The Budding and Fission Yeast Model Systems for Aging Biology

Chapter 30

The Budding and Fission Yeast Model Systems for Aging Biology: Rapid Advancement With New Technologies Kurt W. Runge, Haitao Zhang Lerner College of Medicine at CWRU, Cleveland Clinic, Cleveland, OH, United States

INTRODUCTION It is difficult to overstate the contribution that yeasts have made to understanding of molecular genetics and cell biology, and the past two decades have firmly established these organisms as excellent model systems to study the biology of aging. The budding yeast Saccharomyces cerevisiae has been utilized the most fully owing to the large international research community and that ability to easily manipulate its genome in the lab. The fission yeast also Schizosaccharomyces pombe has a similarly long and stellar history as S. cerevisiae. The tools for using S. cerevisiae and S. pombe are very similar, but the two yeasts are extremely different and each has preserved different biological processes that are conserved in humans (Hedges, 2002; Sipiczki, 2004). S. cerevisiae and S. pombe diverged from each other almost a billion years ago, close to the time when yeast diverged from the last common ancestor of fungi and mammals (Sipiczki, 2004). Thus, the presence of similar pathways in these budding and fission yeasts provide robust evidence for strong evolutionary conservation over a long period, indicating that these conserved pathways are likely to play similar roles in mammals. These yeasts have also become a proving ground for the application next-generation sequencing (NGS) technologies that can leverage the power of microbial genetics, assaying millions of individuals at once. When coupled with the standard genetic crosses that are the hallmark of these model systems, these new approaches further extend contribution of yeasts to biology in general and the biology of aging in particular. Understanding the approaches used to investigate yeast aging will therefore provide a better foundation for interpreting the literature so that these results can be applied to humans. Both S. cerevisiae and S. pombe ferment sugars in the presence of oxygen (Dashko et al., 2014), secrete digestive enzymes into the environment, sense the level of nutrients available, and either import them or synthesize them. Consequently, many of the biosynthetic and metabolic pathways are conserved in both yeasts. However, S. cerevisiae appears to have duplicated its genome in its evolutionary history and then lost many genes and amplified others compared to S. pombe. Both yeasts have similar genome sizes (12.2 MB for S. cerevisiae vs. 12.4 MB for S. pombe, not including the ribosomal RNA gene repeats (Engel et al., 2014; McDowall et al., 2015)), but S. cerevisiae has 16 chromosomes with small centromeres and telomere-associated regions while S. pombe had 3 chromosomes with larger centromeres and telomeres more reminiscent of mammalian chromosomes. S. cerevisiae has amplified some gene families compared to S. pombe (e.g., 10 cyclins for the paralog of mammalian cdk5 in S. cerevisiae vs. 3 in S. pombe) and lost others (e.g., the dimethylation of lysine 9 on histone H3 in heterochromatin is absent in S. cerevisiae but present in S. pombe and mammals) (Engel et al., 2014; McDowall et al., 2015). Consequently, some processes in aging biology such as the formation of senescence-associated heterochromatin foci in mammalian cells, which occurs during aging, are better modeled in S. pombe (Roche et al., 2016). In contrast to these differences in chromatin modification, the most basic differences between the two species, budding in S. cerevisiae versus fission in S. pombe, have made S. cerevisiae the trailblazer in replicative aging, i.e., the number of times a cell can divide prior to death or the replicative life span (RLS). The budding mode of cell division produces a larger mother cell and a smaller daughter cell, allowing one to visually distinguish “newly born” daughters from older mothers. Consequently, one can isolate newly born cells with a micromanipulator to perform aging studies (Fig. 30.1A). These newly born buds are “younger” as they have longer RLSs than the mother cells. This budding mode has other characteristics that have been exploited to construct innovative systems to produce aged cells, as described later in this chapter. In contrast, S. pombe division produces two equal daughters in most cases, making it difficult to know which or if one of these cells Conn’s Handbook of Models for Human Aging. https://doi.org/10.1016/B978-0-12-811353-0.00030-0 Copyright © 2018 Elsevier Inc. All rights reserved.

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FIGURE 30.1  Features of replicative life span (RLS). (A) Budding yeast. Saccharomyces cerevisiae shows a mother–daughter size difference at cell division, with the smaller daughter being the “newly born” cell and the larger mother budding multiple times until death. The mother and daughter have different transcriptional programs for different cell-specific processes. These cell-specific transcriptional programs have been exploited to create systems that can efficiently monitor RLS and produce large amounts of aged cells. (B) Fission yeast. Schizosaccharomyces pombe cells divide by fission to produce two daughters of equivalent size, so it is unclear if one progeny cell is older or younger than the other. When RLS is performed using the micromanipulator assay, cell asymmetry begins to occur within a few divisions. While the mean RLS for most S. cerevisiae lab strains is 20–30 divisions, S. pombe is only 6–10 divisions. As described below in the section on the use of microfluidics, when S. pombe RLS is monitored without micromanipulation, this asymmetric cell morphology is not observed in later divisions.

