Centro de Ciências do Ambiente-Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4719-057 Braga, Portugal1
Imunologia Comparada, Instituto de Biologia Molecular e Celular (IBMC), 4150-171 Porto, Portugal2
Author for correspondence: Manuela Côrte-Real. Tel: +351 253 604314. Fax: +351 253 678980. e-mail: mcortereal{at}bio.uminho.pt
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ABSTRACT |
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Keywords: active cell death, apoptosis, yeast, acid stress
Abbreviations: PI, propidium iodide; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labelling
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INTRODUCTION |
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The reported mechanisms underlying programmed cell death in prokaryotes are different from those involved in apoptosis of animal cells. Examples include mechanisms involved in programmed cell death of plasmid-free segregants, avoiding in this way the survival of bacteria unable to retain the plasmid (Yarmolinsky, 1995 ; Naito et al., 1995
), and programmed cell death of phage-infected Escherichia coli, with the prevention of virus spreading among the bacterial population (Matsuyama et al., 1999
; Skulachev, 1999a
, 2000
). Moreover, programmed cell death in bacteria also plays an important role in developmental processes, such as lysis of the mother cell during sporulation of Bacillus subtilis and lysis of vegetative cells in fruiting body formation of Myxococcus xanthus (Lewis, 2000
). In addition, programmed cell death has been reported in unicellular eukaryotic organisms (Ameisen, 1996
). The yeast Saccharomyces cerevisiae has been used mainly as a tool to study human apoptosis regulatory proteins and it is not generally accepted to have an endogenous programmed cell death process. However, evidence indicating the presence of some basic features characteristic of an apoptotic phenotype in S. cerevisiae was recently reported (Madeo et al., 1997
; Ligr et al., 1998
). The expression of the anti-apoptotic protein Bcl-2 in S. cerevisiae provides antioxidant protection and delays natural death (Longo et al., 1997
). Furthermore, it was recently shown in S. cerevisiae that depletion of glutathione or exposure to low external doses of H2O2 triggered the cell into apoptosis, whereas depletion of reactive oxygen species (ROS) or hypoxia prevented apoptosis (Madeo et al., 1999
). In addition, an intracellular accumulation of ROS was detected in the cell cycle mutant cdc48S565G of S. cerevisiae and in yeast cells expressing mammalian Bax. These results allowed the identification of ROS production as a key cellular event common to the known scenarios of apoptosis in yeast and animal cells (Madeo et al., 1999
). Recently, it was suggested that the Rad9 protein of Schizosaccharomyces pombe could be a member of the Bcl-2 protein family, being the first protein of this family identified in yeast (Komatsu et al., 2000
). The above evidence has suggested that a programmed cell death process occurs in yeasts and shares some conserved aspects with metazoan apoptosis. Nevertheless, yeast cell death processes are far from being clarified. Characteristic features of cell death patterns in yeast need to be identified in order to provide some insights into the cell death mode(s) in this unicellular eukaryote.
Acetic acid is a normal end product of the alcoholic fermentation carried out by S. cerevisiae. This compound is not metabolized by glucose-repressed yeast cells and enters the cell in the undissociated form by simple diffusion. Inside the cell, the acid dissociates and if the extracellular pH is lower than the intracellular pH, this will lead to intracellular acidification, anion accumulation and inhibition of the metabolic cell activity, namely fermentation/respiration (Leão & van Uden, 1986 ; Cássio et al., 1987
; Pampulha & Loureiro, 1989
). Moreover, it was shown that in S. cerevisiae, under certain conditions, acetic acid compromises cell viability and ultimately results in two types of cell death, high and low enthalpy (Pinto et al., 1989
). Previous studies from our laboratory have focused on the assessment by flow cytometry of cell structural and functional changes induced by acetic acid in S. cerevisiae and Zygosaccharomyces bailii (Ludovico, 1999
; Prudêncio et al., 1998
). However, the process by which the yeast cell dies when injured by acetic acid is unknown.
