Correspondence: Alena Pichová, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20, Prague 4, Czech Republic. Tel.: +420 24106 2526; fax: +420 2417 222 57; e-mail: firstname.lastname@example.org
During a 10-day culture ageing, cells of the wild-type Saccharomyces cerevisiae strain JC 482 retain their viability, while mitochondrial function and morphology change. Cell routine and uncoupled respiration rates increase to a maximum on day 4 and then decline to near zero. The decline, which occurs also in mitochondria isolated from cells of different age, is not due to increasing proportion of petites. Rhodamine 123 fluorescence intensity reporting on mitochondrial membrane potential appears to drop slightly for 4 days and then more sharply at the time when respiration rate also decreases. The MitoTracker Green fluorescent signal related to the mitochondrial content per cell also decreases. The branched tubular mitochondrial network of 1-day-old cells dissolves into short fragments; during the first 4 days, this fragmentation is associated with increasing function of mitochondria, while later on, it accompanies functional decline, which is also indicated by the decreasing ratio of Rhodamine 123 fluorescence to MitoTracker Green fluorescence. As shown by cell counting, microscopy and flow cytometry, the cell size distribution in the population broadens, and the population thus becomes more heterogeneous. The changes in respiration rate, mitochondrial membrane potential, mass and structure point to changes in the mitochondrial status during ageing.
The development of a yeast culture is commonly described by a growth curve, which usually deals with the first and very dynamic part of the culture development. Its most remarkable period is naturally the exponential phase where the cells have the shortest generation time. It is followed by diauxic shift and entrance into the stationary phase, during which many cell characteristics dramatically change. These phases of culture growth reflect changes in the types of available nutrients and their gradual depletion from the culture medium, and the ageing of cells under these conditions is called chronological ageing. Chronological lifespan is defined as the length of time a yeast cell can survive in a nondividing state (Fabrizio & Longo, 2003). When these effects of changing and diminishing nutrient supply are eliminated, that is, under conditions of constant sufficient nutrient supply, the cells still display signs of ageing related to the number of divisions which they have undergone – the so-called replicative ageing. The replicative lifespan is defined as the number of daughter cells produced by a mother cell before senescence (Muller et al., 1980; Jazwinski et al., 1989; Kaeberlein et al., 2007). Ageing of yeast cells is thus determined by two main aspects – time spent in ageing culture and the number of divisions individual cell has undergone.
During both chronological and replicative ageing, yeast cells undergo many morphological and functional changes. Morphological changes involve remodelling of the cell wall and subcellular structures such as mitochondria, nucleus and vacuoles. With increasing number of cell divisions, the cell size increases, and the cell wall loses its smoothness (Nestelbacher et al., 1999). Both replicatively and chronologically aged yeast cells exhibit markers of apoptosis (Laun et al., 2001; Herker et al., 2004; Lesur & Campbell, 2004). After staining of nuclei with DAPI, old cells show more diffuse chromatin than young cells, and TUNEL test detects strand breaks in DNA (Madeo et al., 1997; Pichova et al., 1997; Laun et al., 2001; Breitenbach et al., 2003). These changes are accompanied by alteration of the properties and function of the subcellular structures.
Yeast is often used as a suitable model eukaryote for studying ageing and apoptosis (Madeo et al., 1999; Fabrizio et al., 2004; Smith et al., 2008; Büttner et al., 2011). For instance, chronologically ageing yeast from long-term cultivations is used as a model of cells from postmitotic tissues (MacLean et al., 2001).
Mitochondria play a key role in cell energy metabolism. One of the markers of the cellular energy state is the mitochondrial membrane potential, whose changes may reflect changes in the respiration rate, ATP production, ROS generation (Laun et al., 2001), and which plays also an important role in apoptosis as its dissipation is one of the crucial steps of apoptosis (Ricci et al., 2003). It was reported to change during ageing of eukaryotic cells; isolated hepatocytes from old rats were found to exhibit more heterogeneous and lower mitochondrial membrane potential than cells from young rats (Hagen, 1997).
Another important functional marker, the mitochondrial respiration rate, was observed to change in different eukaryotic cell types during ageing. For instance, permeabilized rat cardiac fibres from strain F344 with short lifespan, which were used for respiration rate experiments as a model of ageing human tissue, displayed the mitochondrial defects associated with ageing. The coupled mitochondrial oxidation in old rats was approximately 45% lower than in cells from young adult animals (Lemieux et al., 2010).
