Tài liệu Báo cáo khoa học: A possible role of mitochondria in the apoptotic-like programmed nuclear death of Tetrahymena thermophila Takashi Kobayashi and Hiroshi Endoh docx

A possible role of mitochondria in the apoptotic-like
programmed nuclear death of Tetrahymena thermophila
Takashi Kobayashi and Hiroshi Endoh
Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Japan
Mitochondria are known to play a major role in apop-
tosis or programmed cell death (reviewed in [1,2]).
Multiple cell death-associated factors have been identi-
fied in mitochondria. These factors may be divided into
three categories based on their functions: cyto-
chrome c, Smac ⁄ DIABLO, and Omi ⁄ HtrA2, all of
which are involved in caspase activation [3–7], while
apoptosis-inducing factor (AIF) and endonuclease G
(EndoG) are direct effectors of nuclear condensation
and DNA degradation [8,9]. The pro- and antiapoptotic
members of the Bcl-2 family proteins regulate loss of
mitochondrial inner membrane potential, which results
in the release of these apoptogenic factors [1,10]. The
involvement of mitochondria in apoptosis is common
among metazoans and plants [11]. Homologues of the
aforementioned mitochondrial apoptosis factors have
been identified even in protistans, such as the cellular
slime moulds and kinetoplastids [12,13]. Taking these
discoveries into consideration, the crucial role played
by mitochondria in apoptosis appears to have an early
evolutionary origin.
The ciliated protozoan Tetrahymena thermophila
undergoes a unique process during conjugation, i.e.
programmed nuclear degradation. Unicellular Tetra-
hymena has two morphologically and functionally dif-
ferent nuclei within the same cytoplasm. One is the
germinal micronucleus and the other is the somatic

macronucleus. These nuclei both originate from a ferti-
lized micronucleus (synkaryon) during conjugation
[14,15]. As the new macronuclei differentiate from the
synkaryon via two postzygotic nuclear divisions, the
parental macronucleus begins to degenerate, in a
Keywords
nuclear apoptosis; autophagosome;
endonuclease; mitochondria; Tetrahymena
Correspondence
T. Kobayashi, Institute for Molecular
Science of Medicine, Aichi Medical
University, Yazako, Nagakute, Aichi
480-1195, Japan
Fax: +81 561 63 3532
Tel: +81 561 62 3311 (ext. 2087)
E-mail:
(Received 13 April 2005, revised 19 July
2005, accepted 24 August 2005)
doi:10.1111/j.1742-4658.2005.04936.x
The ciliated protozoan Tetrahymena has a unique apoptosis-like process,
which is called programmed nuclear death (PND). During conjugation, the
new germinal micro- and somatic macro-nuclei differentiate from a zygotic
fertilized nucleus, whereas the old parental macronucleus degenerates,
ensuring that only the new macronucleus is responsible for expression of
the progeny genotype. As is the case with apoptosis, this process encompas-
ses chromatin cleavage into high-molecular mass DNA, oligonucleosomal
DNA laddering, and complete degradation of the nuclear DNA, with the
ultimate outcome of nuclear resorption. Caspase-8- and caspase-9-like
activities are involved in the final resorption process of PND. In this report,
we show evidence for mitochondrial association with PND. Mitochondria

and the degenerating macronucleus were colocalized in autophagosome
using two dyes for the detection of mitochondria. In addition, an endo-
nuclease with similarities to mammalian endonuclease G was detected in
the isolated mitochondria. When the macronuclei were incubated with iso-
lated mitochondria in a cell-free system, DNA fragments of 150–400 bp
were generated, but no DNA ladder appeared. Taking account of the pre-
sent observations and the timing of autophagosome formation, we conclude
that mitochondria might be involved in Tetrahymena PND, probably with
the process of oligonucleosomal laddering.
Abbreviations
AIF, apoptosis-inducing factor; DAPI, 4,6-diamino-2-phenylindole; DePsipher, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-
tetraethylbenzimidazolylcarbocyanine iodide; EndoG, endonuclease G.
5378 FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS
process known as ‘programmed nuclear death’ (PND),
because it is controlled by specific gene expression [16].
Programmed nuclear death resembles apoptosis in cer-
tain aspects: nuclear condensation, chromatin conden-
sation, and DNA laddering are observed during the
destruction of the parental macronucleus [16–18], and
several studies have demonstrated the involvement of
caspase-like enzymes [19,20]. Caspase family proteins
are essential for eukaryotic apoptosis, so it seems likely
that PND and apoptosis are regulated by similar
molecular mechanisms.
Previously, we identified caspase-8- and caspase-9-
like activities, which appear to be involved in the final
resorption of the parental macronucleus during PND
in T. thermophila, and suggested the involvement of
mitochondria in this process [19]. In mammalian apop-
tosis, caspase-8 and caspase-9 are known to be associ-

