Quantitative assessment of changes in cell growth, size and morphology during telomere-initiated cellular senescence in Saccharomyces cerevisiae

Abstract

Telomerase-deficient cells of the budding yeast S. cerevisiae experience progressive telomere shortening and undergo senescence in a manner similar to that seen in cultured human fibroblasts. The cells exhibit a DNA damage checkpoint-like stress response, undergo changes in size and morphology, and eventually stop dividing. In this study, a new assay is described that allowed quantitation of senescence in telomerase-deficient est2 cells with applied statistics. Use of the new technique revealed that senescence was strongly accelerated in est2 mutants that had homologous recombination genes RAD51, RAD52 or RAD54 co-inactivated, but was only modestly affected when RAD55, RAD57 or RAD59 were knocked out. Additionally, a new approach for calculating population doublings indicated that loss of growth capacity occurred after approximately 64 generations in est2 cells but only 42 generations in est2 rad52 cells. Phase contrast microscopy experiments demonstrated that senescing est2 cells became enlarged in a time-dependent manner, ultimately exhibiting a 60% increase in cell size. Progressive alterations in physical properties were also observed, including striking changes in light scattering characteristics and cellular sedimentation rates. The results described herein will facilitate future studies of genetic and environmental factors that affect telomere shortening- associated cell senescence rates using the yeast model system.

1. Introduction

Telomerase is an RNA-dependent DNA polymerase complex that replicates the ends of chromosomes at regions called telomeres in most eukaryotes. It has reverse transcriptase activity that adds short telomeric repeat sequences to the 3’ ends of chromosomal DNA during S phase of the cell cycle [1–5]. The enzyme is critical because it allows cells to overcome the end replication problem, which arises because the major DNA polymerases in the cell cannot completely replicate the ends of linear chromosomes [6–8].

Absence of telomerase results in progressive shortening of telomeres in dividing cells in vitro and also in vivo. Most human cells reduce production of telomerase during embryonic development, leading to shortening of telomeres as humans age. Humans of the same age can have different average telomere lengths and older individuals with the shortest telomeres are at higher risk of developing several age-related diseases [1,8]. Several studies have provided evidence that telomere lengths monitored within blood cells can be affected positively or negatively by lifestyle choices, including activities related to smoking, stress, exercise, and food intake [1,9–14]. Although most human somatic cells do not produce telomerase, approximately 90% of cancer cells have reactivated telomerase expression, which results in immortalization of the cells. This observation has stimulated research into the development of new anti-cancer therapies that target telomerase and/or telomeres to limit the lifespans of cancer cells [15–19].

Primary human fibroblast cells propagated in liquid cell culture mimic the telomere shortening observed in vivo, undergoing approximately 25–40 population doublings before the cells stop dividing. This phenomenon is called telomere-initiated cellular senescence or in vitro cell aging and is due to progressive telomere shortening in the cells caused by absence of telomerase. Many alterations occur in the cells during the advancement of senescence, including accumulation of modifications in nuclear DNA, changes in expression and secretion of different proteins, alteration of cell morphology and cell surface antigens, and activation of cellular stress responses [20–27]. An important concept that has emerged in recent years is that humans and animals may accumulate dysfunctional cells with the characteristics of cultured senescent cells in tissues and organs during normal aging and that these senescent-like cells might become targets of anti-aging therapy [22,24,25,28–34].

Saccharomyces cerevisiae, a unicellular eukaryote referred to as budding yeast, has proven to be an important experimental model to study the senescence process, in large part because of the sophisticated molecular genetics techniques available for investigation of this organism [35–40]. The composition of yeast telomerase is analogous to the telomerase complex of humans, consisting of multiple protein subunits plus an RNA component that serves as template for new DNA synthesis [39–41]. The core yeast telomerase enzyme consists of the Est1, Est2, and Est3 proteins along with TLC1 RNA. Est2 is the catalytic component and is functionally equivalent to hTERT, the reverse transcriptase subunit of human telomerase.

Inactivation of the yeast EST1, EST2, EST3 or TLC1 genes causes progressive telomere shortening and cellular senescence (loss of growth capability) after approximately 60–70 cell cycles, a process that is similar to that seen in primary human cells propagated in liquid culture. Yeast cells undergoing senescence become larger in size and accumulate increasing numbers of G₂ phase cells while undergoing a DNA damage-induced cell cycle checkpoint arrest response that requires some, but not all, of the checkpoint genes associated with a conventional DNA damage response [42–44]. Telomerase reactivation and mating rescue experiments demonstrated that most non-dividing, fully senescent yeast cells do not lose viability, but instead exist in a metabolically active, growth-arrested state [42]. Telomere shortening rates vary among different cells and therefore in vitro cell aging is a stochastic process, whereby individual cells lose their capacity for additional growth at different times. The senescence process has been monitored in telomerase-deficient yeast cells using both solid media plate assays and liquid culture assays [35,37–40,42,45,46]. Plate assays involve streaking cells from single colonies onto the surfaces of fresh plates, allowing the cells to grow into new colonies, and then streaking the cells out again and again from individual colonies until there is a strong reduction in the numbers and the sizes of new colonies that form. Typically, this takes three or four streak plates and a total of approximately 60–70 cell divisions [35,39,42].

