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Vol. 19, Issue 5, 2127-2134, May 2008
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Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071
Submitted November 6, 2007;
Revised January 29, 2008;
Accepted February 27, 2008
Monitoring Editor: Thomas Fox
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Mcd1/Rad21 is a cohesin subunit, and homologues are found from yeast to human (Guacci et al., 1997). The fission yeast cohesin Rad21 is a cell cycle-regulated nuclear phosphoprotein that repairs double-strand DNA breaks, and it is essential for mitotic growth (Birkenbihl and Subramani 1992). In budding yeast (Saccharomyces cerevisiae), Mcd1 is required for chromosome morphogenesis from S phase through mitosis, and it functions in both sister chromatid cohesion and chromosome condensation (Guacci et al., 1997; Michaelis et al., 1997).
During mitotic cell cycle, proteolytic cleavage of the Mcd1 at the metaphase–anaphase transition promotes loss of cohesin, followed by its dissociation from the chromatids (Guacci et al., 1997; Michaelis et al., 1997; Uhlmann et al., 1999). Esp1 and Pds1 are the key regulators of this process. Esp1, a separin or separase, is inactivated by forming a complex with its inhibitor Pds1 (called securin) before anaphase (Ciosk et al., 1998; Uhlmann et al., 1999). During metaphase, degradation of Pds1 mediated by a Cdc20-specified multisubunit ubiquitin ligase termed anaphase-promoting complex/cyclosome (APC/Ccdc20) releases Esp1, which cleaves cohesin Mcd1/Rad21, thereby releasing the sister chromatids (Cohen-Fix et al., 1996; Uhlmann et al., 1999; Nasmyth et al., 2000).
In addition to its function in chromatid cohesion, recent studies show that cohesin plays a role in apoptosis (Chen et al., 2002; Pati et al., 2002). Human Rad21 (hRad21) is found as a nuclear caspase target. Induction of apoptosis by diverse stimuli causes the cleavage of hRad21. The cleaved C-terminal product of hRad21 is translocated from the nucleus to cytoplasm and acts as a nuclear signal for apoptosis. RAD21 is also found to be overexpressed in prostate (Porkka et al., 2004) and breast cancer cells (Atienza et al., 2005). In Caenorhabditis elegans, apoptotic nuclei, as indicated by 4',6-diamidino-2-phenylindole (DAPI) staining, were observed in the gonads of adult evl-14/pds-5 and scc-3 mutants (Wang et al., 2003), suggesting a role of these cohesion-related genes in apoptotic cell death. Apoptotic cell death is also reported in mutation of yeast Pds5, a cohesion-related protein (Ren et al., 2005).
In recent years, yeast has been used successfully as a model system for studying apoptosis (Madeo et al., 2004). The yeast genome encodes many proteins of the basic molecular machinery executing cell death in mammalians, including homologues of apoptosis-inducing factor 1 (AIF1) (Wissing et al., 2004), caspases (Madeo et al., 2002), endonuclease G (Büttner et al., 2007), and HtrA2/Omi (Fahrenkrog et al., 2004). In addition, mitochondria play an important role in cell death both in mammals and yeast (Wang 2001; Eisenberg et al., 2007).
Here, we report that the yeast Mcd1 is cleaved upon induction of apoptosis by hydrogen peroxide (H2O2). The cleaved C-terminal fragment of Mcd1 is further cleaved into smaller fragment and then translocated from nucleus into mitochondria. The translocation causes the decrease of mitochondrial membrane potential (
M) and amplification of cell death in a cytochrome c-dependent manner. We further demonstrate that Esp1, a caspase-like protease, is responsible for the cleavage of Mcd1 during H2O2-induced apoptosis in yeast.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ade2 ADE5 CAN1s cyh2R his7-1 leu2 lys2-2 met13-c trp1-63 tyr1-2 ura3-1). Plasmids of MCD1-GFP, MCD1–6HA, and the temperature-sensitive mcd1-1 mutant were provided by Dr. V. Guacci (Guacci et al., 1997) and integrated into MDY506 and MDY507. N-terminal 6HA tagging of Mcd1 was constructed according to Gueldener et al. (2002) and Gauss et al. (2005). The yca1 deletion was introduced by polymerase chain reaction (PCR)-mediated gene replacement (Wach et al., 1994), replacing the complete sequence of YOR197W with a G418 marker. Plasmid of ESP1 overexpression was provided by Dr. K. Nasmyth (Uhlmann et al., 2000). Plasmids for making Esp1-degron and Pds1-degron were purchased from EUEOSCARF (Frankfurt, Germany; http://web.uni-frankfurt.de/fb15/mikro/euroscarf/), and the two strains were constructed according to Sanchez-Ziaz et al. (2004). Mcd1 C-terminal truncations were constructed according to Gelperin et al. (2005). Different truncations were first amplified by PCR, using MCD1-GFP plasmid as the template. The PCR products were then integrated into vector BG1805 (kindly provided by Dr. E. Grayhack, University of Rochester School of Medicine and Dentistry, Rochester, NY) (Gelperin et al., 2005). Yeast strain BY4742 and its derived deletion strains (aif1, cyc1 and nuc1) was purchased from Open Biosystems (Huntsville, AL).
