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<title>Role of apoptosis-inducing factor (AIF) in programmed nuclear death during conjugation in Tetrahymena thermophila</title>
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Akematsu and Endoh BMC Cell Biology 2010, 11:13
http://www.biomedcentral.com/1471-2121/11/13

RESEARCH ARTICLE

Open Access

Role of apoptosis-inducing factor (AIF) in
programmed nuclear death during conjugation in
Tetrahymena thermophila
Takahiko Akematsu*, Hiroshi Endoh

Abstract
Background: Programmed nuclear death (PND), which is also referred to as nuclear apoptosis, is a remarkable
process that occurs in ciliates during sexual reproduction (conjugation). In Tetrahymena thermophila, when the new
macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the
progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events
are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains
numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes
apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation.
Results: We focused on the role of mitochondrial apoptosis-inducing factor (AIF) during PND in Tetrahymena. The
disruption of AIF delays the normal progression of PND, specifically, nuclear condensation and kilobase-size DNA
fragmentation. AIF is localized in Tetrahymena mitochondria and is released into the macronucleus prior to nuclear
condensation. In addition, AIF associates and co-operates with the mitochondrial DNase to facilitate the
degradation of kilobase-size DNA, which is followed by oligonucleosome-size DNA laddering.
Conclusions: Our results suggest that Tetrahymena AIF plays an important role in the degradation of DNA at an
early stage of PND, which supports the notion that the mitochondrion-initiated apoptotic DNA degradation
pathway is widely conserved among eukaryotes.

Background
Among protists, ciliates have evolved complicated structures for the spatial segregation of the germline and
soma, irrespective of their unicellular organization. One
remarkable feature of ciliates is their nuclear dualism.
Ciliates bear two functionally and morphologically distinct nuclei within the same cytoplasm: a reproductive
somatic macronucleus and a germinal micronucleus.
The polyploid macronucleus is large and supports
almost all vegetative functions through active transcription, whereas the diploid micronucleus is transcriptionally silent [1]. These nuclei both originate from a
fertilized micronucleus (synkaryon) via two successive
postzygotic divisions (PZDs) during a unique form of
sexual reproduction known as conjugation. Programmed
nuclear death (PND), also known as nuclear apoptosis,
* Correspondence: b1sk103@stu.kanazawa-u.ac.jp
Division of Life Science, Graduate School of Natural Science and Technology,
Kanazawa University, Shizenken, Kakuma-machi, Kanazawa, Ishikawa, Japan

is a unique process in ciliates whereby only the parental
macronucleus is eliminated from the cytoplasm of the
progeny during conjugation, while the parental cytoplasm is taken over by the progeny, even after sexual
reproduction. In Tetrahymena thermophila, once the
new macronucleus differentiates from the synkaryon,
the parental macronucleus begins to degenerate. This
degeneration has three distinct stages, beginning with
the degeneration of the macronuclear DNA into large (>
30-kb) fragments. This fragmentation occurs prior to
nuclear condensation and involves Ca 2+-independent,
Zn 2+ -insensitive nuclease activity [2]. In the second
stage, marked changes occur in the degenerating macronucleus, including size reduction and chromatin condensation. During this second stage, the macronuclear
DNA is degraded into smaller fragments, which comprise an oligonucleosome-scale ladder that consists of
~180-bp units [3,4]. Meanwhile, many small autophagosomes approach and engulf the nucleus, resulting in the

© 2010 Akematsu and Endoh; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

Akematsu and Endoh BMC Cell Biology 2010, 11:13
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formation of a large autophagosome with a double
membrane [5]. At this stage, lysosomes are closely associated with the autophagosome without fusion, indicating that the pH of the parental macronucleus is still
neutral. In the third stage, the macronuclear DNA is
degraded completely. Lysosomes fuse with the autophagosomal membrane, releasing their contents into and
acidifying the macronucleus, which is then resorbed
through autophagy in the acidic environment [6].
Kobayashi and Endoh [7] indicated that autophagosomes contain many mitochondria that have lost their
membrane potential. In general, the loss of mitochondrial
membrane potential leads to the release of cytochrome c
and apoptosis-inducing factor (AIF) into the cytosol [8].
Thus, it is reasonable to assume that the mitochondrial
pathway plays a key role in Tetrahymena PND. Indeed,
mitochondria play key roles in a number of apoptotic
and programmed cell death (PCD) processes in animals,
such as morphogenesis, tissue homeostasis, and immunity [9]. In animals, apoptosis involves both caspasedependent and caspase-independent pathways. Cytochrome c participates in the activation of caspases, which
are major effectors of apoptosis, whereas AIF is involved
in the caspase-independent pathway [10,11]. Caspase
activation affects a number of substrates with important
biological functions, leading to the loss of their functional
roles [12]. However, it is unclear whether PCD in plants
and protozoa involves the activation of caspase-like
enzymes. Considering that caspase homologs are not present in fungi, plants, and protists, with the exception of
animals [13], the origins of these activities remain
unknown. Furthermore, isolated mitochondria from T.
thermophila show strong DNase activity, similar to that
of human endonuclease G (EndoG), which mediates the
caspase-independent apoptotic pathway (also referred to
as mitochondrial pathway) [7]. Based on these information, PND looks to occur by the caspase-independent
pathway. However, an EndoG homolog has not been
identified in the Tetrahymena genome database.
AIF is a nuclear-encoded mitochondrial flavoprotein
that possesses NADH oxidase activity in its C-terminal
region. The primary sequence of AIF is highly homologous to those of oxidoreductases from animals, fungi,
plants, eubacteria, and archaebacteria [13,14]. AIF is a
novel, mammalian, caspase-independent death effector
that, upon the induction of apoptosis, translocates from
the mitochondrial intermembrane space to the nucleus
[15]. Once in the nucleus, AIF causes chromatin condensation and large-scale (~50 kb) DNA fragmentation
[8]. AIF-mediated PCD has been observed in roundworms (Caenorhabditis elegans) [16] and in a cellular
slime mold (Dictyostelium discoideum) [17], which suggests that the AIF pathway is a phylogenetically primitive form of apoptosis.