The major goal of the current study was to develop new methods to analyze the kinetics of cellular senescence and to quantify the changes that occur in the morphology and physical properties of the cells. A new plate-based senescence assay is described that allowed cell aging rates to be monitored quantitatively and permitted application of several commonly used statistical measures. In addition, a cell division counting method was developed to calculate the number of generations of growth achieved by the cells. The plate assay and the cell division counting system were subsequently used to quantify the strong acceleration of senescence that occurs in telomerase-deficient cells that are also defective in genes affecting homologous recombination. Other changes occurring in senescing cells were also analyzed, including time- dependent increases in the sizes of the cells, which could be correlated with changes occurring in the physical properties of the cells.

2. Materials and Methods

2.1. Yeast strains and plasmids

All yeast strains used for this project were derived from BY4742 (MATα his3Δ1 leu2 Δ0 lys2Δ0 ura3Δ0) [47]. These strains included YLKL803 (BY4742, est2Δ::HygB^r containing pLKL82Y [CEN/ARS URA3 GAL1-V10p::EST2]) and YLKL807 (est2Δ::HygB^r rad52Δ::G418^r + pLKL82Y), which have been described [42,48]. YLKL961 (est2Δ::HygB^r) is derived from YLKL803 but contains pVL715 [2μ URA3 ADH1p::EST2]) [49] rather than pLKL82Y. Double mutant derivatives of YLKL803 were created by either plasmid or PCR fragment-mediated gene disruption-deletion as described [50]. These strains included YLKL1203 (est2Δ::HygB^r rad51Δ:LEU2), YLKL1204 (est2Δ::HygB^r rad54Δ:LEU2), YLKL1205 (est2Δ::HygB^r rad57Δ:LEU2), YLKL1534 (est2Δ::HygB^r rad55Δ:LEU2), and YLKL1547 (est2Δ::HygB^r rad59Δ:LEU2).

2.2. Yeast cell growth and transformation

Yeast growth media, including YPDA, YPG and synthetic dropout media, were prepared as described [51]. Transformation of DNA into yeast cells was performed using early stationary phase cells and a PEG/LiAc chemical-based method [52]. Synthetic media plates containing 2% glucose and 1% 5-fluoroorotic acid (5-FOA) (Gold Biotechnology) were used for counterselective growth of Ura⁻ cells. Plasmid DNAs were extracted from E. coli DH5a cells grown in liquid TB broth using Qiaprep plasmid miniprep kits. Horizon 11–14 gel rigs were used to perform gel electrophoresis using 0.7% - 0.9% agarose gels in 1 X TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA). Gels were stained in ethidium bromide (0.5 μg/mL) for 15–20 min and images were captured using an Alpha Innotech Red Imaging system.

2.3. Solid media-based senescence assays

New senescence assays involving telomerase-deficient YLKL803 cells employed either rich YPDA or synthetic (defined) plate media and were accomplished by performing a series of streaks using toothpicks. For the first streak, cells were picked from a freshly grown galactose minus uracil stock plate so that cells were producing telomerase from the GAL1-V10p::EST2 plasmid pLKL82Y prior to initiation of the experiment. Cells from a single colony were touched with a toothpick and streaked onto new plates as double columns (shown in Figure 2A). After the first rectangular column was created by repeatedly making overlapping parallel lines with a sterile toothpick, the toothpick was flipped and used to pull cells from the bottom of the first column over to where the second column was to be initiated. A new toothpick was then used to streak a complete second column (rectangle). The second column was always streaked away from the center so that individual colonies formed closer to the outer edges of the plate.

For each plate, four separate double columns were streaked so that single colonies became visible after incubation of the plates for 3–4 days at 30°C (see plate photographs in Figure 2A and 2C). For the second streak, small-to-medium sized (typically 0.8 – 0.9 mm diameter) single colonies from the first streak plate were picked and streaked into new double columns onto new plates. This process was performed again to streak cells from the second plate to the third streak plate. Altogether, four separate double columns were streaked per plate and cells were allowed to grow in the incubator at 30°C for 3–4 days, depending on the growth rate of the colonies. Slow- growing double mutants were sometimes grown for an extra day to ensure that the sizes of the picked colonies were similar for all strains. For the fourth streak, 48 different individual colonies were picked and streaked as single tall rectangular columns onto 6 different plates using a grid pattern placed underneath the plates, creating 8 separate columns on each plate as shown in Figure 2. These plates were incubated at 30°C for 4 days. As cells aged, their growth rates slowed, which is why the last streak plates were always incubated for 4 days. Full columns were defined as complete rectangles with confluent growth throughout, i.e., without internal gaps and without shortening of growth on one end. Examples are presented in Figure 2A. Data from the plate assays were analyzed and mean, median, standard deviation and unpaired Student’s T-test results calculated as appropriate. Results obtained with the new grid-based assay were highly reproducible, as shown by the multiple trials presented in the text and also the similar results that have been observed by students working in the lab over the past 5 years.