Cells were all grown at 30°C, except where noted in the text. For overexpression, cells were first cultured in YPRaf medium (1% yeast extract, 2% peptone, and 2% Raffinose) overnight to reach 1 x 108cells/ml, and then they were transferred to YPG medium (1% yeast extract, 2% peptone, and 2% galactose) for minimum of 1 h. Apoptosis was induced by 5 mM H2O2 (Madeo et al., 1999).
Spotting and Survival Assay
For spotting assay, cells were cultured in liquid medium as desired. Cells were then diluted to 2.5 x 106cells/ml. Four fivefold series dilutions were made, and 5 µl of each dilution was plated on a YPG plate. Cells were grown at 30°C for 2 d. Survival assay was conducted according Mason et al. (2005). Survival rate was calculated as number of colonies divided by the number of colonies in its corresponding control.
Fluorescence Microscopy
For fluorescence microscopy, cells were collected by centrifuge, rinsed with phosphate-buffered saline (PBS), and mounted on a coverslip with anti-fading medium (0.1 M propyl gallate and 50% glycerol in PBS) containing 0.5 µg/ml DAPI. For mitochondrial staining, cells were collected at log phase and washed one time with PBS. Cells were resuspended in 100 µl of PBS containing 2 µM MitoFluor 589 (Invitrogen, Carlsbad, CA) or 1 µM MitoTracker Red (Invitrogen). Cells were mounted on a coverslip, and they were checked immediately. A Nikon TE300 inverted microscope, equipped with a Cascade 650 cooled monochrome digital camera (Roper Scientific, Trenton, NJ) was used for image acquisition.
Western Blot Analysis
Cells were collected by centrifugation and lysed in lysis buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% SDS, and protease inhibitor cocktail. Glass beads (200 µm) were added, and they were vortexed vigorously. Sample was boiled and spun down, and the supernatant was used to run the SDS-gel. Lysates were separated on 10% SDS-polyacrylamide gels, and they were transferred to polyvinylidene difluoride membranes. The membranes were then blocked in 4% nonfat milk and incubated with anti-hemagglutinin (HA) antibody (clone 3F10; Roche Diagnostics, Indianapolis, IN). The membranes were then incubated anti-immunoglobulin G horseradish peroxidase. For caspase inhibitor analysis, 200 µM of a pan-caspase inhibitor zVAD-fmk or 50 µM of Caspase 1 and 8 were added to the cells for 1 h before the addition of H2O2.
Annexin V Staining
Yeast cells were collected and washed twice with sorbitol buffer (0.8 M sorbitol and 2% potassium acetate, pH 7.0), resuspended in sorbitol buffer containing 10 mM dithiothreitol for 10 min, and then digested with 0.4 mg/ml Zymolyase for 30 min. Cells were harvested and resuspended in 50 µl of binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, and 1.2 M sorbitol, pH 7.4). Then, 4 µl of Annexin V conjugate was added to the cell suspension and incubated for 20 min at room temperature in the dark. Cells were rinsed with binding buffer, mounted under a coverslip with anti-fading medium containing 0.5 µg/ml DAPI, and examined with epifluorescence microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To study the potential role of cohesin in yeast apoptosis, H2O2 was used to induce apoptosis in a yeast strain carrying a green fluorescent protein (GFP)-tagged MCD1. When the MCD1-GFP strain was cultured in the absence of H2O2, Mcd1-GFP was exclusively localized in the nucleus (Guacci et al., 1997; Figure 1A). When H2O2 was added to the culture, Mcd1-GFP was "knocked out" from nucleus into cytoplasm, forming small GFP spots, similar to the size of mitochondria (Figure 1A). Counterstaining with DAPI showed that the GFP spots in the cytoplasm contained DNA. To confirm that the Mcd1-GFP colocalizes with mitochondria, MitoFluor 589, a mitochondrial-specific dye, was used to stain mitochondria. As shown in Figure 1B, the GFP spots colocalized with MitoFluor, indicating that the Mcd1-GFP is indeed in mitochondria.