Page 2 of 15

In the present study, we investigated whether the proapoptotic function of AIF is conserved in Tetrahymena
PND. To address this issue, we cloned the Tetrahymena
AIF homolog and performed gene disruption to analyze
its biological functions. We discuss the unique evolution
of apoptotic mechanisms.

Results
PND in T. thermophila

The nuclear events that are typical for conjugation in T.
thermophila and that are specifically involved in nuclear
apoptosis are illustrated schematically in Figure 1.
Although previous studies have reported the details of
these events [2,3], we show the timing of these events
with regard to our experimental conditions.
Identification of the Tetrahymena homolog of AIF

Using the Tetrahymena genome database http://www.
ciliate.org/, we identified two AIF homologs
(TTHERM_00622710 and TTHERM_01104910) that are
similar to human AIF. As described below, one of these
homologs, TTHERM_01104910, had no apparent effect
on mitochondrial nuclease activity (Additional File 1).
Therefore, in the present study, we focused on the role
of the TTHERM_00622710 homolog.
This gene for Tetrahymena AIF lacks introns and
encodes a 70-kDa protein. A primary sequence comparison revealed that residues in the FAD/NAD binding
domain and the oxidoreductase domain of Tetrahymena
AIF are highly conserved in human AIF (GenBank
accession no. AAD16436.1), cellular slime mold AIF
(GenBank; EAL63305.1), and the C. elegans AIF homolog Wah-1 (NCBI; NP_499564.2) (Figure 2A). Tetrahymena AIF is ~24% identical and 45% similar to human
AIF. The putative DNA-binding sites in human AIF,
which are required for its interaction with DNA and the
induction of cell death [18], were identified in each phylum. MitoProt II, which is a prediction server for mitochondrial targeting sequences and cleavage sites,
revealed a candidate mitochondrial localization sequence
(MLS) in the N-terminus of Tetrahymena AIF (residues
1-13). However, the N-terminal portions of the remaining three proteins showed no sequence similarity.
To determine whether endogenous Tetrahymena AIF
is constitutively expressed during conjugation, mRNA
samples extracted from starved cells (just before mixing
the mating types) and conjugating cells were subjected
to RT-PCR analysis. Using AIF-specific primers, a single
340-bp band was detected in the starved cells (Figure
2B, lane 1). The mRNA of conjugating cells was
extracted every 4 h (4 - 20 h) after the initiation of conjugation. AIF was expressed continuously during conjugation, although expression decreased at 4 h (Figure 2B,
lane 2), which corresponded to the meiotic prophase. In

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Page 3 of 15

A

B
micronucleus
Macronucleus

J

C

P rophas e
V egetative

~4h

16~20 h
Meios is

Mac III

I

D
Nuclear E xchange

Mac IIe

H

E

A
m
pMa
Mac IIp

4~8 h

Nuclear development

F ertilization

12~16 h
G

F
Mac I

P ZD

8~12 h

Figure 1 Nuclear events during conjugation of Tetrahymena thermophila. Conjugation in T. thermophila is a complicated process that is
initiated by interaction between complementary mating types, which form a conjugating pair. A. Vegetative phase. B. Meiotic prophase. C.
Meiosis. D. Nuclear exchange. One of four meiotic products mitotically divides, forming two pronucei. Subsequently, one of the pronuclei is
reciprocally exchanged between mating partners. E. Fertilization (synkaryon formation). F. PZD (postzygotic division). Fertilized nucleus
successively divides twice. G. Mac I. Anteriorly-located nuclei differentiate into the new macronuclei, while posterior nuclei remain the
micronuclei. H. Mac IIp. The parental macronucleus migrates posteriorly and begins to degenerate. I. Mac IIe. Pair separates (exconjugants). J. Mac
III. One of two micronuclei is eliminated. Progeny of T. thermophila do not undergo conjugation during the first ~100 vegetative fissions after
conjugation called “immature.” A: macronuclear anlagen. m: micronuclei. pMa: parental macronucleus. The scale bar in photograph indicates 10
μm.

the control experiment, histone h3 (HHT3) was also
found to be expressed during conjugation as shown in
previous study [19].
AIF translocates from the mitochondria to the parental
macronucleus

To examine the subcellular localization of Tetrahymena
AIF, we constructed a plasmid that expresses a fusion
protein composed of AIF and GFP (AIF::GFP) under the

control of the AIF promoter (Figure 3A). The transformants stably expressed AIF::GFP in the presence of 50
μg/ml paromomycin, whereas paromomycin at concentrations >50 μg/ml had detrimental effects on cell
growth. However, the signal was too weak to allow cytological analysis. To solve this problem, we performed
indirect immunofluorescence using GFP-specific polyclonal antibodies, to determine the localization of the
fusion protein. AIF::GFP signals were observed at the

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Page 4 of 15

A

B

T ime(h) 0 4

AIF
HHT3
rRNA

1

2

8 12 16 20

3

4

5

6

Figure 2 Sequence alignment of AIF proteins and analysis of AIF expression. A. CLUSTAL-W was used to generate AIF sequence alignment,
including Homo sapiens, Dictyostelium discoideum, Caenorhabditis elegans and Tetrahymena thermophila. Boxes indicate the NAD/FAD binding
domain and oxidoreductase domain. MLS in N-terminal portion of T.thermohila indicates mitochondrial localization sequence. Asterisks indicate
identical amino acids. Colons and semicolons indicate amino acid similarity. Arrowheads indicate a potential DNA binding site of human AIF. B.
RT-PCR analysis of AIF transcript during conjugation. Histone h3 (HHT3) was used as a control. SSU rRNA was used as a loading control.

