Department of Molecular Biology and Biotechnology, Firth Court, University of Sheffield, Western Bank, Sheffield, S10 2TN, UK
* Author for correspondence (e-mail: k.ayscough{at}sheffield.ac.uk)
Accepted 17 February 2005
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Summary |
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Key words: Cell death, cAMP, CAP/SRV2, Mitochondria, ROS
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Introduction |
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The discovery that yeast cells display apoptosis-like characteristics has validated yeast as a model system in which to study aspects of programmed cell death in higher eukaryotes (Fröhlich and Madeo, 2001; Can and Reed., 2002
; Tissenbaum and Guarente, 2002
). Studies that have expressed certain mammalian genes in yeast have suggested that the basic machinery of apoptosis has been evolutionarily conserved. Expression of the mammalian pro-apoptotic gene Bax can induce cell death in yeast that is accompanied by typical signs of apoptosis (Ligr et al., 1998
). This lethal phenotype can be suppressed by expression of the mammalian anti-apoptotic gene Bcl-2, suggesting that yeast has apoptotic pathways. Markers of apoptosis were also associated with yeast cell death after treatment with acetic acid, H2O2 and ultraviolet irradiation (Ludovico et al., 2001
; Del Carratore et al., 2002
).
The actin cytoskeleton is a dynamic structure whose organization by actin-binding proteins (ABPs) facilitates its participation in a diverse array of cellular processes (Ayscough et al., 1997; Drubin et al., 1988
; Karpova et al., 1995
). There is a close association between actin organization and mitochondrial function. In particular, the actin cytoskeleton has been implicated in the movement of mitochondria along actin filaments via an ARP2/3-dependent propulsion mechanism (Boldogh et al., 2001
). Actin-cytoskeleton function is also important for the correct inheritance of mitochondria from the mother cell to the newly forming bud (Simon et al., 1997
). A link between the actin-regulatory protein gelsolin and mitochondrially induced apoptosis has also been described in mammalian cells (Ohtsu et al., 1997
; Koya et al., 2000
). Koya and colleagues demonstrated that overexpression of gelsolin prevented a loss of mitochondrial membrane potential, cytochrome c release and subsequent apoptosis in Jurkat cells (Koya et al., 2000
).
Recently, we demonstrated that the dynamic state of the actin cytoskeleton is also important for maintaining the membrane potential of mitochondria in yeast (Gourlay et al., 2004). A reduction in actin dynamics caused by mutations in actin itself lead to the development of apoptotic phenotypes such as a loss of mitochondrial membrane potential, elevated ROS levels and DNA fragmentation (Gourlay et al., 2004
). Here, we show that the actin-regulatory proteins Sla1p and End3p (Ayscough et al., 1999
; Benedetti et al., 1994
; Holtzman et al., 1993
) and the ubiquitin ligase Rsp5p are upstream regulators of actin-mediated oxidative stress. Loss of Sla1p or Endp3 function results in a less dynamic cytoskeleton, which in turn promotes a loss of mitochondrial membrane potential and an increase in oxidative stress.
We also report that overproduction of the high-affinity cAMP phosphodiesterase PDE2 suppresses the actin defects and associated oxidative-stress phenotype observed in end3 cells. PDE2 is a negative regulator of the Ras/cAMP signalling pathway, which links stress response and cell growth to nutrient availability (for a review, see Rolland et al., 2002
). Our data provide the first evidence in a eukaryotic system of a physiologically relevant pathway linking nutritional sensing via the Ras/cAMP pathway to actin-mediated apoptosis.
