# Global Transcriptional Analysis of Yeast Cell Death Induced by Mutation of Sister Chromatid Cohesin

Authors: Qun Ren; Hui Yang; Bifeng Gao; Zhaojie Zhang
Journal: Comparative and Functional Genomics
 (2008)

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## Abstract

Cohesin is a protein  complex that regulates sister chromatid cohesin during cell division. Malfunction in chromatid cohesin results in chromosome missegregation and aneuploidy. Here, we report that mutations of MCD1 and PDS5, two major components of cohesin in budding yeast, cause apoptotic cell death, which is characterized by externalization of phosphatidylserine at cytoplasmic membrane, chromatin condensation and fragmentation, and ROS production. Microarray analysis suggests that the cell death caused by mutation of MCD1 or PDS5 is due to the internal stress response, contrasting to the environmental or external stress response induced by external stimuli, such as hydrogen peroxide. A common feature shared by the internal stress response and external stress response is the response to stimulus, including response to DNA damage, mitochondria functions, and oxidative stress, which play an important role in yeast apoptotic cell death.


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## Body

## 1. Introduction

It has been increasingly evident that yeast as well as other unicellular organisms can undergo apoptotic cell death (for recent review, see [1]). The apoptotic cell
death shares many morphological and biochemical features with mammalian
apoptosis, including DNA fragmentation and chromatin condensation, exposure of
phosphatidylserine on the outer membrane surface, leakage of Cytochrome C from
mitochondria [2]. Apoptosis in yeast can be induced by external stresses, such
as hydrogen peroxide [3], acetic acid [4], as well as mutations of certain
essential genes, such as CDC48 [5], ORC2 [6], and PDS5 [7]. The yeast apoptotic
cell death is promoted by proapoptotic genes, including the metacaspase
YCA1/MCA1 [8, 9] and apoptosis-inducing factor AIF1 [10].Cohesin is a protein complex that regulates sister chromatid cohesion and
is required for ordered chromosome segregation [11]. Cohesin physically links
the sister chromatids during replication. This attachment makes it possible for
the kinetochore of each sister chromatid to attach to microtubules from
different poles. During anaphase, cohesin is released from chromosomes, and
proper segregation occurs [12]. Cohesin contains at least four protein subunits:
Smc1, Smc3, Mcd1/Scc1, and Scc3 [13, 14]. In addition to the four subunits,
several additional proteins are required to establish or maintain the chromatid
cohesion but are not part of the cohesin complex,such as Pds5 [14–17], Scc2, and Scc4 [18].
Scc2 and Scc4, for example, are required for loading the cohesin complex onto
chromosomes, but do not colocalize with the complex [18]. Pds5 is essential for both mitosis [15, 16, 19] and meiosis [20]. Pds5 localizes to
chromosomes in a cell cycle-dependent manner
and is required for sister chromatid cohesion, chromosome condensation, DNA
repair, and proper chromosome segregation.In addition to its function in chromatid cohesion, studies show that
cohesin has a role in apoptosis [21, 22]. Human RAD21/MCD1 is reported 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/MCD1
is also overexpressed in prostate [23] and
breast cancer cells [24]. Suppression of RAD21/MCD1 reduces the ability for
cell proliferation and increases the level of apoptosis [24]. In yeast (S. cerevisiae), our recent study
showed that mutation of PDS5 causes cell death in early meiosis [7].Here we report that mutation of MCD1
(mcd1-1) and PDS5 (pds5-1) showed an apoptotic phenotype, including exposure of phosphatidylserine to the outer
membrane, accumulation of DNA breaks, condensation and fragmentation of chromatin, and production of ROS.
Microarray analysis of the global transcript of mcd1-1 and pds5-1 mutants, in
comparison withH2O2 induced apoptosis, showed that the
majority of the differentially expressed genes were upregulated. These genes
were involved in a variety of functions, including response to stimulus, cell
cycle regulation, mitochondrial functions, and cell wall organization. Our data
suggest that the cell death caused by mutation of MCD1 or PDS5 is due to
response to physiological or internal stress, compared to the H2O2-treated
cells, which is considered response to environmental or external stress.


## 2. Results

### 2.1. Mutation of MCD1 and PDS5 Showed Major Markers of Apoptosis

Our previous report [7] showed that mutation of PDS5 caused
apoptotic cell death in yeast during early meiosis. Here, we demonstrated that
mutation of PDS5 and MCD1, two cohesin related genes, caused apoptotic cell
death during mitosis. Figure 1 shows some of the “hallmarks” of apoptosis—the translocation of phosphatidylserine from
the inner leaflet to the outer leaflet of the plasma membrane (Figure 1(a)),
and nuclear fragmentation (Figure 1(b)).
ROS accumulation was also observed in mcd1-1 and pds5-1 mutants, when grown at nonpermissive temperature (Figures 1(c) and 1(d)). Note that the ROS level was higher in mcd1-1 and
pds5-1 mutants even at 21°C (Figure 1(d)), compared to the wild type. Chromatin condensation was also observed by transmission electron microscopy (Figure 2).Apoptotic cell death in mcd1-1 and pds5-1 mutants. WT, mcd1-1, and pds5-1 mutants were shifted from permissive temperature (21°C) to nonpermissive temperature (37°C) for 12 hours, then stained with (a) Annexin V, PI, and DAPI; (b) DAPI for nucleus and (c) Dihydrorhodamine 123 for ROS. (d) Quantitative measurement of ROS production of H2O2-treated cells, mcd1-1 and pds5-1 mutants at permissive, and nonpermissive temperature.
(a)(b)(c)(d)Figure 2
TEM images of mcd1-1 and
pds5-1 mutants, growing at nonpermissive temperature for 2 hours, showing
chromatin condensation (arrow in mcd1-1 and pds5-1)N= Nucleus.


### 2.2. General Characteristics of Gene Expression

In attempt to
understand the mechanism of the cohesin-induced apoptosis in yeast,a global analysis of gene
expression was performed in mcd1-1 and pds5-1 mutants undergoing apoptotic cell
death.H2O2-treated
cells were used as a positive control. The number of up- and downregulated
genes were shown in Figure 3 (the original data is available at http://www.uwyo.edu/microscopy/cohesin.xls). The microarray data were further validated by reverse transcriptase-polymerase
chain reaction (RT-PCR) (Figure 4). 5 different genes from each group were
chosen for RT-PCR analysis. These genes represent genes that are up- and downregulated,
and genes related to cohesin complex, which is of interest for further
analysis. At both time points (see materials and methods for determination of
the time points), more genes were upregulated than downregulated, suggesting a
genetic program was being activated, rather than the general gene expression shutdown
that might be expected in a rapid cell death. At time point 1, pds5-1 had more
genes differentially regulated than the other two, while mcd1-1 had the least.
At time point 2, however, the number of differentially regulated genes in
mcd1-1 surpassed that of pds5-1 or H2O2-treated cells,
especially the number of upregulated genes (Figures 3, 5). Furthermore, while
the number of differentially regulated genes in pds5-1 and H2O2-treated
cells decreased from time point 1 to time point 2, the number of genes in
mcd1-1 increased from time point 1 to time point 2. These results suggest that
cells may have a slower but more profound response at later time point, to the
defect of Mcd1, than to the defect of Pds5, or H2O2-treated
cells.Figure 3
Number of up- and downregulated
genes at time point 1 and point 2 inH2O2-treated cells,
mcd1-1, and pds5-1 cells. *The number of unchanged genes includes genes with
marginal increase and marginal decrease (see Materials and Methods for detail).Figure 4
RT-PCR of 5 different genes from each group of time point 1.Figure 5
Schematic representation of differentially expressed genes in the three groups.T1: time point 1; T2: time point 2.Only a small number of differentially expressed genes overlap
among the three groups in both time points (Figure5). The total number of
overlapped genes were 174 in time point 1 (167 upregulated, and 7 downregulated)
and 349 in time point 2 (323 upregulated and 26 downregulated). This suggests
that these three groups may undergo apoptotic cell death through different
pathways. To better understand the pathways, the gene expression profiles were
analyzed using Netaffx analysis software from Affymetrix (http://www.affymetrix.com). Using Affymetrix’s gene ontology (GO) mining
tool, it was found that in H2O2-treated cells, the most
prominent group of genes (the group with the lowest ρ-value) that were differentially expressed was
“response to stimulus” (Table 2). In mcd1-1 and pds5-1 mutants, however, the
most prominent group is related either to physiological process (mcd1-1), or
regulation of biological process (pds5-1). This may reflect the fact that the H2O2-treated
cells mainly responded to the external stress, termed “environmental stress
response” (ESR) [25], while in mcd1-1 and pds5-1 mutants, the gene
expression responded mainly to biological or physiological changes due to the
malfunction of MCD1 or PDS5.Table 1
Fold changes of each gene expression, corresponding to the RT-PCR in Figure4.
H2O2pds5-1mcd1-1GENEFold changeGENEFold changeGENEFold changeHSP26A+10.6HSP26A+30HSP26A+9.8NHP6A−4NHP6A−2.3NHP6A−2SMC3+8ALD3+52ALD3+12SCC4+9.8REC8+19.7PDS5−1.7ESP1+6.1YKU80+7.5ESP1−1.3ActinControlActinControlActinControlTable 2
The most prominent gene group (the group with the lowest ρ-value) that is differentially expressed in response to theH2O2 treatment, or MCD1 and PDS5 mutations.
Time point 1Time point 2mcd1-1Physiological process (GO ID = 7582)Physiological process (GO ID = 50789)pds5-1Regulation of biological process (GO ID = 50789)Regulation of biological process (GO ID = 50789)H2O2Response to stimulus (GO ID = 50896)Regulation of biological process (GO ID = 50789)All threeResponse to stimulus (GO ID = 50896)Regulation of biological process (GO ID = 50789)