Akematsu and Endoh BMC Cell Biology 2010, 11:13
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surface and in the cytoplasm of each cell (Figure 3B),
similar to the pattern observed with MTG, a mitochondrion-specific fluorescent dye. Therefore, AIF is localized within mitochondria.
To examine the translocation of AIF during conjugation, the localization of the fusion protein (AIF::GFP)
was observed throughout this process. As shown in Figure 3Ca, before conjugation, AIF::GFP was distributed
over the cell surface along the ciliary rows. During
nuclear exchange, the pattern of AIF expression changed, and intense signals were detected in the posterior
region of each cell (Figure 3Cb). Meanwhile, the signals
nearly overlapped (and probably surrounded) the parental macronucleus during the stages that correspond to
PZD to Mac IIe (Figure 3Cc-e, see also Figure 1). Living
cells expressed AIF::GFP during the MAC IIp stage (Figure 3D). Although the AIF::GFP signal in living cells
was weak, as mentioned above, the signal was concentrated and visualized at the posterior region of the cell,
where it overlapped with the parental macronucleus
(indicated by red arrows in the Figure). These results
suggest that AIF is released from mitochondria and
translocates to the parental macronucleus before nuclear
condensation.
AIF plays roles in the growth and PND of T. thermophila

To understand the functions of AIF in T. thermophila
PND, we knocked out AIF by homologous recombination (Figure 4A and 4B). After selection with increasing
concentrations of paromomycin, ΔAIF strains that did
not express AIF were obtained (Figure 4C inset). Similar
to the situation in C. elegans [16], ΔAIF exhibited a
somewhat reduced growth rate compared to the wildtype strain (Figure 4C). As shown in Figure 5A, the
nuclear events occurring during Tetrahymena conjugation could be classified into six stages (A- F). When
ΔAIF strains of different mating types were mixed, they
initiated normal nuclear events (stages A to D; 6-14 h)
with the same frequency as the wild-type strain (Figure
5B). The parental macronuclei in the ΔAIF strains were
reduced in size by 66-85%, whereas those in the wildtype strain were reduced in size by 46-61% at 10-14 h
(Figure 5C). These results suggest that AIF is involved
in the condensation of the parental macronucleus. At
14-16 h, the ΔAIF strain showed a slight delay in transiting from stage C to D (p < 0.01, t-test), thereby
further implicating the AIF protein in nuclear condensation (Figure 5B). Although the reduction in size of the
ΔAIF strain occurred after a delay of 4 h, the peak of
stage E (Mac IIe) was shifted from 20 h (in the wildtype strain) to 22 h (in the ΔAIF strain) (Figure 5B). In
addition, the strains exhibited a delay in stage F (Mac
III), which corresponds to the final resorption of the
parental macronucleus at 20-22 h. Agarose gel

Page 5 of 15

electrophoresis revealed that kilobase-size DNA fragmentation occurred in the wild-type strain at 8-10 h
(Figure 5B). At this time-point, most of the cells were in
stages A to C, suggesting that large-scale DNA fragmentation occurs before nuclear differentiation. However,
DNA fragmentation in ΔAIF began with a 4-h delay. By
12-14 h, most of the ΔAIF cells had undergone nuclear
differentiation; however, the reduction in size of the parental macronucleus was also delayed (Figure 5C). Oligonucleosome-size DNA fragmentation was observed in
both the wild-type and ΔAIF strains 16 h after mating,
which suggests that large-scale DNA fragmentation is
dependent upon AIF in the early stage of PND (Figure
5B). These results indicate that knocking out AIF hinders the first wave of nuclear degradation, including the
condensation of the parental macronucleus and kilobase-size DNA fragmentation.
AIF cooperates with mitochondrial nuclease to promote
DNA degradation

Previously, we demonstrated the presence of strong
DNase activity in mitochondria isolated from Tetrahymena [7]. To investigate whether the putative mitochondrial DNase of Tetrahymena interacts with AIF,
mitochondria were purified from vegetatively growing
wild-type and ΔAIF cells (Figure 6A), and incubated
with a circular plasmid as substrate DNA. Mitochondria
from the wild-type strain showed strong DNase activities, in that they converted the supercoiled DNA into
an open circular form that could be further cleaved into
smaller fragments, yielding a smear of degradation products on the gel (Figure 6B, lane 2). In contrast, no
smear was observed when mitochondrial extracts from
the KO strain were used; instead, only nicking of the
plasmid DNA was observed (Figure 6B, lane 3). In addition, a time-course analysis showed that the level of
DNase activity was drastically reduced in the KO strain,
as compared with the wild-type strain (Figure 6C).
These results are similar to those obtained for C. elegans, in which an interaction between Wah-1 and Cps-6
was detected [16]. When linear DNA was employed as
the substrate, the KO strain showed low DNase activity,
whereas wild-type strain digested completely the substrate DNA (Figure 6D). These results indicate that AIF
interacts with the mitochondrial DNase to affect not
only nicking activity, but also endonuclease activity.
The second Tetrahymena homolog of AIF,
TTHERM_01104910, contains an FAD/NAD binding
domain and an oxidoreductase domain that shares 27%
identity with TTHERM_006222710. The continuous
expression of TTHERM_01104910 was confirmed both
in the vegetative phase and during conjugation. This
expression pattern was in accordance with that reported
in the Tetrahymena Gene Expression Database (TGED;

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Figure 3 Translocation of AIF from mitochondria to parental macronucleus. A. Map of expression vector, named AKgfpTtAIF, with
neomycin resistance cassette (Neor-cassette), 5’UTR and ORF of AIF, codon-optimized GFP (GFP-cassette), replication origin derived from
Stylonychia lemnae and telomeres from Tetrahymena. Neomycin resistance is expressed under control of b-tubulin promoter. Before biolistic
bombardment, the plasmid was digested with SfiI to expose telomere sequences on both ends. B. After biolistic bombardment, cytoplasmic
localization of AIF::GFP was detected using a-GFP. This fluorescent pattern was coincided with MitoTracker Green (MTG). No-bombardment
indicates non-transformed wild-type cell. Scale bar in photograph indicates 10 μm. C. Translocation of AIF::GFP was visualized with a-GFP
antibodies. White arrows indicate parental macronucleus. Overlay represents fusion image of blue (nuclei) and red (AIF::GFP) fluorescence. Scale
bar in photograph indicates 10 μm. D. Fluorescence microscopy of living cells expressing AIF::GFP at the stage of MacIIp. Red arrows indicate
AIF::GFP in parental macronuclei. pMa denotes parental macronucleus.