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Materials and Methods |
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Multicopy suppressor screen
1x105 end3 cells (KAY450) were transformed with 20 µg genomic library constructed in YEp13 [a kind gift from P. Sudbery, originally purchased from the ATCC (http://www.atcc.org/)], plated onto YPD medium containing 1.5 mM H2O2 and incubated at 30°C for 3 days. KAY450 cells exhibit restricted growth under these conditions. Surviving colonies were sequentially replica plated onto YPD medium containing 2 mM H2O2, 2.5 mM H2O2 and then 3 mM H2O2. 16 colonies were isolated on 3 mM H2O2, the plasmids rescued and retransformed into KAY450 cells. All rescued plasmids retransformed into KAY450 cells allowed growth on medium containing 3 mM H2O2.
Reactive oxygen species detection using flow cytometry
Cells were incubated overnight in the presence of 5 µg ml1 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes). Cells were sonicated before analysis; fluorescence was then analysed using a Becton-Dickinson Flow Cytometer. Fluorescence-activated cell sorting (FACS) parameters were set at excitation and emission settings of 304 nm and 551 nm (filter FL-1), respectively.
Viability assays
Assays were carried out as previously described (Gourlay et al., 2004). Briefly, cells were grown in liquid YPD medium. Cell number was determined in triplicate, with a Schärfe Systems TT Cell Counter and Analyser. Serial dilutions were plated onto YPD agar plates and the number of surviving colonies was counted. The viability was determined by dividing the number of surviving colonies by the calculated number of plated colonies and multiplying by 100 to give a percentage.
Fluorescence microscopy
Rhodamine-phalloidin and DAPI staining was performed as previously described for F-actin (Hagan and Ayscough, 2000). Cells were processed for immunofluorescence microscopy as described previously (Ayscough and Drubin, 1998
). Antibodies were used to detect Srv2p (a gift from D. Drubin, University of California at Berkeley, CA) by immunofluorescence microscopy at 1:50 dilution. Secondary antibodies used were fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit antibodies (Vector Laboratories) at 1:100 dilution. Cells were viewed with an Olympus BX-60 fluorescence microscope with a 100 W mercury lamp and an Olympus 100x Plan-NeoFluar oil-immersion objective. Images were captured using a Roper Scientific Micromax 1401E cooled CCD camera using IP lab software (Scanalytics, Fairfax, VA) on an Apple Macintosh G4 computer.
Hydrogen-peroxide- and latrunculin-A-sensitivity halo assays
10 µl overnight culture was added to 2 ml YPD and 2 ml 1% sterile agar. The cells were poured onto a YPAD plate and cooled, and then sterile disks containing 10 µl hydrogen peroxide at concentrations of 1%, 2%, 3% and 5%, or latrunculin-A (LatA) at 2 mM and 5 mM were placed onto each plate. Each plate was performed in triplicate for each strain. Plates were incubated at 30°C for 3 days and then halo radii were measured in mm. To obtain the relative sensitivity to hydrogen peroxide, the logarithm of the hydrogen-peroxide concentration (log[H2O2]) was plotted as a function of halo diameter in mm, curve fits were obtained and the [H2O2] required to give a halo diameter of 21 mm was calculated. These amounts were then normalized to that of the wild type and their inverse was taken to determine the relative apparent sensitivity of each strain to H2O2.
Mitochondrial visualization and inhibition
Mitochondria were visualized using using either 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] at 20 ng/ml as previously described (Simon et al., 1997) or a mitochondrion-targeted green fluorescent protein (GFP) as described previously (Westermann and Neupert, 2000
). The mitochondrial inhibitor antimycin A (Sigma) was added to liquid cultures at a concentration of 0.1 mg ml1. This concentration was sufficient to inhibit the build up of detectable ROS in overnight cultures but did not affect cell number when a assayed using a Schärfe Systems TT Cell Counter and Analyser.
