Budding yeast cells turn back the clock

yeast membrane

Editor's Introduction

Gametogenesis eliminates age-induced cellular damage and resets life span in yeast

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The process by which rejuvenation occurs in new generations has long interested biologists and vexed fairytale stepmothers. In this report, researchers sought to determine whether, in principle, molecular damage caused by aging is eliminated during the production of gametes in sexually reproducing organisms. They chose yeast as a model since it has an inducible sexual lifecycle and a short, measurable lifespan during which it exhibits molecular signs of aging. When during the sexual cycle does elimination of cellular damage occur, and what genes regulate this process? The answer could produce therapies aimed at treating diseases that are acquired more frequently with age.

Paper Details

Original title
Gametogenesis eliminates age-induced cellular damage and resets life span in yeast
Elçin Ünal
Original publication date
Vol. 332 no. 6037 pp. 1554-1557
Issue name


Eukaryotic organisms age, yet detrimental age-associated traits are not passed on to progeny. How life span is reset from one generation to the next is not known. We show that in budding yeast resetting of life span occurs during gametogenesis. Gametes (spores) generated by aged cells show the same replicative potential as gametes generated by young cells. Age-associated damage is no longer detectable in mature gametes. Furthermore, transient induction of a transcription factor essential for later stages of gametogenesis extends the replicative life span of aged cells. Our results indicate that gamete formation brings about rejuvenation by eliminating age-induced cellular damage.


Most, if not all, eukaryotic organisms age; however, the age-induced changes are not transmitted to the progeny. How life span is reset from one generation to the next is not known. We wished to test the hypothesis that resetting of life span occurs during gametogenesis. In budding yeast, gamete formation (sporulation) requires meiosis and includes the generation of new membrane compartments, protein and organelle degradation, and synthesis of a resistant spore wall (1). To determine whether gamete formation causes rejuvenation, we asked whether spores derived from aged cells have reset their life span and are young or whether they inherit the progenitor’s age and remain old. We isolated replicatively aged cells on the basis of biotin labeling of mother cells (2) and induced them to sporulate in the same flask as young cells (fig. S1). Upon sporulation, tetrads were dissected and the replicative life span (RLS) of each spore was measured. We found that the life spans of the spores derived from young and aged cells were indistinguishable in two Saccharomyces cerevisiae strain backgrounds: W303, in which sporulation efficiency decreases with age (34) (Fig. 1B), and A364a, in which sporulation efficiency remains high despite aging (4) (fig. S2). In contrast, aged cells obtained by the same procedure but not induced to sporulate die rapidly (Fig. 1A). The RLS of the four spores from a tetrad produced from young and aged cells is the same; no statistically significant differences are observed (4) (Fig. 1C). This is in contrast to mitosis, where age is asymmetrically inherited between the mother cell and the bud, culminating in the production of a young daughter and an old mother cell (5). Thus, sporulation resets RLS.


Fig. 1. Gametogenesis resets RLS. The average number of cell divisions of the starting cell population is indicated in the keys. The median life span is written next to each curve. Error bars denote standard deviation. (A) RLSs of young and aged wild-type A702 cells, directly after sorting, before sporulation. (B) Postsporulation RLSs of spores from young and aged A702 cells. (C) Age distribution of spores from A702 in individual tetrads from young and aged progenitors, n = 10. The life span of spores from each tetrad is compared with the mean life span of young spores to obtain a P value. The average P value from 10 tetrads is 0.303 for young and 0.642 for aged spores (t test), which indicates no statistically significant difference in replicative age among spores from a given tetrad.

Question Being Asked

Does the process of gametogenesis (sporulation) restore the replicative life span of aged cells to that of young cells?


The researchers use a micromanipulation-equipped light microscope to physically separate daughter cells from mothers. The total number of daughters produced by a mother is defined as its replicative life span.

Aged cells are purified by labeling their cell walls with a molecule called biotin that does not incorporate into daughter cells upon subsequent cell divisions.  A mixture of old and young cells is washed over plastic beads coated in a protein that binds biotin, allowing aged cells to be separated from young cells prior to life span analysis or sporulation.