The aim of the present study was to identify morphological, structural and functional cellular markers that would allow the characterization of the mode(s) of cell death induced by acetic acid in S. cerevisiae.
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METHODS |
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Treatments with acetic acid and inhibition of protein synthesis.
Exponential-phase cells were harvested and suspended (107 cells ml-1) in YEPD (pH 3·0, set with HCl) containing 0, 20, 40, 80, 120, 160 and 200 mM acetic acid. The treatments were carried out for 200 min at 26 °C with magnetic stirring (100 r.p.m.). The inhibition of protein synthesis was performed by adding 100 µg cycloheximide (Merck) ml-1 (Barnett et al., 2000 ) to the cell suspensions at the same time as the different acetic acid concentrations tested. Cycloheximide at this concentration was not cytotoxic after 200 min incubation, as assessed by c.f.u. counts. Viability was determined by c.f.u. counts after 2 d incubation at 26 °C on YEPD agar plates. No further colonies appeared after that incubation period. In all the above experiments, the extracellular pH did not change during the incubations.
Transmission electron microscopy analysis.
Cells from different treatment conditions were washed with phosphate/magnesium buffer (40 mM K2HPO4/KH2PO4, pH 6·5, 0·5 mM MgCl2), suspended in 2·5% (v/v) glutaraldehyde in 40 mM phosphate/magnesium buffer, pH 6·5, and fixed overnight at 4 °C. After fixation, the cells were rinsed twice in 0·1 M phosphate/citrate buffer (pH 5·8) and suspended in this buffer containing 10 U lyticase (Boehringer Mannheim) ml-1 for about 90 min at 37 °C, to digest the cell wall. After cell wall digestion of the pre-fixed yeast cells, protoplasts were washed and postfixed with 2% (w/v) osmium tetroxide (2 h) followed by 30 min incubation with 1% (w/v) aqueous uranyl acetate. Dehydration was performed as described by Byers & Goetsch (1991) for embedding vegetatively grown yeast cells. After 100% ethanol washes, the samples were transferred to 100% propylene oxide, and infiltrated with 50% (v/v) propylene oxide and 50% (v/v) Epon (TAAB Laboratories) for 30 min and with 100% Epon overnight. Cells were transferred to gelatin capsules with 100% Epon and incubated at 60 °C for 48 h before cutting thin sections and staining with uranyl acetate and lead acetate. Micrographs were taken with a Zeiss EM 10C electron microscope. For quantitative assessment of yeast cells with condensed chromatin, at least 50 yeast cells with nuclear profiles were counted per sample.
Terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) and propidium iodide staining.
DNA strand breaks were demonstrated by TUNEL with the In Situ Cell Death Detection Kit, Fluorescein, from Boehringer Mannheim. This technique labels free 3'-OH termini with FITC-labelled deoxyuridine triphosphate (dUTP), which was detected by epifluorescence microscopy. Yeast cells were fixed with 3·7% (v/v) formaldehyde as described by Madeo et al. (1999) and cell walls were digested with lyticase (as above, for 105 min). Cytospins of the cell suspensions were made using a Shandon Cytospin 2 cytocentrifuge, operating at 1500 r.p.m. for 5 min. The slides were rinsed with PBS, incubated in permeabilization solution (0·1%, v/v, Triton X-100 and 0·1%, w/v, sodium citrate) for 2 min on ice, rinsed twice with PBS and incubated with 10 µl TUNEL reaction mixture, containing terminal deoxynucleotidyl transferase and FITC-dUTP, for 60 min at 37 °C. Finally the slides were rinsed three times with PBS and a coverslip was mounted with a drop of anti-fading agent Vectashield (Molecular Probes) and with 2 µl of a propidium iodide (PI; Molecular Probes) working solution (50 µg ml-1) in Tris buffer (10 mM, pH 7·0) with MgCl2 (5 mM) and RNase (0·5 µg ml-1). Observations were carried out using a Zeiss Axioskop epifluorescence microscope equipped with a HBO-100 mercury lamp, filter set 40 (BP360/51, BP485/17, BP560/18) from Zeiss, excitation filter BP 450-490, beam splitter FT510 and emission filter LP520. Images were acquired with a Spot 2 camera (Diagnostic Instruments). For the quantitative assessment of TUNEL staining, 50700 cells were counted per sample.