is newly born (Fig. 30.1B). As described below, this difficulty is being addressed using novel microscopic approaches that monitor S. pombe cell growth without micromanipulation, with some surprising results as to how fission yeast ages. In addition to replicative aging, both yeasts are amenable to chronological aging that measures the length of time cells can remain viable in a stationary phase culture, called the chronological life span (CLS). Cells are grown in flasks through exponential phase to stationary phase where the glucose in the medium is exhausted and then monitored for survival by plating aliquots onto medium to measure the fraction of viable cells or colony-forming units/mL (Fig. 30.2A). While this system is easy to adapt in the lab, a fundamental question is what does this kind of survival actually tell us? Yeast living out in the wild on grapes or other fruit can enter stationary phase and eventually die in a nutrient-starved environment, but one remaining viable cell can regrow in the presence of new nutrients to outcompete many other slower-growing microbes. Consequently, an open question is whether CLS should be measured until almost all cells in culture die or when the viable cells drop to levels of 1% of the starting density to give a curve similar to how aging in groups of mammals and other metazoans are monitored. In budding yeast, a significant amount of cells die and regrow during a CLS experiment as cells approach 0.1% of the remaining viable cells (Fabrizio and Longo, 2006; Longo et al., 2012) (Fig. 30.2B). However, many clever approaches have adapted this assay to yield important information regarding the cellular processes important for how cells prepare to stop rapid growth as nutrients decline and have revealed the importance of some universally conserved

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FIGURE 30.2  Chronological life span (CLS) assays in both yeasts. (A) The basic CLS assays grow cells in a flask until saturation (day 0 or t = 0). Aliquots are then sampled and the fraction of viable cells are determined by plating cells on fresh medium to form colonies and/or staining cells with vital dyes. In the case of looking for mutants that extend life span, the cells that have been regrown from aliquots have amplified the viable cells over the inviable or arrested cells. These regrown cells are then processed to determine the relative proportion of specific mutants, e.g., by determining the relative frequency of the barcodes that tag each mutation in a deletion strain set (Wei et al., 2008). (B) Saccharomyces cerevisiae cells show regrowth after a substantial loss of viability. This regrowth and death cycle can continue for months (Fabrizio et al., 2004). (C) Schizosaccharomyces pombe cells do not show the regrowth of S. cerevisiae cells but uniformly lose viability (Chen and Runge, 2009), which allows for the direct selection of mutants that live longer than other cells (Chen et al., 2013). (D) S. cerevisiae cells in stationary phase form multiple cell types: Mothers that arrest as large cells and small daughters that bud from these mothers as nutrients are exhausted, and the dense Q cells that can survive for long periods are stress resistant (Allen et al., 2006). (E) S. pombe cells starved for reduced nitrogen will arrest as cells of various sizes or lysed cells (in blue) and can also form Q cells that arrest in G0, are small, round, and stress resistant (Su et al., 1996). In contrast to nitrogen starvation, glucose starvation causes growth to cease but does not produce Q cells.

genes (Powers et al., 2006; Wei et al., 2008; McCormick et al., 2015; Anisimov et al., 2011; Fok et al., 2014; Kennedy and Lamming, 2016; Wilkinson et al., 2012). In fission yeast, CLS assays that do not show regrowth have been devised (Fig. 30.2C) and have revealed new gene products that regulate life span in this assay (Chen and Runge, 2009; Chen et al., 2013; Ohtsuka et al., 2008, 2009; Sideri et al., 2014). A major advance in the concept of CLS assays has been the isolation of what could be called a new differentiated form of both yeasts: quiescent cells (Q cells) in the G0 state of the cell cycle. When S. cerevisiae cells in a CLS assay are aged for several days, one can isolate a subpopulation of small, dense cells from the culture (Fig. 30.2D) that can survive for almost 1 year in water, in contrast to the ∼2–3 week CLS of the bulk culture (Fabrizio and Longo, 2006; Allen et al., 2006; Miles and Breeden, 2017). How these cells form from the aging culture and survive in G0 has important implications for understanding the control of cell senescence (Miles and Breeden, 2017). S. pombe cells have a similar G0 state that can survive for months at a time, which is induced by transferring cells to medium that lacks a nitrogen source (Fig. 30.2E). Medium lacking reduced nitrogen (−N medium) is a long-used method in S. pombe for inducing diploids to form haploid spores, but how this laboratory manipulation is related to conditions under which S. pombe and other fission yeast strains evolved is quite unclear. The S. pombe Q cells have not been reported in stationary phase cultures to date as efforts have focused on understanding the transition that can be induced in a tractable fashion (Su et al., 1996; Yanagida, 2009). As described below, these studies in both yeasts should provide complementary information as the control of entry into G0 will most likely reflect the two different modes of cell cycle control, with S. cerevisiae affecting many more controls at the G1 to S phase transition while S. pombe affects more controls at G2 to M phase transition (Bahler, 2005; Huberman, 1996;