Panel A

Aged cells isolated before sporulation show a shorter replicative life span than young cells.

Panel B

In contrast to Panel A, the replicative life spans of spores isolated from aged and young cells are similar.

Panel C

Replicative life spans of haploids from multiple tetrads showing no statistical difference between cells from separate sporulation events.  

We next asked how sporulation affects age-dependent cellular changes, such as increased levels of protein aggregation (6), aberrant nucleolar structures, and increased levels of extra-chromosomal ribosomal DNA (rDNA) circles (ERCs) (78). In budding yeast, protein aggregates associate with Hsp104 and form foci in replicatively aged cells, which can be visualized by Hsp104 joined to enhanced green fluorescent protein (Hsp104-eGFP) (6) (fig. S3, A to D). During vegetative growth, Hsp104-eGFP foci are distributed asymmetrically between the mother and daughter cell; less than 10% of young cells displayed foci, but Hsp104-eGFP foci began to accumulate by generation eight in mother cells and were present in 85% of aged cells (469) (fig. S3D). Throughout sporulation, most aged cells contained Hsp104-eGFP foci [mononucleates (95%), binucleates (90%), and tetranucleates (86%)], but the foci were essentially absent in mature tetrads (3%) (Fig. 2, A and B), which suggests that age-associated protein aggregates are cleared during sporulation. The polarisome is required for the asymmetric distribution of Hsp104-eGFP foci during mitosis (9). Deleting the genes encoding the polarisome components Bud6 or Spa2 did not interfere with aggregate elimination during sporulation (fig. S3, E and F). Proteasome function also appeared dispensable for aggregate clearance. Treatment of cells with the proteasome inhibitor MG132 after the second division prevented neither sporulation nor the clearance of Hsp104 aggregates (10) (fig. S3, G and H). In contrast, treatment of cells with the autophagy inhibitor chloroquine prevented sporulation, and aggregates persisted (11) (fig. S3, G and H), which suggests that an autophagy-dependent process and/or spore formation are required for aggregate clearance.


Fig. 2. Sporulation eliminates age-induced cellular damage. (A) Analysis of Hsp104-eGFP aggregates in aged sporulating A25825 cells. (B) Quantification of Hsp104-eGFP foci in young (1.3 ± 0.6 generations) and aged (6.8 ± 1.3 generations) A25825 cells before meiosis I (mononuc), after meiosis I (binuc), after meiosis II (tetranuc), and in tetrads.(C) rDNA and ERCs in young (1.3 ± 0.5 generations) and aged (15 ± 2.7 generations) A26370 cells. (D) Nucleolar morphology in young (1.4 ± 0.6 generations) and aged (14.2 ± 6.1 generations) A26271 cells.

Question Being Asked

After establishing that life span is reset in spores of aged cells, the researchers wanted to know which biomarkers of aging are eliminated from cells in the process of sporulation. 


The presence of protein aggregates associated with the Hsp104 chaperone protein were assessed by fluorescence microscopy of cells in the process of sporulation expressing green fluorescent protein tagged Hsp104.  

ERCs were detected by Southern blot analysis. Specificity of detection was ensured by treatment of DNA samples with one restriction enzyme known to cut rDNA and one known not to cut followed by electrophoresis and detection.

Learn more about Southern blotting in this video.

Panel A

Representative microscopy images of cells during the stages of sporulation that are expressing an Hsp104-GFP fusion protein. Hsp104-eGFP foci suggest the presence of protein aggregates. 

Panel B

Quantification of the fraction of cells at each stage of sporulation containing Hsp104-eGFP foci. Few young cells contain Hsp104 foci at any stage of sporulation, but a remarkable drop in foci occurs in aged cells between the second meiotic division and final spore packaging into tetrads. 

Panel C

Genomic DNA isolated from young or aged cells before or after sporulation was run on an agarose gel to detect the abundance of extrachromosomal rDNA circles (ERCs) that have been shown to accumulate with age. Decreased detection of the bands labeled ERCs is evident in aged cells only after sporulation.  