### 2.3. Genes Involved in Response to Stimulus

When the 174
overlapped genes from the three groups in time point 1 were analyzed using the
GO mining tool, it was found that the most prominent group of differentially
expressed genes was related to “response to stimulus” (Table2), suggesting
that, like the H2O2-treated cells, the mcd1-1 and pds5-1
mutants, when grown at nonpermissive temperature, were also under stress. An
evidence of the stress was the production of ROS (Figure 1). The changes of
gene expression in mcd1-1 and pds5-1 mutants were referred to “internal stress
response” (ISR), contrasting to the environmental, or external stress response
(ESR). At time point 2, the most prominent change of gene expression in all
types of cells, including H2O2-treated cells, was related
to either physiological process (mcd1-1), or regulation of biological process
(pds5-1 and H2O2-treated), suggesting that the ESR could
eventually become ISR, if the stress continues.Further analysis of
the overlapped genes of the three groups revealed that at time point 1, 29 out
of 174 of the overlapped genes were a response to stimulus, including response
to DNA repair, oxidative stress, heat and nutrition (Table3). Only 3 out of 29
were downregulated and 26 were upregulated.
In time point 2, response to stimulus was one of the top three prominent
groups that are differentially regulated (1st= regulation of
biological process (Table 2), 2nd= development, 3rd= response to stimulus). Among the 58 differentially regulated genes response to
stimulus, 31 (53%) were related to DNA damage (Table 4), suggesting that more
DNA damages were caused by the extended time of internal or external stress.  It has been reported that both Pds5 and Mcd1
are involved in DNA repair [26, 27]. The elevated number of differentially expressed genes response to DNA
damage could be related in part, to the malfunction of mcd1-1 or pds5-1 in DNA
damage  repair.Table 3
List of differentially regulated genes response to stimulus in time point 1.
Response toGeneFold changeDescription+H2O2mcd1-1pds5-1DNA damageRMI110.562.839.85Involved in response to DNA damageRFX18.572.146.50Involved in DNA damage checkpoint pathwayEPL17.462.146.96Component of NuA4, a histone H4/H2A acetyltransferase complexNTG16.962.307.46DNA N-glycosylase and apurinic/apyrimidinic (AP) lyaseYKU806.962.147.46Subunit of the telomeric Ku complex (Yku70p-Yku80p)NHP6A−4.00−2.00−2.30High-mobility group nonhistone chromatin proteinoxidative stressSRX121.113.739.19SulfiredoxinHSP1211.312.468.57Plasma membrane localized protein that protects membranes from desiccationCTT110.564.9217.15Cytosolic catalase TGRE38.572.4613.00Aldose reductaseHeatHSP2610.569.8529.86Small heat shock protein with chaperone activityGRE38.572.4613.00Aldose reductaseSSA38.002.4612.13ATPase involved in protein folding and the response to stressSSA48.002.1411.31Heat shock protein that is highly induced upon stressSSE26.502.008.00Member of the heat shock protein 70 (HSP70) familyNTH213.932.0010.56Putative neutral trehalasenutrition & othersALD319.7012.1351.98Cytoplasmic aldehyde dehydrogenasePTR39.193.0312.13Component of the SPS plasma membrane amino acid sensor systemDDR28.576.5017.15Multistress response proteinFIG28.573.258.57Cell-wall adhesinADR18.573.039.85Carbon source-responsive zinc-finger transcription factorYGP18.003.0310.56Cell-wall-related secretory glycoproteinHAL17.462.836.50Cytoplasmic protein involved in halotoleranceTSL16.964.2913.93Large subunit of trehalose 6-phosphate synthase/phosphatase complexMET46.502.469.19Lecine-zipper transcriptional activatorMPT56.502.009.19Member of the Puf family of RNA-binding proteinsPHO5−2.46−2.14−2.00Repressible acid phosphataseTIR1−2.83−2.64−2.00Cell wall mannoprotein of the Srp1p/Tip1p family of serine-alanine-rich proteinsUnknownYIL169C10.563.7327.86Putative protein of unknown functionYOR019W9.192.4610.56Hypothetical proteinTable 4
List of differentially expressed genes in time point 2 response to DNA
damage (bold = genes differently expressed in both time points).
GeneFold changeDescriptionH2O2mcd1-1pds5-1PSO214.936.969.19Required for a
postincision step in repair of DNA breaksCST913.934.0014.93SUMO E3 ligaseEAF713.002.469.85Subunit of the
NuA4 histone acetyltransferase complexMIG312.134.2914.93Probable
transcriptional repressorEPL112.133.738.57Component of NuA4NHP6A12.133.738.57High-mobility
group non-histone chromatin proteinSCC412.132.309.19Subunit of
cohesin loading factor (Scc2p-Scc4p)SLX111.312.8310.56Subunit of
5'-flap endonuclease complexVID2110.564.5913.00Component of the
NuA4 histone acetyltransferase complexYKU8010.563.259.85Subunit of the
telomeric Ku complex (Yku70p-Yku80p)RAD599.196.509.19Involved in
repair of DNA double-strand breaksHPR59.192.839.19DNA helicase and
DNA-dependent ATPaseSIR49.192.837.46Silent
information regulator that,EAF69.192.467.46Esa1p-associated
factor, subunit of the NuA4 acetyltransferase complexTFB39.192.149.19Subunit of TFIIH
and nucleotide excision repair factor 3 complexesNTG18.573.487.46DNA N-glycosylase
and apurinic/apyrimidinic (AP) lyaseMGT18.572.649.19DNA repair
methyltransferaseSNF58.572.649.19Subunit of the
SWI/SNF chromatin remodeling complexHMI18.572.466.96Mitochondrial
inner-membrane localized ATP-dependent DNA helicaseRTT1078.572.007.46Interacts with
Mms22p and is implicated in Mms22-dependent DNA repairEXO18.002.648.005'-3' exonuclease
and flap-endonucleaseELC18.002.1411.31Elongin CHEX38.002.146.96Protein
containing a RING finger domain that interacts with Slx8pDOA18.002.008.57WD repeat proteinMMS48.002.008.00Subunit of the
structure-specific Mms4p-Mus81p endonucleaseRAD167.465.288.00Recognizes and
binds damaged DNA in an ATP-dependent mannerMEC37.462.836.96DNA damage and
meiotic pachytene checkpoint proteinPIN47.462.469.19Involved in G2/M
phase progression and response to DNA damageCAC27.462.008.57Component of the
chromatin assembly complexLCD16.963.487.46Essential protein
required for the DNA integrity checkpoint pathwaysPAN26.962.3012.13Subunit of the
Pan2p-Pan3p poly(A)-ribonuclease complexPrevious
reports indicate that both Pds5 [27, 28] and Mcd1 [26] have a role in DNA
repair. To explore if the defect in DNA repair is involved in mcd1-1- or pds5-1-induced
apoptosis, wild type, pds5-1- and mcd1-1 cells were exposed to UV radiation
(150 J/m2) using a UV cross-linker (Stratagene, Inc.), and then
incubated for 20 hours at 37°C before being assayed with DAPI. DAPI staining showed that in pds5-1, broken nuclei increased at least 18%, while in
mcd1-1,  broken nuclei increased about 15%, compared to cells without UV
radiation. At the same condition, UV radiation had almost no effect on wild
type (Figure 6(a)). ROS levels were much higher in mcd1-1 and pds5-1 mutants after UV
radiation (Figure 6(b)).These results suggest that (1) Mcd1 and Pds5 participate in DNA break repair, and (2) defect of mcd1-1 or pds5-1 in DNA
break repair plays a role in  apoptosis.UV radiation caused increase
of apoptotic cell death in mcd1-1 and pds5-1 mutants. (a) wild type, pds5-1
cells, and mcd1-1 cells were exposed to UV radiation (150 J/m2) then
incubated for 20 hours at 37°C. Cells were stained with DAPI to identify the
fragmented nuclei. Data are from 3 independent experiments; at least 100 cells were counted for each experiment. (b) ROS
was produced in pds5-1 and mcd1-1 mutants. Wild type and the mcd1-1 and
pds5-1mutants were exposed to UV radiation (150 J/m2) then incubated
for 2 and 18 hours at 37°C. Dihydrorhodamine 123 (Priault et al. pages 684–91) was added into cell culture
and incubated for another 2 hours, ROS production was measured using a
microplate reader.
(a)(b)


### 2.4. Genes Involved Mitochondria Functions

Mitochondria play an
important role in yeast apoptosis [29, 30]. In our current study, microarray
analysis revealed that many differentially expressed genes were localized in
mitochondria.  In time point 1, among the
174 differentially expressed genes in all three groups, 38 were found localized
in mitochondria (Table 5). 26 of the 38 genes were localized only in mitochondria.
Interestingly, 12 of the 38 have unknown functions. The genes that have known
functions were involved in response to stimulus, constituents of mitochondrial
membrane, regulation of ATP syntheses, and other enzymes. Of particular
interest is the upregulation of CYC7, which encodes the isoform 2 of Cytochrome
C. Cytochrome C has been reported to be released from mitochondria to cytoplasm
after induction of apoptosis [31]. The increase of expression level of CYC7 may
also contribute to the release and the abundance of Cytochrome C in cytoplasm
after induction of apoptosis. The upregulation of CYC7 only appeared in time
point 1, not time point 2, suggesting that the release of Cytochrome C is an
early event during apoptosis. At time point 2, 40 (12 localized only
mitochondria; 8 with unknown functions) of the commonly up- or downregulated
genes were found localized in mitochondria. Although the number of genes was
similar to that of time point 1, the majority of genes were different and only
7 appeared in both time points (bold letters in Tables 5 and 6). Of the 7
genes, 3 (ELG1, NTG1, and NTH2) were response to stimulus/DNA repair; 3 (GCV1,
GLT1, and AEP1) were related to amino acid/protein metabolism; and one
(YDR332W) with unknown functions. In general, the mitochondrial genes were
mainly involved in response to stimulus/DNA damage repair, metabolism of DNA,
proteins, and amino acids. BIR1, the gene encodes an antiapoptotic protein, was
found overexpressed in all three cases (Table 6). When checked at time point 1,
BIR1 was also overexpressed in all three cases, but the fold change in mcd1-1
was less than 2, and therefore was filtered out. Because BIR1 is also involved
in coordinating cell cycle events for proper chromosome segregation, it
is unclear that the overexpression of BIR1 is due to its role of cell cycle
control, or  apoptosis.Table 5
List of differentially regulated genes that are related to mitochondria functions in time point 1  (Bold = genes appeared in both time points;*  = only those localized in mitochondria).
Gene nameFold changeDescriptionH2O2mcd1pds5-1Localized only in
mitochondriaCYC713.932.649.85Cytochrome c
isoform 2OM1411.313.2514.93Integral
mitochondrial outer membrane proteinALD49.192.6419.70Mitochondrial
aldehyde dehydrogenaseLSC28.572.309.85Beta subunit of
succinyl-CoA ligaseCEM18.572.3011.31Mitochondrial
beta-keto-acyl synthaseSTF18.572.469.85Involved in
regulation of the mitochondrial F1F0-ATP synthaseRSM198.002.148.00Mitochondrial
ribosomal protein of the small subunitOM458.003.0316.00Major constituent
of the mitochondrial outer membraneCYB28.004.0012.13Cytochrome b2
(L-lactate cytochrome-c oxidoreductase)YME27.462.007.46Integral inner
mitochondrial membrane proteinMRPL197.462.008.57Mitochondrial
ribosomal protein of the large subunitSLM57.462.149.19Mitochondrial
asparaginyl-tRNA synthetaseAEP16.962.007.46Required for
expression of the mitochondrial OLI1 geneFMP466.962.0010.56Putative redox
protein containing a thioredoxin foldMBR16.963.739.85Involved in
mitochondrial functions and stress responseODC16.062.009.19Mitochondrial
inner membrane transporterGLT1−3.48−2.00−2.46NAD(+)-dependent
glutamate synthaseLocalized in mitochondria and nucleus/cytoplasmNTH213.932.0010.56Putative neutral
trehalaseGCV111.312.8312.13T subunit of the
mitochondrial glycine decarboxylase complexGOR19.852.0010.56Glyoxylate
reductaseDIA49.192.308.57Probable
mitochondrial seryl-tRNA synthetaseRSF18.002.308.57Protein required
for respiratory growthYHL009W-B8.002.838.57Retrotransposon
TYA Gag and TYB Pol genesNTG16.962.307.46DNA N-glycosylase
and apurinic/apyrimidinic (AP) lyaseELG16.962.3010.56Required for S
phase progression and telomere homeostasisLRG1−2.46−2.30−3.25Putative
GTPase-activating protein (GAP)Of unknown
functionsTMA1019.707.4613.93Protein of unknown
function that associates with ribosomes*YPL222W12.132.309.85The authentic, nontagged
protein was localized to the mitochondria.*YDR379C-A10.562.8310.56Hypothetical
protein identified by homology.UIP48.572.6419.70Protein of unknown
function that interacts with Ulp1p*YDR070C8.579.8522.63The authentic, nontagged
protein was localized to the mitochondria*YOR205C8.002.009.85The authentic, nontagged
protein was localized to the mitochondria*YHL021C7.462.146.96The authentic, nontagged
protein was localized to the mitochondria*YDR332W7.462.6411.31Hypothetical
proteinYML128C7.466.0622.63Protein of unknown
function*YJL062W-A6.962.148.57Putative protein
of unknown function*YNR040W6.962.308.57Hypothetical
protein*YNL195C6.963.0327.86Hypothetical
proteinTable 6
List of differentially expressed genes that are related to mitochondria functions in time point 2,  (bold = genes appeared in both time points;*  = only those localized in mitochondria).
Gene nameFold changeDescriptionH2O2mcd1-1pds5-1Localized only in
mitochondriaCRC127.8632.009.85Mitochondrial
inner membrane carnitine transporterPUT116.0032.008.57Proline oxidaseHXT1413.009.199.85Hexose transportUBP1611.312.468.57Ubiquitin-dependent
protein catabolismAAC111.318.008.57Mitochondrial
inner membrane ADP/ATP translocatorPGS110.562.837.46Phosphatidylglycerolphosphate
synthaseYHL009W-A10.563.489.19DNA-mediated
transpositionGCV29.192.6412.13P subunit of the
mitochondrial glycine decarboxylase complexAEP19.193.4811.31Protein
biosynthesisHTD29.193.738.00Mitochondrial
3-hydroxyacyl-thioester dehydrataseHMI18.572.466.96Mitochondrial
ATP-dependent DNA helicaseARG27.462.649.85Acetylglutamate
synthaseGLT1−2.64−3.48−2.64NAD(+)-dependent
glutamate synthaseILV5−3.03−2.14−3.48Acetohydroxyacid
reductoisomerasePOR2−3.03−2.00−2.00Ion transportLocalized in
mitochondria and
nucleus/cytoplasmJJJ116.002.1421.11endocytosisHSF112.133.739.19Trimeric heat
shock transcription factorSPC1059.853.037.46Protein required
for accurate chromosome segregationGCV19.858.008.00T subunit of the
mitochondrial glycine decarboxylase complexNTH29.195.289.19Putative neutral
trehalaseVPS158.572.007.46Myristoylated
serine/threonine protein kinaseVPS548.572.008.00Component of the
Golgi-associated retrograde protein complexPTC58.572.008.57Mitochondrially
localized type 2C protein phosphataseLAS18.572.0012.13Essential nuclear
protein possibly involved in morphogenesisBIR18.572.468.57Cell cycle,
anti-apoptoticELG18.572.4612.13Required for S
phase progression and telomere homeostasisNTG18.573.487.46DNA N-glycosylase
and apurinic/apyrimidinic (AP) lyaseAFT28.573.738.57Iron-regulated
transcriptional activatorRIS18.002.467.46Member of the
SWI/SNF family of DNA-dependent ATPasesGYP18.003.038.57Vesicle-mediated
transportSUR7−2.83−2.14−3.48Putative integral
membrane proteinMRH1−2.83−2.00−2.83Protein that
localizes primarily to the plasma membraneOf unknown
functions*YNL130C-A18.388.5712.13Protein of unknown
function*YBR047W13.934.298.57Protein of unknown
function*YLR346C11.3111.3114.93Protein of unknown
function*YLR253W9.852.468.00Protein of unknown
functionTOF29.853.0313.93Protein of unknown
function*YDR332W9.854.927.46Hypothetical
protein*YGL226W8.572.308.00Protein of unknown
function*YOR305W8.002.009.19Protein of unknown
function