ated with the mitochondrial pathway. Active caspase-8
induces the release of mitochondrial apoptosis factors,
in a process that is mediated by tBid (caspase-8-
cleaved Bid) [21]. Thus, cytochrome c is released into
the cytoplasm where it activates caspase-9 [4]. In addi-
tion, mitochondria play a key role in the execution of
apoptosis, which is separate from the caspase pathway
mentioned above. By analogy, it is reasonable to
assume that mitochondria play a key role in PND in
Tetrahymena. Unfortunately, the involvement of mito-
chondria in PND has not been clarified fully. To eluci-
date the role of the mitochondrion as a key effector
we studied the localization of mitochondria during the
death process and the levels of mitochondrial nuclease
activity. Using two different fluorescent dyes, we found
that the mitochondria colocalize with the degenerating
macronucleus in autophagosomes. In addition, we
detected a mitochondrion-derived endonuclease activ-
ity, which may be responsible for degrading DNA dur-
ing PND. A possible role of mitochondria in PND in
Tetrahymena is discussed.
A
A’
B
B’
C
C’
D
D’
E

E’
F
F’
Fig. 1. DePsipher staining of cells during conjugation. The cells are
stained with DAPI (left) and the mitochondrial membrane potential-
dependent dye DePsipher (right). (A) A preconjugating cell. Micro-
nucleus (mic) and macronucleus (Mac) sets are observed by DAPI
staining. Most of the mitochondria show red fluorescence, while
green fluorescence is occasionally visible in cells that are stained
with DePsipher. (B) Nuclear selection-stage cell (6 h after mating
induction). One of four meiotic products is positioned at the paroral
zone. (C) Post-zygotic division I (PZD I)-stage cell (7 h). (D) PZD
II-stage cell (7.5 h). The program for degeneration of the old paren-
tal macronucleus begins at this stage. Degenerating meiotic prod-
ucts are observed in the posterior region of the cells (arrowheads
in C and D). Some of these nuclei are stained green by DePsipher
(white arrowheads), while others are not (yellow arrowheads). (E)
Mac IIp-stage cell (12 h). The degenerating old macronucleus
(dOM) is stained green by DePsipher. The micronuclei and macro-
nuclear anlagen (MA) do not display this staining pattern. (F) Mac
IIe-stage cell (16 h). The dOM also stains green during its degrada-
tion. The scale bar indicates 10 lm.
T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena
FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS 5379
Results
Co-localization of mitochondria and the
degenerating macronucleus in the
autophagosome
Previously we proposed an involvement of mitochon-
dria in PND from the results of preliminary experi-

ments with the DePsipher dye, which is useful for
detecting the loss of membrane potential in mitochon-
dria [19]. The DePsipher dye accumulates in the
multimeric form in the intermembrane spaces of mito-
chondria, and fluoresces bright red when the mito-
chondria retain membrane potential, whereas the dye
disperses throughout the cytoplasm in monomeric
form, and shows a green fluorescent colour when the
mitochondrial membrane potential is lost, as happens
in apoptotic cells. To confirm mitochondrial involve-
ment in PND, more detailed observations were car-
ried out. In nonconjugating cells, the vast majority of
mitochondria showed red fluorescence, and only a
small proportion showed green fluorescence in the
cytoplasm (Fig. 1A). The fluorescence patterns
remained unchanged in the conjugating cells as long
as the parental macronucleus showed no signs of
degeneration (Fig. 1B–D). However, when the paren-
tal macronucleus began to degenerate, the staining
pattern changed drastically, and the nucleus was
stained green (Fig. 1E,F). At this stage, the parental
macronucleus has been taken in autophagosome
[17,22,23]. In contrast, the precondensed parental
macronucleus, the presumptive micronuclei, and the
developing macronuclear anlagen showed no fluores-
cence (Fig. 1A–F). These observations suggest that
many mitochondria are taken into the autophago-
some with the parental macronucleus and have lost
membrane potential. Thereby, DePsipher changed to
the monomeric form (green fluorescence) but would

not have diffused into the cytosol through autophago-
some membrane, resulting in specific localization to
the autophagosome containing degenerating macro-
nucleus. Small spots of green fluorescence, where
some mitochondria are thought to be incorporated
into small autophagosomes for turnover, were sporad-
ically observed, and some of them correspond to the
degenerating meiotic products (Fig. 1C and D; white
arrowheads).
A macronucleus that is committed to degeneration
is initially surrounded by the autophagosome, and is
eventually resorbed [17]. Thus, an autophagosome
that contains a degenerating macronucleus is called
‘the large autophagosome’ here. The large autophago-
some fuses with lysosomes, and becomes acidic in the
final step of PND [22,23]. DePsipher staining of the
macronucleus appeared initially during the stage of
autophagosome formation, and persisted until resorp-
tion of the parental macronucleus (Fig. 1D–F). Based
on these observations, we examined the possibility
that the monomeric forms of DePsipher localize to
the large autophagosome merely in response to low
pH. In order to exclude this possibility, conjugating
cells were stained with acridine orange (AO), which is
an indicator dye for acidic organelles [22]. Numerous
acidic organelles ) stained in orange ) were observed
Fig. 2. Distribution of acidic organelles dur-
ing degeneration of parental macronucleus.
The living cells during conjugation were stai-
ned with AO, which has different staining