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Figure 4 Construction of AIF-deficient strain. A. Map of expression vector, pKoTtAIF, with neomycin resistance cassette (Neor-cassette). It
consists of 5’ and 3’ UTR sequences of AIF and part of its 3’ ORF. The neomycin resistance is expressed under the control of b-tubulin promoter.
Before biolistic bombardment, the plasmid was digested with BamHI. B. Schematic representation of the wild-type (WT) and mutant locus of the
AIF together with the targeting plasmid. Replacement of AIF to neomycin-resistant gene (Neor) in the macronucleus was confirmed by PCR using
10 ng of genomic DNA from isolated macronuclei as template. Small triangles located in the gene loci indicate specific primer pairs for the PCR
amplification. C. Cell growth curve of CU428. Circles and squares indicate cell density of wild-type strain and ΔAIF, respectively. Points and
attached bars correspond to the means of four identical measurements and standard deviations. The inset indicates RT-PCR analysis of the
expression levels of AIF and Neor. b-tubulin (BTU) was used as a control.

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A
S tage B

S tage A

B

100

S tage D

S tage C

S tage E

S tage F

6 8 10 12 14 1618 20 22 24 M M

WT

S tage

(%)

80

Kb

A
B
C
D
E
F

60
40
20

10
5
3

1
0.5

0
6

8

10

12

14

16

18

20

22

24

28

100
AIF

6 8 10 12 14 16 18 20 22 24 MM

S tage

(%)

80
A
B
C
D
E
F

60
40
20

Kb
10
5
3

1
0.5

0
6

8

10

12

14

16

18

20

22

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28

C

R elative S ize of Macronucleus

T ime (h)
1.2
WT
AIF

1
0.8
0.6
0.4
0.2
0
6

8

10

12

14 16
T ime (h)

18

20

22

24

Figure 5 Progression of PND by disruption of AIF. A. Nuclear events during conjugation were divided into 6 stages (stage A ~stage F). B.
Time course analysis of progression of the nuclear events in wild-type and ΔAIF between 6 h and 28 h after initiation of conjugation. The
percentages of the nuclear stages were counted, and were expressed as a percentage of the total number of tested cells (300-400 cells).
Columns and attached bars correspond to the means of four identical measurements and standard deviations. Fragmental DNA was isolated
from the strains every 2 h during conjugation. The black stars between 8-12 h in ΔAIF indicate delay of kb-sized DNA fragmentation. M denotes
kbp-ladder size marker (left) and 100-bp ladder size marker (right). C. Changes in size of parental macronuclei between 6 h and 28 h after
initiation of conjugation. Columns (points) and attached bars correspond to the means of four identical measurements (80-100 cells) and
standard deviations.

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Page 9 of 15

http://tged.ihb.ac.cn/). Knocking out the gene did not
influence the mitochondrial DNase activity during PND
(Additional File 1, 2). Therefore, the second AIF homolog does not appear to be involved in PND.

Figure 6 Mitochondrial nuclease activity. A. Fractionation PCR. A
partial fragment of the mitochondrial large subunit ribosomal RNA
(rRNA) or b-tubulin (BTU) was amplified by PCR, using fraction
samples from wild-type and ΔAIF that contained equal amounts of
DNA. N and Mt indicate nuclei/unbroken cell fraction and
mitochondrial fraction, respectively. No contamination of nuclear
DNA was detected in mitochondrial fraction. B. Purified
mitochondria (2 μg protein) from wild-type (lane 2) and ΔAIF (lane
3) were incubated with 2 μg substrate plasmid DNA with a circular
form for 30 min at 37°C in 30 μl reaction buffer containing 20 mM
KCl and 50 mM MOPS (pH 6.5). Lane 4 (M) and 5 (M) indicate 100bp ladder size marker and lHindIII-digest, respectively. The substrate
DNA appears in the nicked open circular (OC), linear (L), and
supercoiled (SC) forms. C. The nuclease assay was performed under
various incubation times. Lane 2-4, substrate DNA was coincubated
with wild-type mitochondria. Lane 5-7, substrate DNA was
coincubated with ΔAIF mitochondria. Undigested sample is seen in
lane 1. D. Substrate specificity of the activities. End forms of linear
plasmids with 5’- or 3’-overhang or with blunt ends are indicated at
the left of gel.

Discussion
In unicellular ciliates, the parental cell-derived cytoplasm is taken over by the progeny nucleus after sexual
reproduction. Therefore, the development of PND (i.e.,
the selective elimination of the parental macronucleus)
may have been inevitable when the first ciliate established the spatial differentiation of the germinal and
somatic nuclei. PND occurs in a limited area of the
cytoplasm and is uncoupled from the plasma membrane
events associated with PCD (programmed cell death),
for example, Fas ligand- Fas receptor binding; however,
PND involves mitochondrial apoptotic effectors, such as
EndoG-like DNase activity [7]. Thus, ciliates have developed a novel mechanism for executing PND in which
part of the intrinsic machinery (i.e., AIF) used for PCD
appears to have been adapted for a specialized form of
apoptosis.
The primitive mechanism of apoptosis may have been
established as a product of the interaction between an
ancestral host cell and its endosymbiotic primitive mitochondria [13]. One of the major pathways of apoptosis,
the caspase-dependent pathway, appears to have been
independently established in animals later during eukaryotic evolution, given that fungi, plants, and protists
commonly lack caspase homologs. Caspase-independent
pathways function in mammalian and C. elegans apoptosis, as evidenced by the finding that apoptosis can occur
in the presence of caspase inhibitors [16,20]. AIF, which
is assumed to be evolutionarily ancient because it has
been identified in various organisms, ranging from protists to animals, is localized within the intermembrane
mitochondrial space [13,21]. Disturbances in AIF can
delay several major apoptotic events in the nucleus,
including nuclear condensation, chromatin digestion,
and DNA loss [10,16,17]. These AIF-mediated events
resemble those that occur in the early stages of Tetrahymena PND.
Involvement of AIF in PND