### 2.5. Genes Involved in Cell Cycle Regulation

Deregulation of cell cycle has been reported in yeast cells
undergoing apoptotic cell death [32, 33]. The apoptotic aging cells, for
example, were described as to attempt the second budding cycle without the
completion of the first one. Irregular expression of cell cycle related genes
was also reported in old cells and orc2-1 cells that underwent apoptosis [33].
In our current study, we found that 62 genes that were differentially expressed
in all three groups at time point 2 were related to cell cycle regulation
(Table 7), while only 12 in time point 1 were related to cell cycle, suggesting
that this deregulation occurs at the late stage of apoptosis. Cell cycle
interruption was also observed at time point 1. In mcd-1-1 mutant, for example,
SEM images showed the daughter cell was unable to bud off the mother cell even
when the size of the daughter cell was similar to the size of the mother cell
(Figure 7(a)). The 62 all upregulated genes (Table 7) were involved in both
mitotic and meiotic cell cycles. 6 genes were
involved in mitotic spindle checkpoint (MAD2, MAD3, DMA2, SGO1, IBD2, and
MPS1); 3 in DNA damage checkpoint (PIN4, MEC3, LCD1) and two with unknown
functions. Of the two genes with unknown functions, YOR019W interacts with ribosomes,
and SPR6 was involved in sporulation. Stress, especially external
(environmental, nutritional) stress, is the major cause of meiosis or
sporulation [34]. The fact that 23 out of the 62 genes (Table 7) are involved
in meiotic cell cycle suggests that yeast cells under the external (H2O2),
or internal (mc1-1 and pds5-1) stress may attempt to initiate meiotic division.
REC8, for example, is a meiotic specific gene that is only expressed during
early meiosis. Its expression, however, was detected under all three conditions
(Table 7). On the mitotic side, many of the differentially expressed genes are
involved in checkpoints of G1/S or G2/M transitions, such as SWI4, a DNA
binding component of the SBF complex (Swi4p-Swi6p) that regulates late
G1-specific transcription of targets, including cyclins, and genes required for
DNA synthesis and repair. Another example is SFI1, a Centrin (Cdc31p)-binding
protein, required for progression through G2/M  transition.Table 7
List of differentially regulated genes that are related to cell cycle in time point 2 (note that all genes are upregulated).
Gene nameFold changeDescriptionH2O2mcd1-1pds5-1CSM425.9913.0013.93Meiotic
chromosome segregationREC810.1014.908.60Meiotic sister
chromatid cohesionSPR318.3818.3813.00Cellular morphogenesis
during conjugation with cellular fusionREC11417.154.9222.63Meiotic
recombinationMER117.152.8318.38Regulation of
nuclear mRNA splicingCST913.934.0014.93Synaptonemal
complex formationSWI413.933.739.19G1/S transition
of mitotic cell cycleSPS413.004.597.46DNA metabolismOKP113.002.148.57Chromosome
segregationSFI112.133.7311.31G2/M transition
of mitotic cell cycleEPL112.133.738.57Regulation of
transcription from RNA polymerase II promoterMPS312.132.8310.56Nuclear migration
during conjugation with cellular fusionSCC412.132.309.19Double-strand
break repairMAD212.132.0011.31Mitotic spindle
checkpointFKH211.313.4813.00G2/M-specific
transcription in mitotic cell cycleISC1011.312.6414.93Protein required
for sporulationMPC5410.5621.1117.15Spore wall
assemblyDMA210.564.299.85Establishment of
mitotic spindle orientationREC10710.563.259.85Meiotic
recombinationIBD210.563.0317.15Mitotic spindle
checkpointMPT510.562.6412.13Reentry into
mitotic cell cycle after pheromone arrestMAD310.562.649.85Mitotic spindle
checkpointCLN310.562.309.85G1 cyclin
involved in cell cycle progressionREC1049.8514.939.85Meiotic
recombinationKEL29.855.667.46Regulation of
exit from mitosisKAR39.854.9210.56Mitotic sister
chromatid cohesionBCK29.853.259.19Regulation of
progression through cell cycleRTT1019.852.839.85Negative
regulation of DNA transpositionSLK199.852.467.46Meiosis/mitotic
spindle organizationLRS49.852.3010.56Protein involved
in rDNA silencingMND19.1968.596.96Meiotic
recombinationHOP29.194.9214.93Meiotic
recombinationKIP19.193.037.46Mitotic spindle
assembly and chromosome segregationSLD29.192.469.19DNA strand
elongationTFB39.192.149.19Regulation of
transcriptionTAF29.192.149.19G1-specific
transcription in mitotic cell cycleSPO118.577.468.57DNA metabolismPRP468.573.7311.31Nuclear mRNA
splicingUME68.573.259.85G2/M transition
of mitotic cell cycleELG18.572.4612.13Regulation of DNA
transpositionCDC458.572.469.19Prereplicative
complex formation and maintenanceBIR18.572.468.57Mitotic spindle
elongationCHL18.572.468.00Mitotic sister chromatid
cohesionSGO18.572.308.57Sister chromatid
segregationSLD38.572.149.19DNA unwinding
during replicationCEF18.003.7310.56Nuclear mRNA
splicingMPS18.003.258.57Mitotic spindle
assemblySET38.003.257.46Histone deacetylationSPC728.002.648.00Mitotic spindle
elongationCDC268.002.4618.38Protein
ubiquitinationMCM228.002.309.85Chromosome
segregationMMS48.002.008.00Involvement in recombination and DNA repairNIP1007.464.929.85Establishment of
mitotic spindle orientationDBF47.463.259.19Regulation of DNA
replicationMEC37.462.836.96DNA damage
checkpointPIN47.462.469.19G2/M transition
of mitotic cell cycleGIC27.462.149.19Regulation of
exit from mitosisIME16.9617.1516.00Regulation of
transcriptionLCD16.963.487.46DNA damage
checkpointULP16.962.467.46Ubl
(ubiquitin-like protein)-specific proteaseYOR019W13.938.5711.31Protein of
unknown functionSPR610.562.309.19Protein of
unknown function(a) Scanning electron microscopy images of wild type,H2O2-treated
(4 hours) cells, mcd1-1 and pds5-1 cells at nonpermissive temperature for 2 hours.
(b) rhodamine-phalloidin staining of actin of wild type (with or without H2O2 for 4 hours), mcd1-1 and pds51-1 mutants grown at permissive or nonpermissive
temperature for 2 hours.
(a)(b)