characteristics. Green and red fluorescence
correspond to DNA and acidic organelles,
respectively. (A) Prezygotic division III (6 h).
Many lysosomes are observed. Yellow fluor-
escence (merged green and red colours)
represents the degenerating meiotic prod-
ucts (dmic). (B) PZD II (7.5 h). The precon-
densed parental macronuclei are still not
stained yellow. (C) Mac IIp (12 h). (D) Mac
IIe (16 h). The condensed parental macro-
nucleus displays yellow fluorescence, which
indicates the beginning of lysosome fusion.
Mac, Macronucleus; mic, micronucleus;
dmic, degenerating meiotic products; dOM,
degenerating old macronucleus. The scale
bar indicates 10 lm.
Mitochondria in nuclear death of Tetrahymena T. Kobayashi and H. Endoh
5380 FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS
in the cytoplasm of the conjugating cells, while intact
macro- and micronuclei were stained green with AO
(Fig. 2). The localization of the acidic organelles
(Fig. 2) is clearly different in distribution and in num-
ber from that of the green fluorescent signals of the
DePsipher dye seen in Fig. 1C and D, indicating that
there is no interaction between DePsipher monomers
and acidic organelles. When the extrameiotic products
(Fig. 2A) and the parental macronucleus (Fig. 2C and
D) began to degenerate, they were stained in yellow
(merged colour of green and orange), resulting from
the fusion of the nuclei and lysosomes, as reported

previously [22].
Green fluorescence of DePsipher did not directly
show the localization of mitochondria in the autophag-
osome, as the red fluorescence corresponding to intact
mitochondria was not observed in the area. Therefore,
to confirm further the localization, the MitoTracker
Green ) a dye that accumulates in the lipid environ-
ment of mitochondria ) was used. With this dye, mito-
chondria can be easily localized, irrespective of
membrane potential. In the nonconjugating cells, the
mitochondria were arranged mainly along ciliary lows
(Fig. 3A). Similar staining patterns were observed for
conjugating cells (Fig. 3B–E). MitoTracker stained the
degenerating parental macronucleus, but not the other
nuclei (Fig. 3C–E). Moreover, the density of staining
was high around the degenerating macronucleus, pre-
sumably corresponding to the space between the
autophagosomal membrane and nuclear envelope
(Fig. 3C–E). In a previous study, mitochondria were
not observed in or outside the large autophagosome
using the electron microscope [17]. Considering this
report and our observations of the monomeric form of
DePsipher in the autophagosome together, the mito-
chondria taken in the autophagosome might be broken,
once they were incorporated into the autophagosomes.
These observations led us to an idea that the appar-
ently dead mitochondria (or broken membrane frag-
ments) that have lost membrane potential, together
with the degenerating parental macronucleus, are taken
up preferentially by the autophagosome. This, in turn,

suggests that some molecules released from the incor-
porated broken mitochondria may play a role in the
execution of the death program.
Mitochondrion-derived nuclease activities
The uptake of mitochondria coincides with nuclear
condensation and oligonucleosomal DNA laddering
[19]. The hypothesis that mitochondria are associated
with nuclear condensation and ⁄ or DNA degradation
in PND is linked with the notion of mitochondrial
nuclease activities. In order to examine whether the
mitochondria in Tetrahymena have any nuclease activ-
ity, the mitochondria were purified from vegetatively
growing cells and incubated with a circular plasmid as
the substrate DNA. The substrate plasmid DNA was
AA’
BB’
C
C’
D
D’
E
E’
Fig. 3. Mitochondrial staining by a membrane potential-independent
dye. The cells were stained with DAPI (left) or MitoTracker Green
(right). (A) A preconjugating cell. (B) Conjugant during meiotic divi-
sion II (6 h after mating induction). (C and D) Mac IIp-stage (12 h).
The MitoTracker fluorescence is localized around the degenerating
old parental macronucleus (dOM). (E) Mac IIe-stage cell (14 h).
Scale bar ¼ 10 lm.
T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena

FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS 5381
coincubated with the isolated mitochondria at neutral
pH, and an experimental condition was surveyed
(Fig. 4A). All of the following experiments were car-
ried out in the following conditions: 200 lL reaction
containing 20 lg protein, incubated for 120 min at
30 °C. The putative DNase had an optimum pH of
6.0–6.5 for the digestion of circular DNA (Fig. 4B,
lane 3 and 4). The divalent cation requirement for the
mitochondrial DNase activity was investigated
(Fig. 4C). As shown by inhibition with EDTA
(Fig. 4C, lanes 6–8), the mitochondrial nuclease activ-
ity required divalent cations. However, higher concen-
trations (5 and 10 mm)ofMg
2+
inhibited the DNA
cleavage activity (Fig. 4C, lane 4 and 5) and weak inhi-
bition was observed even in 1 mm of Mg
2+
(compare
lane 2 and 3 in Fig. 4C), indicating a different nature
from most other DNases. On the other hand, nicking
activity was unaffected by Mg
2+
, as shown by the
increased amounts of open circular DNA (Fig. 4C,
lanes 4 and 5). The addition of Mn
2+
and Ca
2+

gave
similar inhibition results (data not shown). In the pre-
sent experiment, which involved mixing mitochondria
with plasmid DNA, the low levels of endogenous diva-
lent cations carried across with the mitochondria may
have been sufficient to support nuclease activity. Zinc
(Zn
2+
) ions, which are strong inhibitors of DNases,
inhibited completely the nuclease activity (Fig. 4C,
lanes 9–11). The presence of the DNase activity in
mitochondria is reminiscent of mammalian mitochond-
rial EndoG, which mediates the caspase-independent
pathway of apoptosis.
A
B
C
Fig. 4. Mitochondrial nuclease activity. Purified mitochondria were incubated with plasmid DNA under various conditions. (A) Basic assay for
mitochondrial nuclease activity. The assay was performed under various conditions. Lanes 1–5: isolated mitochondria (approximately 0–20 lg
protein) and 2 lg substrate DNA were coincubated for 120 min at 30 °C in 200 lL reaction buffer (50 m
M Hepes ⁄ NaOH pH 7.0, 10 mM KCl,
1m
M MgCl
2
). The DNA was then purified and electrophoresed. Lanes 6–10, mitochondria (20 lg protein) and substrate DNA were coincubated
in reaction buffer at 30 °C for 0–120 min. Lanes 11–16, the assay was carried out for 120 min at 0–50 °C. PH (preheated sample) denotes the
mixtures that were preincubated at 90 °C for 5 min before the reaction. The substrate DNA appears in the nicked open circular (OC), linear (L),
and supercoiled (SC) forms. (B) Optimal pH of the nuclease activity. The assay was performed at various pH values. The reaction mixtures con-
tained 50 m
M sodium citrate (pH 5.0 or 5.5), Mops (pH 6.0 or 6.5) or Hepes (pH 7.0, 7.5, 8.0), and 20 mM KCl. (C) Divalent cation requirement

of the mitochondrial nuclease activity. Reaction mixtures that contained 50 m
M Mops (pH 6.5) and 10 mM KCl, together with 1, 5, and 10 mM
MgCl
2
(lanes 3, 4, and 5, respectively), 1, 5, and 10 mM EDTA (lanes 6, 7, and 8, respectively), and 0.1, 1, and 5 mM ZnCl
2
(lanes 9, 10, and 11,
respectively) were assayed at 30 °C for 120 min. A standard reaction (S) was performed with 50 m
M Mops (pH 6.5) and 10 mM KCl (lane 2).
The undigested sample (U) was similar to the standard reaction, but contained no test sample (lane 1).
A
B
Fig. 5. (A) Fractionation PCR. A partial fragment of the mitochond-
rial large subunit ribosomal RNA (23S rRNA) was amplified by PCR,
using fraction samples that contained equal amounts of protein.
Lane, 1 pre-mitochondrial fraction; lane 2, mitochondrial fraction;
lane 3, post-mitochondrial fraction 1; lane 4, post-mitochondrial frac-
tion 2; lane 5, cytosolic fraction. PCR products were observed in
fractions 1–3 (lanes 1–3). (B) The nuclease activities of the fractions
under two different pH conditions. The reaction mixtures (200 lL)
contained 50 m
M sodium acetate (pH 5.0) or Mops (pH 6.5), 10 mM
KCl, 20 l g plasmid DNA as substrate, and 20 lg protein from each
fraction. The isolation of each fraction is described in Experimental
procedures. Lanes 1 and 6, pre-mitochondrial fraction; lanes 2 and
7, mitochondrial fraction; lanes 3 and 8, post-mitochondrial fraction
1; lanes 4 and 9, post-mitochondrial fraction 2; lanes 5 and 10, cyto-
solic fraction.
Mitochondria in nuclear death of Tetrahymena T. Kobayashi and H. Endoh
5382 FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS

Lysosomal contamination of the mitochondrial frac-
tion used in this study was unavoidable. To confirm
that the nuclease activity was derived from mitochon-
dria, we prepared pre- and postmitochondrial fractions
for testing in the DNase assay (see Experimental pro-
cedures). The relative ratios of mitochondria and lyso-
somes in each fraction were compared by using PCR
analysis for the mitochondria and acid phosphatase
assays for the lysosomes (Fig. 5A, Table 1). Fraction 2
was used as the mitochondrial fraction in the above
experiments (Fig. 5A, lane 2). Although mitochondria
were also detected in fractions 1 (the premitochondrial
fraction) and 3 (postmitochondrial fraction 1) by PCR
amplification, they were not detected in fractions 4 and
5, and mitochondria were most abundant in fraction 2
(Fig. 5A). On the other hand, acid phosphatase activ-
ity was higher in fractions 3 and 4 than in fraction 2
(Table 1). These results indicate that fraction 2 con-
tains a significant number of mitochondria, and that
fraction 3 is the main lysosomal fraction. The DNA-
cleavage activities in each fraction were compared at
pH 5.0 and pH 6.5 (Fig. 5B). Under somewhat acidic
conditions (pH 5.0) the nuclease activity was consider-
ably inhibited and there was no significant difference
between the fractions (Fig. 5B lane 1–5), suggesting
that the lysosomal nuclease might be activated only
under more acidic conditions. As expected, fraction 2
had the highest DNA-cleavage activity at pH 6.5
(Fig. 5B, lane 7), although fraction 1 (premitochon-
drial faction) and the two postmitochondrial fractions

(3 and 4) also showed nuclease activities, probably due
to low-level contamination with mitochondria and ⁄ or
the lysosomal enzyme itself (Fig. 5B, lanes 6, 8, 9).
Taking these results into consideration, it can be
judged that the DNase activity was derived mainly
from mitochondria rather than lysosomes.
To determine whether chromatin-associated DNAs,
as opposed to naked DNAs, are degraded by this
DNase the mitochondria were incubated with isolated
macronuclei as the substrate (Fig. 6). Under the pre-
sent experimental conditions of low osmotic pressure
and ⁄ or freeze–thawing of the mitochondrial fraction,
mitochondria are usually burst, resulting in the release
of the putative DNase as well as divalent cations. Pro-
longed incubation enhanced DNA cleavage, thereby
generating fragments of approximately 150–400 bp
(Fig. 6 lanes 3–5). Although the chromatin-sized lad-
ders were not identified, their sizes corresponded
roughly to the monomeric and dimeric forms of the
DNA ladder, as demonstrated previously for Tetra-
hymena [16,19].
Discussion
In the ciliated protozoan Tetrahymena, apoptosis-like
cell death is known to occur following treatment with
staurosporine [24], C
2
ceramide [25], or Fas-ligand
[26]. On the other hand, PND is a process in which
only the parental macronucleus is removed from the
cytoplasm of the next generation. This degradative

process occurs in a restricted area of the cytoplasm
and does not affect other nuclei that are located within
the same cytoplasm. Since they are unicellular, this
process must have been developed in a ciliate ancestor
that evolved spatial differentiation of the germline and
soma. Factors that resemble those operating in apop-
tosis also participate in nuclear death, which suggests
that PND is a modified form of apoptosis. In this
study, a possible involvement of mitochondria in PND
was suggested, as shown by the simultaneous uptake
of mitochondria and the parental macronucleus in
autophagosomes. This finding leads us to hypothesis
that some of the mitochondria are taken into the large
autophagosome, and the incorporated mitochondria
subsequently lose membrane potential or break down,
as indicated by the staining with two different dyes,
which leads to the release of mitochondrial factors into
a limited space, without affecting other organelles
within the same cytoplasm. Alternatively, mitochond-
Table 1. Acid phosphatase activities of Tetrahymena cell fractions.
Fractions
AP activity
(mAÆmin
)1
Ælg
)1
protein)
Relative
value
1 Pre-mitochondrial 0.8958 ± 0.1802 1.24

2 Mitochondrial 0.7196 ± 0.0435 1.00
3 Post-mitochondrial 1 3.1093 ± 0.1531 4.32
4 Post-mitochondrial 2 2.0750 ± 0.2412 2.88
5 Cytoplasmic 0.4356 ± 0.2008 0.61
Fig. 6. Nuclear DNA degradation by mitochondrial nucleases. The
isolated nuclei were incubated with mitochondria. The reaction was
carried out for 0 min (lane 1), 30 min (lane 2), 60 min (lane 3),
90 min (lane 4), and 120 min (lane 5). M represents the 100-bp
DNA ladder.
T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena
FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS 5383
rial degeneration may play a crucial role in autophago-
some formation, as the scattered small autophago-
somes shown by green fluorescence are probably
formed prior to the formation of the large autophago-
some (Fig. 1C and D). In either case, the autophago-
some can acquire some key molecule from the
sequestered mitochondria. This notion is supported by
the presence of a nuclease activity in the mitochondria
of Tetrahymena.
DNase activities of isolated mitochondria
In general, mitochondria have signalling pathways that
involve either AIF or EndoG, in which these molecules
execute apoptosis in a caspase-independent manner [2].
To identify mitochondrial factors in Tetrahymena,we
focused on EndoG-like enzyme activities, as EndoG is
a nuclease and AIF is not. In this study, we detected
strong nuclease activities in isolated mitochondria
(Fig. 4). This activity required divalent cations and
was strongly inhibited by the addition of Zn