Phosphatidylserine exposure
Phosphatidylserine (PS) exposure was assayed using FITC/annexin-V as previously described (Madeo et al., 1997). Briefly, 1x106 cells were washed twice in 200 µl digestion buffer (1.2 M sorbitol, 0.5 mM MgCl2, 35 mM K2HPO4, pH 6.8) and resuspend in 15 U ml1 lyticase, 2% glusulase. Cells were incubated at 30°C for 2 hours and washed twice in 200 µl annexin-V binding buffer (Roche) containing 1.2 M sorbitol. Cells were then resuspend in 39 µl annexin-V binding buffer containing 1 µl FITC/annexin-V (Roche) and incubated at room temperature for 30 minutes. Cells were washed twice in binding buffer and analysed by flow cytometry. Using a Becton-Dickinson Flow Cytometer. FACS parameters were set at excitation and emission settings of 304 nm and 551 nm (filter FL-1), respectively.
Caspase activity
In vivo staining of caspase activity by flow cytometry was carried out using a staining solution containing FITCVAD-fmk (CaspACETM, Promega) as previously described (Madeo et al., 2002a).
Glycogen staining and heat-shock sensitivity
Iodine staining of yeast cells as an assay for glycogen content was carried out as previously described (Care et al., 2004). Heat-shock sensitivity was carried out as previously described (Sass et al., 1986
).
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Results |
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end3 and sla1
118-511 cells show elevated ROS levels and H2O2 sensitivity
A reduction in mitochondrial membrane potential is often associated with an increase in cytoplasmic ROS accumulation. To investigate this, end3 and sla1
118-511 cells were grown in the presence of the indicator dye H2DCFDA and assessed by flow cytometry. Wild-type cells grown overnight to early stationary phase typically displayed two peaks of fluorescence representing populations with low (M1) and high (M2) ROS levels (Fig. 3A). Most wild-type cells were contained in the M1 population. In
end3 and sla1
118-511 cells, a dramatic shift was observed to a high-ROS-accumulating M2 population. Quantification of this event revealed that the average proportion of wild-type cells in M2 was 21±2% (Fig. 3B). In
end3 and sla1
118-511 cells, the M2 population was 83±4% and 97±1%, respectively (Fig. 3B). In agreement with this increase in ROS accumulation,
end3 and sla1
118-511 cells both displayed an increased sensitivity to H2O2 (Fig. 3C). Using a halo assay approach, it was calculated that
end3 and sla1
118-511 mutants had 1.7 and 2.0 times greater sensitivity than wild-type cells, respectively (Fig. 3C).
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A loss of mitochondrial membrane potential and an increase in ROS have been linked to the onset of apoptosis. An early marker for the apoptotic process is the exposure of phosphatidylserine on the surface of cells (Madeo et al., 1997). Phosphatidylserine can be assayed in yeast cells that have had their cell walls disrupted using a FITC-labelled annexin-V substrate. The addition of sublethal amounts of H2O2 to cultures grown to log phase was used to assay the effects on phosphatidylserine exposure using FITC/annexin-V (Fig. 3E). This experiment was carried out in triplicate and a representative data set is presented. Although no PS exposure was detected in wild-type cells over a 0.0-0.6 mM range, both
end3 and sla1
118-511 showed a dose-dependent increase in the number of cells displaying a high fluorescence, suggesting that a greater proportion of the population was entering apoptosis. This data suggests that
end3 and sla1
118-511 cells undergo apoptosis in response to low levels of H2O2, in agreement with their relative sensitivity as observed by the halo assay approach (Fig. 3C).
