Hypothesis: cell volume limits cell divisions

Mammalian somatic cells and also cells of the yeast Saccharomyces cerevisiae are capable of undergoing a limited number of divisions. Reaching the division limit is referred to, apparently not very fortunately, as replicative aging. A common feature of S. cerevisiae cells and fibroblasts approaching the limit of cell divisions in vitro is attaining giant volumes. In yeast cells this phenomenon is an inevitable consequence of budding so it is not causally related to aging. Therefore, reaching a critically large cell volume may underlie the limit of cell divisions. A similar phenomenon may limit the number of cell divisions of cultured mammalian cells. The term replicative (generative) aging may be therefore illegitimate.     

酿酒酵母衰老机制研究进展

酿酒酵母衰老机制的研究对解析高等真核生物衰老的分子机制具有重要意义。酿酒酵母有两种衰老形式：时序衰老（chronologicalaging）和复制衰老（replicative aging）。酿酒酵母衰老研究中通常使用的寿命定义有两种：世代寿命和时序寿命。前者是指单个酿酒酵母细胞在死亡之前的分裂次数;后者是指一定数量的酵母细胞在后二次生长和稳定期的存活时间。本文分别综述了这两种衰老形式的分子机制及两者的相同点和不同点。     

A mother's sacrifice: what is she keeping for herself?

Individual cells of the budding yeast, Saccharomyces cerevisiae, have a limited life span and undergo a form of senescence termed replicative aging. Replicative life span is defined as the number of daughter cells produced by a yeast mother cell before she ceases dividing. Replicative aging is asymmetric: a mother cell ages but the age of her daughter cells is 'reset' to zero. Thus, one or more senescence factors have been proposed to accumulate asymmetrically between mother and daughter yeast cells and lead to mother-specific replicative senescence once a crucial threshold has been reached. Here we evaluate potential candidates for senescence factors and age-associated phenotypes and discuss potential mechanisms underlying the asymmetry of replicative aging in budding yeast.     

Intestinal crypt properties fit a model that incorporates replicative ageing and deep and proximate stem cells

A model of intestinal crypt organization is suggested based on the assumption that stem cells have a finite replicative life span. The model assumes the existence in a crypt of a quiescent ('deep') stem cell and a few more actively cycling ('proximate') stem cells. Monte Carlo computer simulation of published intestinal crypt mutagenesis data is used to test the model. The results of the simulation indicate that stabilization of the crypt mutant phenotype following treatment with external mutagen is consistent with a stem cell replicative life span of about 40 divisions for mouse colon and 90-100 divisions for mouse small intestine, corresponding to a deep stem cell cycle time of about 3.9 and 8.5 weeks for colon and small intestine, respectively. Simulation of the data obtained for human colorectal crypts suggests that the proximate stem cell cycle time is about 80 h, assuming a replicative life span of 50-150 divisions, and that the deep stem cell divides approximately every 30 weeks.     

2-micron circle plasmids do not reduce yeast life span

Extrachromosomal rDNA circles (ERCs) and recombinant origin-containing plasmids (ARS-plasmids) are thought to reduce replicative life span in the budding yeast Saccharomyces cerevisiae due to their accumulation in yeast cells by an asymmetric inheritance process known as mother cell bias. Most commonly used laboratory yeast strains contain the naturally occurring, high copy number 2-micron circle plasmid. 2-micron plasmids are known to exhibit stable mitotic inheritance, unlike ARS-plasmids and ERCs, but the fidelity of inheritance during replicative aging and cell senescence has not been studied. This raises the question: do 2-micron circles reduce replicative life span? To address this question we have used a convenient method to cure laboratory yeast strains of the 2-micron plasmid. We find no difference in the replicative life spans of otherwise isogenic cir⁺ and cir⁰ strains, with and without the 2-micron plasmid. Consistent with this, we find that 2-micron circles do not accumulate in old yeast cells. These findings indicate that naturally occurring levels of 2-micron plasmids do not adversely affect life span, and that accumulation due to asymmetric inheritance is required for reduction of replicative life span by DNA episomes.     