In the present study, we provide the first evidence that
AIF is involved in Tetrahymena PND. AIF translocates
from the mitochondria to the parental macronucleus
before nuclear differentiation (Figure 3B-D), and interacts with the mitochondrial DNase, thereby triggering
the initial DNA degradation by the DNase, the optimal
pH of which is about neutral, indicating a role for AIF
in the early stage of PND. Taking these observations
into consideration, AIF appears to function as a suicide
factor in the parental macronucleus. However, the

Akematsu and Endoh BMC Cell Biology 2010, 11:13
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knocking out of the AIF gene in the parental macronucleus only slowed by up to 4 h the early stages of PND,
including nuclear condensation and kilobase-size DNA
fragmentation (Figure 5B and 5C), and did not completely inhibit the progression of PND. Indeed, by the end
of conjugation, the AIF-deficient cells were delayed only
approximately 1 h, as compared with the wild-type controls (Figure 5B). Is there a mechanism that compensates for the deficiency of AIF, thereby allowing the
appropriate execution of the death program? After
translocation of AIF into the parental macronucleus,
new macronuclei differentiate somewhat later and initiate gene expression immediately. Gene expression from
the zygotic macronucleus is indispensable for the completion of the final resorption by autophagy [3]. This
delay can be interpreted in different ways. One possibility is that when the AIF mRNA is transcribed in the
developing macronuclear anlage and the zygotic AIF
protein becomes available, the DNA in the parental
macronucleus begins to degrade behind schedule, resulting in the recovery of PND progression. It seems most
likely that the time lag in gene expression from the
zygotic macronucleus is a major cause of the delay in
the early stage of PND. Another possibility is that other
DNases exist in the Tetrahymena mitochondria (E.
Osada, personal communication), as identified using
SDS-DNA PAGE [22]. Although these DNases are unlikely to either interact with AIF or to be major effectors
of PND, they may contribute to the retarded DNA
degradation, resulting in the delayed progression of
PND.
At the stage of nuclear differentiation, two types of
macronucleus, the parental macronucleus and the new
zygotic macronucleus, co-exist for a period of time in
the same cytoplasm. Through collaboration with the
mitochondrial DNase, AIF may prevent simultaneous
gene expression from the two different macronuclei
with different genotypes through initial digestion of the
parental macronuclear DNA.
Interaction between AIF and the mitochondrial DNase

In mammals, DNA binding by AIF is required for
nuclear condensation and initial DNA cleavage [18]. In
contrast, neither FAD/NAD binding ability nor oxidoreductase activity is required for apoptogenesis [11,15]. It
remains unclear as to how AIF induces DNA fragmentation during apoptosis. One possibility is that AIF exerts
its function by interacting with downstream effectors.
AIF and EndoG are two of the many apoptogenic proteins that are released from mitochondria during apoptosis in animals [23]. In C. elegans, Wah-1/AIF cooperates with Cps-6/EndoG to promote DNA degradation in vitro. In addition, wah-1 (RNAi) strains and cps6 mutants display similar defects in cell death and DNA

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degradation, and both Wah-1 and Cps-6 are localized to
mitochondria [16]. These findings strongly suggest that
the mitochondrial PCD pathway is evolutionarily conserved. Previously, an endonuclease activity was identified in the mitochondria of T. thermophila [7]. This
activity required divalent cations and was strongly inhibited by Zn 2+ , which is a strong inhibitor of most
DNases. In addition, the optimal pH for this endonuclease activity was pH 6.5, while activity was inhibited at
pH 5.0, suggesting that the DNase and lysosomal
enzymes function in different steps of PND. These characteristics are reminiscent of mammalian mitochondrial
EndoG, which mediates the caspase-independent pathway of apoptosis [24]. 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+ [24]. Our plasmid cleavage
assay demonstrated that the mitochondria of the ΔAIF
strains had significantly reduced DNase activity (Figure
6B-D), indicating an interaction between Tetrahymena
AIF and the DNase. Notably, this result is similar to the
aforementioned situation in C. elegans [16]. The cooperative action of these two proteins implies that the
mitochondrial DNase is an important executor that is
activated by AIF. Thus, AIF and mitochondrial DNase
appear to constitute a widely conserved DNA degradation pathway that acts in the early stage of apoptosis.
However, no sequence homologous to EndoG was
detected in the Tetrahymena database, raising the possibility that ciliates have independently developed a mitochondrial DNase during the course of ciliate evolution.
Indeed, mitochondrial proteomic analysis of Tetrahymena has shown that 45% of the proteins are specific to
Tetrahymena or ciliates [25]. The roles of AIF and mitochondrial DNase are illustrated schematically in Figure
7.
The biochemical and morphologic features of apoptosis have been highly conserved throughout evolution,
even in unicellular organisms, such as cellular slime
molds, kinetoplastids, dinoflagellates, ciliates, and heterokonts [17,26,27]. A recent study suggested that any
protein that has previously been implicated in apoptosis
must have a phylogenetically conserved apoptosis-unrelated vital function [28]. For example, AIF serves as a
redox-active enzyme in respiratory chain complex I.
AIF-deficient mouse embryonic stem cells fail to form a
viable embryo [10]. As in C. elegans, for which the wah1 (RNAi) strains are viable but exhibit a reduced growth
rate [16], our ΔAIF strains exhibited a slower growth
rate, as compared to that of the wild-type strain (Figure
4C). Therefore, AIF may be bifunctional, serving as both
a vital protein and a death effector. Among ciliates,
apoptosis-like nuclear degradation has been observed
during resting cyst formation leading to change in