2+
.In
addition, the optimal pH of this activity was pH 6.5,
while the activity was inhibited at lower pH (5.0;
Fig. 4B), suggesting that the DNase and lysosomal
enzymes function in different steps of PND. These
characteristics suggest similarities with the mammalian
EndoG. Indeed, the mammalian EndoG also requires
divalent cations, such as Mg
2+
and Mn
2+
, exhibits
biphasic pH optima of 7.0 and 9.0, and is potently
inhibited by Zn
2+
[27]. Digestion using the cell-free
system, in which isolated macronuclei and mitochon-
dria were mixed, generated nucleosome-sized DNA
fragments, although a laddering pattern was not
observed (Fig. 6). In Arabidopsis, the mitochondria
alone can induce large-sized DNA fragments (30 kb)
and chromatin condensation, whereas an additional
cellular factor is required for DNA laddering in the
cell-free system [28]. An additional factor would be
insufficient for ladder formation in the present study.
However, our findings imply that the nuclease activity
is involved in the process of DNA laddering (as is the
case with EndoG) rather than in the production of
large-sized DNA fragments, considering the timing of

uptake of the mitochondria in the autophagosome, as
discussed below.
Mitochondria as a possible executor of PND
The process of DNA degradation during PND can be
divided into three different steps, based on the sizes of
the DNA fragment generated [16–19]: (a) initial gen-
eration of high-molecular-weight (30-kb) DNA frag-
ments, followed by (b) oligonucleosome-sized ladder
formation, and (c) eventual complete degradation of
the DNA. The initial higher-order DNA fragmentation
precedes nuclear condensation [18]. Moreover, this
DNA fragmentation is a prerequisite for nuclear con-
densation. An as yet unidentified enzyme has been sug-
gested to act as a Ca
2+
-independent, Zn
2+
-insensitive
nuclease [18]. In mammalian apoptosis, AIF is known
to act as a caspase-independent death effector that
localizes to the mitochondrial intermembrane space
and translocates to the nucleus after its release from
mitochondria. Apoptosis-inducing factor causes chro-
matin condensation and degrades DNA into fragments
of sizes > 50 kb. To date, there has been no evidence
of an association between mitochondria and Tetrahym-
ena cell death, and mitochondrial homologues of mam-
malian apoptosis factors, such as AIF, have not been
identified in the Tetrahymena genome, despite the
ongoing Tetrahymena genome sequencing project.

Therefore, it seems likely that the putative mitochond-
rial apoptosis factor is not involved in the initial DNA
fragmentation step. Following the initial stage des-
cribed above, the DNA is degraded to oligonucleo-
some-sized ( 180-bp) fragments. The uptake of
mitochondria into the large autophagosome is
observed at this stage (Figs 1 and 3). According to the
observation made by Lu and Wolfe [23], who used a
combination strategy of 4,6-diamino-2-phenylindole
(DAPI) staining for the detection of DNA and Azo
dye staining for the identification of acid phosphatase
activity, lysosomal bodies approach the condensed
macronucleus prior to the formation of the large
autophagosome. It seems likely that the lysosomal
bodies incorporate some mitochondria, as indicated by
the dispersed small green fluorescence (Fig. 1). As the
nucleus becomes more condensed, many lysosomal
bodies fuse with each other, thereby forming lamellar
vesicles. Eventually, the macronucleus is completely
enveloped by a lamellar vesicle, which then corres-
ponds to the large autophagosome. Despite the enclo-
sure of the nucleus within the lamellar vesicle, acid
phosphatase activity is restricted to the lamellar vesicle
at this stage, which indicates that the lysosomal
enzyme is not localized inside the nucleus. In this
instance, the intranuclear pH should still be close to
neutral. As mentioned above, the putative mitochond-
rial nuclease presented here has an optimal pH of 6.5.
During the second period of PND, the nuclease that is
released from mitochondria is transported selectively