Panel D

Representative images showing diffuse Fob1-GFP localization in aged but not young cells, an indicating loss of DNA organization in the nucleolus. Compact Fob1-GFP localization in nuclei of aged cells following sporulation resembles young cells. 

Aged cells are also defective in rDNA metabolism, displaying fragmented nucleoli and forming extra chromosomal rDNA circles (ERCs) (78). ERCs decreased considerably during sporulation in aged cells, reaching levels similar to those of young cells (Fig. 2C and fig. S4). In aged cells, the rDNA structure also underwent dramatic changes as judged by the localization of Fob1-GFP, a nucleolar protein that binds to the rDNA (12). In 60% of aged cells, the Fob1-GFP appeared enlarged and discontinuous, which probably reflects rDNA condensation defects and nucleolar fragmentation, respectively. After aged cells sporulate, more than 90% of the tetrads contained a single Fob1-GFP focus per spore and displayed a morphology indistinguishable from that of young cells (Fig. 2D). Together, our results demonstrate that gamete formation eliminates age-induced protein aggregation and nucleolar aberrations.

To determine which aspects of gametogenesis are necessary for RLS resetting, we deleted two transcription factors that trigger different stages of sporulation and asked whether RLS was reset. Such studies are possible because in budding yeast, sporulating cells can resume vegetative growth (return to growth) (4), provided that the sporulation-inducing cue, nutrient deprivation, is withdrawn. We first analyzed the RLSs of young (1.4 ± 0.6 generations) and aged (16.2 ± 4 generations) cells from a strain that lacked Ime1. Without Ime1, yeast cells are unable to initiate sporulation but still sense nutrient deprivation (13). To ensure that only cells that responded to the sporulation-inducing cues were included in the analysis, we used a pIME1:mCherry reporter construct (fig. S5A) (14). Aged ime1Δ cells lost viability rapidly, with a median life span of four generations (Fig. 3A). Similar results were obtained with wild-type cells that had responded to sporulation cues as judged by pIME1:mCherry expression but had not yet entered the sporulation program (fig. S5B) Thus, the initiation of sporulation driven byIME1 is required to reset RLS. Furthermore, nutrient deprivation and other sporulation signals are insufficient to promote RLS resetting.


Fig. 3. IME1 and NDT80, but not the meiotic nuclear divisions, are required for life-span resetting. (A) RLSs of young and aged A23998 (ime1Δ) cells. (B) RLSs of young and aged A24074 (ndt80Δ) cells. The median life span of the aged cells is 0; therefore, the average is shown. (C) The life spans of young and aged A27377 (spo12Δ) spores. (D) The life spans of young and aged A24142 (cdc5-mn) spores.

Question Being Asked

Can we learn which events in the process of sporulation are required to reset life span by studying mutants that disrupt completion of specific stages? 

Panels A and B

Replicative life spans of aged cells lacking the meiosis transcription factor IME1 (Panel A) or NDT80 (Panel B) following the sporulation cue.  

Panel C

Replicative life span of cells lacking SPO12 that produce two diploid spores as a result of only undergoing the first nuclear division of meiosis. 

Panel D

Replicative life span of cells that do not undergo meiotic divisions resulting from inactivation of regulatory protein Cdc5 but form a single diploid spore following the sporulation cue.  

In the absence of the transcription factor Ndt80, yeast cells complete premeiotic DNA replication, initiate recombination, and arrest in pachytene (15). We used Zip1-GFP to identify the ndt80Δ cells arrested in pachytene (fig. S5C) (16) and found that young cells (1.3 ± 0.6 generations) resumed vegetative growth and divided an average of 21.6 ± 7.9 times (median RLS = 21). In contrast, almost 50% of the aged cells (16.8 ± 4.4 generations) lost viability within the first mitotic division. The remaining cells underwent significantly fewer divisions compared with young cells (3.4 ± 4.9 generations) (Fig. 3B). We conclude that NDT80-induced processes are necessary for RLS resetting, and that the events before NDT80 function, such as premeiotic DNA replication and recombination, are insufficient to promote rejuvenation.