2.4. Determination of the number of cell divisions completed during senescence on plate media

The method used for calculation of the number of generations of growth within colonies was conceptually similar to the approach used by Joseph and Hall [53], but details had to be worked out empirically to ensure that all cells within the colonies were harvested and counted. Initial experiments established that the bore sizes of conventional glass Pasteur pipettes were not large enough to core entire colonies reliably. Testing of several alternatives led to the determination that a 1 mL graduated plastic pipette with an attached electronic pipette aid could be used to core all cells from typical small-to-medium sized colonies. The electronic pipette aid was used to pull the colony, along with the plate agar beneath it, up into the 1 mL pipette (photos explaining the process are presented in Figure 4A). After expulsion of the plug of agar containing the colony into water, tests demonstrated that it was important to rinse the plastic pipette 3–4 times with water from the recipient microfuge tube, which contained either 500 μL or 1000 μL of sterile deionized water. The tube was mixed using a vortexer adjusted to its highest setting for 10 s to separate the cells from the agar and then sonicated for 10 s at amplitude 24 with a Sonics and Materials VCX130 sonicator. A total of 12 μL of cells were transferred onto a hemocytometer and analyzed using a United Scope Model M837T video camera-linked phase contrast microscope to determine cell density. For each assay, 7 to 10 colonies were harvested and used to calculate averages and standard deviations. The average number of cells per colony was used to calculate how many cell divisions were completed by the cells on each streak plate. Defining the variable y as the number of doublings undergone by a single cell to form a colony, the total number of cells in a colony was calculated as 2^y (e.g., after one cell divides twice it will form 4 cells and this can be represented as 2² = 4 total cells; other examples are shown in Figure 4B). Similarly, if 6 cells are present, then 2.5 generations of growth have occurred, corresponding to two complete doublings plus half of a third doubling.

2.5. Measurement of senescent cell sizes

Cells from each senescence assay streak plate were diluted into dH₂O, sonicated 15 s and analyzed by phase contrast microscopy. The image capture program ScopeImage DynamicPro was used to visualize fields of cells at 400×-1000× with a length scale overlaid on each field as shown in Figure 5B. The largest diameter of each unbudded cell and the maximum diameter of the mother cell within each budded cell was determined. A total of 10 to 15 cells were measured for each stage of senescence and the results were averaged.

2.6. Analysis of light scattering and cell sedimentation rates

Cells from each senescence streak were harvested from a YPDA plate, rinsed with dH₂O, sonicated, counted by hemacytometer, and diluted to 1 × 10⁷ cells/mL in 0.5 mL dH₂O. Solutions were placed into standard minicuvettes, mixed, and light scattering immediately assessed at 600 nm. Five replicates were analyzed for each sample. Cells were also analyzed at 1 × 10⁶ cells/mL (not shown), which produced OD₆₀₀ values of 0.12 and 0.15 for WT and senescent cells, respectively.

For sedimentation rate tests, cells were sonicated and counted as before and diluted to 1 × 10⁷ cells/mL in 2 mL dH₂O. The cells were then re-sonicated for 15 s, followed by aliquotting of 450 μL into four standard minicuvettes (height: 4.5 cm). After mixing briefly by pipetting up and down in all cuvettes, an initial OD₆₀₀ was measured that corresponded to T=0. Subsequently, OD₆₀₀ values were recorded at 10 min intervals for 70 min using a Bio-Rad Smartspec Plus spectrophotometer. All initial OD₆₀₀ readings were between 1.0 and 1.5. Four replicates were analyzed for each strain and averages and standard deviations are presented in each graph.

3. Results

3.1. Development of a new assay for measuring senescence kinetics

Wildtype yeast cells are routinely grown on Petri dishes, where they form colonies that can be picked with a sterile toothpick or loop and restreaked to form new colonies essentially indefinitely. By contrast, telomerase-deficient cells, i.e., est1, est2, est3 or tlc1 mutants, undergo progressive telomere shortening and can be restreaked only a limited number of times before the cells cease dividing. This form of in vitro cell aging is a stochastic process and therefore the number of cell cycles that each cell can complete before losing the capacity to continue dividing is variable. Because of this variability, it has proven difficult to perform quantitative comparisons of senescence kinetics among different mutants or under different environmental conditions using plate assays.

We have previously described a yeast strain (YLKL803) that has been engineered to allow expression of Est2, the catalytic subunit of telomerase, to be modulated using the galactose-inducible GAL1-V10 promoter [42,48] (Figure 1A). The cells produce Est2 and grow normally when propagated on media containing galactose as carbon source, but upon transfer to glucose media the cells undergo telomere shortening and subsequent senescence. Thus, the cells grown in glucose can be picked from colonies and re-streaked to new plates only a limited number of times. A typical senescence assay is shown in Figure 1B, in which cells were initially picked from a single colony on a synthetic galactose plate lacking uracil and streaked to a glucose-containing YPDA plate, shown as the 1^st streak in the figure. New colonies formed after incubation of the plates for 3 days at 30°C. A single colony from the 1^st streak plate was then picked and streaked to a 2^nd plate and the process was repeated two more times. The cells readily grew into small-to-medium sized colonies on the first three streak plates but show reduced colony-forming ability on the 4^th streak. Although the progression depicted on the left side of Figure 1B is similar to those that are commonly reported, the numbers and the sizes of colonies observed on 4^th streak plates are variable in practice; several additional 4^th streak outcomes are shown on the right side of the figure to illustrate typical variations.