### 2.6. Genes Involved in Cell Wall Organization

Scanning electron microscopy was used to investigate the cell
morphology under the external and internal stresses. Consistent with the
microarray data, the cell surface structure of the pds5-1- and theH2O2-treated
cells was similar, in which cell wall sunk, forming a hole on the cell surface (Figure 7(a)).The mcd1-1 cells
appeared to be more damaged than pds5-1- or H2O2-treated
cells. More cells collapsed, and cell surface was not as smooth as the other
two, suggesting a potential cell composition change. The mcd1-1 cells were
generally larger than the wild type or the other two. Further examination of
the gene expression related to cell wall revealed that only 7 genes in time
point 1 and 5 genes in time point 2 were differentially expressed in all three
cases (Table 8), suggesting that the cytoskeleton, rather than the cell wall
composition or organization, may contribute more to the morphological changes observed
by SEM. To test the possibility of the involvement of cytoskeleton in cell-morphology change, rhodamine-conjugated
phalloidin was used to stain actin. As shown in Figure 7(b), for the wild type
and mutants grown at permissive temperature, actin appeared to be in both
polymerized (filaments) and nonpolymerized (monomers) forms. The filamentous
actin was usually oriented parallel to the longitudinal axis of the cell, as
well as along the cell wall. In H2O2-treated cells, and
mutants grown at nonpermissive temperature, actin was present mostly as
monomers, indicating the damage of the cytoskeleton.Table 8
List of differentially expressed genes in all three groups that are related to cell wall.
Gene nameFold changeDescriptionH2O2mcd1-1pds5-1Time point 1SSP210.563.0310.56Spore wall
assemblySPI19.195.6619.70Cell-wall protein
of unknown functionFIG28.573.258.57Cellular
morphogenesisYGP18.003.0310.56Amino acid
metabolism (response to stress/nutrient)GAS47.462.147.46Carbohydrate
metabolismPHO5−2.46−2.14−2.00Phosphate
metabolismTIR1−2.83−2.64−2.00Cell-wall mannoprotein
of the Srp1p/Tip1p familyTime point 2SPR318.3818.3813.00Cell-wall organization and biogenesisSSP216.009.199.85Spore-wall
assemblyGAS411.314.598.57Carbohydrate
metabolismLRE16.962.147.46Cell-wall
organization and biogenesisSUR7−2.83−2.14−3.48Endocytosis


### 2.7. Apoptotic Cell Death at Nonpermissive Temperature

Temperature shift in several temperature-sensitive mutants, includes
CDC48 [5], ORC2 [6], and PDS5 [7], showed apoptotic cell death. We speculate
that all cells undergo apoptotic cell death at the nonpermissive temperature.
To test this speculation, wild type cells were incubated overnight at 42°C, a
nonpermissive temperature for wild type cells. Apoptotic cell death was
revealed in majority of wild-type cells.
More than 90% of wild-type cells showed positive Annexin V staining,
nuclei fragmentation, and chromatin condensation. About 50% of cells showed positive TUNEL staining, while none
showed positive TUNEL staining at permissive temperature (Figure 8). ROS level
was much higher than cells grown at 30°C. Wild type showed no significant
difference from the mcd1-1 or pds5-1 mutants when both incubated at 42°C
(data not shown). This result suggests that high temperature stress causes
apoptosis in yeast and can be used as an apoptosis inducer for studying the
mechanism of apoptotic cell death in yeast.Figure 8
TUNEL staining of wild-type cells at permissive (30°C)
and nonpermissive temperature (42°C), showing DNA breaks at nonpermissive temperature.


## 2.1. Mutation of MCD1 and PDS5 Showed Major Markers of Apoptosis

Our previous report [7] showed that mutation of PDS5 caused
apoptotic cell death in yeast during early meiosis. Here, we demonstrated that
mutation of PDS5 and MCD1, two cohesin related genes, caused apoptotic cell
death during mitosis. Figure 1 shows some of the “hallmarks” of apoptosis—the translocation of phosphatidylserine from
the inner leaflet to the outer leaflet of the plasma membrane (Figure 1(a)),
and nuclear fragmentation (Figure 1(b)).
ROS accumulation was also observed in mcd1-1 and pds5-1 mutants, when grown at nonpermissive temperature (Figures 1(c) and 1(d)). Note that the ROS level was higher in mcd1-1 and
pds5-1 mutants even at 21°C (Figure 1(d)), compared to the wild type. Chromatin condensation was also observed by transmission electron microscopy (Figure 2).Apoptotic cell death in mcd1-1 and pds5-1 mutants. WT, mcd1-1, and pds5-1 mutants were shifted from permissive temperature (21°C) to nonpermissive temperature (37°C) for 12 hours, then stained with (a) Annexin V, PI, and DAPI; (b) DAPI for nucleus and (c) Dihydrorhodamine 123 for ROS. (d) Quantitative measurement of ROS production of H2O2-treated cells, mcd1-1 and pds5-1 mutants at permissive, and nonpermissive temperature.
(a)(b)(c)(d)Figure 2
TEM images of mcd1-1 and
pds5-1 mutants, growing at nonpermissive temperature for 2 hours, showing
chromatin condensation (arrow in mcd1-1 and pds5-1)N= Nucleus.


## 2.2. General Characteristics of Gene Expression

In attempt to
understand the mechanism of the cohesin-induced apoptosis in yeast,a global analysis of gene
expression was performed in mcd1-1 and pds5-1 mutants undergoing apoptotic cell
death.H2O2-treated
cells were used as a positive control. The number of up- and downregulated
genes were shown in Figure 3 (the original data is available at http://www.uwyo.edu/microscopy/cohesin.xls). The microarray data were further validated by reverse transcriptase-polymerase
chain reaction (RT-PCR) (Figure 4). 5 different genes from each group were
chosen for RT-PCR analysis. These genes represent genes that are up- and downregulated,
and genes related to cohesin complex, which is of interest for further
analysis. At both time points (see materials and methods for determination of
the time points), more genes were upregulated than downregulated, suggesting a
genetic program was being activated, rather than the general gene expression shutdown
that might be expected in a rapid cell death. At time point 1, pds5-1 had more
genes differentially regulated than the other two, while mcd1-1 had the least.
At time point 2, however, the number of differentially regulated genes in
mcd1-1 surpassed that of pds5-1 or H2O2-treated cells,
especially the number of upregulated genes (Figures 3, 5). Furthermore, while
the number of differentially regulated genes in pds5-1 and H2O2-treated
cells decreased from time point 1 to time point 2, the number of genes in
mcd1-1 increased from time point 1 to time point 2. These results suggest that
cells may have a slower but more profound response at later time point, to the
defect of Mcd1, than to the defect of Pds5, or H2O2-treated
cells.Figure 3
Number of up- and downregulated
genes at time point 1 and point 2 inH2O2-treated cells,
mcd1-1, and pds5-1 cells. *The number of unchanged genes includes genes with
marginal increase and marginal decrease (see Materials and Methods for detail).Figure 4
RT-PCR of 5 different genes from each group of time point 1.Figure 5
Schematic representation of differentially expressed genes in the three groups.T1: time point 1; T2: time point 2.Only a small number of differentially expressed genes overlap
among the three groups in both time points (Figure5). The total number of
overlapped genes were 174 in time point 1 (167 upregulated, and 7 downregulated)
and 349 in time point 2 (323 upregulated and 26 downregulated). This suggests
that these three groups may undergo apoptotic cell death through different
pathways. To better understand the pathways, the gene expression profiles were
analyzed using Netaffx analysis software from Affymetrix (http://www.affymetrix.com). Using Affymetrix’s gene ontology (GO) mining
tool, it was found that in H2O2-treated cells, the most
prominent group of genes (the group with the lowest ρ-value) that were differentially expressed was
“response to stimulus” (Table 2). In mcd1-1 and pds5-1 mutants, however, the
most prominent group is related either to physiological process (mcd1-1), or
regulation of biological process (pds5-1). This may reflect the fact that the H2O2-treated
cells mainly responded to the external stress, termed “environmental stress
response” (ESR) [25], while in mcd1-1 and pds5-1 mutants, the gene
expression responded mainly to biological or physiological changes due to the
malfunction of MCD1 or PDS5.Table 1
Fold changes of each gene expression, corresponding to the RT-PCR in Figure4.
H2O2pds5-1mcd1-1GENEFold changeGENEFold changeGENEFold changeHSP26A+10.6HSP26A+30HSP26A+9.8NHP6A−4NHP6A−2.3NHP6A−2SMC3+8ALD3+52ALD3+12SCC4+9.8REC8+19.7PDS5−1.7ESP1+6.1YKU80+7.5ESP1−1.3ActinControlActinControlActinControlTable 2
The most prominent gene group (the group with the lowest ρ-value) that is differentially expressed in response to theH2O2 treatment, or MCD1 and PDS5 mutations.
Time point 1Time point 2mcd1-1Physiological process (GO ID = 7582)Physiological process (GO ID = 50789)pds5-1Regulation of biological process (GO ID = 50789)Regulation of biological process (GO ID = 50789)H2O2Response to stimulus (GO ID = 50896)Regulation of biological process (GO ID = 50789)All threeResponse to stimulus (GO ID = 50896)Regulation of biological process (GO ID = 50789)


