Yeast cultures grown on liquid media will divide reaching a certain cell density and then will enter the postdiauxic phase. Cells maintained in this condition will survive for a variable time ranging from a few days to several weeks. Factors affecting the survival time include incubation temperature, nutrient availability, pH, acetic acid concentration (Werner-Washburne et al., 1996; Burtner et al., 2009; Mirisola & Longo, 2012), and genotype (Fig. 1). It is commonly believed that CLS mimics the hypometabolic starvation condition of higher eukaryotes (Fabrizio et al., 2005); however, this is incorrect as cells grown on synthetic dextrose media reach their maximum cell density within 48 h but remain in a high metabolic state (Gray et al., 2004) for the subsequent three to five days and only then begin to die (50% survival is normally reached after 6–7 days by the widely used DBY746 yeast strain; Fabrizio et al., 2001). It is important to note that wild-type cells do not die because they starve; in fact, both the intracellular glycogen and the extracellular ethanol (and other nutrients) remain high for the entire survival period (Fabrizio et al., 2005). It has instead been demonstrated that chronologically aging yeast cells die exhibiting apoptotic markers (Herker et al., 2004). Yeast survival in the wild may have taken advantage of the ability of yeast to cope with huge changes in nutrient availability, including fluctuations of nitrogen and carbon sources. In unfavorable natural conditions, apoptosis may have been an important adaptive mechanism that was selected to ensure the survival of some members of the clonal population. The substances released by the dying cells enhance the survival of the clones in the population by providing nutrients (Herker et al., 2004), thus these findings are consistent with the existence of an altruistic aging program (Fabrizio et al., 2004).
Many genotypic variations have been associated with chronological extension of life span. Although some of them can be considered specific for yeasts or fungi, some have also been found in higher eukaryotes, including mammals. Briefly, there are two major prochronological aging pathways discovered in yeast that have been confirmed in mammals. One is the Tor/S6K pathway (Fabrizio et al., 2001), which responds to amino acid and glucose availability, the second one is the Ras/adenylate cyclase (AC)/PKA pathway (Longo et al., 1999; Fabrizio & Longo, 2003), which senses glucose but is also influenced by other nutrient availabilities (Fontana et al., 2010). These two pathways have been demonstrated to converge on the Rim15 kinase and on the transcription factors Msn2/4 and Gis1 (Reinders et al., 1998; Pedruzzi et al., 2000, 2003; Cameroni et al., 2004; Wei et al., 2008, 2009). Interestingly, in addition to stress responsive genes, these transcription factors control both intracellular and extracellular carbon source balances (Bonawitz et al., 2007; Wei et al., 2008). It must be noted that the histone deacetylase Sir2, originally discovered as a modulator of RLS, also converges on Msn2/4 activity regulation (Fabrizio et al., 2005; Medvedik et al., 2007; Smith et al., 2007). These findings suggest that metabolic regulation by Msn2/4 plays a pivotal role longevity regulation.
Some tips to assess the CLS
The population mean and maximal survival are usually determined by measuring the clonogenic survival of yeast cultures upon chronological aging. Here, the ability of yeast cells to form new colonies on rich media can be quantified as colony-forming units (CFU). Survival is then reported as the percentage of CFU measured at day 3 (conventionally referred to as the 100% survival point). Complementary approaches to assess yeast cell survival, such as the FUN-1 assay measuring the metabolic activity of yeast cells (see below), are used to confirm the age-dependent decline in clonogenic survival (Fabrizio & Longo, 2003).
CLS measures clonogenic survival in the high metabolic postdiauxic phase. It is well known that both the mean and the maximum survival time are affected by the medium used and the strain tested. For instance, rich media normally ensure a much longer viability than synthetic media [SDC, synthetic dextrose complete, (Werner-Washburne et al., 1996)]. This is probably due to the prolonged slow growth phase after the YPD-based cultures have reached the high-density status. In addition, YPD cultures tend to be more permissive to the ‘adaptive regrowth’ phenomenon, that is, the restart of growth of a few surviving cells in a chronologically old culture (Fabrizio et al., 2004), and are therefore more prone to artifacts. For these reasons, SDC medium is the preferred choice. The experiments start by streaking the strain of interest from a frozen stock onto YPD-rich medium. After 2 days of growth, some cells are transferred onto YPGly medium to be sure that the strain still has functional mitochondria. Petite strains, which have lost functional mitochondria, are not capable of growing on nonfermentable carbon sources. This is an important step to perform as the lack of functional mitochondria profoundly affects CLS. Cells from the YPGly plate are then inoculated into 1 mL of SDC medium for an overnight incubation. This preculture is then diluted to an initial density of 1–2 × 106 cells mL−1 (corresponding to an OD600 of 0.1–0.2) in 10–50 mL of synthetic complete medium containing 2% glucose (SDC). Yeast cultures are incubated (30 °C, volume/medium ratio of 5 : 1, shaking at 220 r.p.m), and after c. 10 h of growth, the glucose concentration in the medium drops and yeast switches to a respiration-based metabolism. After this ‘diauxic shift’, yeast starts using the ethanol produced during the fermentative phase through mitochondrial oxidative phosphorylation. In the postdiauxic phase, metabolic rates remain high until day 5–6. The highest optical density, which is obtained around day 3, varies from strain to strain from 7 to 15 OD600. Different yeast strains may have a different mean as well as maximum life span. For example, mean survival of wild-type strains ranges from 6–7 days (DBY746/SP1) to 15–20 days (S288C/BY4700) in SDC medium (Fabrizio et al., 2005).