Progression through sporulation up to pachytene is not sufficient for resetting of RLS. Thus, later stages of sporulation, such as the meiotic nuclear divisions and/or spore formation, must be required. To determine whether both meiotic divisions are necessary for RLS resetting, we deleted SPO12. The resulting spo12Δ cells undergo a single nuclear division and form two diploid spores (17). We found that young (1.3 ± 0.6 generations) and aged (14 ± 1.8 generations) spo12Δ cells had indistinguishable RLSs, which suggests that two consecutive meiotic divisions are not a prerequisite for rejuvenation (Fig. 3C). The life span of spores within individual two-spored asci is very similar (fig. S6) (for the young, P = 0.15; for the aged, P = 0.29, n = 30, Wilcoxon signed-rank test). To test if resetting of RLS can occur in the absence of any nuclear divisions, we inactivated the pololike kinase Cdc5 during sporulation (cdc5-mn) (18). Cells lacking Cdc5 do not undergo any meiotic divisions and form single spores. Like spo12Δ spores, cdc5-mn spores obtained from aged cells regained their replicative potential (Fig. 3D). We conclude that the meiotic divisions per se are dispensable for RLS resetting and note that our findings exclude a model where halving of the genome or diluting aging factors brings about the resetting of RLS (4).

NDT80-regulated genes that mediate spore formation could be required for rejuvenation. As NDT80 expression is sufficient to induce the expression of mid- and late-sporulation genes in vegetative cells (1920), we determined whether Ndt80 could extend the life span of vegetative cells. We expressed NDT80 from the GAL1-10 promoter (GAL-NDT80), whose expression can be regulated by a Gal4–estrogen receptor fusion (Gal4.ER) (2122) (fig. S7, A and B). Expression of NDT80 significantly extended the life span of mitotic cells (fig. S7, C and D) (P < 0.0001,Z score = 4, Mann-Whitney test). To test whether induction of NDT80 in replicatively aged cells also extends life span, we transiently induced Ndt80 with β-estradiol in young and aged cells and followed their RLSs in the absence of β-estradiol. Aged cells that transiently expressed NDT80 lived significantly longer than aged cells treated in the same manner but lacking the GAL-NDT80 fusion (Fig. 4A) (P < 0.0001, Z score = 8.23, Mann-Whitney test). Transient expression of NDT80 even led to an extension of life span in young cells (Fig. 4A) (P < 0.0001, Z score = 4.85, Mann-Whitney test). Similar results were obtained in experiments comparing cell divisions in liquid culture, excluding the possibility that transient expression of NDT80 extends life span because it causes cells to become more resistant to the micromanipulations involved in the pedigree analysis (Fig. 4, B and C). Thus, a transient induction of NDT80 is sufficient to extend the life span of replicatively aged cells.


Fig. 4. Transient NDT80 expression extends the life span of vegetatively growing aged cells. (A) Life span of young and aged cells from A25823 (GAL4.ERNDT80) and A25824 (GAL4.ERGAL-NDT80). (B) (Top) Description of the experiment; (bottom) aged cells from A27507 (GAL4.ERNDT80) and A27484 (GAL4.ER,GAL-NDT80) labeled before and after β-estradiol. (C) The number of cell divisions after β-estradiol treatment was calculated by the difference between the green- and red-labeled bud scars. The distribution of n = 60 cells is shown for A27507 (GAL4.ER,NDT80) and n = 100 cells for A27484 (GAL4.ERGAL-NDT80). (D) Fob1-GFP in young and aged cells from strains A27507 (GAL4.ERNDT80) and A27484 (GAL4.ERGAL-NDT80) after a 6-hour β-estradiol treatment. (E) Percentage of cells with enlarged Fob1-GFP from A27507 (GAL4.ERNDT80) and A27484 (GAL4.ERGAL-NDT80) following β-estradiol treatment. The average of two independent experiments is shown. 100 to 200 cells were counted for each time point; error bars display the range.