Mitochondrial morphology changes during cell ageing. Mitochondria frequently undergo fission and fusion that regulate their shape, size and number. They are very dynamic especially in the exponential phase of yeast growth where fission and fusion events occur every few minutes (Nunnari et al., 1997). The two processes must be balanced for optimal mitochondrial function. It was observed that replicatively older cells (10–12 generations) contain mainly fragmented mitochondrial network, while the tubular morphology dominates in younger cells (Scheckhuber et al., 2007). Mitochondrial fission plays a role in response to new energy needs and in dividing cell, facilitating the separation of mother and daughter mitochondrial networks during cytokinesis (Hermann & Shaw, 1998). Tubular filaments of mitochondria split into small isolated compartments by fission, which occurs within the tubule or at a branching point (Nunnari et al., 1997) and is highly controlled, for example, by dynamin-related GTPase Dnm1p (Otsuga et al., 1998; Shaw & Nunnari, 2002). Mitochondrial fusion maintains the tubular mitochondrial network. It occurs when a free mitochondrial tip touches another mitochondrial tip or tubule side (Nunnari et al., 1997). Complete fusion of all mitochondrial organelles occurs during yeast mating and sporulation (Nunnari et al., 1997; Hermann & Shaw, 1998), and it is regulated by a transmembrane GTPase Fzo1p (Hermann & Shaw, 1998; Rapaport et al., 1998). Mutations in corresponding genes affect both processes. In ∆dnm1 cells mitochondrial fission is blocked, but fusion continues, resulting in the formation of complicated net-like mitochondrial structures. The fusion ∆fzo1 mutant exhibits extensive and rapid fragmentation of mitochondrial tubules (Shaw & Nunnari, 2002).
This report is concerned with changes in the function and structure of mitochondria during ageing – respiration rate, mitochondrial membrane potential, morphology and quantity.
Materials and methods
Yeast strain and growth conditions
Cells of the strain Saccharomyces cerevisiae JC 482 (MAT α, ura3-52, leu2, RAS2, his4-539, from J. Cannon, University of Missouri) were grown in YPD medium containing 1% yeast extract, 2% peptone and 2% d-glucose (all in w/v). In some experiments, the buffering capacity of the medium was increased with phosphate. Two hundred and fifty millilitre flasks containing 50 mL of medium were inoculated from an overnight culture to the final concentration of 7.5 × 106 cells mL−1 fresh medium, and the cells were grown at 30 °C on a rotary shaker at 160 r.p.m.
Determination of cell concentration and size
The exact cell concentration in suspension was measured by CASY TT Cell Counter (Roche Diagnostics Ltd., Switzerland), which operates on the principle of a pulsed low-voltage electric field of 1 MHz frequency applied to a measuring pore via two platinum electrodes. The electrolyte-filled pore has a defined electrical resistance, and the passing of intact cells (isolators, which displace the electrolyte) increases this resistance according to their volume. The response of the electric field to the size of passing cells can also be used for determining the cell size and thus distribution of cell sizes in the ageing culture. The cell size distribution profiles in cultures of different age were also assessed from the forward scatter data obtained on BD LSRII flow cytometer (Becton Dickinson) and by direct microscopic measurement of cell size performed with the Olympus IX81 fluorescent microscope, in which approximately 200 cells were measured on each day.
Calcofluor White staining
Cell wall and bud scars were visualized using the Calcofluor White (FB 28; Sigma-Aldrich) staining under the fluorescence microscope with an appropriate filter set (Streiblová & Beran, 1963).
Isolation of mitochondria
Mitochondria were isolated from mechanically homogenized protoplasts by differential centrifugation according to Kováč et al. (1968).
Cell viability assay and quantification of petite mutants
Cell viability was determined by a plating test. Approximately, 100–200 cells were plated on YPD plates (2% of agar) and cultivated for 48 h at 30 °C.
Detection and quantification of petite mutants was performed by the tetrazolium (TTC) agar overlay technique (Ogur et al., 1957), which allows to distinguish respiring red (grand) and nonrespiring white (petite) colonies. A plate with colonies was overlaid with agar containing 10 g L−1 of 2,3,5-triphenyltetrazolium chloride (Sigma), and the plates were incubated for 30 min at 30 °C. The percentage of nonrespiring colonies was counted.
Alternatively, formation of petites was monitored through their inability to grow on nonfermentable medium using replica plating technique on YPG plates that contained 3% (v/v) glycerol instead of d-glucose (Treco & Lundblad, 2001).