Up-regulation of the Cdc42 GTPase limits the replicative life span of budding yeast

Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.     

Resveratrol Modulates Mitochondria Dynamics in Replicative Senescent Yeast Cells

Mitochondria form a reticulum network dynamically fuse and divide in the cell. The balance between mitochondria fusion and fission is correlated to the shape, activity and integrity of these pivotal organelles. Resveratrol is a polyphenol antioxidant that can extend life span in yeast and worm. This study examined mitochondria dynamics in replicative senescent yeast cells as well as the effects of resveratrol on mitochondria fusion and fission. Collecting cells by biotin-streptavidin sorting method revealed that majority of the replicative senescent cells bear fragmented mitochondrial network, indicating mitochondria dynamics favors fission. Resveratrol treatment resulted in a reduction in the ratio of senescent yeast cells with fragmented mitochondria. The readjustment of mitochondria dynamics induced by resveratrol likely derives from altered expression profiles of fusion and fission genes. Our results demonstrate that resveratrol serves not only as an antioxidant, but also a compound that can mitigate mitochondria fragmentation in replicative senescent yeast cells.     

Effect of superoxide dismutase deficiency on the life span of the yeast Saccharomyces cerevisiae. An oxygen-independent role of Cu,Zn-superoxide dismutase

Effects of the absence of Cu,Zn-superoxide dismutase (CuZnSOD) on the replicative life span of the yeast Saccharomyces cerevisiae were studied under different oxygen conditions. In both strains, replicative life span and the rate of cell divisions were found to be similar under the atmosphere of air and under hypoxic (3% oxygen) and anoxic conditions. These results indicate that deleterious consequences of the lack of CuZnSOD are not limited to elevation of superoxide concentration and involve function(s) other than superoxide scavenging.     

Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae

Increased replicative longevity in Saccharomyces cerevisiae because of calorie restriction has been linked to enhanced mitochondrial respiratory activity. Here we have further investigated how mitochondrial respiration affects yeast life span. We found that calorie restriction by growth in low glucose increased respiration but decreased mitochondrial reactive oxygen species production relative to oxygen consumption. Calorie restriction also enhanced chronological life span. The beneficial effects of calorie restriction on mitochondrial respiration, reactive oxygen species release, and replicative and chronological life span could be mimicked by uncoupling agents such as dinitrophenol. Conversely, chronological life span decreased in cells treated with antimycin (which strongly increases mitochondrial reactive oxygen species generation) or in yeast mutants null for mitochondrial superoxide dismutase (which removes superoxide radicals) and for RTG2 (which participates in retrograde feedback signaling between mitochondria and the nucleus). These results suggest that yeast aging is linked to changes in mitochondrial metabolism and oxidative stress and that mild mitochondrial uncoupling can increase both chronological and replicative life span.     

Telomere Recombination Accelerates Cellular Aging in Saccharomyces cerevisiae

Telomeres are nucleoprotein structures located at the linear ends of eukaryotic chromosomes. Telomere integrity is required for cell proliferation and survival. Although the vast majority of eukaryotic species use telomerase as a primary means for telomere maintenance, a few species can use recombination or retrotransposon-mediated maintenance pathways. Since Saccharomyces cerevisiae can use both telomerase and recombination to replicate telomeres, budding yeast provides a useful system with which to examine the evolutionary advantages of telomerase and recombination in preserving an organism or cell under natural selection. In this study, we examined the life span in telomerase-null, post-senescent type II survivors that have employed homologous recombination to replicate their telomeres. Type II recombination survivors stably maintained chromosomal integrity but exhibited a significantly reduced replicative life span. Normal patterns of cell morphology at the end of a replicative life span and aging-dependent sterility were observed in telomerase-null type II survivors, suggesting the type II survivors aged prematurely in a manner that is phenotypically consistent with that of wild-type senescent cells. The shortened life span of type II survivors was extended by calorie restriction or TOR1 deletion, but not by Fob1p inactivation or Sir2p over-expression. Intriguingly, rDNA recombination was decreased in type II survivors, indicating that the premature aging of type II survivors was not caused by an increase in extra-chromosomal rDNA circle accumulation. Reintroduction of telomerase activity immediately restored the replicative life span of type II survivors despite their heterogeneous telomeres. These results suggest that telomere recombination accelerates cellular aging in telomerase-null type II survivors and that telomerase is likely a superior telomere maintenance pathway in sustaining yeast replicative life span.     