Fig. 1.

Method employed to monitor senescence in the yeast model system. (A) Strain YLKL803 contains the plasmid pLKL82Y, which expresses EST2 from a mutant GAL1 promoter that has reduced basal activity. (B) Example of a traditional senescence streak assay revealing the reduced but variable growth observed on the last streak.

After testing several potential improvements, a new single column growth assay was developed in order to make the senescence assay more quantifiable. In the standard version of the new assay, cells are restreaked from colonies as shown in Figure 1B for the first three streak plates. However, for the 4^th plate, individual colonies from the 3^rd streak plate are picked and streaked as equal-sized rectangular columns using a grid placed under the plate as a guide (Figure 2A). The plates are then incubated at 30°C for 3 or 4 days. In a typical experiment, streaks 1, 2 and 3 are performed eight times on two plates each, picking from a separate colony for each new streak (Figure 2B). Then, for the 4^th streak, 48 single colonies from the 3^rd streak plates are streaked into rectangular columns on six new plates using the grids, creating eight columns per plate. The number of columns showing confluent growth are then scored. Examples of full columns formed by wildtype cells are shown in Figure 2A (right side) and examples of each step in the assay for the est2 strain YLKL803 are shown in Figure 2C. An important advantage of this approach is that the 4^th streak plates essentially represent 48 separate scorable assays of growth capability and this large number allows for the application of statistics.

Fig. 2.

Illustration of new method for quantitative analysis of senescence rates. (A) Cells are streaked repeatedly from single colonies to form new isolated colonies in the traditional manner for the first three streak plates. For the final senescence plate, colonies are streaked into single rectangular columns using a grid has been placed under the plate. In the example shown, YLKL803 (GAL1-V10::EST2) was grown on galactose plates, making the cells Est2⁺, which resulted in 8 full columns of growth on the 4^th streak. (B) A key element of this approach is that colonies are picked and streaked to new plates multiple times (two plates for each strain with four double columns per plate). This procedure generates many colonies that are individually streaked into 48 single columns on the 4^th streak plate, in essence creating 48 separate senescence tests. (C) Examples of senescence assay plates 1, 2, 3 and 4 resulting from repeatedly streaking YLKL803 cells to YPDA plates. In this typical example, several incomplete columns are observed on the 4^th streak plate.

To investigate the reproducibility of this approach to quantitation of senescence, three separate assays were performed weeks apart from each other by streaking YLKL803 cells from galactose minus uracil plates, ensuring that cells were Est⁺ initially, onto YPDA glucose plates (rich growth media). Three additional assays were performed by streaking cells from the galactose minus uracil plates to less nutrient-rich synthetic glucose complete plates, which is the other major type of growth media used in yeast research laboratories (Figure 3A). A control experiment was performed by streaking the cells repeatedly to galactose minus uracil (Gal-Ura) plates, keeping EST2 expression on throughout the assay. As expected, almost all cells that were grown continuously on galactose plates were able to form full columns on the 4^th streak plates (46 out of 48) and there was an average of 7.6 full columns out of 8 total columns on each plate. By contrast, YLKL803 cells propagated on YPDA plates only formed 1.8, 2.0 and 1.8 full columns per 4^th streak plate (Figure 3A). Similarly, cells repeatedly streaked to synthetic glucose plates formed only 3.3, 3.0 and 3.5 confluent columns per plate on the 4^th streak. Application of the Student’s T-test indicated that the results of each glucose assay were strongly different from the control galactose assay (p < .005). However, comparing the trials on different media to each other (YPDA-trial 1 vs synthetic glucose complete-trial 1, etc.) yielded p values of 0.31, 0.36 and 0.15 for trials 1, 2 and 3, indicating that the differences seen on rich versus synthetic plates were not significant. Median values were close to the corresponding means, suggesting minimal skewing of the distribution of values in each assay. The standard deviation calculated for the control galactose assay was low (< 10% of the mean), but was high relative to each mean for the senescent cells (Figure 3A), which is a consequence of the stochastic nature of the process. Overall, these findings are an indication that the senescence occurring in the cells could be quantified with good reproducibility.

Fig. 3.

Quantitation of senescence in est2 and est2 rad52 cells using the column assay method. (A) Results obtained from analysis of 4^th streak plates. Senescence assays were performed using YLKL803 cells propagated on either YPDA or synthetic complete glucose plates. Results from three separate assays (trials) conducted weeks apart are shown. (B and C) Results of assays performed by streaking cells from individual colonies into single columns on the 3^rd streak rather than on the 4^th streak using YPDA plates. As expected, most est2 cells formed full columns on the 3^rd streak plate, but est2 rad52 cells did not. (D) Plate senescence assays demonstrating the consequences of inactivating several RAD52 epistasis group genes involved in homologous recombination. (E) A different senescence assay strain (YLKL961) containing pVL715 (ADH1p::EST2 URA3) produces results similar to YLKL803 (GAL1-V10::EST2). Cells were streaked to synthetic glucose plates containing 5-FOA initially to select for cells that had lost the plasmid, followed by repeated streaking of the plasmidless cells to glucose complete plates.