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Figure 7 Schematic representation of PND and a possible role of AIF, based on information described in [6]and from the present
study.

macronuclear DNA content in Colpoda [29]. In this
case, the macronuclear chromatin is extruded into the
cytosol, and the degradation of the extrusion body is
accompanied by a reduction in the size of the nucleus,
oligonucleosome-size DNA cleavage, and nuclear acidification by lysosomes. This observation indicates that ciliates may have repeatedly adapted their mitochondrial
pathway not only for sexual reproduction, but also for
cyst formation. Alternatively, it is possible that Tetrahymena PND is superficially similar to, but entirely different from animal apoptosis, although AIF participates in

this phenomenon. Although no endonuclease G homolog was identified in a survey of the macronuclear genome of Tetrahymena, EndoG is present in some other
protists, such as the kinetoplastid Trypanosoma [30],
Leishmania [31] and apicomplexan Cryptosporidium
[32]. In addition, phosphorylation of histone H2AX,
which is linked to DNA fragmentation during mammalian apoptosis [33-35], has not been demonstrated in the
degenerating macronucleus of Tetrahymena [36]. These
observations argue for this interpretation, as claimed
previously [36]. In conclusion, there are now two

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Page 12 of 15

incompatible interpretations of the origin of Tetrahymena PND: 1) PND developed independently and
merely utilized AIF as a suicide factor; and 2) PND
shares a common origin with other forms of apoptosis.
Identification of the nuclease(s) localized in the mitochondria is needed to elucidate the origin of PND.

BTU-F-NotI (5’-gcggccgcTCCACAGAGACACTAAA-3’)
and BTU-R-EcoRI (5’-gaattcTTTTAATTGCTTAAAGGAGTGA-3’). The PCR program included 5 min at 94°
C followed by 30 cycles of 94°C for 1 min, 55°C for 1
min, and 72°C for 1 min. The resulting 809-bp product
was cloned into pT7 blue T-vector.

Conclusions
Mitochondrion of Tetrahymena contains AIF and yetunidentified DNase similar to mammalian and C. elegans endonuclease G. When new macronuclei are differentiated, AIF translocates from mitochondria to the
parental macronucleus in the posterior region of cell.
Knockout of AIF showed delayed progression of PND,
that is, delay of nuclear condensation and kb-sized DNA
fragmentation, corresponding to the initial stage of the
nuclear apoptosis. Furthermore, in vitro assay using
AIF-deficient mitochondria revealed that mitochondrial
DNase acitivity was drastically reduced, suggesting that
mitochondrial DNase activity depends upon the presence of AIF. From the results, we presently conclude
that mitochondrial AIF might have a major role for
executing the nuclear apoptosis of Tetrahymena in a
simple and primitive fashion, implying its ancient origin.

Cloning of the neomycin resistance gene

Methods
Culture methods and the induction of conjugation

T. thermophila strains CU428 and B2086 (wild-type),
were purchased from the National Tetrahymena Stock
Center (Cornell University). The cells were cultured at
room temperature in 2% proteose peptone (Difco), 1%
yeast extract (Difco), and 0.5% glucose. To induce mating, the cells were incubated in 0.25% proteose peptone,
0.25% yeast extract, and 4% glucose at room temperature. At mid-log phase, the cells were washed with 10
mM Tris-HCl (pH 7.2) and incubated overnight. To
induce conjugation, equal numbers of both strains were
mixed and kept at room temperature.
Cloning of the T. thermophila AIF gene and b-tubulin
promoter

The
T.
thermophila
AIF
homolog
(TTHERM_00622710), including the 1-kb 5’- and 3’untranslated regions (UTRs), was amplified from CU428
genomic DNA using the following primers: AIF-F (5’GGTGTTGGTTTGTAGTTC-3’) and AIF-R (5’-CACCCAATSGTGAACTGA-3’). Polymerase chain reaction
(PCR) was carried out using the following program: 5
min at 94°C followed by 30 cycles of 94°C for 1 min, 46°
C for 1 min, and 72°C for 5 min. The resulting 3,966-bp
product was cloned into pT7 blue T-vector (Novagen)
as a backbone for construction of the knock-out (KO)
plasmid. The b-tubulin promoter was amplified from
CU428 genomic DNA using the following primers:

The neomycin resistance gene and the MTT1 3’-UTR
(corresponding to the poly-A signal) were obtained from
pTTMN [37]. This region (Neo r ) was amplified using
the following primers: Neo-F-EcoRI (5’-gaattcAAACTTAAAATAATGGCAAG-3’) and Neo-R-XhoI (5’ctcgagCCGGGCTGCAGCAATTC-3’). The PCR program included 5 min at 94°C followed by 30 cycles of
94°C for 1 min, 55°C for 1 min, and 72°C for 1 min.
The resulting 1,338-bp product was cloned into pT7
blue T-vector.
Construction of the KO plasmid

Inverse PCR was performed using the AIF backbone
plasmid as template with the following primers: AIF-FNotI
(5’-gcggccgcGTGATTCCTCTTGCGAACAGTTCTT-3’) and AIF-R-XhoI (5’-ctcgagCTTCTCATCCCGATGT-3’). The start codon of AIF was
destroyed by changing TAC to GAC in the forward primer. The PCR program included 5 min at 94°C followed
by 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°
C for 6 min. The resulting 5,517-bp product was selfligated and cloned. The plasmid was then digested with
NotI and XhoI and integrated into the b-tubulin promoter (NotI/EcoRI-digested fragment) and Neo r (EcoRI/
XhoI-digested fragment) sites to express Neo r under
control of the b-tubulin promoter (Neor-cassette). The
resultant plasmid (pKoTtAIF) was linearized with
BamHI before biolistic bombardment.
Construction of a GFP-tagged AIF expression plasmid