## 2.3. Genes Involved in Response to Stimulus

When the 174
overlapped genes from the three groups in time point 1 were analyzed using the
GO mining tool, it was found that the most prominent group of differentially
expressed genes was related to “response to stimulus” (Table2), suggesting
that, like the H2O2-treated cells, the mcd1-1 and pds5-1
mutants, when grown at nonpermissive temperature, were also under stress. An
evidence of the stress was the production of ROS (Figure 1). The changes of
gene expression in mcd1-1 and pds5-1 mutants were referred to “internal stress
response” (ISR), contrasting to the environmental, or external stress response
(ESR). At time point 2, the most prominent change of gene expression in all
types of cells, including H2O2-treated cells, was related
to either physiological process (mcd1-1), or regulation of biological process
(pds5-1 and H2O2-treated), suggesting that the ESR could
eventually become ISR, if the stress continues.Further analysis of
the overlapped genes of the three groups revealed that at time point 1, 29 out
of 174 of the overlapped genes were a response to stimulus, including response
to DNA repair, oxidative stress, heat and nutrition (Table3). Only 3 out of 29
were downregulated and 26 were upregulated.
In time point 2, response to stimulus was one of the top three prominent
groups that are differentially regulated (1st= regulation of
biological process (Table 2), 2nd= development, 3rd= response to stimulus). Among the 58 differentially regulated genes response to
stimulus, 31 (53%) were related to DNA damage (Table 4), suggesting that more
DNA damages were caused by the extended time of internal or external stress.  It has been reported that both Pds5 and Mcd1
are involved in DNA repair [26, 27]. The elevated number of differentially expressed genes response to DNA
damage could be related in part, to the malfunction of mcd1-1 or pds5-1 in DNA
damage  repair.Table 3
List of differentially regulated genes response to stimulus in time point 1.
Response toGeneFold changeDescription+H2O2mcd1-1pds5-1DNA damageRMI110.562.839.85Involved in response to DNA damageRFX18.572.146.50Involved in DNA damage checkpoint pathwayEPL17.462.146.96Component of NuA4, a histone H4/H2A acetyltransferase complexNTG16.962.307.46DNA N-glycosylase and apurinic/apyrimidinic (AP) lyaseYKU806.962.147.46Subunit of the telomeric Ku complex (Yku70p-Yku80p)NHP6A−4.00−2.00−2.30High-mobility group nonhistone chromatin proteinoxidative stressSRX121.113.739.19SulfiredoxinHSP1211.312.468.57Plasma membrane localized protein that protects membranes from desiccationCTT110.564.9217.15Cytosolic catalase TGRE38.572.4613.00Aldose reductaseHeatHSP2610.569.8529.86Small heat shock protein with chaperone activityGRE38.572.4613.00Aldose reductaseSSA38.002.4612.13ATPase involved in protein folding and the response to stressSSA48.002.1411.31Heat shock protein that is highly induced upon stressSSE26.502.008.00Member of the heat shock protein 70 (HSP70) familyNTH213.932.0010.56Putative neutral trehalasenutrition & othersALD319.7012.1351.98Cytoplasmic aldehyde dehydrogenasePTR39.193.0312.13Component of the SPS plasma membrane amino acid sensor systemDDR28.576.5017.15Multistress response proteinFIG28.573.258.57Cell-wall adhesinADR18.573.039.85Carbon source-responsive zinc-finger transcription factorYGP18.003.0310.56Cell-wall-related secretory glycoproteinHAL17.462.836.50Cytoplasmic protein involved in halotoleranceTSL16.964.2913.93Large subunit of trehalose 6-phosphate synthase/phosphatase complexMET46.502.469.19Lecine-zipper transcriptional activatorMPT56.502.009.19Member of the Puf family of RNA-binding proteinsPHO5−2.46−2.14−2.00Repressible acid phosphataseTIR1−2.83−2.64−2.00Cell wall mannoprotein of the Srp1p/Tip1p family of serine-alanine-rich proteinsUnknownYIL169C10.563.7327.86Putative protein of unknown functionYOR019W9.192.4610.56Hypothetical proteinTable 4
List of differentially expressed genes in time point 2 response to DNA
damage (bold = genes differently expressed in both time points).
GeneFold changeDescriptionH2O2mcd1-1pds5-1PSO214.936.969.19Required for a
postincision step in repair of DNA breaksCST913.934.0014.93SUMO E3 ligaseEAF713.002.469.85Subunit of the
NuA4 histone acetyltransferase complexMIG312.134.2914.93Probable
transcriptional repressorEPL112.133.738.57Component of NuA4NHP6A12.133.738.57High-mobility
group non-histone chromatin proteinSCC412.132.309.19Subunit of
cohesin loading factor (Scc2p-Scc4p)SLX111.312.8310.56Subunit of
5'-flap endonuclease complexVID2110.564.5913.00Component of the
NuA4 histone acetyltransferase complexYKU8010.563.259.85Subunit of the
telomeric Ku complex (Yku70p-Yku80p)RAD599.196.509.19Involved in
repair of DNA double-strand breaksHPR59.192.839.19DNA helicase and
DNA-dependent ATPaseSIR49.192.837.46Silent
information regulator that,EAF69.192.467.46Esa1p-associated
factor, subunit of the NuA4 acetyltransferase complexTFB39.192.149.19Subunit of TFIIH
and nucleotide excision repair factor 3 complexesNTG18.573.487.46DNA N-glycosylase
and apurinic/apyrimidinic (AP) lyaseMGT18.572.649.19DNA repair
methyltransferaseSNF58.572.649.19Subunit of the
SWI/SNF chromatin remodeling complexHMI18.572.466.96Mitochondrial
inner-membrane localized ATP-dependent DNA helicaseRTT1078.572.007.46Interacts with
Mms22p and is implicated in Mms22-dependent DNA repairEXO18.002.648.005'-3' exonuclease
and flap-endonucleaseELC18.002.1411.31Elongin CHEX38.002.146.96Protein
containing a RING finger domain that interacts with Slx8pDOA18.002.008.57WD repeat proteinMMS48.002.008.00Subunit of the
structure-specific Mms4p-Mus81p endonucleaseRAD167.465.288.00Recognizes and
binds damaged DNA in an ATP-dependent mannerMEC37.462.836.96DNA damage and
meiotic pachytene checkpoint proteinPIN47.462.469.19Involved in G2/M
phase progression and response to DNA damageCAC27.462.008.57Component of the
chromatin assembly complexLCD16.963.487.46Essential protein
required for the DNA integrity checkpoint pathwaysPAN26.962.3012.13Subunit of the
Pan2p-Pan3p poly(A)-ribonuclease complexPrevious
reports indicate that both Pds5 [27, 28] and Mcd1 [26] have a role in DNA
repair. To explore if the defect in DNA repair is involved in mcd1-1- or pds5-1-induced
apoptosis, wild type, pds5-1- and mcd1-1 cells were exposed to UV radiation
(150 J/m2) using a UV cross-linker (Stratagene, Inc.), and then
incubated for 20 hours at 37°C before being assayed with DAPI. DAPI staining showed that in pds5-1, broken nuclei increased at least 18%, while in
mcd1-1,  broken nuclei increased about 15%, compared to cells without UV
radiation. At the same condition, UV radiation had almost no effect on wild
type (Figure 6(a)). ROS levels were much higher in mcd1-1 and pds5-1 mutants after UV
radiation (Figure 6(b)).These results suggest that (1) Mcd1 and Pds5 participate in DNA break repair, and (2) defect of mcd1-1 or pds5-1 in DNA
break repair plays a role in  apoptosis.UV radiation caused increase
of apoptotic cell death in mcd1-1 and pds5-1 mutants. (a) wild type, pds5-1
cells, and mcd1-1 cells were exposed to UV radiation (150 J/m2) then
incubated for 20 hours at 37°C. Cells were stained with DAPI to identify the
fragmented nuclei. Data are from 3 independent experiments; at least 100 cells were counted for each experiment. (b) ROS
was produced in pds5-1 and mcd1-1 mutants. Wild type and the mcd1-1 and
pds5-1mutants were exposed to UV radiation (150 J/m2) then incubated
for 2 and 18 hours at 37°C. Dihydrorhodamine 123 (Priault et al. pages 684–91) was added into cell culture
and incubated for another 2 hours, ROS production was measured using a
microplate reader.
(a)(b)


## 2.4. Genes Involved Mitochondria Functions

Mitochondria play an
important role in yeast apoptosis [29, 30]. In our current study, microarray
analysis revealed that many differentially expressed genes were localized in
mitochondria.  In time point 1, among the
174 differentially expressed genes in all three groups, 38 were found localized
in mitochondria (Table 5). 26 of the 38 genes were localized only in mitochondria.
Interestingly, 12 of the 38 have unknown functions. The genes that have known
functions were involved in response to stimulus, constituents of mitochondrial
membrane, regulation of ATP syntheses, and other enzymes. Of particular
interest is the upregulation of CYC7, which encodes the isoform 2 of Cytochrome
C. Cytochrome C has been reported to be released from mitochondria to cytoplasm
after induction of apoptosis [31]. The increase of expression level of CYC7 may
also contribute to the release and the abundance of Cytochrome C in cytoplasm
after induction of apoptosis. The upregulation of CYC7 only appeared in time
point 1, not time point 2, suggesting that the release of Cytochrome C is an
early event during apoptosis. At time point 2, 40 (12 localized only
mitochondria; 8 with unknown functions) of the commonly up- or downregulated
genes were found localized in mitochondria. Although the number of genes was
similar to that of time point 1, the majority of genes were different and only
7 appeared in both time points (bold letters in Tables 5 and 6). Of the 7
genes, 3 (ELG1, NTG1, and NTH2) were response to stimulus/DNA repair; 3 (GCV1,
GLT1, and AEP1) were related to amino acid/protein metabolism; and one
(YDR332W) with unknown functions. In general, the mitochondrial genes were
mainly involved in response to stimulus/DNA damage repair, metabolism of DNA,
proteins, and amino acids. BIR1, the gene encodes an antiapoptotic protein, was
found overexpressed in all three cases (Table 6). When checked at time point 1,
BIR1 was also overexpressed in all three cases, but the fold change in mcd1-1
was less than 2, and therefore was filtered out. Because BIR1 is also involved
in coordinating cell cycle events for proper chromosome segregation, it
is unclear that the overexpression of BIR1 is due to its role of cell cycle
control, or  apoptosis.Table 5
List of differentially regulated genes that are related to mitochondria functions in time point 1  (Bold = genes appeared in both time points;*  = only those localized in mitochondria).
Gene nameFold changeDescriptionH2O2mcd1pds5-1Localized only in
mitochondriaCYC713.932.649.85Cytochrome c
isoform 2OM1411.313.2514.93Integral
mitochondrial outer membrane proteinALD49.192.6419.70Mitochondrial
aldehyde dehydrogenaseLSC28.572.309.85Beta subunit of
succinyl-CoA ligaseCEM18.572.3011.31Mitochondrial
beta-keto-acyl synthaseSTF18.572.469.85Involved in
regulation of the mitochondrial F1F0-ATP synthaseRSM198.002.148.00Mitochondrial
ribosomal protein of the small subunitOM458.003.0316.00Major constituent
of the mitochondrial outer membraneCYB28.004.0012.13Cytochrome b2
(L-lactate cytochrome-c oxidoreductase)YME27.462.007.46Integral inner
mitochondrial membrane proteinMRPL197.462.008.57Mitochondrial
ribosomal protein of the large subunitSLM57.462.149.19Mitochondrial
asparaginyl-tRNA synthetaseAEP16.962.007.46Required for
expression of the mitochondrial OLI1 geneFMP466.962.0010.56Putative redox
protein containing a thioredoxin foldMBR16.963.739.85Involved in
mitochondrial functions and stress responseODC16.062.009.19Mitochondrial
inner membrane transporterGLT1−3.48−2.00−2.46NAD(+)-dependent
glutamate synthaseLocalized in mitochondria and nucleus/cytoplasmNTH213.932.0010.56Putative neutral
trehalaseGCV111.312.8312.13T subunit of the
mitochondrial glycine decarboxylase complexGOR19.852.0010.56Glyoxylate
reductaseDIA49.192.308.57Probable
mitochondrial seryl-tRNA synthetaseRSF18.002.308.57Protein required
for respiratory growthYHL009W-B8.002.838.57Retrotransposon
TYA Gag and TYB Pol genesNTG16.962.307.46DNA N-glycosylase
and apurinic/apyrimidinic (AP) lyaseELG16.962.3010.56Required for S
phase progression and telomere homeostasisLRG1−2.46−2.30−3.25Putative
GTPase-activating protein (GAP)Of unknown
functionsTMA1019.707.4613.93Protein of unknown
function that associates with ribosomes*YPL222W12.132.309.85The authentic, nontagged
protein was localized to the mitochondria.*YDR379C-A10.562.8310.56Hypothetical
protein identified by homology.UIP48.572.6419.70Protein of unknown
function that interacts with Ulp1p*YDR070C8.579.8522.63The authentic, nontagged
protein was localized to the mitochondria*YOR205C8.002.009.85The authentic, nontagged
protein was localized to the mitochondria*YHL021C7.462.146.96The authentic, nontagged
protein was localized to the mitochondria*YDR332W7.462.6411.31Hypothetical
proteinYML128C7.466.0622.63Protein of unknown
function*YJL062W-A6.962.148.57Putative protein
of unknown function*YNR040W6.962.308.57Hypothetical
protein*YNL195C6.963.0327.86Hypothetical
proteinTable 6
List of differentially expressed genes that are related to mitochondria functions in time point 2,  (bold = genes appeared in both time points;*  = only those localized in mitochondria).
Gene nameFold changeDescriptionH2O2mcd1-1pds5-1Localized only in
mitochondriaCRC127.8632.009.85Mitochondrial
inner membrane carnitine transporterPUT116.0032.008.57Proline oxidaseHXT1413.009.199.85Hexose transportUBP1611.312.468.57Ubiquitin-dependent
protein catabolismAAC111.318.008.57Mitochondrial
inner membrane ADP/ATP translocatorPGS110.562.837.46Phosphatidylglycerolphosphate
synthaseYHL009W-A10.563.489.19DNA-mediated
transpositionGCV29.192.6412.13P subunit of the
mitochondrial glycine decarboxylase complexAEP19.193.4811.31Protein
biosynthesisHTD29.193.738.00Mitochondrial
3-hydroxyacyl-thioester dehydrataseHMI18.572.466.96Mitochondrial
ATP-dependent DNA helicaseARG27.462.649.85Acetylglutamate
synthaseGLT1−2.64−3.48−2.64NAD(+)-dependent
glutamate synthaseILV5−3.03−2.14−3.48Acetohydroxyacid
reductoisomerasePOR2−3.03−2.00−2.00Ion transportLocalized in
mitochondria and
nucleus/cytoplasmJJJ116.002.1421.11endocytosisHSF112.133.739.19Trimeric heat
shock transcription factorSPC1059.853.037.46Protein required
for accurate chromosome segregationGCV19.858.008.00T subunit of the
mitochondrial glycine decarboxylase complexNTH29.195.289.19Putative neutral
trehalaseVPS158.572.007.46Myristoylated
serine/threonine protein kinaseVPS548.572.008.00Component of the
Golgi-associated retrograde protein complexPTC58.572.008.57Mitochondrially
localized type 2C protein phosphataseLAS18.572.0012.13Essential nuclear
protein possibly involved in morphogenesisBIR18.572.468.57Cell cycle,
anti-apoptoticELG18.572.4612.13Required for S
phase progression and telomere homeostasisNTG18.573.487.46DNA N-glycosylase
and apurinic/apyrimidinic (AP) lyaseAFT28.573.738.57Iron-regulated
transcriptional activatorRIS18.002.467.46Member of the
SWI/SNF family of DNA-dependent ATPasesGYP18.003.038.57Vesicle-mediated
transportSUR7−2.83−2.14−3.48Putative integral
membrane proteinMRH1−2.83−2.00−2.83Protein that
localizes primarily to the plasma membraneOf unknown
functions*YNL130C-A18.388.5712.13Protein of unknown
function*YBR047W13.934.298.57Protein of unknown
function*YLR346C11.3111.3114.93Protein of unknown
function*YLR253W9.852.468.00Protein of unknown
functionTOF29.853.0313.93Protein of unknown
function*YDR332W9.854.927.46Hypothetical
protein*YGL226W8.572.308.00Protein of unknown
function*YOR305W8.002.009.19Protein of unknown
function