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Humphrey and Pearce, 2005; McInerny, 2011). Understanding the mechanisms that allow the formation, maintenance, and return to growth of Q cells will have a major impact on how we consider chronological aging and the function of terminally differentiated cells in mammals. As the yeast aging fields continue to advance rapidly, the goals of this chapter will be to provide the reader with background knowledge of the recent advances regarding how life span is studied and how newer technologies are affecting this understanding. It is hoped that with this approach, the reader will gain an understanding of how “we know what we know”—what kinds of experiments form the foundation of the current models and how these models may be impacted by the current direction of research. In this regard, it is important to note some of the existing models for regulation of yeast RLS and CLS, most of which have been extensively reviewed (Longo et al., 2012; Kaeberlein, 2010; Stanfel et al., 2009; Lin and Austriaco, 2014). Major pathways include regulation by the TOR (target of rapamycin) kinase, whose activity controls protein synthesis and protein degradation by autophagy in the presence of nutrients, and Sir2/rDNA (Steinkraus et al., 2008). The array of ribosomal RNA genes or rDNA has a classic mode of aging where one or more of the ∼9 kB repeat units recombines out as a circle to form an episomal plasmid that replicates apart from the chromosome. The extrachromosomal rDNA circles (ERCs) can build up to large numbers in aging cells. The formation of these circles is governed by the nicotine adenine dinucleotide–dependent deacetylase Sir2 and the Fob1 protein that binds to rDNA and prevents the collision of DNA replication and RNA polymerase forks (Defossez et al., 1999; Kaeberlein et al., 1999). It is now clear that aging is much more than ERCs and nutrient sensing for the TOR pathway, and that TOR, Sir2, and the rDNA can affect life span in unknown ways. However, these earlier models are always considered when new approaches identify the old players such as Sir2 or the rDNA repeats to determine whether the new discoveries are something new. One of the major advantages of the yeast system is the large array of genetic tools that allow high-throughput analysis of phenotypes and assays of gene function. While these and other aspects of systems biology have been evolving rapidly in both yeasts (Engel et al., 2014; McDowall et al., 2015; Forsburg, 2005), the application of these techniques to replicative and chronological aging was initially more challenging. The initial difficulties with these assays have been solved in multiple ways for S. cerevisiae and include both brute force approaches and the production of genetically modified strains that convert the labor-intensive RLS assay to one that can be applied in a high-throughput manner to cells grown in culture. Finally, the application of microlithography and microfluidics combined with microscopy has allowed researchers to track replicatively aging S. cerevisiae and S. pombe, which has resulted in conclusions for S. pombe in marked contrast to the micromanipulation assay.

REPLICATIVE LIFE SPAN IN SACCHAROMYCES CEREVISIAE The S. cerevisiae community has created a number of impressive, publically available resources that are distributed to most labs at an affordable cost. The most useful in aging research has been the deletion strain set, a set of strains in 96-well plates where each gene’s open reading frame is deleted and replaced with the G418 resistance marker and one or two defined nucleotide “barcodes” (Winzeler et al., 1999; Kim et al., 2010). The strains include those deletions that are viable as haploids stored in a series of 96-well plates, making it possible to isolate individual mutants and mass screen cells for growth phenotypes in a variety of contexts (Bandyopadhyay et al., 2010; Collins et al., 2007, 2010; Guenole et al., 2013; Roguev et al., 2008; Tong et al., 2004). These strains have been used in a number of genome-wide studies in different ways. A very complete database of the RLS of 4698 viable deletion strains was constructed using the micromanipulator assay with 5 cells per strain, followed up by more standard assays of 20 cells per strain in cells of both mating types (McCormick et al., 2015). The 238 long-lived strains that were identified were clustered into several groups that have notable overlap with life span-extending genes in metazoans, with the identification of ribosomal protein genes and genes affecting energy metabolism (tricarboxylic acid cycle, mitochondrial translation), which were or have become important candidates for regulators of aging (Howitz and Sinclair, 2008; Johnson et al., 1999; Sinclair et al., 1998; Sinclair, 2005; Sohal and Weindruch, 1996). The link with protein translation is one of the validations of the entire approach as the life span-extending effects of eliminating one of the duplicate genes for ribosomal proteins had been previously established (Labunskyy et al., 2014; Steffen et al., 2008), and the TOR kinase–pathway inhibitor rapamycin that acts as a life span-extending drug in mice is currently being tested in dogs (Steinkraus et al., 2008; Dog Aging Project, 2016). This study also identified tRNA import into the nucleus as a novel pathway for life span extension: loss of a tRNA exporter (Los1) or overproduction of a tRNA importer both extended life span to a significant degree (McCormick et al., 2015). Previous work had shown that nuclear localization of the tRNA export protein Los1 was under the control of the DNA damage-activated kinase Rad53, which was also true in the strain used to assay life span (McCormick et al., 2015; Ghavidel et al., 2007). Rad53 has all of the characteristics expected for Los1 regulator, but the genetics of RLS with this rad53 and los1 mutants are complicated.

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This complication may stem from the fact that Rad53 has multiple functions important for DNA replication and the DNA damage response in addition to nuclear localization of Los1. Dissecting the potential link between the DNA damage response and life span regulation should prove to be a fascinating new area in the coming years.