To obtain the GFP sequence, pTub-tel3 GFP4 [38],
which contains codon-optimized GFP based on Paramecium caudatum codon usage and the Paramecium tubulin poly-A signal, was used. This cassette (GFP-cassette)
was amplified using the following primers: GFP-FBamHI (5’-ggatccAGAAAGGGAGAAGAATTGT-3’)
and GFPpolyA-R (5’-CTCGAGCGGCCGCCAGT-3’).
The PCR program included 5 min at 94°C followed by
30 cycles of 94°C for 1 min, 48°C for 1 min, and 72°C
for 1 min. The resulting 1,010-bp product was cloned
into pT7 blue T-vector. The open reading frame (ORF)
of AIF and the 1-kb 5’-UTR carrying the AIF promoter
were amplified from CU428 genomic DNA using the
following primers: AIF-F-XhoI (5’-ctcgagCACCCAATSGTGAACTGA-3’) and AIF-R-BamHI (5’-ggatccAATTTTAGCAGATTAAGAAGC-3’). The PCR

Akematsu and Endoh BMC Cell Biology 2010, 11:13
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program included 5 min at 94°C followed by 30 cycles
of 94°C for 1 min, 46°C for 1 min, and 72°C for 3 min.
The resulting 3,017-bp product (AIF-cassette) was
cloned into pT7 blue T-vector. The backbone of the
expression plasmid used in our laboratory contains the
Tetrahymena telomere sequence and the Stylonychia
replication origin [39]. This plasmid was digested with
NotI and EcoRI for integration of the Neo r -cassette
(NotI/XhoI-digested fragment), AIF-cassette (XhoI/
BamHI fragment), and GFP-cassette (BamHI/EcoRI
fragment). The resultant plasmid (pAKgfpTtAIF) was
linearized with SfiI before biolistic bombardment.

Page 13 of 15

lysates were centrifuged at 12,000 rpm for 10 min, and
the supernatants were incubated with 0.2 mg/ml RNase
for 30 min at 37°C. Proteinase K (0.2 mg/ml) was then
added to all samples, which were incubated for 1 h at
37°C. Next, 0.5 M NaCl and 50% 2-propanol were
added, and the samples were incubated overnight at -20°
C. Fragmented DNA was recovered by centrifugation at
12,000 rpm for 20 min, and the precipitate was dissolved in TAE buffer. Ten micrograms of each DNA
sample were then electrophoresed on a 1% agarose gel
in TAE and stained with ethidium bromide.
Indirect immunofluorescence

Tetrahymena transformation

For Tetrahymena transformation, mid-log phase cells
were harvested by centrifugation and incubated overnight in 10 mM Tris-HCl (pH 7.2). The cells were then
centrifuged and packed in 1 ml of 10 mM Tris-HCl at a
final concentration of 1 × 107 cells/ml. A 100- μl aliquot
was then spread on a sterile 2-cm circular piece of filter
paper. Transformation was achieved using a Biolistic
PDS-1000/He Particle Delivery System (Bio-Rad). Gold
particles 0.6 μm in size (10 mg/ml in sterile H2O) were
coated with 5 μg linearized DNA/50 μl particles. Cells
were bombarded with the DNA-coated gold particles at
650 psi. Following bombardment, the cells were re-suspended in culture medium and incubated for 6 h. The
transformants were screened with 50 μg/ml paromomycin. After three days, the paromomycin-resistant cells
were grown in culture medium containing increasing
concentrations of paromomycin (from 100 to 1,200 μg/
ml) to support the allelic assortment process.
Reverse transcription (RT)-PCR analysis

Total RNA was extracted from approximately 1 × 105
cells using Sepasol-RNA1 Super (Nacalai Tesque). Five
micrograms of total RNA were used for RT with ReverTra Ace (Toyobo). A 340-bp AIF-specific product was
produced using the primers AIF-RT-F (5’AAATCTCTCCACTACACT-3’) and AIF-RT-R (5’AATTTTAGCAGATTAAGAAGC-3’). The program
included 1 min at 94°C followed by 30 cycles of 94°C
for 1 min, 48°C for 1 min, and 72°C for 30 s.
Fragmented DNA isolation and agarose gel
electrophoresis

Fragmented DNA, such as kb-sized and oligonucleosome-sized DNA, was extracted from the cells at various
times during conjugation. In the following procedure,
high-molecular-weight DNA is not generally recovered.
Cells (1 × 105) were collected by centrifugation (12,000
rpm for 1 min) and re-suspended in cold lysis buffer
containing 10 mM EDTA, 0.5% Triton-X 100, and 10
mM Tris-buffer (pH 7.2). After 10 min at 4°C, the

To image GFP-tagged AIF, cells were fixed in 50% cold
methanol and kept on ice for 30 min. After washing
with PBS, the cells were blocked in 1% bovine serum
albumin (BSA) and incubated for 2 h at room temperature with rabbit polyclonal anti-GFP antibodies (BioReagents) diluted 1:200 in PBS, 1% BSA, and 0.1%
Tween20. The cells were washed to remove excess primary antibodies and then incubated with goat anti-rabbit rhodamine-conjugated antibodies (Biomedical
Technologies Inc.) for 2 h at room temperature. Excess
secondary antibodies were then removed and nuclei
were stained with 0.01 μg/μl DAPI for 10 min.
Preparation of the mitochondria