Cell enlargement: One possible mechanism underlying cellular senescence

We previously demonstrated an inverse relationship between the G1 volume of human diploid fibroblast-like (HDFL) cells obtained from foreskin tissue and clonal replicative potential. On the basis of these results, we suggested that one process underlying in vitro senescence is a progressive increase in the mean cell volume of successive progeny within clonal lineages. We now report that the size of HDFL cells, as well as of chick embryo fibroblasts, can be increased in the virtual absence of cell division by culturing at low density and at low serum concentration (0.1-1.0%). Consequent to an increase in cell size, the replicative potential of the cells is reduced to the level of later-passage cells of similar size. By clonal analysis, the populations of enlarged cells contain up to three times as many nondividing cells as do controls. In the enlarged populations, the proportion of cells producing attenuated clones (four or fewer progeny) increases by about 30%, whereas the proportion of cells yielding >32 cells declines by a similar percentage. These observations lead us to propose that replicative potential may be limited by cell size, which in turn may be regulated by a kinetic relationship between cellular growth and cell division cycles.     

The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective mitochondrial segregation in old mother cells

Prohibitin proteins have been implicated in cell proliferation, aging, respiratory chain assembly and the maintenance of mitochondrial integrity. The prohibitins of Saccharomyces cerevisiae, Phb1 and Phb2, have strong sequence similarity with their human counterparts prohibitin and BAP37, making yeast a good model organism in which to study prohibitin function. Both yeast and mammalian prohibitins form high-molecular-weight complexes (Phb1/2 or prohibitin/BAP37, respectively) in the inner mitochondrial membrane. Expression of prohibitins declines with senescence, both in mammalian fibroblasts and in yeast. With a total loss of prohibitins, the replicative (budding) life span of yeast is reduced, whilst the chronological life span (the survival of stationary cells over time) is relatively unaffected. This effect of prohibitin loss on the replicative life span is still apparent in the absence of an assembled respiratory chain. It also does not reflect the production of extrachromosomal ribosomal DNA circles (ERCs), a genetic instability thought to be a major cause of replicative senescence in yeast. Examination of cells containing a mitochondrially targeted green fluorescent protein indicates this shortened life span is a reflection of defective mitochondrial segregation from the mother to the daughter in the old mother cells of phb mutant strains. Old mother phb mutant cells display highly aberrant mitochondrial morphology and, frequently, a delayed segregation of mitochondria to the daughter. They often arrest growth with their last bud strongly attached and with the mitochondria adjacent to the septum between the mother and the daughter cell.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm2. The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm2 containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm2 containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell.     

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice ¹. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage ². Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.Open in a separate windowClick here to view.^{(75M, flv)}     

Mnsod overexpression extends the yeast chronological (G(0)) life span but acts independently of Sir2p histone deacetylase to shorten the replicative life span of dividing cells

Studies in Drosophila and Caenorhabditis elegans have shown increased longevity with the increased free radical scavenging that accompanies overexpression of oxidant-scavenging enzymes. This study used yeast, another model for aging research, to probe the effects of overexpressing the major activity protecting against superoxide generated by the mitochondrial respiratory chain. Manganese superoxide dismutase (MnSOD) overexpression increased chronological life span (optimized survival of stationary (G₀) yeast over time), showing this is a survival ultimately limited by oxidative stress. In contrast, the same overexpression dramatically reduced the replicative life span of dividing cells (the number of daughter buds produced by each newly born mother cell). This reduction in the generational life span by MnSOD overexpression was greater than that generated by loss of the major redox-responsive regulator of the yeast replicative life span, NAD+-dependent Sir2p histone deacetylase. It was also independent of the latter activity. Expression of a mitochondrially targeted green fluorescent protein in the MnSOD overexpressor revealed that the old mother cells of this overexpressor, which had divided for a few generations, were defective in segregation of the mitochondrion from the mother to daughter. Mitochondrial defects are, therefore, the probable reason that MnSOD overexpression shortens replicative life span.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

Summary The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm². The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm² containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm² containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell. This work was supported by IIT Research Institute.     