## 2.5. Genes Involved in Cell Cycle Regulation

Deregulation of cell cycle has been reported in yeast cells
undergoing apoptotic cell death [32, 33]. The apoptotic aging cells, for
example, were described as to attempt the second budding cycle without the
completion of the first one. Irregular expression of cell cycle related genes
was also reported in old cells and orc2-1 cells that underwent apoptosis [33].
In our current study, we found that 62 genes that were differentially expressed
in all three groups at time point 2 were related to cell cycle regulation
(Table 7), while only 12 in time point 1 were related to cell cycle, suggesting
that this deregulation occurs at the late stage of apoptosis. Cell cycle
interruption was also observed at time point 1. In mcd-1-1 mutant, for example,
SEM images showed the daughter cell was unable to bud off the mother cell even
when the size of the daughter cell was similar to the size of the mother cell
(Figure 7(a)). The 62 all upregulated genes (Table 7) were involved in both
mitotic and meiotic cell cycles. 6 genes were
involved in mitotic spindle checkpoint (MAD2, MAD3, DMA2, SGO1, IBD2, and
MPS1); 3 in DNA damage checkpoint (PIN4, MEC3, LCD1) and two with unknown
functions. Of the two genes with unknown functions, YOR019W interacts with ribosomes,
and SPR6 was involved in sporulation. Stress, especially external
(environmental, nutritional) stress, is the major cause of meiosis or
sporulation [34]. The fact that 23 out of the 62 genes (Table 7) are involved
in meiotic cell cycle suggests that yeast cells under the external (H2O2),
or internal (mc1-1 and pds5-1) stress may attempt to initiate meiotic division.
REC8, for example, is a meiotic specific gene that is only expressed during
early meiosis. Its expression, however, was detected under all three conditions
(Table 7). On the mitotic side, many of the differentially expressed genes are
involved in checkpoints of G1/S or G2/M transitions, such as SWI4, a DNA
binding component of the SBF complex (Swi4p-Swi6p) that regulates late
G1-specific transcription of targets, including cyclins, and genes required for
DNA synthesis and repair. Another example is SFI1, a Centrin (Cdc31p)-binding
protein, required for progression through G2/M  transition.Table 7
List of differentially regulated genes that are related to cell cycle in time point 2 (note that all genes are upregulated).
Gene nameFold changeDescriptionH2O2mcd1-1pds5-1CSM425.9913.0013.93Meiotic
chromosome segregationREC810.1014.908.60Meiotic sister
chromatid cohesionSPR318.3818.3813.00Cellular morphogenesis
during conjugation with cellular fusionREC11417.154.9222.63Meiotic
recombinationMER117.152.8318.38Regulation of
nuclear mRNA splicingCST913.934.0014.93Synaptonemal
complex formationSWI413.933.739.19G1/S transition
of mitotic cell cycleSPS413.004.597.46DNA metabolismOKP113.002.148.57Chromosome
segregationSFI112.133.7311.31G2/M transition
of mitotic cell cycleEPL112.133.738.57Regulation of
transcription from RNA polymerase II promoterMPS312.132.8310.56Nuclear migration
during conjugation with cellular fusionSCC412.132.309.19Double-strand
break repairMAD212.132.0011.31Mitotic spindle
checkpointFKH211.313.4813.00G2/M-specific
transcription in mitotic cell cycleISC1011.312.6414.93Protein required
for sporulationMPC5410.5621.1117.15Spore wall
assemblyDMA210.564.299.85Establishment of
mitotic spindle orientationREC10710.563.259.85Meiotic
recombinationIBD210.563.0317.15Mitotic spindle
checkpointMPT510.562.6412.13Reentry into
mitotic cell cycle after pheromone arrestMAD310.562.649.85Mitotic spindle
checkpointCLN310.562.309.85G1 cyclin
involved in cell cycle progressionREC1049.8514.939.85Meiotic
recombinationKEL29.855.667.46Regulation of
exit from mitosisKAR39.854.9210.56Mitotic sister
chromatid cohesionBCK29.853.259.19Regulation of
progression through cell cycleRTT1019.852.839.85Negative
regulation of DNA transpositionSLK199.852.467.46Meiosis/mitotic
spindle organizationLRS49.852.3010.56Protein involved
in rDNA silencingMND19.1968.596.96Meiotic
recombinationHOP29.194.9214.93Meiotic
recombinationKIP19.193.037.46Mitotic spindle
assembly and chromosome segregationSLD29.192.469.19DNA strand
elongationTFB39.192.149.19Regulation of
transcriptionTAF29.192.149.19G1-specific
transcription in mitotic cell cycleSPO118.577.468.57DNA metabolismPRP468.573.7311.31Nuclear mRNA
splicingUME68.573.259.85G2/M transition
of mitotic cell cycleELG18.572.4612.13Regulation of DNA
transpositionCDC458.572.469.19Prereplicative
complex formation and maintenanceBIR18.572.468.57Mitotic spindle
elongationCHL18.572.468.00Mitotic sister chromatid
cohesionSGO18.572.308.57Sister chromatid
segregationSLD38.572.149.19DNA unwinding
during replicationCEF18.003.7310.56Nuclear mRNA
splicingMPS18.003.258.57Mitotic spindle
assemblySET38.003.257.46Histone deacetylationSPC728.002.648.00Mitotic spindle
elongationCDC268.002.4618.38Protein
ubiquitinationMCM228.002.309.85Chromosome
segregationMMS48.002.008.00Involvement in recombination and DNA repairNIP1007.464.929.85Establishment of
mitotic spindle orientationDBF47.463.259.19Regulation of DNA
replicationMEC37.462.836.96DNA damage
checkpointPIN47.462.469.19G2/M transition
of mitotic cell cycleGIC27.462.149.19Regulation of
exit from mitosisIME16.9617.1516.00Regulation of
transcriptionLCD16.963.487.46DNA damage
checkpointULP16.962.467.46Ubl
(ubiquitin-like protein)-specific proteaseYOR019W13.938.5711.31Protein of
unknown functionSPR610.562.309.19Protein of
unknown function(a) Scanning electron microscopy images of wild type,H2O2-treated
(4 hours) cells, mcd1-1 and pds5-1 cells at nonpermissive temperature for 2 hours.
(b) rhodamine-phalloidin staining of actin of wild type (with or without H2O2 for 4 hours), mcd1-1 and pds51-1 mutants grown at permissive or nonpermissive
temperature for 2 hours.
(a)(b)


## 2.6. Genes Involved in Cell Wall Organization

Scanning electron microscopy was used to investigate the cell
morphology under the external and internal stresses. Consistent with the
microarray data, the cell surface structure of the pds5-1- and theH2O2-treated
cells was similar, in which cell wall sunk, forming a hole on the cell surface (Figure 7(a)).The mcd1-1 cells
appeared to be more damaged than pds5-1- or H2O2-treated
cells. More cells collapsed, and cell surface was not as smooth as the other
two, suggesting a potential cell composition change. The mcd1-1 cells were
generally larger than the wild type or the other two. Further examination of
the gene expression related to cell wall revealed that only 7 genes in time
point 1 and 5 genes in time point 2 were differentially expressed in all three
cases (Table 8), suggesting that the cytoskeleton, rather than the cell wall
composition or organization, may contribute more to the morphological changes observed
by SEM. To test the possibility of the involvement of cytoskeleton in cell-morphology change, rhodamine-conjugated
phalloidin was used to stain actin. As shown in Figure 7(b), for the wild type
and mutants grown at permissive temperature, actin appeared to be in both
polymerized (filaments) and nonpolymerized (monomers) forms. The filamentous
actin was usually oriented parallel to the longitudinal axis of the cell, as
well as along the cell wall. In H2O2-treated cells, and
mutants grown at nonpermissive temperature, actin was present mostly as
monomers, indicating the damage of the cytoskeleton.Table 8
List of differentially expressed genes in all three groups that are related to cell wall.
Gene nameFold changeDescriptionH2O2mcd1-1pds5-1Time point 1SSP210.563.0310.56Spore wall
assemblySPI19.195.6619.70Cell-wall protein
of unknown functionFIG28.573.258.57Cellular
morphogenesisYGP18.003.0310.56Amino acid
metabolism (response to stress/nutrient)GAS47.462.147.46Carbohydrate
metabolismPHO5−2.46−2.14−2.00Phosphate
metabolismTIR1−2.83−2.64−2.00Cell-wall mannoprotein
of the Srp1p/Tip1p familyTime point 2SPR318.3818.3813.00Cell-wall organization and biogenesisSSP216.009.199.85Spore-wall
assemblyGAS411.314.598.57Carbohydrate
metabolismLRE16.962.147.46Cell-wall
organization and biogenesisSUR7−2.83−2.14−3.48Endocytosis


## 2.7. Apoptotic Cell Death at Nonpermissive Temperature

Temperature shift in several temperature-sensitive mutants, includes
CDC48 [5], ORC2 [6], and PDS5 [7], showed apoptotic cell death. We speculate
that all cells undergo apoptotic cell death at the nonpermissive temperature.
To test this speculation, wild type cells were incubated overnight at 42°C, a
nonpermissive temperature for wild type cells. Apoptotic cell death was
revealed in majority of wild-type cells.
More than 90% of wild-type cells showed positive Annexin V staining,
nuclei fragmentation, and chromatin condensation. About 50% of cells showed positive TUNEL staining, while none
showed positive TUNEL staining at permissive temperature (Figure 8). ROS level
was much higher than cells grown at 30°C. Wild type showed no significant
difference from the mcd1-1 or pds5-1 mutants when both incubated at 42°C
(data not shown). This result suggests that high temperature stress causes
apoptosis in yeast and can be used as an apoptosis inducer for studying the
mechanism of apoptotic cell death in yeast.Figure 8
TUNEL staining of wild-type cells at permissive (30°C)
and nonpermissive temperature (42°C), showing DNA breaks at nonpermissive temperature.