As shown in Figures 1B and 2C, growth of colonies is typically robust on the third streak plate, after which the cells have undergone approximately 60 generations of growth (assuming approximately 20 cell divisions are required to form a small-to-medium sized colony on a plate; this assumption is addressed more fully below). To determine if senescence might be detectable on the 3^rd streak plate, new experiments were performed in which cells were streaked for colonies on plates 1 and 2 but were streaked into scorable columns using grids on the 3^rd plate. Two trials using YLKL803 produced 42 and 47 full columns out of 48 total (Figure 3B). The strong growth suggests that senescence is not yet measurable at this stage of the process in the est2 single mutants.

3.2. Analysis of cell senescence in telomerase-deficient cells that are also defective in homologous recombination

It is well established that est2 rad52 double mutants, which are telomerase-deficient and also strongly defective in homologous recombination, undergo loss of growth potential earlier than est2 single mutants, primarily due to faster telomere shortening in cells that cannot restore telomere lengths by unequal strand exchange [39,40,42,46,54]. In conventional plate senescence assays this characteristic of est2 rad52 cells manifests as a qualitative reduction in colony numbers and sizes on the 3^rd streak plate rather than the 4^th plate, but with variability in growth on this streak plate as described above. New assays were performed using est2 rad52 double mutants (YLKL807), whereby cells were streaked for colonies normally on plates 1 and 2 but were streaked into scorable single columns using grids on the 3^rd plate. Colonies were always allowed to grow to the same approximate size on the first and second streak plates, which required an extra day of growth for the slower growing est2 rad52 double mutants. The recombination-deficient mutant cells produced only 6 and 7 full columns among 48 total column assays (Figure 3C), a strong reduction compared to the 42–47 columns seen with est2 single mutants (Figure 3B). Analysis of the double mutant strain data using unpaired Student’s t-tests to compare trial 1 and trial 2 in Figures 3B and 3C revealed strong differences with p < 0.005.

In addition to Rad52, several other members of the RAD52 epistasis group of homologous recombination proteins are also involved in genetic exchange between chromosomes [55–57]. New tests were performed to assess the impacts of other members of this group on senescence. Growth of est2, est2 rad51, est2 rad52, est2 rad54, est2 rad55, est2 rad57 and est2 rad59 strains was analyzed using 3^rd streak plates as for Figures 3B and 3C and results are presented in Figure 3D. Inactivation of RAD51, RAD52 and RAD54 had the greatest impact on senescence, as these mutants generated only 1.2, 0.7 and 1.0 full columns per 3^rd streak plate versus 7.5 full columns for est2 single mutants. Growth of all of the recombination mutants was reduced relative to est2 cells with p < 0.05, though the results with est2 rad59 cells barely achieved significance at p = 0.03 (Figure 3D).

3.3. The new assay produces similar results using an alternative yeast assay strain

The new assay was further assessed using a different senescence strain system. An est2 strain was constructed (YLKL961) that contains the plasmid pVL715 (ADH1p::EST2 URA3) and therefore constitutively produces Est2 protein from the ADH1 promoter. This strain was maintained on glucose plates lacking uracil so that all cells were Est2⁺ prior to the assay. The cells were then streaked onto synthetic glucose plates containing 5-FOA (streak #1) using the pattern shown in Figure 2C to select for cells that had spontaneously lost the plasmid and therefore become Est2⁻. Colonies on the 5-FOA plates were subsequently picked and streaked for single colonies two more times (streak plates 2 and 3) onto glucose complete plates and then into grid-based columns on the 4^th streak plates. The average number of full columns formed after growth for 4 days at 30°C on the 4^th streak was 3.3 out of 8 total on each plate (Figure 3E). This result is similar to the values of 3.3, 3.0 and 3.5 measured with the original assay system that also used synthetic glucose plates (Figure 3A, Glu-complete media).