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; Pati et al., 2002). When a pan-caspase inhibitor (zVAD-fmk) was added before the addition of H2O2, no apparent decrease of Mcd1 level was observed (Figure 2A), suggesting that caspase or caspase-like protease may be responsible for the Mcd1 decrease. We then knocked out YCA1, which encodes the only identified caspase-like protease in yeast, to see whether Yca1 is involved. Although yca1 delayed, if not abolished apoptosis in yeast (Madeo et al., 2002), no effect was observed on the decrease of Mcd1 in the yca1 background (Figure 2A), suggestive of other enzyme being responsible for the decrease of Mcd1.
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). The 48-kDa band was similar to that cleaved by Esp1 during mitotic division (Uhlmann et al., 1999). The presence of the 48- and 83-kDa bands suggests that there should have a C-terminal band of Mcd1, which has a molecular mass of 
113 kDa. It was likely that the C-terminal fragments were unstable and further digested into small fragments by the N-end rule pathway (Rao et al., 2001); therefore, they could not be detected when using the HA antibody at C terminus. Please note that the molecular mass of Mcd1 shown here is bigger than what is predicted (www.yeastgenome.org). This difference has been reported previously (Uhlmann et al., 1999; 2000). It is unclear the cause(s) of the difference. Apparently, the addition of the HA tag increases the retardation in gel migration in addition to the actual increase in molecular mass (Uhlmann, personal communication).
H2O2 Causes the Translocation of the Truncated Mcd1 C-Terminal Fragment into Mitochondria
It was clearly seen that the Mcd1 was translocated from nucleus to mitochondria in the presence of H2O2 (Figure 1). The translocated Mcd1 was most likely a C-terminal fragment of Mcd1, because 1) the GFP observed in Figure 1 was tagged at the C terminus of Mcd1, and 2) Mcd1 was shown to be cleaved at the presence of H2O2 (Figure 2). Because no C-terminal fragments could be detected, possibly due to its small size, a series of C-terminal truncations of Mcd1 (Figure 3) were created and overexpression of the different truncations were tested. No mitochondrial target sequence in Mcd1 was predicted using MultiLoc (Hoeglund et al., 2006) and other software. The truncations were therefore determined arbitrarily, starting with 20 amino acids from C terminus of Mcd1, except for Mcd1-C95aa, which has a relatively high probability of mitochondrial location (Table 1). The predicted molecular masses of the C-terminal truncations range from 2 to 11 kDa. The plasmid carrying GFP only was used as a control. Overexpression of the various C-terminal Mcd1 truncations revealed that all truncations localized in cytoplasm in the absence of H2O2. When treated with H2O2, the GFP signal remained in cytoplasm, except for truncation Mcd1-C40aa, which formed small GFP dots around edge of the cells (Table 1; Figure 4A), similar to the endogenous Mcd1-GFP when treated with H2O2 (Figure 1). MitoTracker staining revealed that these small spots colocalized with mitochondria (Figure 4B), indicating Mcd1-C40aa was translocated from cytoplasm into mitochondria at the presence of H2O2.
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) was used to compare the survival rate of wild-type and Mcd1-C40aa–overexpressed cells. As shown in Figure 5A, the survival rate of Mcd1-C40aa overexpressed cells decreased significantly, compared with the wild type, or cells carrying the GFP vector. This result suggests that the small fragment of C-terminal Mcd1 amplifies cell death induced by H2O2.
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) from Open Biosystems (data not shown).
Overexpression of Mcd1 C-Terminal Truncation Causes the Decrease of M
Previous studies show that mitochondria play an important role in the regulation of apoptotic cell death. The collapse of M is an irreversible hallmark of early apoptosis (Karbowski and Youle 2003). To further investigate the role of the Mcd1-C40aa truncation in H2O2-induced apoptosis, the Mcd1-C40aa truncation was overexpressed with or without the presence of H2O2, and the M was examined using MitoTracker Red CMRos (Invitrogen), which stains mitochondria in a M-dependent manner (Pozniakovsky et al., 2005). The MitoTracker specifically stains mitochondria when the M is high, but stains in a diffused pattern when M is lost. As shown in Figure 6, when treated with H2O2, 70% of the wild-type cells showed a diffused MitoTracker staining, but almost all Mcd1-C40aa overexpressed cells showed a diffused staining, indicating that overexpression of Mcd1-C40aa caused more M loss.