## 3. Discussion

Sister
chromatid cohesin plays a crucial role in accurate chromosomal segregation in
the eukaryotic cell cycle. Recent studies indicate that cohesin proteins are
also involved in apoptosis. Two studies showed that the Rad21 of mammalian
cells was cleaved during apoptosis induced
by diverse stimuli. The C terminal of Rad21 cleavage product was translocated
to cytoplasm and caused amplification of the cell-death signal [21, 22]. In
human breast cancer cells, expression level of RAD21 is higher than normal
cells. Inhibition of RAD21 lows the survival rate of breast cancer cells and
induces apoptotic cell death [24]. In S. cerevisiae, mutation of PDS5 causes apoptotic-like cell death
in early meiosis [7]. When treated with H2O2, which
causes apoptosis in yeast [3], the Mcd1 level was decreased (Yang & Zhang,
unpublished data), suggesting a role of MCD1 in peroxide-induced apoptosis. Our
current study further suggests that certain cohesin proteins, such as Mcd1 or
Pds5, are required for cell proliferation. Malfunction of these cohesin
proteins causes apoptotic cell death in yeast, as well as in mammalian cells.Yeast cells show an apoptotic phenotype under a variety of environmental
stresses, such as hydrogen peroxide [3, 4]. Temperature shift in several
temperature-sensitive mutants includes CDC48 [5], ORC2 [6], and PDS5 [7], and also shows apoptotic
cell death. Our current study suggests that the cell death caused by nonpermissive
temperature is due to the internal stress response (ISR), compared to external
stress response (ESR). A common feature shared by the ISR and ESR is response
to stimulus, including response to DNA
damage, oxidative stress, and heat. Our data also suggest that ESR may
become ISR that ultimately causes the cell death. Since all the genes in the
temperature sensitive mutants are essential genes, it would be interesting to
see if cell death caused by the malfunction of all essential genes is apoptotic
and due to ISR. Another commonality of the yeast cell death is the apoptotic
nature of cell death at nonpermissive temperature, because all yeast strains,
including the wild type die at their nonpermissive temperature, that is, above
37°C.
Our result shows that even the wild-type cells undergo apoptotic cell death at
their nonpermissive temperature (Figure 8).Several studies have shown that DNA damage
stimulates apoptosis in budding yeast. Mutation in CDC13, for example, triggers
apoptotic cell death due to the accumulation of DNA damage [35].UV radiation
also induces some features of apoptosis [36].These findings suggest that yeast
may share some of the apoptotic pathways induced by DNA damage in mammals, especially
those p53-independent pathways. Sister chromatid cohesin also appears to be
involved in DNA repair. In fact, the cohesin subunit Rad21 was first identified
in Schizosaccharomyces pombe by
its sensitivity to UV or ionizing radiation, suggesting a role in DNA repair
[37]. A recent study showed that downregulation of RAD21 impaired DNA double
strand break repair and cells were more sensitive to etoposide and bleomycin,
which induce double strand breaks [24]. In fission yeast, mutants of PDS5 lost
the proliferative ability after arrest in G2 and were hypersensitive
to DNA damage [27]. Our current study further confirms that DNA damage plays an
important role in yeast cell death due to the fact that (1) a large amount of
genes related to DNA damage repair are differentially expressed during
apoptosis by either H2O2 treatment or by mutation of the
cohesin protein Mcd1 or Pds5; and (2) UV radiation caused significant increase
of cell death in mcd1-1 and pds5-1 mutants. We speculate that this defect in DNA
repair may trigger the signaling pathway that ultimately leads to the cell
death.


## 4. Materials and Methods

### 4.1. Yeast Strains and Culture Conditions

The wild-type strain used in this study isMATa ADE2 ade5can1RCYH2s his7 2 leu1-d lys2-1 met13-d trp1-63 tyr1-1 ura3-13. Plasmids of mcd1-1 and pds5-1 were provided by Dr. V Guacci and transformed
into the wild-type strain. The construction and characterization of both mcd1-1
and pds5-1 were described previously [15, 38]. Both mcd1-1 and pds5-1 are
temperature sensitive mutants, growing normally at 21°C, but unable to grow at
37°C.
Cells were grown in YPDA medium (1% Yeast
Extract, 2% Peptone,
2% Dextrose, 0.001% Adenine) on a mechanical
shaker at specified temperature.


### 4.2. Annexin V Staining

Exposed
phosphatidylserine was detected by Alexa Fluor-488 conjugated annexin V (Invitrogen,
Inc.).1×105 yeast cells were collected and washed twice with sorbitol buffer (0.8 M
sorbitol, 2% potassium acetate, pH 7.0), resuspended in sorbitol buffer
containing 10 mM dithiothreitol for 10
minutes, then digested with 0.4 mg mL−1 Zymolyase 100 T (ICN
Biomedicals, Inc.) for 30 minutes. Cells were then harvested and resuspended in
50 μL binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, 1.2 M
Sorbitol, pH 7.4). 4 μL of annexin V conjugate, 2 μl of 1 μg mL−1 propidium iodide (PI) were added to the cell suspension and incubated for 20
minutes at room temperature in dark. Cells were rinsed with binding buffer,
mounted under a coverslip with antifading medium (0.1 M propyl gallate, 50% glycerol in
PBS) containing 0.5 μg mL−1 4’, 6-diamidino-2-phenylindole (DAPI). A
Nikon-TE300-inverted microscope, equipped with a Cascade 650-cooled CCD
monochrome camera (Roper Scientific) was used for image acquisition.


### 4.3. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) Assay

DNA
breaks were detected by the ApoAlert DNA Fragmentation Assay Kit (Clontech
Laboratories, Inc.) following the manufacturer’s user manual. Briefly, yeast
cells were digested with 0.1 mg/mL Zymolyase 100 T for 5 minutes, then
spread on polylysine-coated
slides. The slides were fixed with 4% paraformaldehyde for 30 minutes,
and then rinsed with PBS. Cells were permeabilized by 0.2% Triton X-100 in PBS for
5 minutes on ice. DNA breaks were detected by TUNEL reaction using fluorescein
conjugated dUTP. Positive control is conducted by incubation of cells with
DNase I and negative control by omitting the terminal deoxynucleotidyl
transferase.


### 4.4. Transmission Electron Microscopy

TEM sample
preparation was conducted as described previous [34]. Briefly, cells
were fixed with 2.5% (v/v) glutaraldehyde in phosphate buffered saline (PBS)
(pH=7.2) for 40 minutes at room temperature. Cells were further fixed with 2%
potassium permanganate in water for 1 hour at room temperature, then stained
with 2% uranyl acetate for 90 minutes. Fixed cells were dehydrated with 30%,
50%, 75%, 85%, 95%, and 100% ethanol, then embedded in Spurr resin (Electron
Microscopy Sciences, Pa). 60 nm ultrathin sections were cut with a diamond
knife, stained with lead citrate for 30 minutes, and examined using a Hitachi
H-7000 electron microscope, equipped with a high-resolution (4K×4K)-cooled
CCD digital camera (Gatan, Inc.).


### 4.5. Detection of Reactive Oxygen Species (ROS)

ROS was detected with dihydrorhodamine 123 (Sigma Chemical
Co.). Dihydrorhodamine 123 (50 μM, final concentration) was added into cell
culture and incubated for 2 hours. Cells were then mounted on microscope slides
and viewed with fluorescence microscope using a rhodamine optical filter. A
GEMINI XS fluorescence microplate reader (Molecular Devices, CA) was used for
the quantitative measurement of ROS.H2O2 treated cells,
mcd1-1, pds5-1 mutants were stained with Dihydrorhodamine 123 and placed in a
96 well plate. Fluorescence reading was perfumed with the wavelength of 570 nm
for excitation and 615 nm for emission.


### 4.6. Microarray Analysis

For pds5-1 cells and mcd1-1 mutants, cells were inoculated
into fresh YPDA medium and cultured at21°C overnight to a density
of 1.0×107 cells/ml, then shifted to 37°C for 2 or 12 hours and
harvested for RNA isolation. The 2- and 12-hour time points were chosen because
these time points represented cells with an apoptosis rate of 1–5% and 50–60%,
respectively. These two time points were referred as time point 1 (2 hours) and
time point 2 (12 hours). For control, wild-type cells were treated as the same
as above and RNA was isolated at the two same time points.H2O2-treated wild-type
cells were used as a positive control. Wild-type cells were inoculated into
fresh medium and incubated at 37°C until the cultures reached a density of 5×106 cells/mL. 5 mM H2O2 was
added to the culture and the cells were continually cultured at 37°C for
4 or 10 hours before harvested for total RNA isolation. The 4- and 10-hour
treatment of H2O2 represents an apoptosis rate of 3–5%
and 50–60%, respectively, and as referred to time point 1 and time point 2, corresponding to
mcd1-1 and pds5-1. The control sample was treated as same as above, except with
the omission of H2O2.Total RNA was extracted using the enzymatic
lysis protocol of Qiagen RNeasy minikits (Qiagen; Valencia, Calif) with the
following modification. Buffer Y1 was replaced with sorbitol buffer (0.8 M
sorbitol, 2% potassium acetate, pH 7.0). 0.5 mg/mL (final concentration) of
Zymolyase-100 T (MP Biomedicals, Aurora, Ohio) was used for cell-wall removal and the
enzyme digestion time was 5 minutes. RNA labeling and hybridization procedures
were performed according to protocols from the Microarray Core at the University
of Colorado Health Science Center.At each time point, total RNA was extracted
from 5–10×107 cells using 2 Qiagen columns. The quality of the purified total RNA was
evaluated by Agilent bioanalyzer and NanoDrop Spectrophotometer. 2.5 μg
of high-quality total RNA was used as template for cDNA synthesis by reverse
transcription. Affymetrix one-cycle cDNA Synthesis Kit, cDNA Sample Cleanup
Module, and IVT Labeling Kit were
used to produce biotinylated cRNA target. The Agilent bioanalyzer and NanoDrop
Spectrophotometer were used to determine the cRNA yield, purity, and size
distribution. Approximately, 20 μg of cRNA were used for fragmentation with the
buffer from Affymetrix Hybridization Control Kit. The fragmented biotin-labeled
cRNA was quantitated, and hybridized to the Affymetrix GeneChip
Yeast Genome 2.0 Array, which includes approximately 5 744 probe sets for 5 841
of the 5 845 genes present in S. cerevisiae.Microarray data was first analyzed using
Affymetrix’s GeneChip Operating Software. The expression level of the cohesin
mutants andH2O2-treated cells at different time points
was compared with that of the corresponding control cells, using the software’s
default parameters (Alpha1 = 0.05, Alpha2 = 0.065, Tau = 0.015, Gamma1H = 0.002,
Gamma1L = 0.002, Gamma2H = 0.002667, Gamma2L = 0.002667, Perturbation = 1.1). A call to
each gene indicating the change in transcript level between the baseline
(control) and the experiment array was assigned with one of the following
values: Increase (I), Decrease (D), No Change (NC), Marginal Increase (MI), or Marginal Decrease
(MD). Genes with a value of NC, MI, or MD were all regarded as no change of
transcription level, and only the genes with a value I or D were selected. Fold
change of the differentially expressed genes was calculated based on the signal-log ratio. Only
the genes with a fold change equal to or greater than 2 were selected to ensure
the gene expression was truly differential. Microarray data were derived from
two independent experiments. Values were average of the two-independent
experiments


### 4.7. Scanning Electron Microscopy (SEM)

Cells at time point 1 were fixed with 2.5% glutaraldehyde in
phosphate buffer (pH 7.3), then dehydrated with ethanol (30, 50, 75, 85, 95,
100%, 5 minutes each). Cells were dried with a critical point dryer; sputter
coated with gold, and examined using a JEOL JSM5800LV SEM, operated at 20 Kv.