Increased aerobic metabolism is essential for the beneficial effects of caloric restriction on yeast life span

Calorie restriction is a dietary regimen capable of extending life span in a variety of multicellular organisms. A yeast model of calorie restriction has been developed in which limiting the concentration of glucose in the growth media of Saccharomyces cerevisiae leads to enhanced replicative and chronological longevity. Since S. cerevisiae are Crabtree-positive cells that present repression of aerobic catabolism when grown in high glucose concentrations, we investigated if this phenomenon participates in life span regulation in yeast. S. cerevisiae only exhibited an increase in chronological life span when incubated in limited concentrations of glucose. Limitation of galactose, raffinose or glycerol plus ethanol as substrates did not enhance life span. Furthermore, in Kluyveromyces lactis, a Crabtree-negative yeast, glucose limitation did not promote an enhancement of respiratory capacity nor a decrease in reactive oxygen species formation, as is characteristic of conditions of caloric restriction in S. cerevisiae. In addition, K. lactis did not present an increase in longevity when incubated in lower glucose concentrations. Altogether, our results indicate that release from repression of aerobic catabolism is essential for the beneficial effects of glucose limitation in the yeast calorie restriction model. Potential parallels between these changes in yeast and hormonal regulation of respiratory rates in animals are discussed. G. A. Oliveira and E. B. Tahara contributed equally to this work.     

The impact of brewing yeast cell age on fermentation performance,attenuation and flocculation

Individual cells of the yeast Saccharomyces cerevisiae exhibit a finite replicative lifespan, which is widely believed to be a function of the number of divisions undertaken. As a consequence of ageing, yeast cells undergo constant modifications in terms of physiology, morphology and gene expression. Such characteristics play an important role in the performance of yeast during alcoholic beverage production, influencing sugar uptake, alcohol and flavour production and also the flocculation properties of the yeast strain. However, although yeast fermentation performance is strongly influenced by the condition of the yeast culture employed, until recently cell age has not been considered to be important to the process. In order to ascertain the effect of replicative cell age on fermentation performance, age synchronised populations of a lager strain were prepared using sedimentation through sucrose gradients. Each age fraction was analysed for the ability to utilise fermentable sugars and the capacity to flocculate. In addition cell wall properties associated with flocculation were determined for cells within each age fraction. Aged cells were observed to ferment more efficiently and at a higher rate than mixed aged or virgin cell cultures. Additionally, the flocculation potential and cell surface hydrophobicity of cells was observed to increase in conjunction with cell age. The mechanism of ageing and senescence in brewing yeast is a complex process, however here we demonstrate the impact of yeast cell ageing on fermentation performance.     

Relief of oxidative stress by ascorbic acid delays cellular senescence of normal human and Werner syndrome fibroblast cells

Primary human cells have a definite life span and enter into cellular senescence before ceasing cell growth. Oxidative stress produced by aerobic metabolism has been shown to accelerate cellular senescence. Here, we demonstrated that ascorbic acid, used as an antioxygenic reagent, delayed cellular senescence in a continuous culture of normal human embryonic cells, human adult skin fibroblast cells, and Werner syndrome (WS) cells. The results using human embryonic cells showed that treatment with ascorbic acid phospholic ester magnesium salt (APM) decreased the level of oxidative stress, and extended the replicative life span. The effect of APM to extend the replicative life span was also shown in normal human adult cells and WS cells. To understand the mechanism of extension of cellular life span, we determined the telomere lengths of human embryonic cells, both with and without APM treatment, and demonstrated that APM treatment reduced the rate of telomere shortening. The present results indicate that constitutive oxidative stress plays a role in determining the replicative life span and that suppression of oxidative stress by an antioxidative agent, APM, extends the replicative life span by reducing the rate of telomere shortening.