Annexin V staining.
Phosphatidylserine exposure was detected by an FITC-coupled annexin V reaction with the ApoAlert Annexin V Apoptosis kit (CLONTECH Laboratories), essentially as described by Madeo et al. (1999) . Cells were harvested and washed with sorbitol buffer (1·2 M sorbitol, 0·5 mM MgCl2, 35 mM K2HPO4, pH 6·8). Cell walls were digested with 15 U lyticase (Sigma) ml-1 and 2% (v/v) glusulase (NEE-154 Glusulase; NEN) in sorbitol buffer for about 30 min at 28 °C; digestion with the two enzymes was monitored by phase-contrast microscopy in order to prevent damage to the unfixed protoplasts. Cells were then washed twice with binding buffer (10 mM HEPES/NaOH, pH 7·4, 140 mM NaCl, 2·5 mM CaCl2; CLONTECH Laboratories) containing 1·2 M sorbitol. To 38 µl cell suspension in binding/sorbitol buffer were added 2 µl annexin V (20 µg ml-1) and 2 µl of a PI working solution (as described for the TUNEL reaction) and incubated for 20 min at room temperature. The cells were then washed and resuspended in binding/sorbitol buffer. Finally the slides with coverslips were mounted with the cell suspensions. Microscope settings and image acquisitions were as described above for the TUNEL technique. For quantitative assessment of annexin VPI staining, at least 150 yeast cells were counted per sample.
Reproducibility of the results.
All the experiments were repeated at least three times. The data reported for c.f.u. counts are mean values and standard deviations. Quantitative data for annexin V and TUNEL staining and for chromatin condensation are from one representative experiment; while absolute data were not comparable in these experiments performed on different days, the trends described were fully consistent in the independent experiments.
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RESULTS |
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Extensive chromatin condensation along the nuclear envelope in cells treated with 2080 mM acetic acid (shown in Fig. 2b for 40 mM) was revealed by quantitative transmission electron microscopy analysis (Table 1
), whereas nuclei of untreated cells were homogeneous in shape and density (Fig. 2a
). Only a low percentage of cells treated with 120 mM acid, and no cells treated with 160 or 200 mM, exhibited chromatin condensation (Table 1
). Consistent with the enhancement by cycloheximide of the survival of S. cerevisiae to 4080 mM acetic acid treatment, a decrease in the number of cells with chromatin condensation along the nuclear envelope was evident in the presence of cycloheximide (Table 1
).
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In yeasts, as in mammalian cells, phosphatidylserine has an asymmetric distribution in the lipid bilayer of the cytoplasmic membrane (Cerbón & Calderón, 1991 ). The exposure of phosphatidylserine at the outer surface of the cytoplasmic membrane occurs at the early stages of apoptosis (Martin et al., 1995
) when membrane integrity is still retained. Therefore, with the technique used in the present study, which includes simultaneous staining with FITC-coupled annexin V and the membrane impermeant fluorochrome PI, those early apoptotic cells only stain green, from FITC, indicating the presence of phosphatidylserine at the outer surface of the plasma membrane [annexin V (+), PI (-), shown in Fig. 3a
for 20 mM acetic acid]. Numbers of annexin V (+), PI (-) cells were maximal at 4080 mM acetic acid (Table 2
). Yeast cells in more advanced apoptotic stages, or necrotic cells, stain green and red due to the inability of the cell membrane to exclude PI [annexin V (+), PI (+), shown in Fig. 3b
for 120 mM acid]. Numbers of annexin V (+), PI (+) cells were maximal at 80 and 120 mM acetic acid (Table 2
). In the 120 mM sample, most of the annexin-V-negative cells were PI-positive [annexin V (-), PI (+)]. Untreated cells did not exhibit a positive annexin V reaction or PI staining [annexin V (-), PI (-)].