GENERATION OF SACCHAROMYCES CEREVISIAE CELL POPULATIONS FROM LATE IN THE REPLICATIVE LIFE SPAN In an exponentially growing population, half of the yeast have just been “born” as new daughters that have never budded, and the number of cells at the end of their RLS (20 divisions or more) is quite low. The process of how budding yeast asymmetrically divides has been exploited in a variety of ways to isolate populations of old cells including direct isolation of old cells and new ways to allow the survival of the older mother cell but not the younger daughter cell. The first method relied on the fact that the cell wall of the mother cell stayed with the mother at cell division. Consequently, proteoglycans that make up the cell wall could be cross-linked to biotin. These biotinylated cells can then be grown in culture and the older cells recovered by means of streptavidin beads (e.g., see Sinclair, 2013). While this method has been effective for isolating older cells, exponential growth makes it cumbersome to isolate larger numbers of cells for biochemistry. Ten cell divisions will increase total cell number by 1000-fold, so growing even 107–1010 cells requires almost a 1-L culture, and the purification must be repeated to reach the 20 or 30 cell divisions for most strains to reach the middle of the RLS. An additional drawback of this approach is that screening for drugs that increase life span or observing aging cells continuously is not possible because of the large number of young cells born each division. Systems that allow the isolation of large amounts of aged mother cells have relied on a second consequence of budding: the mother and daughter cells each possesses specific transcripts that distinguish them. Different labs have made use of these genes to either ensure the early death of the daughter cells or the survival of the mother cells to enrich for the mother cells. These systems are inducible: a large population of cells can be grown up and the inhibition of the daughter cell growth is then induced, so old mother cells can be isolated (Afonso et al., 2010; Jarolim et al., 2004; Lindstrom and Gottschling, 2009). The increase in cell number due to newly born cells and their progeny is drastically reduced, making it much easier to follow the fate of the mother cells or isolate substantial numbers of them for biochemical analysis. The DeaD assay, short for Death of Daughters, is based on controlling the transcription of CDC6, a gene essential for DNA replication (Jarolim et al., 2004). In this system, the normal CDC6 gene is deleted, and protein is produced from two different promoters: one that is inducible and expressed in all cells and one that is only expressed in mother cells (Fig. 30.3A). Cell populations are grown up in the presence of the inducer. When the inducer is removed (e.g., by washing cells) the mother cells have a major growth advantage over the daughter cells, as only the mother cells can produce Cdc6 protein (Jarolim et al., 2004). Because the Cdc6 protein can be segregated at division, some daughter cells will inherit this essential replication factor, which allows the daughter cells to survive for one or two divisions. Validation of the assay showed that some of the mutations that affect aging in wild-type cells had a similar effect in the DeaD assay. However, the average life span of the mother cells was reduced to 4–10 divisions compared to ∼20 in wild-type cells (Jarolim et al., 2004). A major use of the DeaD assay has been to screen for RLS-extending drugs (Goldfarb, 2014; Sun et al., 2013, 2014; Klionsky et al., 2012; Xiang et al., 2011; Timmermann et al., 2010). The DeaD assay is highly amenable to screening in 96-well plates because the rate of growth is largely dependent on how fast the mother cells divide. As mother cells near the end of their life span, the amount of time between cell divisions greatly increases. An RLS-extending drug would therefore increase the number of rapid, “youthful” divisions and cell number and the well containing the drug will increase more rapidly than control wells. As might be expected from the success of this system, some of the major advances with the system that identify over 100 RLS-extending compounds are in patents and the published literature. Readers interested in following advances with this system will quickly find that the DeaD assay is not a unique search term, and using the name of the DeaD assay strain, K6001, may be better place to start. The daughter arrester assay is based on killing daughter cells with a conditional poison (Afonso et al., 2010) (Fig. 30.3B). Cells contain a synthetic gene transcribed from a daughter cell-specific promoter. Key components of the synthetic fusion protein are an enzyme that converts an added drug to a toxin (Ura3 in this case), a yellow fluorescent protein to monitor expression, a G1/S degradation tag to limit the lifetime of the protein to one cell cycle, and both nuclear export and import signals to shuttle the protein from the cytoplasm (where the toxin is made) to the nucleus where the degradation tag is active. Like the DeaD assay, the system has the advantage of severely limiting growth once the drug is added. In this case, the drug is 5-fluoroorotic acid, which is a well-characterized tool for killing cells by the synthesis of 5-fluorouracil. The drug is largely inert in cells that lack the conversion pathway (Boeke et al., 1987). An advantage of this kind of system is that cells are grown and then the drug is added to the cells as opposed to harvesting the cells away from the inducer in the DeaD assay. The daughter arrester was presented as a proof-of-principle genetic device that “integrates

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FIGURE 30.3  Systems to produce replicatively aged Saccharomyces cerevisiae cells by preserving the mother or eliminating the daughter cells. (A) The Death of Daughters (DeaD) assay eliminates the production of a protein required for DNA replication. All cells can grow on medium with galactose as the sole carbon source, but only mother cells can continue to grow on medium where glucose is the carbon source (Jarolim et al., 2004). (B) The daughter arrester assay uses a synthetic construct to transiently produce an enzyme in daughter cells that can convert an added compound to a toxin. DegTag is a G1/S degradation tag, NES is a nuclear export signal, NLS is a nuclear localization signal, YFP is yellow fluorescent protein to tract the production and location of the protein, and killer enzyme in this case is the product of the URA3 gene. Conversion of 5-fluoroorotic acid (5-FOA) by Ura3 enzyme allows the eventual production of a toxic base that is incorporated into RNA and DNA (Afonso et al., 2010). (C) The mother cell enrichment (MEP) assay. Daughter cells produce a Cre–estrogen receptor fusion that is inactive in the cytoplasm until estradiol is added. On addition of estradiol, the activated Cre fusions enter the nucleus and inactivate one or two genes required for DNA replication by catalyzing the recombination between two loxP sites. Loss of either gene is lethal and daughter cells can only grow for one or two divisions until their supply of Cdc20 or Ubc9 protein is exhausted (Lindstrom and Gottschling, 2009).

information from the endogenous cell cycle and an external stimulus.” While other papers using this system have yet to be published, it has potential. The mother cell enrichment program (or MEP) similarly uses a daughter cell-specific promoter to prevent the growth of daughter cells (Lindstrom and Gottschling, 2009). In MEP, a daughter-specific promoter drives the expression of a Cre recombinase–estrogen receptor variant fusion protein. In the absence of estradiol, the recombinase is sequestered in the cytoplasm and both mother and daughter cells survive and grow. Once estradiol is added, the activated recombinase can move to the nucleus and catalyze the excision of loxP sites that eliminate the function of two essential genes: UBC9 and CDC20 (Fig. 30.3C). Consequently, daughter cells and their progeny cannot grow. The system does not affect RLS as determined by the micromanipulation assay and responds to mutations known to affect RLS (Lindstrom and Gottschling, 2009). One issue with the MEP is that estradiol-specific mutants can arise in the population and will quickly takeover the culture. Analysis of the rates that these mutants occur indicated that recessive mutations in multiple genes could give rise to this phenotype. Consequently, the MEP is performed in diploid cells where the rate of production of estradiol-resistant mutants is greatly reduced. The MEP has been used to examine different features of aging, including the genomic instability associated with aging and the characteristics of old cells (Henderson et al., 2014; Hughes and Gottschling, 2012; Hughes et al., 2016; Lindstrom et al., 2011; Thayer et al., 2014). The latter approach has led to several exciting advances in the aging field. By using stable