Reduction in actin dynamics leads to apoptosis in end3-1 cells
As yeast cells exit the log phase of growth, they have to change their metabolic strategy from a primarily fermentative one to aerobic respiration. This metabolic change is referred to as the diauxic shift. In the experiments described above, the accumulation of ROS in end3 and sla1
118-511 cells was assessed in cultures grown overnight to stationary phase. Therefore, the increase in mitochondrial activity upon entry into stationary phase, coupled with reduced mitochondrial membrane potential, might give rise to ROS accumulation. To assess whether a reduction in actin dynamics can lead to apoptosis in cells in the log phase of growth, we analysed cells expressing the temperature-sensitive end3-1 allele (Benedetti et al., 1994
). At 24°C, end3-1 cells display a wild-type actin-cytoskeleton phenotype at log phase, with punctate actin patches polarized to sites of cell growth (Fig. 4A) (Warren et al., 2002
). When shifted to 37°C for 2 hours, the actin cytoskeleton closely resembles that of
end3 cells. Cortical actin structures become fewer and larger, suggesting a slowing down of cytoskeletal dynamics (Fig. 4A) (Warren et al., 2002
). To test for a respiratory defect, end3-1 mutant cells were plated on media containing either glucose or glycerol as the sole carbon source at 24°C or 37°C (Fig. 4B). Cells expressing the end3-1 allele grew at 24°C and 37°C on glucose-containing medium but only at 24°C on glycerol-containing plates. The inability of end3-1 cells to grow at 37°C on glycerol-containing medium suggests a temperature-sensitive respiratory defect. Wild-type,
end3 and sla1
118-511 cells were also tested and all grew at both temperatures on glucose plates. As expected,
end3 and sla1
118-511 cells could not grow at either temperature on glycerol-containing medium (Fig. 4B).
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In order to examine the nature of the temperature-sensitive respiratory defect, wild-type and end3-1 mutant cells were grown to log phase at 24°C before being shifted to 37°C for 2 hours. We used a mitochondrion-targeted GFP to examine morphological changes in response to the temperature shift. In wild-type cells, temperature shift had a small effect on mitochondrial morphology, with cells showing a more punctate appearance than observed at 24°C (Fig. 4C). end3-1 cells incubated at 37°C for 2 hours displayed a similar increase in mitochondrial membrane disorganization to those incubated at 24°C. At 24°C, mitochondrial membranes were clearly visible in log-phase cells when stained with DiOC6 (Fig. 4D). Strikingly, after 2 hours at 37°C, end3-1 cells displayed a severe reduction in mitochondrial membrane staining, demonstrating a rapid reduction in mitochondrial membrane potential (Fig. 4D).
At 24°C, both log-phase wild-type and log-phase end3-1 cells showed low levels of ROS accumulation, with most of the population appearing in the M1 peak (Fig. 5A). No significant accumulation of ROS was detected in wild-type cells when shifted to 37°C for 2 hours (Fig. 5A). However under the same conditions, a significant accumulation of ROS was observed in end3-1 cells (Fig. 5A). Interestingly the loss of mitochondrial membrane potential and ROS accumulation in end3-1 cells at the restrictive temperature was also accompanied by significant activation of yeast caspase, as assayed using FITCVAD-fmk (Fig. 5B) (Madeo et al., 2002a). Wild-type cells showed no increase in caspase activation when shifted to 37°C for 2 hours (Fig. 5B). To demonstrate the physiological significance of mitochondrial membrane potential loss, ROS accumulation and caspase activation, wild-type and end3-1 cells were assayed for viability using the same temperature shift and culture conditions (Fig. 5C). Wild-type cells showed no significant loss in viability on temperature shift. By contrast, although end3-1 viability was similar to that of the wild type at 24°C, it fell dramatically after incubation at 37°C for 2 hours (Fig. 5C). These data support a pathway that leads from a reduction of actin dynamics to the development of apoptotic markers and cell death.
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Rsp5p-dependent targeting of Sla1p to the cell cortex
Because RSP5 overexpression led to the rescue of many of the phenotypes associated with the deletion of END3, we considered that high levels of ubiquitination might allow some functions of End3p to be bypassed. One previously identified role of End3p is the targeting of Sla1p to cortical-containing structures (Warren et al., 2002). Fewer punctate Sla1p-GFP spots were observed in
end3 cells than in wild type cells and there is a significant level of cytoplasmic staining (Fig. 7A,B) (Warren et al., 2002
). As sown in Fig. 7C, overexpression of RSP5 in
end3 cells can restore Sla1p localization to cortical patches and reduce its presence in the cytosol compared with the wild type, and there is a significant reduction in the level of cytoplasmic staining (Fig. 7A,B) (Warren et al., 2002
). This suggests a role for Rsp5p in targeting Sla1p to the membrane.