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M loss from H2O2 treatment.
Cell Death Amplified by the Mcd1 C-Terminal Fragment Is Dependent on Cyc1 But Not Aif1 and Nuc1
Mitochondrial proteins such as apoptosis-inducing factor, endonuclease G, and cytochrome c play important roles in apoptosis via their release from mitochondria to nucleus or to cytoplasm in mammalian and yeast (Wang 2001; Wissing et al., 2004; Büttner et al., 2007). We next asked whether the translocation of Mcd1 C-terminal fragment into mitochondria is related to the function of mitochondrial proapoptotic proteins. The Mcd1-C40aa truncation was overexpressed in the knockout strains of CYC1, AIF1, and NUC1, separately. Spotting and plating assays were used to analyze the survival rate for each strain. As shown in Figure 7, overexpression of Mcd1-C40aa caused more cell death in nuc1 and aif1 mutants, similar to the overexpression of Mcd1-C40aa in wild type, suggesting that the cell death amplified by the overexpression is independent of Aif1 and Nuc1. In cyc1 strain, however, survival rate was increased when Mcd1-C40aa was overexpressed, indicating that Cyc1 is required for the cell death amplified by the C-terminal fragment of Mcd1.
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, 2000). Sequence comparison revealed that the S. cerevisiae Esp1 has a similar sequence to human caspase-1 at their conserved proteolysis catalytic sites (Uhlmann et al., 2000). To see whether Esp1 is responsible for cleaving Mcd1p during H2O2-induced apoptosis, Esp1 was overexpressed under a GAL1 promoter. After 6 h of overexpression, 20% of cells showed markers of apoptotic cell death (Figure 8A). Interestingly, when Esp1 was overexpressed in a MCD1-GFP strain, Mcd1-GFP was translocated from nucleus in mitochondria, similar to H2O2-treated cells (Figure 8B).
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). Our result revealed that 20% of the cells were ROS positive after 6 h of ESP1 overexpression. When N-tert-butyl-a-phenylnitrone (PBN), a ROS scavenger, was added, the ROS-positive cells decreased from 20% to 5%. The percentage of cells with Mcd1-GFP translocated to mitochondria also dropped dramatically, compared with cells without PBN treatment. These results suggest that ROS plays a major role in translocating the Mcd1 fragment from cytoplasm to mitochondria.
A previous report (Uhlmann et al., 2000) suggests that Esp1 resembles the function of caspase-1. If this were the case, caspase-1 inhibitor should block the decrease of Mcd1 when treated with H2O2. Caspase-1 and caspase-8 inhibitors were used to test this possibility. As shown in Figure 8C, caspase-1 inhibitor inhibited the decrease of Mcd1, similar to the pan-caspase inhibitor zVAD-fmk (Figure 2A). Caspase-8 inhibitor slowed down the decrease, but it did not totally block the decrease (compare with the wild type in Figure 2A). This result suggests that a caspase-1–like protease, possibly Esp1, is responsible for the cleavage of Mcd1 in H2O2-induced apoptotic cell death.
To further test the function of Esp1 as a caspase-like protease, mutational analysis of Esp1 was performed using a heat-inducible Esp1-degron strain (Sanchez-Diaz et al., 2004). Western blot revealed that the Esp1 was completely depleted after 1 h of induction at 37°C (data not shown). As shown in Figure 8D, at 24°C, when Esp1 was not depleted, Mcd1-GFP was cleaved and translocated into mitochondria at presence of H2O2. When shifted to 37°C for 1 h, Mcd1-GFP remained exclusively inside the nucleus, similar to the control cells. No obvious Mcd1 decrease was observed in the presence of H2O2 when Esp1 was depleted (Figure 8E), further confirming the role of Eps1 in the cleavage of Mcd1.
