A possible role of mitochondria in the apoptotic-like programmed nuclear death of Tetrahymena...

A possible role of mitochondria in the apoptotic-likeprogrammed nuclear death of Tetrahymena thermophilaTakashi 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: [email protected]

(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


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

A A’

B B’

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.



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) of Mg2+ inhibited the DNA

cleavage activity (Fig. 4C, lane 4 and 5) and weak inhi-

bition was observed even in 1 mm of Mg2+ (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 Mg2+, as shown by the

increased amounts of open circular DNA (Fig. 4C,

lanes 4 and 5). The addition of Mn2+ and Ca2+ 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

(Zn2+) 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 mM Hepes ⁄NaOH pH 7.0, 10 mM KCl,

1 mM MgCl2). 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 mM 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 mM Mops (pH 6.5) and 10 mM KCl, together with 1, 5, and 10 mM

MgCl2 (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 ZnCl2 (lanes 9, 10, and 11,

respectively) were assayed at 30 �C for 120 min. A standard reaction (S) was performed with 50 mM 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 mM sodium acetate (pH 5.0) or Mops (pH 6.5), 10 mM

KCl, 20 lg 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.



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 ⁄orthe 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], C2 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)1Ælg)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.



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 Zn2+. 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 Mg2+ and Mn2+, exhibits

biphasic pH optima of 7.0 and 9.0, and is potently

inhibited by Zn2+ [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 Ca2+-independent, Zn2+-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-



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 lgÆmL)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 lL 1%

Nonidet P-40 (NP-40). The mixture was incubated at 65 �Cfor 10 min, followed by 92 �C for 3 min, and 10 lL of

10 · PCR buffer (Promega Inc., Madison WI), 10 lL of

25 mm MgCl2, 2 lL of 10 mm dNTPs, 2 lL of each primer

(100 pmol), 1 U Taq polymerase (Promega), and 60 lL H2O

were added, to give a total reaction volume of 100 lL. PCRwas performed as follows: 25 cycles of 92 �C for 30 s, 50 �Cfor 45 s, and 72 �C for 20 s. The following oligonucleotides

were used to amplify the partial sequence of the mitochond-



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 lL 5 mm 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 lg of

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 lL of

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 MgCl2, 3 mm CaCl2, 25 mm

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

MgCl2, 3 mm CaCl2, 25 mm 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 MgCl2)

and resuspended in solution 3 to a concentration of

0.5 · 106 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 lL of

stop solution (100 mm Tris ⁄HCl pH 7.5, 50 mm EDTA,

2% SDS, 0.2 mgÆmL)1 proteinase K, 100 lgÆmL)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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A possible role of mitochondria in the apoptotic-like programmed nuclear death of Tetrahymena...

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