To isolate mitochondria from wild-type and AIF-deficient strains, mid-log phase cells were harvested by
centrifugation and washed with 10 mM Tris-HCl (pH
7.2). The washed cell pellets were then re-suspended
in cold lysis buffer containing 250 mM sorbitol, 0.2%
BSA, 5 mM iodoacetamide, 1 mM EDTA, and 10 mM
MOPS-KOH (pH 7.2), and homogenized using Physcotron (Microtec Co., Ltd.) on ice. To remove nuclei and
unbroken cells, the lysates were then centrifuged for 5
min at 1,000 × g; the supernatants were decanted into
Corex centrifuge tubes, followed by centrifugation at
8,000 × g for 5 min. Each crude mitochondrial pellet
was re-suspended in 500 μl of SEM buffer containing
250 mM sucrose, 1 mM EDTA, and 10 mM MOPSKOH (pH 7.2). The mitochondria were then purified
on discontinuous sucrose gradients consisting of 1.6 M
(4 ml) and 1.15 M (7 ml) sucrose in SEM buffer in 13
PET centrifuge tubes. The crude mitochondrial suspensions were layered onto the sucrose gradients and
centrifuged at 22,500 rpm for 1 h at 4°C using an
RPS40T rotor in an SCP70H ultracentrifuge. The mitochondrial bands were carefully recovered from the
interface and transferred into Eppendorf tubes. Mitochondria were collected by centrifugation at 8,000 × g
for 10 min, the supernatants discarded, and the mitochondrial pellets suspended in SEM buffer. To confirm
no contamination of nuclear fraction into

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mitochondrial fraction, PCR analysis was carried out
using specific primers. The promers used are as follows: Mitochondrial large subunit rRNA (mtLSUrRNA)
gene; mtLSU-3 (5’-TACAACAGATAGGGACCAA-3’)
and mtLSU-4 (5’-CCTCCTAAAAAGTAACGG-3’), and
b-tubulin; BTU-F (5’-TCCACAGAGACACTAAA-3’)
and BTU-R (5’-ATGCGGTGAGTGCAGAA-3’).
Agarose gel assay for mitochondrial nuclease activity

Substrate plasmid DNA (2 μg of pT7Blue T-vector) was
coincubated with isolated mitochondria (2 μg of protein)
in 30 μl of reaction buffer containing 20 mM KCl and
50 mM MOPS (pH 6.5) at 37°C. To prepare three types
of liner formed DNA which have 3’ overhang, blunt-end
and 5’ overhang, plasmid DNA was digested with KpnI,
SmaI and BamHI, respectively, prior to incubation with
mitochondria. To quench the reaction, 2% SDS and 10
mM MgCl2 were added, and the mixture was incubated
at 50°C for 60 min. DNA samples were loaded onto
1.5% agarose gels, electrophoresed, and visualized by
staining with ethidium bromide.
Additional file 1: Mitochondrial nuclease activity. A. Purified
mitochondria (2 μg protein) from ΔTTHERM_01104910 (a: lane 1) and
ΔTTHERM_006222710 (b: lane 2) were incubated with 2 μg substrate
plasmid DNA for 30 min at 37°C in 30 μl reaction buffer containing 20
mM KCl and 50 mM MOPS (pH 6.5). Lane 4 and 5 indicate 100-bp ladder
size marker and lHindIII-digest, respectively. The substrate DNA appears
in the nicked open circular (OC), linear (L), and supercoiled (SC) forms. B.
The nuclease assay was performed under various incubation times. Lane
2-4 (a), substrate DNA was coincubated with ΔTTHERM_01104910
mitochondria. Lane 5-7 (b), substrate DNA was coincubated with
ΔTTHERM_006222710 mitochondria. Lane 1 shows undigested sample.
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1471-2121-1113-S1.DOC ]
Additional file 2: Cloning of TTHERM_01104910 and construction of
the KO plasmid. A. One of AIF homologs (TTHERM_01104910), including
the 1-kb 5’ - and 3’-untranslated regions (UTRs), was amplified from
CU428 genomic DNA using the following primers: A4910-F (5’TTACCCTTCACTCAAGCC-3’) and A4910-R (5’-ATGGTTGTGCTCGTAGTG-3’).
Polymerase chain reaction (PCR) was carried out using the following
program: 5 min at 94°C followed by 30 cycles of 94°C for 1 min, 53.5°C
for 1 min, and 72°C for 5 min. The resulting 5,281-bp product was
cloned into pT7 blue T-vector (Novagen) as a backbone for construction
of the knock-out (KO) plasmid. Inverse PCR was performed using the
backbone plasmid as template with the following primers: A4910-F-NotI
(5’-gcggccgcGATCGACTCCAAGAGTCGAA-3’) and A4910-R-XhoI (5’ctcgagCTACTTACTTTGCCGC-3’). The start codon of this gene was
destroyed by changing TAC to GAC in the forward primer. The PCR
program included 5 min at 94°C followed by 30 cycles of 94°C for 1 min,
55°C for 1 min, and 72°C for 8 min. The resulting 7,217-bp product was
self-ligated and cloned. The plasmid was then digested with NotI and
XhoI and integrated into the b-tubulin promoter (NotI/EcoRI-digested
fragment) and Neor (EcoRI/XhoI-digested fragment) sites to express Neor
under control of the b-tubulin promoter (Neor-cassette). B. The resultant
plasmid (pKoTtA4910) was linearized with BamHI before biolistic
bombardment.
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1471-2121-1113-S2.DOC ]

Page 14 of 15

Acknowledgements
We thank to Y. Takenaka for kindly supplying the plasmid (pTub-tel3 GFP4)
carrying codon-optimized GFP, M. A. Gorovsky for kindly supplying the
plasmid (pTTMN) carrying neomycin resistance gene, T. Nishiuchi for
technical support of biolistic bombardment and E. Osada for technical
support of preparation of Tetrahymena mitochondria. Thanks are also due to
T. Kobayashi, H. Sugimoto, Y. Fukuda, H. Hasegawa and N. Kitada for your
helpful discussions and encouragements. This study was supported by
Grant-in-Aid for JSPS Fellows (21-2589) to T. A.
Authors’ contributions
TA carried out all experiments and drafted the manuscript. HE critically
revised the manuscript and supervised this project. All authors read and
approved the final manuscript.
Received: 14 May 2009
Accepted: 11 February 2010 Published: 11 February 2010
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Tetrahymena thermophila. BMC Cell Biology 2010 11:13.

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