3.4. Calculation of population doublings occurring during senescence

The goal of the next experiment was to develop a method to quantify the number of generations (i.e., cell divisions or population doublings) undergone by the senescing cells before they stopped dividing. This was accomplished by harvesting whole YLKL803 strain colonies from streak plates 1, 2 and 3, counting the total number of cells per colony, and then calculating how many generations of growth from a single cell led to that number of total cells. The final method that was devised involved coring each colony using a long 1 mL pipette connected to an electronic pipette controller, transfer of the cells into water followed by vortexing and sonicating, and counting by hemacytometer in a phase contrast microscope (see caveats and details in Materials and Methods). The steps of the procedure are depicted in Figure 4A. The number of generations achieved was calculated from the total number of cells in each colony. After being streaked to a plate surface, each individual cell divides into 2 cells and then 4 and 8 and so on multiple times to form a single colony of more than a million cells (Figure 4B). The total number of cells present in a single colony can be calculated by using the formula: 2^y = total number of cells. The variable y is the number of cell divisions that have occurred during formation of the colony. As an example, Figure 4B shows that a colony that has 1,048,576 cells has undergone exactly 20 cell divisions. A caveat to this type of analysis is that there may be some cells in the colony that have lost viability and so these numbers should be considered best estimates. YLKL803 colonies formed on rich YPDA plates and on synthetic glucose plates were analyzed in this manner and results are shown in Figures 4C and 4D. The senescing cells underwent a total of 64.7 and 63.3 generations of growth on YPDA and synthetic plates, respectively. Note that these numbers do not take into account the small, variable number of cell divisions that may occur on the subsequent 4^th streak plates. We have previously demonstrated that cells harvested from 3^rd streak plates exhibit a low average plating efficiency of 0.5%, i.e. only about 1 in 200 cells are able to form colonies when spread onto YPDA plates [42].

Fig. 4.

Determination of the number of population doublings (generations of growth) achieved by telomerase-deficient cells during senescence. (A) Photo array depicting the use of a long 1 mL pipette and pipette controller to core individual colonies from senescence assay plates. (B) Relationship between generations of growth from a single cell and total numbers of cells within the resulting colonies. (C and D) Average cells per colony and cell cycles completed for est2 cells grown on YPDA or synthetic complete glucose plates. (E and F) Cells per colony and cell cycles completed were determined for est2 rad52 cells.

In contrast to the est2 single mutants, est2 rad52 double mutants completed only 42.3 and 42.7 generations on YPDA and synthetic glucose plates before undergoing loss of growth capability (Figure 4E and 4F). These findings and those resulting from the earlier column assays are important because they show that a difference between yeast strains that has previously been characterized qualitatively using plate media can be analyzed in a more quantitative manner.

3.5. Cell sizes increase progressively during senescence

Senescence leads to changes in size and morphology among aging yeast cells and also in cells of higher eukaryotes [39,43,44]. An example of this phenomenon is depicted in Figure 5A, where the senescent cell population is seen to have larger cells and also more large-budded cells. A phase contrast microscope equipped with a length scale (Figure 5B) was used to measure the average diameters of wildtype cells and est2 cells harvested at different stages of the senescence process. The cells were taken from YPDA streak plates 1, 2, 3 and 4 (with streaks performed as in Figure 2C), which corresponded to approximately 21, 42, 63 and >63 generations of growth (numbers taken from Figure 4C). Because unbudded G₁ cells tend to be smaller than budded S and G₂/M cells, these two types of cells were measured separately. The maximum diameter of the slightly oval unbudded cells was determined and the largest diameter of the mother cell was assessed in budded cells. A progressive increase in cell size was observed for both cell types (Figure 5C and 5D). Unbudded cell sizes increased from 5.2 μm to 8.2 μm, a 58% gain, and budded cell diameters progressed from 6.3 μm to 10.1 μm, a 60% increase, by the 4^th streak. A similar trend was observed in est2 rad52 double mutants, i.e., unbudded and budded cells got progressively larger during the course of senescence (Figure 5E and 5F). The magnitude of the increase was reduced in the double mutants, possibly because the est2 rad52 cells were larger than normal before senescence began, e.g., sizes of the non-senescent double mutants were 7.5 and 8.3 mm while the est2 single mutants were only 5.2 and 5.7 μm (compare first columns of Figure 5E and 5F vs. first columns of 5C and 5D).

Fig. 5.

Cell size increases progressively during senescence. (A) Phase contrast images of wildtype and senescent (4^th streak plate) cells. (B) Example of method used to measure the maximum diameters of cells. (C and D) Average sizes of unbudded and budded cells assessed at different stages of senescence. (E and F) Average sizes of unbudded and budded est2 rad52 cells. WT, telomerase-proficient BY4742 cells. Stk1, Stk2, Stk3 and Stk4 refer to the 1^st, 2^nd, 3^rd and 4^th streak plates. Error bars indicate standard deviations.

3.6. Senescence also produces changes in the physical properties of the cells

The large alterations in morphology seen in senescent est2 cells suggested that there might also be measureable changes in the physical properties of the cells, a phenomenon that has not been explored previously. To test this idea, the ability of the cells to scatter visible light (a characteristic of particles suspended in aqueous solutions) and to settle quickly to the bottom of tall cuvettes was examined. Wildtype and senescent cells taken from 4^th streak YPDA plates were sonicated, counted by hemacytometer, diluted to 1 × 10⁷/mL, and their ability to scatter light at 600 nm was assessed. The senescent cells exhibited a 33% increase in light scattering (Figure 6A).

Fig. 6.