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isotope labeling of cells and then activating the MEP system, the Gottschling lab was able to identify ∼135 proteins that are retained by the mother cell throughout life span, 15% of which remain as full-length proteins (Thayer et al., 2014). One of these proteins, Pma1, appears to play a key role in one aspect of aging. As cell age, the membrane potential and function of the mitochondria degrade in wild-type cells and in MEP cells (Hughes and Gottschling, 2012). This degradation could be suppressed by overexpression of components of the vacuolar ATPase, which maintains the low pH required for the function of the yeast vacuole (the equivalent of the mammalian lysosome). This reduced acidity of the vacuole appears to cause a deficit of neutral amino acids in the cytoplasm, as overexpression of a vacuolar neutral amino acid transporter can suppress the mitochondrial defects in aging cells (Hughes and Gottschling, 2012). The cause of the loss of vacuolar acidity is related in part to the retention of Pma1 by mother cells: Pma1 pumps protons out of the cytoplasm to medium and raises cytoplasmic pH and antagonizes the acidification of the vacuole (Henderson et al., 2014). Consequently, the MEP has yielded a number of exciting advances in our understanding of S. cerevisiae aging with potential ramifications for mammalian cells. The demonstrated ability to isolate biochemical amounts of aged cells will be of major use to many yeast labs in the aging field.

QUANTITATIVE TRAIT LOCUS ANALYSIS IN YEAST REPLICATIVE LIFE SPAN ANALYSIS Quantitative trait locus (QTL) analysis is a statistical genetic method to identify genes that affect the outcome of complex traits such as aging and life span (Miles and Wayne, 2008; Liti and Louis, 2012). QTL analysis involves breeding strains with different life span phenotypes to identify genes that cause this difference. Yeast have major advantages in this area as the genomes of many distantly related, interbreeding strains have been sequenced. Consequently, one can breed two strains with sequence variation, e.g., single nucleotide polymorphisms, throughout the genome, which means that the genetic cross contains thousands of markers that will identify the parental source of each chromosomal region. Because yeast are assayed as haploids, the offspring of this first cross can be immediately tested for the desired phenotype (e.g., long life span), whereas diploid organisms must go through second round of breeding. Once a large number of offspring with desired phenotype have been identified, one can determine which alleles from which parent are disproportionately represented in the offspring with a desired phenotype (Liti and Louis, 2012; Steinmetz et al., 2002). Past approaches have used oligonucleotide microarray hybridization to distinguish the differential hybridization of the lab strain and the distantly related strain (Steinmetz et al., 2002; Jiang et al., 2016; Stumpferl et al., 2012), but current methods employ NGS technology to sequence the entire genomes of many recombinants at once (Pais et al., 2014; Jeffares et al., 2015; Hu et al., 2015). One difference between QTL mapping and previous work using the deletion strain set is that the assay is done on strains bearing multiple missense mutations as opposed to absence of a gene. This approach therefore has the potential to identify missense mutations in essential genes that cannot be deleted and produce a viable cell, and to identify genes with altered levels of function in the recombinant strain. QTL mapping for genes affecting S. cerevisiae RLS generally uses one of two sets of crosses to generate recombinants for mapping. Mating is usually done between a well-characterized laboratory strain and either a yeast strain isolated from a vineyard, RM11-1a, or one derived from a clinical isolate in an immune-compromised patient, YJM789 (Liti and Louis, 2012; Steinmetz et al., 2002). A study by Kwan et al. (2013) mated a laboratory yeast strain BY4716 (with the S288c genetic background) with a vineyard yeast strain RM11-1a. This approach represented a departure from standard QTL mapping as the two strains have very similar mean life spans of 27 and 29 divisions, while QTL experiments usually involve strains with larger phenotypic differences. However, in keeping the complex regulation of life span, RLS analysis of 88 meiotic segregants gave a continuum mean RLS values from 12 to more than 40 divisions, consistent with control by multiple genes. Heritability of the length of life span was >80%, and genome-wide linkage and subsequent fine structure analysis revealed that increased longevity was linked to the 1.2 MB locus containing the repeated ribosomal RNA genes (the rDNA or RDN1 locus). Standard backcrossing was used to place the RM11-1a locus in an otherwise BY4716 genetic background and produced a 45% increase in life span compared to BY4716 parental strain. Surprisingly, the genes best known to affect budding yeast RLS through the rDNA repeats, SIR2 and FOB1 (Kaeberlein et al., 1999; Kim et al., 1999), were not responsible for this difference. Instead, the RLS difference mapped to the weaker origin of DNA replication within each ribosomal RNA gene repeat (Kwan et al., 2013). The Kwan et al. study makes several points about the QTL approach to dissecting the genetic control of life span. First, even though the parental strains had similar life spans, their multiple genetic variations effectively “balanced out” the life span-extending and shortening effects of these differences. Presumably the competition for survival that led to evolution of the RM11-1a strain balanced life span with other traits, and these traits were separated in the cross with laboratory strain. Therefore, dissection of highly complex traits can be accomplished using two distantly related strains with similar phenotypes. Second, the continuum of RLS in the segregants and the idea that different long- and short life span genes balance