As already mentioned, yeast cell cultures, as well as bacterial cultures, may start dividing again when the number of survivors drops to 1% (of day 3, 100% value), giving rise to phenomena called adaptive regrowth and gasping, respectively (Zambrano & Kolter, 1996; Fabrizio et al., 2004). As this may lead to misinterpretation of the results, especially for the calculation of the maximum life span, two alternative protocols have been developed. The first one is water incubation. Briefly, after the cultures have reached their maximal density by standard protocol, the cells are harvested by centrifugation, and after a washing step with water, they are incubated in water. This procedure is repeated every 2 days. In this way, the metabolites released by the dead cells are washed away and cannot be used by the survivors. This strategy effectively eliminates the regrowth; however, as it corresponds to an extreme calorie restriction (CR), it is useless when the effects of nutrients or of certain metabolic pathways are the focus. Alternatively, in situ viability may be used (Hu et al., 2013). Briefly, aliquots of 2-day-old liquid culture of, for example, a trp− strain are plated on many SDC plates lacking tryptophan. The plates are incubated at 30 °C but, as the essential amino acid tryptophan is missing, no growth is observed. In a timely manner (e.g. every other day), two of these plates are removed from the incubator, supplemented with tryptophan and put back in the incubator. As all the auxotrophies are now complemented, cells can start dividing. CFUs are scored after 2 days of additional incubation. The survival curve is calculated as the percentage of CFU with respect to CFU at day 2–3, the latter as usual, referred to as 100% survival. Alternatives include amino acids for other auxotrophies (typically leucine, histidine), or plating on agar plates lacking carbon and nitrogen sources (thus mimicking the extreme starvation condition resulting from water incubation) and then adding YPD or SDC instead of just one amino acid every other day. As amino acid availability may affect the survival, the comparison of yeast strains with different auxotrophies must be carefully evaluated (Gomes et al., 2007). One practical possibility to avoid possible artifacts is to equalize the auxotrophies between the strains under analysis transforming yeast cells with an empty plasmid carrying the missing markers, thus balancing any difference in auxotrophies (Hu et al., 2013).
CR, the only known intervention capable of positively affecting the life span in a wide range of species, can be effectively mimicked in these experiments by reducing the glucose concentration from 2% to 0.5–0.1%, or even to 0% (Fabrizio & Longo, 2007). In these conditions of caloric restriction from mild to extreme, yeasts will live longer (extended CLS), entering a state of low metabolic activity. Incubation in water has the additional advantage of reducing the possibility of the adaptive regrowth, as discussed above.
Mutation rate detection during CLS
As with RLS, CLS is found to be associated with various mutations, and this work has led to the discovery of the roles of the oncogene homologue Sch9 and superoxide in age-dependent mutations (Madia et al., 2008, 2009). One method used to assess appearance of mutations as a function of CLS is based on the selection of spontaneous inactivation of the permease CAN1 gene (Chen et al., 1998). This permease is responsible for the uptake of extracellular arginine and its toxic analogues, such as canavanine. Incubation of yeast cells in the presence of canavanine (60 mgL−1) will not allow survival unless a mutation impairing that permease occurs (Guthrie & Fink, 1991). As canavanine and arginine compete for the same transport system, the selective medium must lack arginine, which is a nonessential amino acid. For this reason, arginine auxotrophs cannot be used in this test. From a practical point of view, the experiment is performed at the same time as a standard CLS measurement. Whenever a small amount of the culture is serially diluted to measure the CFU (normally with a bi-daily frequency), a larger aliquot containing c. 2 × 107 cells is collected, washed with sterile water, and plated on media lacking arginine, but containing 60 mgL−1 of canavanine. The mutation frequency is then calculated as the number of Canr colonies with respect to the total number of viable cells scored each plating day in the absence of canavanine (YPD plates). When Chen and coworkers sequenced the mutated CAN1 locus obtained in this way, they found point mutations (65%), frameshifts (25%), and other more complex events (10 %) (Chen et al., 1998).
When point mutations are the main focus of the research, it is possible to measure the reversion rate of stop codons. One of the most commonly used methods is based on the ability to acquire tryptophan prototrophy. DBY746, a widely used aging research yeast strain, has a C-T mutation in codon 403 of the TRP1 gene, leading to a stop amber codon that results in tryptophan auxotrophy (Capizzi & Jameson, 1973). The procedure is quite similar to the inactivation of Can1 method, but in this case, only the few point mutations that revert the amber stop codon to a coding one will be detected, and for this reason, a much higher number of cells must be plated (108) onto SDC-Trp plates.
For detecting frameshifts mutations, lysine prototrophy of an engineered Lys− yeast strain is used. The lys2ΔBglII allele, obtained by Klenow treatment at the BglII restriction site, is inserted at the lys2 locus resulting in a lysine auxotrophy that is only reverted by mutations restoring the original reading frame (Chen et al., 1998).
Finally, gross chromosomal rearrangements are monitored by replacing the HXT13 gene (a member of hexose transporter family) with the URA3 gene, in an otherwise Ura− strain (Chen & Kolodner, 1999). The inserted URA3 gene is therefore positioned on the same chromosome as CAN1, but 7.5 kb more telomeric. 108 cells are harvested and plated on synthetic plates lacking arginine in the presence of both canavanine (60 mgL−1) and 5-fluoro-orotic acid (5-FOA, 1 mgL−1). The only cells capable of surviving in this condition are those that have lost the entire region containing both the CAN1 and the HXT13 loci (Chen & Kolodner, 1999).