### 4.8. Rhodamine-Phalloidin Staining of Actin

Cells at time point 1 were fixed in PBS buffer with 4% formaldehyde for 1 hour.
After washing with PBS buffer three times, fixed cells were resuspended in 1 mL
PBS containing 1% Triton X-100 and incubated at room temperature for 3 minutes
for cell permeabilization. Permeabilized cells were washed with PBS buffer,
resuspended in 20 μl rhodamine-phalloidin (Invitrogen) (final concentration =
160 nM) for 1 hour. Cells were mounted on a glass slide and viewed with a
rhodamine filter.


## 4.1. Yeast Strains and Culture Conditions

The wild-type strain used in this study isMATa ADE2 ade5can1RCYH2s his7 2 leu1-d lys2-1 met13-d trp1-63 tyr1-1 ura3-13. Plasmids of mcd1-1 and pds5-1 were provided by Dr. V Guacci and transformed
into the wild-type strain. The construction and characterization of both mcd1-1
and pds5-1 were described previously [15, 38]. Both mcd1-1 and pds5-1 are
temperature sensitive mutants, growing normally at 21°C, but unable to grow at
37°C.
Cells were grown in YPDA medium (1% Yeast
Extract, 2% Peptone,
2% Dextrose, 0.001% Adenine) on a mechanical
shaker at specified temperature.


## 4.2. Annexin V Staining

Exposed
phosphatidylserine was detected by Alexa Fluor-488 conjugated annexin V (Invitrogen,
Inc.).1×105 yeast cells were collected and washed twice with sorbitol buffer (0.8 M
sorbitol, 2% potassium acetate, pH 7.0), resuspended in sorbitol buffer
containing 10 mM dithiothreitol for 10
minutes, then digested with 0.4 mg mL−1 Zymolyase 100 T (ICN
Biomedicals, Inc.) for 30 minutes. Cells were then harvested and resuspended in
50 μL binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, 1.2 M
Sorbitol, pH 7.4). 4 μL of annexin V conjugate, 2 μl of 1 μg mL−1 propidium iodide (PI) were added to the cell suspension and incubated for 20
minutes at room temperature in dark. Cells were rinsed with binding buffer,
mounted under a coverslip with antifading medium (0.1 M propyl gallate, 50% glycerol in
PBS) containing 0.5 μg mL−1 4’, 6-diamidino-2-phenylindole (DAPI). A
Nikon-TE300-inverted microscope, equipped with a Cascade 650-cooled CCD
monochrome camera (Roper Scientific) was used for image acquisition.


## 4.3. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) Assay

DNA
breaks were detected by the ApoAlert DNA Fragmentation Assay Kit (Clontech
Laboratories, Inc.) following the manufacturer’s user manual. Briefly, yeast
cells were digested with 0.1 mg/mL Zymolyase 100 T for 5 minutes, then
spread on polylysine-coated
slides. The slides were fixed with 4% paraformaldehyde for 30 minutes,
and then rinsed with PBS. Cells were permeabilized by 0.2% Triton X-100 in PBS for
5 minutes on ice. DNA breaks were detected by TUNEL reaction using fluorescein
conjugated dUTP. Positive control is conducted by incubation of cells with
DNase I and negative control by omitting the terminal deoxynucleotidyl
transferase.


## 4.4. Transmission Electron Microscopy

TEM sample
preparation was conducted as described previous [34]. Briefly, cells
were fixed with 2.5% (v/v) glutaraldehyde in phosphate buffered saline (PBS)
(pH=7.2) for 40 minutes at room temperature. Cells were further fixed with 2%
potassium permanganate in water for 1 hour at room temperature, then stained
with 2% uranyl acetate for 90 minutes. Fixed cells were dehydrated with 30%,
50%, 75%, 85%, 95%, and 100% ethanol, then embedded in Spurr resin (Electron
Microscopy Sciences, Pa). 60 nm ultrathin sections were cut with a diamond
knife, stained with lead citrate for 30 minutes, and examined using a Hitachi
H-7000 electron microscope, equipped with a high-resolution (4K×4K)-cooled
CCD digital camera (Gatan, Inc.).


## 4.5. Detection of Reactive Oxygen Species (ROS)

ROS was detected with dihydrorhodamine 123 (Sigma Chemical
Co.). Dihydrorhodamine 123 (50 μM, final concentration) was added into cell
culture and incubated for 2 hours. Cells were then mounted on microscope slides
and viewed with fluorescence microscope using a rhodamine optical filter. A
GEMINI XS fluorescence microplate reader (Molecular Devices, CA) was used for
the quantitative measurement of ROS.H2O2 treated cells,
mcd1-1, pds5-1 mutants were stained with Dihydrorhodamine 123 and placed in a
96 well plate. Fluorescence reading was perfumed with the wavelength of 570 nm
for excitation and 615 nm for emission.


## 4.6. Microarray Analysis

For pds5-1 cells and mcd1-1 mutants, cells were inoculated
into fresh YPDA medium and cultured at21°C overnight to a density
of 1.0×107 cells/ml, then shifted to 37°C for 2 or 12 hours and
harvested for RNA isolation. The 2- and 12-hour time points were chosen because
these time points represented cells with an apoptosis rate of 1–5% and 50–60%,
respectively. These two time points were referred as time point 1 (2 hours) and
time point 2 (12 hours). For control, wild-type cells were treated as the same
as above and RNA was isolated at the two same time points.H2O2-treated wild-type
cells were used as a positive control. Wild-type cells were inoculated into
fresh medium and incubated at 37°C until the cultures reached a density of 5×106 cells/mL. 5 mM H2O2 was
added to the culture and the cells were continually cultured at 37°C for
4 or 10 hours before harvested for total RNA isolation. The 4- and 10-hour
treatment of H2O2 represents an apoptosis rate of 3–5%
and 50–60%, respectively, and as referred to time point 1 and time point 2, corresponding to
mcd1-1 and pds5-1. The control sample was treated as same as above, except with
the omission of H2O2.Total RNA was extracted using the enzymatic
lysis protocol of Qiagen RNeasy minikits (Qiagen; Valencia, Calif) with the
following modification. Buffer Y1 was replaced with sorbitol buffer (0.8 M
sorbitol, 2% potassium acetate, pH 7.0). 0.5 mg/mL (final concentration) of
Zymolyase-100 T (MP Biomedicals, Aurora, Ohio) was used for cell-wall removal and the
enzyme digestion time was 5 minutes. RNA labeling and hybridization procedures
were performed according to protocols from the Microarray Core at the University
of Colorado Health Science Center.At each time point, total RNA was extracted
from 5–10×107 cells using 2 Qiagen columns. The quality of the purified total RNA was
evaluated by Agilent bioanalyzer and NanoDrop Spectrophotometer. 2.5 μg
of high-quality total RNA was used as template for cDNA synthesis by reverse
transcription. Affymetrix one-cycle cDNA Synthesis Kit, cDNA Sample Cleanup
Module, and IVT Labeling Kit were
used to produce biotinylated cRNA target. The Agilent bioanalyzer and NanoDrop
Spectrophotometer were used to determine the cRNA yield, purity, and size
distribution. Approximately, 20 μg of cRNA were used for fragmentation with the
buffer from Affymetrix Hybridization Control Kit. The fragmented biotin-labeled
cRNA was quantitated, and hybridized to the Affymetrix GeneChip
Yeast Genome 2.0 Array, which includes approximately 5 744 probe sets for 5 841
of the 5 845 genes present in S. cerevisiae.Microarray data was first analyzed using
Affymetrix’s GeneChip Operating Software. The expression level of the cohesin
mutants andH2O2-treated cells at different time points
was compared with that of the corresponding control cells, using the software’s
default parameters (Alpha1 = 0.05, Alpha2 = 0.065, Tau = 0.015, Gamma1H = 0.002,
Gamma1L = 0.002, Gamma2H = 0.002667, Gamma2L = 0.002667, Perturbation = 1.1). A call to
each gene indicating the change in transcript level between the baseline
(control) and the experiment array was assigned with one of the following
values: Increase (I), Decrease (D), No Change (NC), Marginal Increase (MI), or Marginal Decrease
(MD). Genes with a value of NC, MI, or MD were all regarded as no change of
transcription level, and only the genes with a value I or D were selected. Fold
change of the differentially expressed genes was calculated based on the signal-log ratio. Only
the genes with a fold change equal to or greater than 2 were selected to ensure
the gene expression was truly differential. Microarray data were derived from
two independent experiments. Values were average of the two-independent
experiments


## 4.7. Scanning Electron Microscopy (SEM)

Cells at time point 1 were fixed with 2.5% glutaraldehyde in
phosphate buffer (pH 7.3), then dehydrated with ethanol (30, 50, 75, 85, 95,
100%, 5 minutes each). Cells were dried with a critical point dryer; sputter
coated with gold, and examined using a JEOL JSM5800LV SEM, operated at 20 Kv.


## 4.8. Rhodamine-Phalloidin Staining of Actin

Cells at time point 1 were fixed in PBS buffer with 4% formaldehyde for 1 hour.
After washing with PBS buffer three times, fixed cells were resuspended in 1 mL
PBS containing 1% Triton X-100 and incubated at room temperature for 3 minutes
for cell permeabilization. Permeabilized cells were washed with PBS buffer,
resuspended in 20 μl rhodamine-phalloidin (Invitrogen) (final concentration =
160 nM) for 1 hour. Cells were mounted on a glass slide and viewed with a
rhodamine filter.


## 5. Conclusion

Our data suggests that the cell death caused by
mutation of MCD1 or PDS5 is due to the internal stress response,contrasting to
the environmental or external stress response induced by external stimuli, such
as hydrogen peroxide. A common feature shared by the internal stress response
and external stress response is the response to stimulus, including response to
DNA damage, mitochondria functions,
and oxidative stress, which play an important role in yeast apoptotic cell
death.



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*Source: 634283-2008-06-09.xml*