Assessment of time-dependent changes in the physical properties of cells during senescence. (A) Light scattering at 600 nm is increased in senescent cells. Wildtype and senescent (4^th streak plate) cells were sonicated and set to a titer of 1 × 10⁷ cells per mL. Five replicates of each strain were analyzed. (B) Diagram illustrating the concept underlying cell sedimentation rate studies, i.e., larger cells tend to fall faster than smaller cells and the process can be monitored as a reduction in OD₆₀₀ over time. (C) Time course of cell sedimentation in wildtype vs. fully senescent cells propagated on rich YPDA plates. (D) Progressive changes in sedimentation rates harvested from 1^st, 2^nd, 3^rd and 4^th streak YPDA plates. (E) Sedimentation of wildtype and fully senescent cells grown on synthetic glucose plates. (F) Sedimentation results for cells harvested from 1^st, 2^nd, 3^rd and 4^th streak synthetic glucose plates. (G) Time course of sedimentation of non-senescent rad52 cells (EST2 rad52) and for 1^st, 2^nd and 3^rd streak plates containing est2 rad52 cells. Error bars indicate standard deviations. Glu-com, synthetic glucose complete media.

Yeast cells, like other eukaryotic cells, will slowly settle to the bottom of aqueous solutions over time. We hypothesized that the larger sizes of senescent cells would cause them to sediment faster than wildtype cells and that this phenomenon could be quantified spectrophotometrically by monitoring the time-dependent decrease in OD₆₀₀ of cell suspensions (illustrated in Figure 6B). Both wildtype and senescent cells propagated on rich YPDA media were harvested, washed, counted and suspended in water at 1 × 10⁷ cells per mL. Sonicated cell aliquots were placed into four cuvettes, mixed, and then OD₆₀₀ values were recorded at 10 min intervals. The senescent cells fell out of solution more quickly than wildtype cells (Figure 6C). The OD₆₀₀ of senescent cells grown on YPDA plates was reduced to 50% after 20 min, while a similar decrease in WT cells required 50 min. Analysis of senescing cells harvested from 1^st, 2^nd, 3^rd, and 4^th streak YPDA plates revealed a continuous increase in sedimentation rates for cells taken from each of the first 3 streak plates, though with only a small change from the 3^rd to the 4^th streak (Figure 6D). Qualitatively similar results were observed when cells were propagated on synthetic glucose complete plates, where the OD₆₀₀ of the senescent cells was decreased by 50% within 25 min, but a similar reduction in WT cells required 38 min (Figure 6E and 6F). Analysis of sedimentation of est2 rad52 double mutants grown on YPDA media revealed a different, more compressed pattern than est2 single mutants grown on YPDA plates (compare Figure 6G vs. 6D). This is likely due to the smaller differences in size between senescent and non-senescent est2 rad52 double mutants described above. In aggregate, these spectrophotometry results demonstrate additional, previously uncharacterized properties of senescent est2 cells that are strikingly different from wildtype cells.

4. Discussion

Yeast cells have proven to be an important and versatile model system for analyzing genetic factors that affect in vitro cell aging. In the current study we have improved the usefulness of this model by developing and testing new assays that allow quantitation of several of the progressive changes that occur in the cells during senescence. Telomerase-deficient cells lose telomere sequences during each cell cycle and cease growing after some number of their telomeres reach critically short lengths, but the precise number of cell divisions required to reach this state varies. This stochastic nature of senescence has made it difficult to quantify the process using plate assays. The single column assay method described here was found to give reproducible results in multiple trials when employed using either YPDA plates or synthetic glucose plates. In addition, similar results were observed with a strain that modulated telomerase expression using a GAL1-V10p::EST2 cassette and a different strain in which a URA3 plasmid containing a constitutive ADH1p::EST2 promoter fusion was removed by selection on 5-FOA plates.

Lundblad et al. have previously described another approach to analyzing senescence using plate assays that, like the one described here, attempted to reduce the subjectivity of the analysis [35,58,59]. In that method, telomerase-deficient cells were streaked from single colonies onto new plates a total of three times, streaking/diluting the cells each time so that new colonies formed on the streaked plates. The amount of colony growth visible on each plate was then analyzed in a genotype-blind manner, whereby the viewer did not know what specific strain was being analyzed, and growth extent was then classified into one of either 5 or 6 different categories ranging from approximately no growth to nearly wildtype levels of growth. Both this earlier approach and the system developed and tested in the current study are superior to other plate-based assays because of their use of many replicates for the analysis and their ability to generate numerical growth scores for different strains. A potential advantage of the full column scoring method described here is that it reduces subjectivity even further, i.e., one simply determines whether or not the cells streaked from an individual colony grew into a confluent full column.

A major advantage of the new plate assay is its ability to compare senescence progression in mutant strains with altered telomere shortening rates. For example, it has been known for many years that reduction of homologous recombination between telomeres accelerates cellular senescence [35,39,42,45,46]. Using the new method, the faster kinetics of senescence in homologous recombination-deficient est2 rad52 cells could be readily expressed in numbers, i.e., est2 cells formed 42 columns out of 48 tested under conditions where est2 rad52 double mutants generated only 6 full columns (Figure 3B and 3C, trial 1). A more critical assessment of this approach was performed by analyzing senescence in a series of isogenic telomerase-deficient cells that also contained mutations in several other RAD52 group genes. The experiments demonstrated that senescence was accelerated in all of the recombination-deficient mutants and that inactivation of RAD51, RAD52 and RAD54 had the most severe impacts. This result is in accord with past studies showing that ionizing radiation resistance and some types of recombination events are more strongly reduced in rad51, rad52 and rad54 mutants than in rad55, rad57 and rad59 cells [55,56,60,61–65]. The results are also largely consistent with those of a prior study that used liquid culture-based senescence assays to analyze growth of tlc1 strains lacking Rad51, Rad52, Rad54 or Rad57 [66]. In that work, growth in liquid broth was strongly reduced in all four recombination-deficient double mutants, with tlc1 rad52 and tlc1 rad54 cells showing the largest effects.