Pds1 deletion was also performed to test the cleavage of Mcd1 by Esp1. Pds1 is an anaphase inhibitor and associated with the Esp1. At early anaphase, Pds1 is degraded by the APC, releasing Esp1. The released Esp1 then acts as a separin for the cleavage of cohesin, causing the separation of sister chromatid (Cohen-Fix et al., 1996; Agarwal and Cohen-Fox, 2002). We speculate that Esp1 has to be released from Pds1 in order for the cleavage of Mcd1 during H2O2-induced apoptosis. As shown in Figure 8E, when Pds1 was depleted by degron, Mcd1-GFP was translocated from nucleus into mitochondria in the absence of H2O2, further suggesting that Esp1 is responsible for the cleavage of Mcd1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and yeast (Eisenberg et al., 2007). On induction of apoptosis, mitochondria release several mitochondrial proteins, such as cytochrome c, endonuclease G, and apoptosis-inducing factor to promote cell death, by either activating caspases, or by neutralizing cytosolic inhibitors of cell death. In mammalian cells, it has been shown that the Bcl-2 family proteins are critical regulators of mitochondrial apoptosis (Jiang and Wang 2004). Some Bcl-2 proteins (antiapoptotic), such as Bcl-xL inhibit cell death, whereas others (proapoptotic) promote apoptosis. Interestingly, both antiapoptotic and proapoptotic Bcl-2 proteins are directly targeted to mitochondria, in which they inhibit or promote the release of apoptotic proteins from mitochondria. One of the well-characterized Bcl-2 proteins is the mammalian Bid, which localizes in cytoplasm in living cells (Li et al., 1998; Luo et al., 1998). On induction of cell death, Bid is cleaved by caspase-8, producing an 11-kDa N-terminal and a 15-kDa C-terminal fragment. The truncated C-terminal fragment (tBid) is translocated from cytosol to mitochondria, causing the release of cytochrome c. In yeast, little is known about the upstream mechanism and regulation of mitochondrial apoptosis. Our current study addresses this issue. Similar to the mammalian Bid, yeast cohesin protein, Mcd1 is cleaved by Esp1, a caspase 1-like protease, upon induction of apoptosis. The truncated C-terminal fragment of Mcd1 is translocated from nucleus to mitochondria, causing the amplification of cell death in a cytochrome c-dependent manner. Notably, the translocation of the Mcd1 fragment to mitochondria requires apoptosis stimulus, such as H2O2. This explains why no cell death occurs during a normal cell cycle, when Mcd1 is also cleaved by Esp1 at the stage of metaphase/anaphase transition. It remains unknown how the truncated Mcd1 is translocated to mitochondria. In tBid, recent studies in both mammals (Lutter et al., 2000) and yeast (Gonzalvez et al., 2005) indicated that the translocation is dependent on cardiolipin, a phospholipid associated with mitochondrial membrane (Hoch 1998). It is also not clear, especially in yeast, how the cell death is affected by the release of cytochrome c. A study Silva et al. (2005) suggests that the release of cytochrome c is involved in activation of caspase in yeast. It is also possible that the release of cytochrome c can halt the electron transfer, which in turn causes the loss of mitochondria membrane potential and increase the ROS production.
Esp1, also called separase, is a cysteine protease-related caspase. Separase is thought to be a repressed protease, cleaving only a few substrates and in a very controlled manner. During a normal cell cycle, Esp1 is bound and inactivated by Pds1, an inhibitory protein (Yanagida 2000). At metaphase/anaphase transition, the securin Pds1 is degraded by APC, releasing Esp1. Here, we showed a second role of Esp1, acting in an apoptosis-specific manner. This provides direct evidence of the caspase function of the separase Eps1 (Figure 9). Furthermore, Mcd1 may not be the only substrate of Esp1. A recent report (Sullivan et al., 2001) shows that Esp1 also cleaves a kinetochore/spindle protein called Slk19. A search of S. cerevisiae genome database yielded 31 putative substrates of Esp1, including Mcd1 (Uhlmann et al., 2000).
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65-kDa C-terminal fragment, which is then translocated to cytoplasm. Overexpression of the C-terminal fragment amplifies apoptotic cell death. It is not clear how cell death is enhanced by the cytoplasm-located C-terminal fragment of hRad21. In yeast, our study indicates that the C-terminal fragment is much smaller, possibly by the further degradation ubiquitin–proteasome pathway (Uhlmann et al., 2000). The much smaller fragment is translocated to mitochondria, rather than cytoplasm. The apparent differences between human and yeast, however, should not underestimate the importance of using yeast as a model system to elucidate the pathways and regulation of apoptosis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Zhaojie Zhang (zzhang{at}uwyo.edu)
Abbreviations used: M, mitochondrial membrane potential; APC/C, anaphase-promoting complex or cyclosome; DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; ROS, reactive oxygen species, zVAD-fmk, N-benzoylcarbonyl-Val-Ala-Asp fluoromethyl ketone.
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