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each other imply that there are a number of other RLS-modifying loci in this cross that the standard QTL analysis did not identify. An independent QTL analysis of S. cerevisiae RLS conducted by Stumpferl et al. used the laboratory strain S96 (also with the S288c background) and the clinical isolate YJM789 (Stumpferl et al., 2012). The RLS of YJM789 was not determinable using the standard micromanipulator assay as this strain displays filamentous growth that disrupts the normal mother–daughter cell division cycle. These workers isolated 39 segregants from separate tetrads amenable to the RLS assay, which also showed a continuum of RLS values, and identified 5 significant QTL associated with altered life span. Two of the QTL that had the largest effect on RLS were further characterized. One locus mapped to the SIR2 gene on chromosome IV and the other identified the rDNA locus on chromosome XII. The SIR2 gene from YJM789 was associated with longer life span when placed in the S96 strain, while the rDNA from this strain was associated with shorter life span. The SIR2 genes in S96 and YJM789 are expressed at similar levels in these yeasts and when the YJM789 SIR2 gene is substituted for S96 gene, indicating that the five amino acid differences between these two alleles were likely the cause of the increased RLS. Sequencing of a YJM789 ribosomal RNA gene unit revealed multiple polymorphisms compared to S96. The effects of the different Sir2 and rDNA polymorphisms on life span have not been reported. This independent QTL study by Stumpferl et al. thus highlights how a different outbred strain can identify different loci. Given that over 100 different S. cerevisiae genomes have been sequenced and many would be amenable to QTL analysis, this approach has an important future in that analysis of yeast aging. While S. cerevisiae researchers have led in the application of QTL analysis to aging, it is important to note that QTL mapping is also an active field in S. pombe, which has a similar range of diverse genetic strains for these applications. S. pombe has spread from Africa across the world with human migration, and these physically isolated strains have been shown to have sufficient genetic variation for QTL mapping (Jeffares et al., 2015). A recent work sequencing 161 different S. pombe strain genomes revealed a five clustered, interbreeding populations with over 200 potential QTL. An important observation is that only a small number of these traits could be linked or “stratified” to the structure of these five populations, indicating that the majority of traits can be mapped in genome-wide association studies without a significant bias. The take-home lesson from these studies is that the S. pombe field has substantial resources for QTL analysis of aging. Early work with S. pombe QTL mapping has moved to “bulk segregation analysis” in which the progeny of a cross between two distant strains are sorted by their phenotypes, and all of the genomes of the selected progeny are sequenced and compared with the parental genomic sequences. Bulk segregation analysis was recently used to map a rare maltose utilization deficiency found in a wild S. pombe strain isolated from grapes, so the tools for this approach are established (Pais et al., 2014; Hu et al., 2015). Thus, the QTL mapping approach is well poised for application to S. pombe aging.

MICROSCOPY AND MICROFLUIDICS: SURPRISES FOR SCHIZOSACCHAROMYCES POMBE REPLICATIVE LIFE SPAN AND CLOSER SCRUTINY FOR SACCHAROMYCES CEREVISIAE As noted earlier in Fig. 30.1, S. pombe RLS is associated with acquisition of asymmetric cell by the older cell in the micromanipulator assay (Erjavec et al., 2008; Nystrom and Liu, 2014; Barker and Walmsley, 1999). Like many aging assays in S. pombe, these first experiments were replicas of methods pioneered in S. cerevisiae to determine how this distantly related organism behaved. The chief problem with S. pombe is that the first division of a mitotically growing cell cannot be established as it can in S. cerevisiae with a newly budded cell. The closest experiment would be to germinate individual spores and attempt to follow the oldest cells throughout their life span, which has not been done and may be complicated by the process of spore germination. Consequently, while some kind of RLS assay can be done, its application in S. pombe has been complicated. The application of microfluidics and lithography to aging biology has been successful for nematodes, where multiple microscope chambers for single worms have been designed to allow single worm aging assays with many worms on a slide (Hulme et al., 2007, 2010; Lockery et al., 2012). The advantage of these microscopic devices is that one can follow individual organisms maintained in an otherwise normal state to observe the changes that occur during aging. When coupled with the increasing automation of microscopes for data collection, a significant amount of data can be collected to reveal individual differences in aging. These types of experiments revealed that, in contrast to the micromanipulator assay, S. pombe do not recapitulate the gradual decline in function seen in the original assays and in S. cerevisiae, leading to the statement that S. pombe cells do not age before dying. The first observations of S. pombe RLS independent of the extensive micromanipulation were pedigree analyses that simply followed the growth of isolated cells over eight generations during the formation of a microcolony (Coelho et al., 2013). Surprisingly, the previously observed “classic” phenotypes of replicative aging, including changes in cell morphology and

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FIGURE 30.4  The fission yeast life span microdissector (FYLM) as an example of a microfluidic platform to monitor replicative life span. The -diagram shows a single channel of a fabricated microdevice that contains thousands of such channels. The entire device fits on a microscope slide that can image over 200 channels at once. Media with Schizosaccharomyces pombe cells flow over the tops of the channels, and S. pombe are pushed through the drain channel. Cells are trapped in the chamber but cannot exit the smaller drain channel. S. pombe ends or poles are distinct: one end is older and the other is newly made. If the old pole is oriented toward the drain channel, one of the daughters will grow out of the channel and eventually be washed away from the continuous flow of media in the media channel. The cell with the old pole stays trapped in the channel. By taking continuous movies of the process, one can count the number and timing of divisions for the cell trapped in the channel until it dies (lyses or stops growing). The system also allows the use of fluorescently tagged proteins to follow their segregation at division and presence during the life span. Similar microfluidic devices that trap Saccharomyces cerevisiae have also been devised that allow the simultaneous imaging of many cells with fluorescently tagged proteins (see text).