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PDE2 overexpression rescues the oxidative-stress phenotype of end3 cells and restores viability
In order to identify upstream regulators of actin-mediated oxidative stress, we carried out a multicopy suppressor screen to identify suppressors of the H2O2 sensitivity observed in end3 cells. Of the 16 plasmids that were isolated, eight contained multiple open reading frames and await further analysis. The other eight contained a single open reading frame encoding Pde2p, the yeast high-affinity cAMP phosphodiesterase (Sass et al., 1986
), and upstream sequence. PDE2 is a negative regulator of the Ras/cAMP signalling cascade and an important component of stress management and cell growth in response to nutrient depletion (Sass et al., 1986
). The effect of PDE2 overexpression on H2O2 sensitivity in wild-type and
end3 cells is shown in Fig. 8A. In order to establish whether the increased H2O2 tolerance observed in
end3 cells overexpressing PDE2 correlated with a reduction in ROS accumulation, wild-type and
end3 cells containing an empty YEp13 vector or YEp13 containing PDE2 were grown to early stationary phase, stained with H2DCFDA and visualized by fluorescence microscopy (Fig. 8B). Cells lacking end3 displayed a marked increase in ROS accumulation compared with the wild type, as indicated by the elevated levels of fluorescence. Strikingly, the overexpression of PDE2 resulted in a substantial reduction in ROS levels in
end3 cells (Fig. 8B). We tested whether PDE2 overexpression led to an improvement in the survival of early-stationary-phase cultures and found that
end3 cells possessed a large proportion of nonviable cells compared with the wild type (Fig. 8C). Interestingly, the overexpression of PDE2 and the concomitant reduction in oxidative stress in
end3 cells correlated with a restoration of viability to wild-type levels. A small but significant increase in culture viability was also observed in wild-type cells overexpressing PDE2 (Fig. 8C).
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Actin dynamics are linked to oxidative stress via a Pde2p-dependent mechanism
One hypothesis for our data and that of others is that the Ras/cAMP pathway links environmental stress to F-actin reorganization (Ho and Bretscher, 2001; Hubberstey and Mottillo, 2002). We therefore tested whether the overexpression of PDE2 in
end3 cells in early stationary phase had an effect on F-actin organization. At this stage of growth, all
end3 cells displayed aggregated F-actin (Fig. 9A). Overexpression of PDE2 in these cells resulted in a significant reduction in the formation of F-actin aggregates. To examine whether the reduction in F-actin aggregation correlated with a restoration of cytoskeletal dynamics, we examined the sensitivity of these strains to the actin-monomer-sequestering drug LatA. Cells lacking End3p displayed an increased resistance to LatA, indicating a reduction in actin dynamics (Fig. 9B). PDE2 overexpression restored LatA sensitivity to
end3 cells, indicating an increase in cytoskeletal dynamics. It has previously been demonstrated that the action of Pde2p to lower cAMP levels has an inhibitory effect on cAMP-dependent protein kinase A (PKA) activity. Inhibition of PKA activity has been shown to lead to the accumulation of storage carbohydrates such as glycogen and an increased resistance to heat shock. We wished to examine whether the rescue of oxidative stress by Pde2p in
end3 cells was a result of PKA downregulation. Cells lacking end3 fail to accumulate glycogen (Care et al., 2004
) (Fig. 9C), this phenotype is surprisingly not restored by PDE2 overexpression. Similarly, the heat-shock sensitivity exhibited by
end3 cells was not rescued by PDE2 expression (Fig. 9D). As expected, PDE2 overexpression in wild-type cells conferred a greater resistance to heat shock. These data suggest that the restoration of actin dynamics and rescue of oxidative stress by PDE2 is achieved by a PKA-independent pathway.