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Preliminary results have shown that the apoptotic markers referred to above are present, in a concentration-dependent way, in samples exposed to 20120 mM acetic acid for time periods shorter than the 200 min used in the present study.
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DISCUSSION |
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The mode of programmed cell death under acetic acid stress conditions was further characterized by the examination of cell death markers that are typical of apoptosis. The evidence obtained indicates that acetic acid, like H2O2 (Madeo et al., 1999 ), can trigger an apoptotic phenotype in S. cerevisiae. The following concentration-dependent changes were observed after treatment with acetic acid: chromatin condensation along the nuclear envelope, exposure of phosphatidylserine at the outer surface of the yeast cytoplasmic membrane and formation of DNA strand breaks. The nuclear ultrastructural changes were significantly reduced when the treatment was carried out in the presence of cycloheximide, reinforcing our interpretation about the active nature of cell death induced by the lower concentration of acetic acid.
Even though a very small population with apoptotic markers was detected at 120 mM, the present results suggest that a non-active (necrotic) process is the predominant cell death mechanism in samples exposed for 200 min to acetic acid at concentrations above 80 mM. This interpretation is based on the following points: (i) mortality at 120 mM (or more) is not significantly inhibited by cycloheximide; (ii) the percentage of cells with apoptotic markers, induced by increasing concentrations of acid, increases with treatments up to 80 mM acid and then decreases; (iii) the percentage of cells with ultrastructural alterations typical of necrosis is highest and very extensive in samples treated with 120160 mM acid. An apoptotic or necrotic phenotype was also reported for S. cerevisiae exposed for 200 min to low or high concentrations of H2O2, respectively (Madeo et al., 1999 ).
Our data regarding the structural/morphological markers of apoptosis showed that the proportion of yeast cells with chromatin condensation induced by 40 mM acetic acid was higher than the proportion of cells exhibiting surface exposure of phosphatidylserine [annexin V (+), PI (-)] and DNA cleavage (TUNEL-positive). This observation indicates that, in our model, nuclear chromatin alteration precedes both phosphatidylserine exposures at the cytoplasmic membrane surface and significant endonuclease DNA cleavage. Similar observations have been reported in other models of apoptosis, i.e. chromatin condensation preceding DNA cleavage (Sun et al., 1994 ) and phosphatidylserine exposure (Darzynkiewicz et al., 1997
).
The yeast S. cerevisiae has been used as a host to express proteins of the Bcl-2 family and hence to study the mechanisms underlying apoptosis in higher eukaryotes. Up to now, only one instance of an active cell death process in yeasts, independent of heterologous expression of pro-apoptotic proteins, has been reported, namely programmed cell death due to exposure of S. cerevisiae to H2O2 (Madeo et al., 1999 ). In the present work, we have shown that the apoptotic phenotype in S. cerevisiae can also be induced by acetic acid, pointing to the possibility that this mode of cell death may be more generalized in yeasts and extended to other stress agents. It is known that ROS play a central role in the regulation of apoptosis at various levels (Madeo et al., 1999
). Further studies are in progress in order to evaluate ROS production in S. cerevisiae under the conditions where programmed cell death induced by acetic acid was detected. This would show if commitment of S. cerevisiae to a programmed cell death process in response to acetic acid is also mediated through a ROS-dependent apoptotic pathway.
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ACKNOWLEDGEMENTS |
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Received 29 January 2001;
revised 26 March 2001;
accepted 14 May 2001.