gradual extension of cell division time, were not observed. Instead, cell shape and rate of division were relatively constant until death occurred. The rate death was 0.3% per cell division or 1 in 333 divisions. This rate is significantly higher than that observed in S. cerevisiae, where an average RLS of 20 divisions gives rise to 210 cells, translating to under 1 death in 1,000,000 divisions. Using Hsp104-GFP as a marker for protein aggregates, Coehle et al. provided evidence that the inheritance of large amounts of aggregates is correlated with death. When protein aggregation is induced by exposure to heat or oxidative stress, the large aggregate was inherited by one of the daughter cells, and those daughter cells subsequently aged and died over a few divisions. The cells inheriting the large aggregates aged similarly to the micromanipulation assays, with increasing division times and changes in cell shape. Thus, death under optimal conditions appeared to be a stochastic event caused by the accumulation of damaged proteins, which occurs in the absence of aging, while death under stressful conditions appears to have a similar cause but the increased amount of damaged material causes the phenotypes of aging. The evolution of fission yeast appears to use a strategy of sacrificing some cells to keep damaged materials from the rest of the population, allowing rapid growth in the absence of aging under optimal conditions. These observations have been supported by experiments using a microfluidic device called a fission yeast life span microdissector, which captures S. pombe cells in small chambers so that cells are trapped (Spivey et al., 2014, 2017). S. pombe cells grow at the poles, so when the old pole is the one stuck furthest into the chamber, cells growing at the new pole will be pushed out of the chamber and washed away and divisions of the cell trapped in the chamber can be counted (Fig. 30.4). By recording movies of ∼160 trapped S. pombe cells at a time, it is possible to determine when cells die and examine the division times and other events prior to death. This assay also revealed an absence of aging phenotypes prior death, and that treatment with the TOR inhibitor rapamycin that increases life span in budding yeast and mice also increases life span in S. pombe (Spivey et al., 2014, 2017). The accumulating evidence therefore suggests that replicative aging in the fission yeast has fundamental differences compared to budding yeast. Microfluidics are also being harnessed to examine S. cerevisiae RLS (Lee et al., 2012; Xie et al., 2012; Liu et al., 2015; Jo et al., 2015). These systems recapitulate much of what has been observed in the micromanipulator assay. Their added benefit is both the single cell analysis and the addition of fluorescently tagged proteins that can be followed during the aging process as markers for cell events. Discoveries so far have revealed the heterogeneity of cell morphologies at the end of RLS (Lee et al., 2012), that the level of Hsp104 is a good predictor of cell life span (Xie et al., 2012), and that the effects of different carbon sources on S. cerevisiae life span are related to the level of induced oxidative stress (e.g., where galactose creates more stress than the preferred carbon source, glucose) (Liu et al., 2015). These systems show great promise for investigating the processes that occur at the level of individual cells as aging proceeds.