into the enclosed nucleus, where the second step of
DNA degradation occurs, resulting in DNA laddering.
Evidence for this stage is provided by the observation
showing the localization of mitochondria at the
circumference of the nucleus (Fig 3.C–E). This hypo-
Mitochondria in nuclear death of Tetrahymena T. Kobayashi and H. Endoh
5384 FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS
thesis is consistent with our previous finding that the
initial degradation of DNA into the chromatin-sized
ladder is suspended once for a few hours, after which
period final DNA loss occurs rapidly [19]. In the final
stage, during which the macronucleus is resorbed, acid
phosphatase activity becomes localized deeper inside
the nucleus, as supported by acridine orange staining,
which reveals that the most highly condensed macro-
nuclei are acidic [22]. In addition, the caspase-8- and
caspase-9-like activities increase dramatically just
before this stage [19].
These three steps of DNA degradation are similar to
those seen in the apoptotic nucleus [29,30]. The large-
fragment-size DNA fragmentation and DNA laddering
are characteristics of the apoptotic nucleus, and the
final DNA degradation step in the autophagosome may
correspond to the phagocytosis of apoptotic bodies by
macrophages. The machinery for apoptosis may have
originated in the era of unicellular protistans, whereas
the apoptotic function of mitochondria is thought to
have evolved relatively recently. For instance, the
nematode Caenorhabditis elegans seems to have no
pathway for caspase activation by cytochrome c.In

contrast, homologues of mitochondrial caspase-inde-
pendent apoptosis effectors, as well as caspase homo-
logues (paracaspases and metacaspases), have been
identified in certain plants, fungi, and protistans, such
as Dictyostelium and Leishmania [11–13]. Indeed, the
role of AIF in apoptosis is widely conserved in phylo-
genetically distant eukaryotes, such as the cellular slime
mould [12] and nematode [31]. More advanced mecha-
nisms may have evolved independently in each eukary-
otic lineage. In this context, it is likely that PND in
Tetrahymena is the simplest and most primitive form of
apoptosis.
Experimental procedures
Stock strains, culturing methods, and induction
of conjugation
Tetrahymena thermophila strains CU813 and CU428.2,
which were kindly supplied by P. Bruns (Cornell Univer-
sity, Ithaca, NY), were used for all experiments. Conditions
for cell culture and mating induction have been described
previously [32].
Cytological analysis
The DePsipher Kit (Trevigen Inc., Gaithersburg, MD) was
used to detect changes in mitochondrial membrane poten-
tial. Conjugated cells were transferred to 5 l gÆmL
)1
DePsipher (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢ -tetraethylbenzimi-
dazolylcarbocyanine iodide) in 10 mm Tris ⁄ HCl pH 7.5
along with stabilizer solution, and incubated for 1.5–2 h at
26 °C. The cells were then transferred to 10 mm Tris ⁄ HCl
pH 7.5 with stabilizer solution. Cells were observed imme-

diately under a fluorescence microscope with fluorescein
isothiocyanate (FITC) and green filters. For photography,
the cells were fixed with formalin (final concentration
0.5%) and stained with DAPI (4,6-diamino-2-phenylindole)
to visualize the nucleus. Acridine orange staining was per-
formed as described in Mpoke and Wolfe (1997) [22]. Mito-
Tracker Green (Molecular Probes Inc., Eugene, OR) stain-
ing has been described previously [33].
Subcellular fractionation
The late log phase cells were harvested by centrifugation at
1000 g for 5 min and washed with cold 10 mm Tris ⁄ HCl
pH 7.5. The washed cells were resuspended in a cold solu-
tion of 0.35 m sucrose, 10 mm Tris ⁄ HCl pH 7.5, 2 mm
EDTA (MIB; mitochondria isolation buffer), and homo-
genized using a Polytron homogenizer. To remove nuclei
and unbroken cells, the homogenate was centrifuged twice
at 1000 g for 5 min, and the precipitate was used as frac-
tion 1. To sediment the mitochondria, the supernatant
(fraction 1; premitochondrial fraction) was centrifuged at
8700 g for 10 min. To increase the purity, the crude mito-
chondria were resuspended in MIB that contained 10%
Percoll (Amersham Pharmacia Biotech AB, Uppsala, Swe-
den) and centrifuged at 5300 g for 5 min. The purified
mitochondria were washed once to remove Percoll and re-
suspended in MIB (fraction 2; mitochondrial fraction). The
supernatant of the crude mitochondrial fraction was centri-
fuged at 10 700 g for 10 min, and then the obtained super-
natant was further centrifuged at 18 100 g for 10 min. Both
precipitates were resuspended in MIB (fraction 3 designated
as postmitochondrial fraction 1, and fraction 4 as designa-