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Discussion |
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A key factor in this proposed role for actin in apoptosis signalling is its close association with the mitochondrial network. Functional links between the two systems have been noted both in yeast (Boldogh et al., 1998; Simon et al., 1997
; Simon et al., 1995
) and in cells of higher eukaryotes (Morris and Hollenbeck, 1995
). However, the studies in yeast have mostly focused on the role of actin in facilitating mitochondrial movement and inheritance. We have discovered that certain proteins that regulate the dynamics of the actin cytoskeleton during remodelling at the cell cortex are also important factors in the release of ROS from mitochondria. The stabilization of actin in sla1
118-511,
end3 or end3-1 cells correlates with a decrease in mitochondrial membrane potential, an increase in oxidative stress and the development of apoptotic markers. We also demonstrated that the oxidative-stress burden originates from these mitochondria, because mutant strains that exhibit stabilized F-actin but lack mitochondrial DNA (rho0) do not accumulate ROS. Furthermore, the addition of sublethal amounts of H2O2 to
end3 and sla1
118-511 cultures resulted in a dose-dependent increase in detection of the early apoptotic marker PS. This suggests that
end3 and sla1
118-511 cells exist close to an apoptotic threshold and implicates the cortical remodelling of actin as a mechanism that directly influences cell death in yeast.
An important question raised by this work is whether all mutations that reduce actin dynamics or perturb the cytoskeleton lead to mitochondrial dysfunction and ROS accumulation. We have analysed the ability of several strains deleted for cortical actin-regulatory proteins to grow using a glycerol carbon source and only those used in our detailed studies have a defect in this function. In addition, a mutation in the cofilin gene (cof1-22) that decreases actin dynamics by reducing the depolymerization rate of F-actin (Lappalainen and Drubin, 1997) does not have a defect in its ability to grow on glycerol, does not accumulate ROS and contains mitochondria with normal morphology and membrane potential (data not shown). These additional observations add to the idea that only a subset of actin-regulatory proteins are involved in the pathway that links actin dynamics to the functioning of the mitochondria and the release of ROS. This also argues that the effect investigated in this study are not due simply to a general reduction in actin dynamics from pleiotropic activities.
The deletion of SLA1 or END3 results in a similar actin-clumping phenotype and elicits the same effect of inducing oxidative stress. This implies that these proteins influence actin dynamics by a similar mechanism. Sla1p is thought to be involved in inhibiting activation of the Arp2/3 complex and so increasing depolymerization of actin at sites where remodelling is required (Rodal et al., 2003). One of the main reported roles of End3p is as a protein required for cortical recruitment of Sla1p (Warren et al., 2002
). The targeting of Sla1p by End3p to sites of actin remodelling explains the similarity in phenotypes observed when the function of either protein is lost. The loss of both Sla1p or End3p function results in defects in endocytosis (Ayscough et al., 1999
; Benedetti et al., 1994
), although the effect of the
end3 mutation is more severe. This suggests that End3p has roles additional to the recruitment of Sla1p.
Another form of regulation that affects the function of Sla1p is ubiquitination. Although Sla1p has not been shown to be ubiquitinated directly, it has recently been reported that the ubiquitin ligase Rsp5p can bind directly to Sla1p (Stamenova et al., 2004). We show that overexpression of RSP5 can rescue many of the defects associated with the loss of End3p. Indeed, RSP5 overexpression rescues the ability of end3-null cells to grow on glycerol, reduces oxidative stress, increases viability and enhances the organization of the actin cytoskeleton. The loss of End3p targeting results in a reduction of Sla1p localization to cortical patches, which then accumulates in the cytosol. The overexpression of RSP5 results in a marked increase in the amount of Sla1p localized to cortical patches. Our data suggest that the increased level of ubiquitination at the cell cortex acts to facilitate the formation of new binding sites for Sla1p, thereby bypassing the role for End3p in Sla1p recruitment. The relocalization of Sla1p then allows it to function relatively normally and to rescue the actin, mitchondrial and oxidative-stress phenotypes with which its mutation is associated.