The respiration rate of cells and isolated mitochondria was measured using high-resolution respirometer Oroboros Oxygraph-2k, and the data were processed using datlab4 software (Oroboros Instruments, Innsbruck, Austria). The measurement was carried out in two 2-mL chambers under physiological conditions (stirring speed 750 r.p.m., temperature 30 °C) in YPD medium with reduced concentration of glucose (0.1%) for whole cells and in MIR-05 medium for isolated mitochondria (0.5 mM EDTA, 3 mM MgCl2·6H2O, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, 1 g L−1 BSA, pH 7.1). The experiment started with the measurement of routine respiration in medium without additional substrates or effectors. Doses of 2.5–10 μL 5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2 μL 1 M KCN were then added in this sequence. The uncoupling agent CCCP was added gradually in 2.5 μL volumes to determine the highest rate of uncoupling. The oxygen consumption (pmol O2 s−1 mL−1) was divided by cell concentration measured by CASY TT Cell Counter or by mg of mitochondrial protein content measured by the bicinchoninic acid (BCA) method using bovine serum albumin as standard (Smith et al., 1985).
Visualization and quantification of mitochondria
Mitochondria were stained with the fluorescent dye MitoTracker Green FM (Molecular Probes) according to the manufacturer's protocol. The procedure was modified for yeast staining. The cells were harvested by centrifugation, diluted to approximately 1 × 106 cells mL−1 and stained for 50 min with 500 nM MitoTracker Green at 30 °C. Stained cells were observed under the fluorescence microscope and analysed by the flow cytometer with a 488 nm filter. For flow cytometry, the stained cells were treated with propidium iodide (PI) to detect and exclude impaired cells from analysis because of unspecific binding of the dye. A total of 10 000 cells per sample were measured. Flow cytometry data were analysed using the flowjo software.
Measurement of mitochondrial membrane potential
Mitochondrial membrane potential was measured by flow cytometry using the fluorescent potential-sensitive dye Rhodamine 123 (Molecular Probes). The assay, taken from Ludovico et al. (2001) with slight modification, was performed as follows: washed cell suspension at a concentration of 1 × 106 cells mL−1 was stained by 50 nM Rhodamine 123 for 10 min at room temperature in the dark. The flow cytometry data analysis comprised a subtraction of intensity of control cells from the intensity of stained cells. PI-positive cells were excluded from the measurement. The resulting value was divided by forward scatter signal to exclude the effect of cell size on measured fluorescence intensity.
Unless otherwise specified, all experiments were performed at least three times with consistent results, and the average value of them was calculated. As a rule, error bars indicate standard deviation.
Determining cell parameters during ageing
The measurements of individual parameters were performed only during the first 10 days of cultivation, because on day 10, the cell respiration rate was near zero, the Rhodamine 123 signal signifying mitochondrial membrane potential was low, the mitochondria were poorly visible, and it was not possible to isolate metabolically functional mitochondria from the cells. Detailed studies were carried out on days 1, 4, 7 and 10.
Cell count, size and viability
The time profile of cell count in the culture during 10-day ageing of strain JC 482 (Fig. 1a) shows that, as expected, the cell count rose markedly on the first day, and the number of cells in the culture on subsequent days changed only little. As shown in Fig. 1b, the average cell size on individual days did not pronouncedly change. Because this overall picture of cell sizes does not provide information on the proportions of smaller and larger cells during ageing, we measured the distribution of cell sizes in the culture on the selected days by cell counter (Fig. 1c). The results show that in 1-day-old culture, most cells are in the 4–6 μm size bracket, fewer larger cells (≥ 6 μm) representing most probably budding cells plus some larger cells, and smaller cells being present only marginally (about 1.4% of the total). On subsequent days, the range of cell sizes becomes progressively broader. Compared with day 1, there are more large (≥ 6 μm) cells, but their number on days 4–10 does not further increase. The percentage of small (≤ 4 μm) cells tends to increase steadily; these cells, when stained with Calcofluor White, appeared to be without bud scars, those with buds being very rare. They may fail to undergo budding probably because of unfavourable conditions in the culture and because of cell size control in G1 period of the cell cycle. Apart from these small cells, there are also shrunken cells, and their fragments present in this fraction of the smallest particles. Flow cytometric data, that is, the forward scatter value recorded in a culture during ageing (Fig. 1e), also corroborated the gradual broadening of the cell size distribution with increasing culture age. Direct microscopic measurements of cell diameter confirmed the results of the cell size distribution obtained by cell counter and flow cytometer (data not shown).
Throughout the 10-day experiment, cell viability was about 90% (Fig. 1d). It started to decrease after 12 days of cultivation, reaching 50% after about 18 days (A. Švenkrtová, unpublished data). After transfer to a fresh medium, the aged cells gave rise to fully functional progeny with functional mitochondria.