Question Being Asked

Since events that follow activation of NDT80 are required for life span to be reset, can merely expressing this transcription factor in old cells suffice to rejuvenate them? 


NDT80 was expressed outside the context of sporulation by placing this gene under control of a promoter that is activated in response to addition of β-estradiol to the growth media.

An alternative to micromanipulation for scoring the number of divisions involves labeling biotin-labeled bud scars with red fluorophore, allowing cells to divide, then labeling both old and new bud scars with green fluorophore. The difference in number of green to number of red bud scars is the number of new divisions.

Nucleolar morphology was assessed by fluorescence microscopy of cells expressing GFP-tagged Fob1 throughout NDT80 induction. 

Panel A

Replicative life spans of young and aged cells after induced expression of transcription factor Ndt80 for a six hour period.

Control young and aged cells did not express Ndt80 during this time. 

Panel B

Explanation of experimental procedure for counting divisions following Ndt80 induction in aged cells shown in Panel C.

Visualizing bud scars allows one to count the number of divisions a cell has undergone. 

Panel C

Histogram of the number of cell divisions following a six-hour induction of Ndt80 in aged cells compared to cells unable to induce Ndt80

Panel D

Representative images of young and aged cells highlighting size differences in Fob1-GFP nucleolar distribution after six-hour induction of Ndt80 (bottom) or control cells (top). 

Panel E

Quantification of the portion of cells with enlarged Fob1-GFP distribution following induction of Ndt80 for 0-7 hours. 

To address how transient induction of Ndt80 extends RLS, we monitored age-dependent cellular changes after NDT80 induction. Neither ERCs nor Hsp104-eGFP aggregates were reduced after NDT80 induction, although it is possible that they were reduced at later time points (fig. S8, A and B). Furthermore, NDT80 expression extended life span in the absence of the autophagy gene ATG1 (fig. S8C). Together these findings suggest that life-span extension can occur in the absence of ERC and Hsp104-aggregate elimination. Although ERCs and Hsp104-eGFP aggregates were not affected by transient NDT80 expression, nucleolar morphology was. The percentage of aged cells with enlarged nucleolar morphology decreased after NDT80 induction (Fig. 4, D and E, and fig. S8D). Thus, transient induction of NDT80 causes a change in nucleolar and/or rDNA structure, which reverts to a state that resembles the morphology of young cells.

We do not yet know whether NDT80- and sporulation-induced RLS resetting use the same mechanism(s). The findings that NDT80 is necessary for life-span extension during sporulation and sufficient for life-span extension during vegetative growth and that nucleolar morphology is altered under both circumstances suggest that at least some processes are shared. Irrespective of the relation between NDT80- and sporulation-induced RLS resetting, we note that resetting of RLS provides an opportunity to dissect the molecular causes of aging. For instance, elimination of Hsp104 aggregates and ERCs seem unlikely to be required for NDT80-dependent life-span extension, but changes in nucleolar function and/or structure may be important. Intriguingly, rDNA instability and not ERCs per se appear to cause aging in yeast (23), and budding yeast cells eliminate most of the nucleolar material during spore packaging (24).

It will be interesting to investigate whether our findings extend to other species. In Caenorhabditis elegans, a number of longevity mutants exhibit a soma-to-germline transformation that contributes to their enhanced survival (25). In mice, reintroduction of telomerase rescues the age-related phenotypes of telomerase-deficient mice (26), which suggests that age-dependent cellular damage can be repaired. Our studies suggest that a transient induction of the gametogenesis program in somatic cells removes age-dependent cellular damage and extends life span. Determining how gametogenesis causes the resetting of life span will provide insights into the mechanisms of aging and could facilitate the development of strategies for longevity.

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Acknowledgments: We thank B. Alpert, M. Boselli, A. Thompson, and J. Chen for technical help; D. Koshland for reagents; and T. Orr-Weaver, F. Solomon, and members of the Amon laboratory for comments on the manuscript. Research was supported by NIH grant GM62207 to A.A. A.A. is also an HHMI investigator. E.Ü. is a fellow of the Jane Coffin Childs Memorial Fund. The authors declare no competing financial interests.