The mechanism by which actin dynamics gives rise to ROS accumulation is unclear. One possibility is that F-actin filaments are involved in the regulation of the mitochondrial voltage-dependent anion channel (VDAC). It has been demonstrated in mammalian systems that the actin-regulatory protein gelsolin can protect against apoptosis by closing VDAC pores (Kusano et al., 2000). It has also been shown that in neuronal cells the antiapoptotic activity of gelsolin is actin dependent (Harms et al., 2004
). In vitro studies have also demonstrated that actin can influence VDAC closure in Neurospora crassa (Xu et al., 2001
). However, in our studies, the overexpression of the antiapoptotic proteins Bcl-2 or Bcl-xL, which are known to regulate VDAC closure, in
end3 cells have no effect on H2O2 sensitivity or culture viability (C.W.G. and K.R.A., unpublished). This suggests that ROS accumulation in
end3 cells occurs by a VDAC-independent mechanism.
Another alternative is that ROS build up occurs as a consequence of dysfunctional regulation of a stress-response pathway. In yeast, the Ras/cAMP pathway plays an important role in the control of growth and metabolism in response to nutritional status. In the presence of high glucose levels, Ras1/2p and CAP/Srv2p activate adenylate cyclase (Cyr1p) to catalyse cAMP production (for a review, see Rolland et al., 2002). The production of cAMP leads to PKA activation, which signals to the nucleus to inhibit a stress response and ensure G1 cell-cycle progression. Pde2p acts as a negative regulator of this pathway by hydrolysing cAMP, preventing PKA activation and derepressing the stress response. Our identification of Pde2p as a suppressor of actin aggregation and oxidative stress lends support to the idea that actin-cytoskeleton regulation is involved in the Ras/cAMP signalling cascade. In support of this link, it has been demonstrated that Ras2p is required for the regulation of actin in response to mild heat stress (Ho and Bretscher, 2001
). The observation that CAP/Srv2p significantly colocalizes with cortical F-actin patches during log-phase but not stationary-phase growth might reflect an important regulatory event. Sequestration of CAP/Srv2p with F-actin aggregates in
end3 stationary-phase cells might in turn lead to perturbation of Ras/cAMP signalling and to some of the phenotypes observed in these cells. Interestingly, cells carrying the constitutively active ras2val19 mutation display several phenotypic similarities to
end3 and sla1
118-511 cells. These include a failure to accumulate glycogen, a lack of adaptation to heat or nutritional stress (Sass et al., 1986
; Care et al., 2004
) (this study) and high levels of oxidative stress (Hlavata et al., 2003
) (this study). This might indicate that
end3 and sla1
118-511 exhibit elevated Ras/cAMP pathway activity. In support of this, the overexpression of the Ras/cAMP negative regulator Pde2p was able to rescue ROS accumulation and viability in
end3 and sla1
118-511 cells. However, because PDE2 overexpression in
end3 cells was unable to rescue glycogen-accumulation and heat-tolerance defects, this suggests that ROS accumulation occurs via a PKA-independent mechanism. Interestingly, Hlavata and colleagues also proposed (Hlavata et al., 2003
) that ras2val19 cells function via a PKA-independent pathway to generate ROS. We propose that there is a PKA-independent pathway in which F-actin remodelling is responsive to cAMP levels, with cytoskeletal aggregation acting as a trigger for ROS generation (Fig. 11). Further to this, we suggest that actin dynamics can act as biosensor of cellular health, linking environmental signalling via the Ras/cAMP pathway to apoptosis. In aged or genetically compromised cells, reduced actin dynamics would affect the ability of some cells to respond to environmental stress and to initiate an apoptotic response, which would eliminate them from the population and would promote the survival of the colony.
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Acknowledgments |
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References |
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