Because the culture pH has recently been shown to affect the cell survival during chronological ageing (Murakami et al., 2011), and medium acidification is also known to promote yeast apoptosis, we buffered the YPD medium and set the initial pH to 6.0 instead of the usual 5.5. Like in the unbuffered medium, the pH gradually dropped during the 10-day cultivation, the final pH of the 10-day-old culture reaching 5 instead of the 3.9 determined in the unbuffered culture. This treatment had no effect on cell survival and mitochondrial functions.
Cell respiration rate
Figure 2a shows the time course of routine cell respiration measured without adding any compound that would influence respiration, the uncoupled respiration after addition of uncoupler CCCP showing the maximum capacity of electron transport chain and the inhibited respiration after addition of respiratory chain complex IV inhibitor KCN, which reflects the nonmitochondrial oxygen consumption during measurement.
The rate of both routine and uncoupled respirations increases to a maximum on day 4 and then decreases to nearly zero. The relative increase in respiration rate after adding an uncoupler, the uncoupled control ratio (Fig. 2b), also peaks on day 4 and then gradually decreases. Hence, on day 4, there is the largest reserve of respiration, the biggest difference between the total capacity and the actually used capacity of respiratory chain. The uncoupled control ratio seems to be directly proportional to the routine respiration rate.
Throughout the experiment, the nonrespirative oxygen consumption varied between 0–2 pmol O2 s−1 (106 cells)−1 and was nearly constant, with a slight decrease from the middle of this period. The ratio of nonrespirative oxygen consumption to total routine respiration increases during ageing, only half of the oxygen being consumed by enzymes of mitochondrial electron transport chain on day 10.
Figure 3a shows the mean signal of MitoTracker Green fluorescence representing the content of mitochondria per cell on different days. The mitochondrial content gradually decreased during the 10 days; however, the correction of respiration for mitochondrial mass did not dramatically alter the time profile of the cell respiration rate.
Respiration rate of isolated mitochondria
The trend of respiration rate of mitochondria isolated from variously aged cells (Fig. 3b) is similar to that found in intact cells (Fig. 2a), but the drop of respiration after day 4 is more pronounced. Under our experimental conditions, the cell respiration rate on day 10 was close to zero, and the respiration of mitochondria isolated from these cells was not measurable.
Formation of petite mutants
One of the possible causes of decreasing respiration is formation of petite mutants. The cell cultures were checked for petite formation. As determined by two methods, the relative proportion of petites decreased during the course of ageing (Fig. 3c) from some 15 ± 3% on day 1 to 3 ± 2% on day 10, presumably due to their being gradually overgrown by respiration-competent cells in the culture. The decrease of respiration rate during ageing is thus not caused by an increased ratio of petite mutants in the culture.
The petites were isolated, and their properties were verified by vital staining of mitochondria and respiration rate measurement. Clear tubular mitochondrial structures were not visualized by MitoTracker Green staining; instead, we observed a diffuse signal of the dye within the cells. Their routine respiration rate was not different from the nonrespirative oxygen consumption of nonpetite, grand cells, that is, close to zero (data not shown).
Mitochondrial membrane potential
The mitochondrial membrane potential was estimated by staining the cells with the fluorescent potential-sensitive dye, and the fluorescence was evaluated by a flow cytometer. The Rhodamine 123 staining slightly decreased during the first 4 days, and then, it dropped more strongly (Fig. 3d). Like MitoTracker Green (see below), Rhodamine 123 staining visualized the mitochondrial network. To obtain an approximate estimation of the functional state of the mitochondria during ageing, the Rhodamine 123 fluorescence signal for cultures of given age was divided by the intensity of the MitoTracker Green signal for the same cultures (Fig. 3a and d). From day 4 on, this measure of ‘mitochondrial membrane potential per unit mitochondrial mass’ decreased with proceeding culture age. This may indicate that not only the mitochondrial mass but also the functional ability of the mitochondria decreases during this period.
During the 10-day ageing, the mitochondrial morphology changed (Fig. 4). On day 1, the mitochondria form long filaments creating a branched tubular network. They are not distributed uniformly within the cell. The large central area free of mitochondria is obviously the vacuole, as checked by fluorescent vacuolar staining with FM4-64 (data not shown). Throughout the experiment, the cells showed as a rule only one vacuole. On day 4, the mitochondrial network starts to dissolve, with some filaments remaining and many mitochondria looking like short network segments.
An extensive fragmentation of mitochondrial network was observed on day 7. The mitochondrial filaments disappear. The population also features more often dead cells that unspecifically accumulate the mitochondrial dye throughout the cell volume. On day 10, the fragmentation further proceeds, and the stained entities appear as small dots or larger patches, usually with weak fluorescent signal; frequently, the dye is again distributed throughout the cell volume.