ted postmitochondrial fraction 2, respectively). The final
supernatant was used as the cytosolic fraction (fraction 5).
Each fraction was stored at )80 °C until use.
PCR
To assess the amount of mitochondria in each fraction, we
used a modified whole-cell PCR method [34]. Aliquots of
each fraction (4 lg protein in 5 lL) were added to 5 lL1%
Nonidet P-40 (NP-40). The mixture was incubated at 65 °C
for 10 min, followed by 92 °C for 3 min, and 10 lLof
10 · PCR buffer (Promega Inc., Madison WI), 10 lLof
25 mm MgCl
2
,2lLof10mm dNTPs, 2 lL of each primer
(100 pmol), 1 U Taq polymerase (Promega), and 60 lLH
2
O
were added, to give a total reaction volume of 100 lL. PCR
was performed as follows: 25 cycles of 92 °C for 30 s, 50 °C
for 45 s, and 72 °C for 20 s. The following oligonucleotides
were used to amplify the partial sequence of the mitochond-
T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena
FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS 5385
rial large subunit rRNA (mtLSUrRNA) gene: mtLSU-3, 5¢-
TACAACAGATAGGGACCAA-3¢; and mtLSU-4, 5¢-
CCTCCTAAAAAGTAACGG-3¢. The PCR products were
cloned into the pBluescript II SK– vector (Stratagene Inc.,
La Jolla, CA) and sequenced using the SQ-5500 DNA
sequencer (Hitachi, Tokyo, Japan).
Acid phosphatase assay
Acid phosphatase activities were assayed using p-nitrophe-

nol phosphate [35,36]. Each fraction sample (10 lL) was
mixed with 190 lL5mm p-nitrophenol phosphate dissolved
in 50 mm sodium acetate buffer (pH 5.0), and the mixture
was incubated at 30 °C for 60 min. To stop the reaction,
1 mL 0.4 m NaOH was added. The amount of liberated
p-nitrophenol was determined spectrophotometrically
at 410 nm.
Agarose gel assay for mitochondrial nuclease
activity
The standard nuclease reaction (200 lL) contained 20 lgof
the protein in the subcellular fraction, 2 lg substrate DNA
[pT7Blue (R) vector; Novagen Inc., San Diego, CA],
50 mm Hepes ⁄ NaOH pH 7.0, 10 mm KCl. The reaction
was incubated at 30 °C for 120 min. To stop the reaction,
300 lL of stop solution (100 mm Tris ⁄ HCl pH 7.5, 50 mm
EDTA, 2% SDS, 0.2 mgÆmL
)1
proteinase K) was added to
the reaction, and the mixture was incubated at 50 °C for
60 min. The stopped reaction was deproteinized with phe-
nol ⁄ chloroform (1 : 1), and the DNA was precipitated with
an equal volume of isopropanol. The precipitated DNA
was washed with 70% ethanol and diluted with 50 lLof
TE buffer (pH 8.0). The DNA samples (10 lL) were loaded
onto a 1% agarose gel, electrophoresed, and visualized by
staining with ethidium bromide.
In vitro nuclear apoptosis
Tetrahymena nuclei were isolated by the modified method
of Mita et al. [37]. Late log phase cells were harvested, and
washed with cold solution 1 (0.25 m sucrose, 10 mm

Tris ⁄ HCl pH 7.5, 10 mm MgCl
2
,3mm CaCl
2
,25mm
KCl). The packed cells were resuspended in 9 vols solution
1. To lyse the cells, 1 ⁄ 5 volumes of 1% NP-40 in solution 1
were added, and the mixture was homogenized using a
magnetic stirrer. The cell lysate was placed on 2 vols solu-
tion 2 (0.33 m sucrose, 10 mm Tris ⁄ HCl pH 7.5, 10 mm
MgCl
2
,3mm CaCl
2
,25mm KCl), and centrifuged at
1200 g for 5 min. The pellet was resuspended in solution 1,
and washed three times using the sucrose superposition
method described above. The nuclear pellet was washed
three times in solution 1 with centrifugation at 400 · g for
10 min. Finally, the nuclear pellet was washed with solution
3 (0.25 m sucrose, 10 mm Tris ⁄ HCl pH 7.5, 1 mm MgCl
2
)
and resuspended in solution 3 to a concentration of
0.5 · 10
6
macronucleiÆmL
)1
.
The isolated nuclei (approximately 10 000 macronuclei)

were incubated with mitochondrial fractions (20 lg pro-
teins) in 200 lL of reaction buffer (50 mm Mops pH 6.5,
10 mm KCl) at 30 °C. To stop the reaction, 300 lLof
stop solution (100 mm Tris ⁄ HCl pH 7.5, 50 mm EDTA,
2% SDS, 0.2 mgÆmL
)1
proteinase K, 100 lg ÆmL
)1
RNase
A) was added to the reaction, and the mixture was incu-
bated at 50 °C for 60 min. The stopped reaction was de-
proteinized with phenol ⁄ chloroform (1 : 1), and the DNA
was precipitated with an equal volume of isopropanol.
The precipitated DNA was washed with 70% ethanol
and diluted in TE buffer (pH 8.0). The DNA samples
were loaded onto a 2% agarose gel, electrophoresed, and
visualized by staining with ethidium bromide.
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