., 2009). In short, virgin daughter cells were isolated from each strain and then allowed to grow into mother cells while their corresponding daughters were microdissected and counted until the mother cell could no longer divide. YEP agar plates (1% yeast extract, 2% bacto-peptone, 2% agar) containing 2% glucose were utilized and strains were grown at 30°C except for dietary restriction experiments where the glucose was kept at 0.05%. Daughter cells were removed from each mother cell each 1-2 hours by micromanipulation. Terminal morphology was defined as the budded state of the mother cells upon senescence. Cells were scored as senescent when they had failed to divide for at least eight hours of incubation at 30°C. The data shown here were pooled across multiple experiments and were generally obtained in 20 cell sets. All experiments were performed by a team of dissectors working in shifts who were blinded to the identity of the strains under examination in any given experiment. Statistical significance for differences in median lifespan was determined using the Wilcoxon Rank-Sum test. Budded and unbudded states were determined visually for each mother cell assayed and statistical comparisons of budding rates utilized Fischer's Exact two-tailed test.
We thank Junko Oshima for her helpful comments on the manuscript. This work was supported by NIH Grant R01AG039390 to MK. JRD and GLS were supported by NIH Training Grant T32AG000057. JS and BMW were supported by NIH Training Grant T32ES007032. MK is an Ellison Medical Foundation New Scholar in Aging.
J.D. and M.K. jointly conceived this study. B.O. contributed to large scale data analysis. All except B.O., B.K.K.and M.K. performed experiments. J.D., A.C., B.K.K. and M.K. wrote the manuscript.