Yeast has been intensively studied because of its suitability as a convenient model of eukaryotic cell. One of the frequently studied processes is yeast ageing, which has been explored in terms of the cellular energy state (Werner-Washburne et al., 1993, 1996; Lin et al., 2001), genome changes (Lesur & Campbell, 2004), signalling pathways and stress responses, retrograde response or autophagy (Herman, 2002; Kaeberlein et al., 2007), transcriptional regulators playing a role in it (Sobering et al., 2002) and its association with apoptosis (Herker et al., 2004).
An important role in most of these aspects is played by mitochondria. Our study of the changes in collective features of mitochondrial morphology and function during 10-day chronological culture ageing showed that both routine and uncoupled respiration rates peak on day 4, that is, late in the postdiauxic or early stationary growth phase (Herman, 2002) and then gradually decline to some 10% on day 10. This holds also for mitochondria isolated from cells of different age. This indicates that the factors causing the drop in respiration rate are either intrinsic to the mitochondria or, if caused by the intracellular environment of the ageing cell, persist even after the removal of mitochondria from this environment. The continuous drop of mitochondrial membrane potential points to a change in the energy state of the mitochondria that takes place throughout the culture.
The changes observed for the first 4 days in the initially rich and branched mitochondrial network of 1-day-old cells, that is, its gradual dissolution into smaller isolated organelles, point to dominance of mitochondrial fission over fusion accompanying the increase in respiration. By contrast, the progressing mitochondrial fragmentation on subsequent days is accompanied by a functional decline that apparently affects both respiration and membrane potential. Thus, this functional remodelling of mitochondria is accompanied by a steady trend to fragmentation over the whole period.
These changes can be related to changes in metabolism during the transition from postdiauxic to stationary phase. It usually occurs on days 4–7 of cultivation depending on the yeast strain and composition of growth medium. In rich media like the YPD medium, this transition may occur even after day 7 (Herman, 2002). The cultivations in our experiments were performed in a rich medium, and the slightly increasing cell count profile was linear and almost constant. Also, the size distribution showed an increasing proportion of small cells in the culture during ageing. The decrease in cell respiration rate and mitochondrial membrane potential may thus reflect the state of the cells arrested in the G1 phase of the mitotic cell cycle but not in the fully resting state (Herman, 2002), which begin to prepare for the true stationary phase by reducing their energy expenditure.
The appearance of small cells exhibiting almost no scars is in agreement with an observation by Fabrizio & Longo (2003) – a partial regrowth of cells occurs after death of old cells in rich YPD medium. It creates a mixed population of young and old cells in an aged culture. Chronological ageing is assumed to reflect the gradually increasing accumulation of oxidative injuries, which progressively impairs the function and efficiency of mitochondrial enzymes and DNA and causes a decrease of respiration rate (Lemieux et al., 2010).
One of the factors that may play a role in ageing population is the fact that aged cultures may contain a small fraction of cells that have started an apoptotic pathway. Studies of cell ageing performed in a synthetic complete medium (Herker et al., 2004) showed that chronologically ageing cells exhibit an increasing number of apoptotic markers over time, although the onset and percentage might vary according to the strain and cultivation conditions. Weinberger et al. (2010) noted that in YPD medium, the decrease of cell viability during chronological ageing is reduced. Also, our data show that the negative effect of culture acidification on cell viability described during ageing in SD medium (Weinberger et al., 2010; Murakami et al., 2011) is low during ageing in rich YPD.
Although the majority of cells in our cultures retained their viability or were immediately replaced by new ones during the 10-day cultivation, the population changed, exhibiting an increasing heterogeneity of cells. According to cell counter and microscopic measurement, older cultures contained more both smaller and larger cells than the almost uniform young culture. The flow cytometric measurement brought a very similar observation, although it works on very different principle than the previous methods. This heterogeneity makes it difficult to describe the cells from older cultures as a whole. These cultures can contain cells whose particular characteristics are different from those of the majority of cells. As mentioned by Allen et al. (2006), older yeast cultures consist of distinct subpopulation of quiescent and nonquiescent cells, which differ in some fundamental characteristics, for instance viability and reproduction ability.
In conclusion, we have observed a series of changes in mitochondrial functions and structure during ageing of a yeast culture. They are connected with changes in the metabolism of the batch culture and the effects of chronological ageing.
The work was supported by the Czech Science Foundation 301/07/0339, internal institutional project RVO 61388971, Research Center 1M0570 and grant ME 09043 of the CR Ministry of Education, Youth and Sports.