The number of cell cycles completed by senescing cells was guaged using a colony harvesting technique developed for this study (Figure 4A). The method allowed determination of the number of generations of growth completed by est2 versus est2 rad52 cells prior to the last streak plate, on which colony growth is poor and variable. The analysis showed that est2 cells completed 64.7 cycles in YPDA plate assays under conditions where est2 rad52 cells completed only 42.3 cycles. This analysis of the number of population doublings represents a second approach, in addition to the plate assays described above, that was used to measure the accelerated senescence of telomerase-deficient rad52 mutants quantitatively.

Several past studies have analyzed yeast cell senescence using liquid culture assays, a method that can potentially also be employed to calculate the number of generations of growth achieved during senescence [39,42–45]. In this method, cells are diluted to a low cell titer in liquid broth and then shaken overnight. The dense cell cultures formed on the following day are diluted to a low titer into fresh broth and shaken overnight again and the process is repeated for several days until a strong reduction in growth is observed. The density of each overnight culture decreases due to senescence over the course of several days, and then begins to go back up again as individual cultures accumulate faster-growing survivor mutants that utilize an alternative, telomerase-independent mechanism for maintaining telomere lengths [37,39,40,67]. In practice, the number of generations and/or days that occur before the cell titer of the overnight culture reaches a minimum density and begins to increase due to accumulation of survivor mutants shows strong variability. Examples of this variability among independent cultures analyzed in a single study can be found in the reports by Ijpma and Greider, Azam et al. and Chang et al. [44,45,68]. The generations of growth completed before reaching a minimum in telomerase- deficient tlc1 cells (equivalent to the est2 mutants studied here) ranged from approximately 60 to 85. Thus, the results of liquid-based assays that reported generation numbers are in general agreement with those obtained in the current study. Although survivor mutants accumulate and eventually take over liquid cultures of telomerase-deficient cells, the plate senescence assays are largely unaffected by this process. Large colonies that may contain fast-growing survivor mutant cells are occasionally observed within 4^th streak plate columns, but their appearance is rare and does not interfere with analysis of growth within the 48 individual assays.

Yeast cells become larger than normal during the senescence process [43,44,69]. We calculated maximum cell diameters at each stage of the plate senescence process and observed a time-dependent increase in size. The increases were found to be substantial, as final average sizes were elevated by 60% among both unbudded and budded cells. Consistent with these findings, there were strong changes in the physical properties of the cells. Using simple spectrophotometric assays, we observed that senescent cells induced light scattering more strongly than normal cells. The ability of eukaryotic cells to scatter light has been demonstrated to be dependent on multiple factors [70–72], but it is likely that the greater overall sizes and the elevated fraction of large-budded (dumbbell-shaped) cells among the senescent cells are major contributors. We also observed that the older cells sedimented out of solution at a faster rate than wildtype cells and that this rate increased substantially as the cells progressed from early to late senescence. The faster sedimentation effect was strongest in cells that had been propagated on rich YPDA plates, where the time course suggested that 90% of senescent cells had fallen to the bottom of the tubes after 30 min, while only 10% of wildtype cells had sedimented at that time (Figure 6C). It is likely that this phenotype is also a result of the increased cell sizes and the larger number of large-budded G₂ cells present in senescent cell populations caused by activation of the telomere shortening-associated DNA damage checkpoint response [39,42–44]. The changes in cellular metabolism and the loss of growth capability that occur in cells undergoing telomere shortening have been linked to changes occurring in the chromosomes. However, cells that have become enlarged, regardless of the underlying cause of the enlargement, display defects in cell cycling that are in part to dilution of cytoplasmic components [73]. It is therefore possible that progressive cell enlargement also contributes to the gradual loss of growth capability observed in telomerase-deficient cells.

In summary, this study has utilized several new quantitative metrics for monitoring the kinetics of cell senescence, the number of cell cycles completed by WT cells versus mutants with accelerated telomere-shortening rates, and time-dependent changes that occur in both the morphological and physical properties of the cells. These results will facilitate future studies investigating genes and metabolic pathways that control cell aging as well as tests of environmental factors and chemicals that influence telomere shortening rates.

Highlights (Ghanem et al.).

New techniques allowed quantitative analysis of senescence in the yeast model system Loss of recombination genes RAD51, RAD52 or RAD54 strongly accelerated senescence Senescence was only modestly affected by inactivation of RAD55, RAD57 or RAD59 High resolution microscopy revealed 60% increases in cells’ sizes during senescence New assays revealed time-dependent changes in physical properties of senescent cells