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HIGH-THROUGHPUT ADVANCES FOR CHRONOLOGICAL LIFE SPAN AND NEW ATTENTION FOR QUIESCENT CELLS CLS in S. cerevisiae and S. pombe uses similar assays to determine how long cells can survive in liquid culture after the glucose and various other nutrients have been exhausted (Longo et al., 2012; Chen and Runge, 2009, 2012; Fabrizio and Longo, 2003; Zuin et al., 2008, 2010). Because cells arrest by starvation, nutrient sensing and degradation of internal protein components by autophagy play important roles in CLS (Sideri et al., 2014; Yamaguchi and Otsu, 2012; Alvers et al., 2009a,b; Aris et al., 2012, 2013). The design of CLS assays require that some key points be kept in mind. First, if the CLS assay is to be model for other organisms, then the assay should have the same effects on yeast as the analogous assay does on other models. For most organisms, these characteristics include life span extension by caloric restriction, and these long-lived cells are frequently resistance to stress (e.g., Fabrizio et al., 2001). While the conditions were established for S. cerevisiae long ago, the primary synthetic medium used in the S. pombe field, called Edinburgh Minimal Medium (EMM or EMM2), does not recapitulate these conserved features of life span and caloric restriction (Chen and Runge, 2009). Instead, S. pombe requires either rich YES medium (yeast extract (YE) and supplements (S) and carbon source) or a medium called SD (a synthetic chemically derived medium (S) plus dextrose (D)) with different levels of components than EMM (Chen and Runge, 2009; Zuin et al., 2010). Second, the assay should incorporate information about the model organism. S. cerevisiae and S. pombe can rapidly grow entire populations from a single cell, something that cannot be done with humans or mice. Yeasts, therefore, may regulate their core machineries differently as most cells in the culture die (e.g., going from ∼108 to 10 cells/mL) compared to when cell number drops to 1% of the original level (from ∼108 to 106 cells/ mL). In fact, S. cerevisiae behave in a complex manner in a CLS assay, as when cells drop to ∼0.1% of the original density of live cells per ml in a CLS assay, a fraction of cells will begin to regrow (Fabrizio et al., 2004) (Fig. 30.2B). The cells also poison themselves by secreting small organic acids (e.g., acetic acid) into the medium (Burtner et al., 2009). This complex behavior necessitates that most S. cerevisiae CLS assays do not go much past a loss in viability below 1%. In contrast, the S. pombe CLS assay can show a drop in viability by 5–6 orders of magnitude without regrowth, which allows one to more easily isolate long-lived cells (Chen and Runge, 2009; Chen et al., 2013). Both S. cerevisiae and S. pombe have been used to screen their respective genome-wide deletion strain sets for mutants with extended CLS. Each gene deletion is tagged by a unique DNA sequence or barcode. These CLS assays have included survival in microtiter plates (Powers et al., 2006) or the standard assay using the barcodes associated with the deletions to identify the mutants associated with different life spans. The barcode frequencies have been determined using microarray hybridization (Wei et al., 2008) or barcode sequencing approaches (Chen et al., 2013; Sideri et al., 2014). Additional genes that extend CLS on overexpression have also been identified (Ohtsuka et al., 2009; Miwa et al., 2011). While the list of genes that regulated life span will not be reviewed here, it is important to note that TOR has emerged as an important life span regulator in both yeasts (Powers et al., 2006; Wei et al., 2008; Sideri et al., 2014). In general, the standard CLS assays have yielded a great deal of information that highlights the importance of a set of evolutionarily conserved pathways that control aging (Longo et al., 2012). The results of the CLS assays present some puzzles. Why are S. cerevisiae so variable at the end of CLS? Why do S. pombe have such a short CLS (14 days)? While the answers to these questions are not known, one possibility is that the assays are missing unappreciated evolutionary features in both species. One such feature that is usually not considered is another differentiated state of yeast that occurs in CLS assays, the quiescent G0 cell or Q cell. When S. cerevisiae grow to saturation and remain in culture, a fraction of cells differentiate into small, dense cells that can be isolated away from the rest of the population (Allen et al., 2006) (Fig. 30.2D). More recent studies have shown Q cells can be sorted away from the other cells by fluorescence-activated cell sorting for analysis (Miles and Breeden, 2017). These cells have remarkable properties in terms of CLS. They can survive for almost 1 year in water, are highly stress resistant, and synchronously reenter the cell cycle when nutrients are added to them (Allen et al., 2006; Miles and Breeden, 2017). The fact that they form a few days after a CLS begins indicates that they are likely playing a role in the analysis of CLS cells. Whether the now-appreciated presence of Q cells in the S. cerevisiae CLS assays makes any difference to the previous conclusions drawn from them is currently unclear. The Breeden lab has analyzed S. cerevisiae Q cells intensely in recent years, and transcriptional repressors and chromatin remodelers (e.g., Xbp1 and Rpd3) have been shown to play key roles in this process (Miles and Breeden, 2017; Miles et al., 2013; McKnight et al., 2015). Proteins known to play roles in the G1/S transition of the cell cycle also have roles in the transition to quiescence (e.g., the SBF transcription complex), and these core complexes are adapted to quiescence induction by the acquisition of other factors (e.g., Msa1 and Msa2) (Miles et al., 2016). Thus, by leveraging information from cycling cells, the core cellular machinery that governs entry into G0 is being elucidated. As identifying such core machineries are a major goal of aging research, this work represents an important step forward. An important future effort will require determining what other cellular processes these conserved pathways control.

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Quiescence in S. pombe is somewhat different. When S. pombe are mated in the laboratory, two cells of opposite mating types are placed together on medium that lacks reduced nitrogen (−N medium). The lack of nitrogen induces a signal that starts the mating program and leads to sporulation to produce haploid spores. However, if a mating partner is not present, cells slowly enter a G0 state characterized by the rod-shaped S. pombe, becoming small, round cells. These cells can survive for months and reenter the cell cycle when nitrogen is added back to the medium and are thus Q cells for this species (Su et al., 1996; Yanagida, 2009). Importantly, these Q cells are not observed in cultures that stop growing from exhausting their glucose supplies (reviewed in Yanagida, 2009). As glucose exhaustion is what causes arrest during S. pombe CLS (Fig. 30.2E); the small round Q cells seem unlikely to play a major role in these assays. A substantial amount of transcriptome analysis and characterization has been done to determine what genes are required for making S. pombe Q cells. The result has been the identification of genes for proteins that act in almost every cellular compartment (Yanagida, 2009; Shimanuki et al., 2007). A major challenge is to determine how these many players are coordinated to govern the establishment, maintenance, and disassembly of the Q-cell state, in effect the opposite of the situation in S. ­cerevisiae, where parts of the core machinery are known. Future work will determine how well information from these two yeasts can help fill our gaps in understanding the Q-cell state and if fundamental differences exist as is seen in replicative aging.

CONCLUSION The yeast model systems have continued to advance our understanding and exploration of the biology of aging. Both S. cerevisiae and S. pombe are well poised to make maximal use of new technologies in DNA sequencing and highthroughput screening. The development of innovative systems to isolate and characterize aged cells, as well as monitoring the process in real time, has provided exciting new discoveries that involve evolutionarily conserved processes. In addition to the stream of discoveries that can be expected from these systems in the future, a remaining challenge will be the application of this new knowledge to metazoan systems. Mammalian cells may share features with one or both systems in addition, to the systems that are mammalian specific. The most successful path forward will likely involve the continuous development of both systems, so the results from mammalian cells can be further tested in yeasts, and new yeast results can prompt new work in mammals.

ACKNOWLEDGMENTS This work was supported by NIH RO1 AG051601 to KWR. HZ is also supported by a Lerner Research Institute Chair’s Innovation award.

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430  SECTION | III  Cellular Models and Invertebrates

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