Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

1

Review

Programmed nuclear death and its relation to
apoptosis and autophagy during sexual reproductionin Tetrahymena thermophila
Takahiko AKEMATSU1*, Takashi KOBAYASHI2, Eriko OSADA3, Yasuhiro
FUKUDA1,4, Hiroshi ENDOH3 and Ronald E. PEARLMAN1G
1

Department of Biology, York University, Toronto M3J 1P3, CanadaGG
Geisel School of Medicine at Dartmouth, Dartmouth College, Lebanon NH 03756, USAGG
3
Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 9201192, JapanGG
4
Department of Biodiversity Science, Division of Biological Resource Science, Graduate
School of Agricultural Science, Tohoku University, Oosaki 989-6711, JapanG
2

SUMMARY
The ciliated protozoan Tetrahymena thermophila has evolved remarkable nuclear dualism thatinvolves spatial segregation of the polyploid somatic macro- and canonical diploid germinal micronucleus in a single cytoplasm. Programmed nuclear death (PND), also referred to as nuclear apoptosis, isa remarkable catabolic process that occurs during conjugation to finish the lifespan of parental soma, inwhich only the parental macronucleus is eliminated from the cytoplasm, but other co-existing nuclei areunaffected. We found that PND involves unique aspects of autophagy, which differ from mammalian or
yeast macroautophagy. When PND starts, the envelope of the parental macronucleus changes its nature
as if it is an autophagic membrane, without the accumulation of other membranous structures from thecytoplasm. The alteration of the parental macronuclear membrane involves exposing certain sugars andphosphatidylserine on the envelope, which are members of a class of “eat-me” signals found on thesurface of apoptotic cells, that are not found on other types of nuclei. Subsequently, small autophagic
vesicles that contain mitochondria and lysosomes fuse with the nuclear envelope stepwise and release
their contents into the nucleus at distinct stages. Mitochondria of Tetrahymena contain apoptosisinducing factor (AIF) and endonuclease G-like DNase, which are responsible for the nuclear
condensation and kb-sized DNA fragmentation, corresponding to the early stage of the nuclear apoptosis.
On the other hand, acidic lysosomal enzymes are responsible for final resorption of the nucleus. Theseelaborate mechanisms, unique to ciliates, ultimately achieve specific macronuclear elimination.
Key words: Nuclear dimorphism, Nuclear degradation, Caspase, Mitochondria, AIF, EndoG,
Autophagosome, Lysosome, Death commitment, Nuclear differentiationG*Corresponding author
Tel: +1-416-736-5241/Fax: +1-416-736-5698GE-mail: taka@yorku.caG

Received: 09 August 2012; Accepted: 12 September
2012.

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Programmed
nuclear death in TetrahymenaG

INTRODUCTION
Ciliates are large unicellular protozoans (upto ~ 400 ȝm in length), which belong to alveolates.
The alveolates consist of three different phyla;
dinoflagellates, apicomplexans, and ciliates. The
most remarkable and virtually unique feature ofciliates is that they maintain stably differentiatedgerm-line and somatic nuclear genomes within asingle cytoplasm (Orias et al., 2011), which iscalled nuclear dualism or nuclear dimorphism. The
canonical diploid germ-line genome is housed in
the micronucleus, while the polyploid somatic
genome is housed in the macronucleus. The micronuclear genome, which is transcriptionaly silent in
the vegetative, asexual stage, is the store of genetic
information for sexual progeny. The macronuclear
genome is the primary source of gene transcripts inthe cell, and its active working maintains the lifeof the cell and provides the phenotype. The microand macronuclei have distinct nucleoporins on
their surface, which characterize their functionaland morphological features (Iwamoto et al., 2009;
Iwamoto, 2011).GT. thermophila (hereafter referred to as Tetrahymena) is an exceptional model ciliate that has
been involved in two studies recognized as theNobel Prize, on catalytic RNA and ribozymes(Cech, 1990) and telomeres and telomerase(Greider and Blackburn, 1985). The macronuclear
genome containing ~ 25,000 known or predicted
protein coding genes, a number comparable to thatof the human genome, has been sequenced (Eisen
et al., 2006; Coyne et al., 2008). The microarraygene expression prolife (Miao et al., 2009), somegene networks (Xiong et al., 2011) and some organelle proteomics have also been analyzed (Smith etal., 2005; Jacobs et al., 2006; Smith et al., 2007;
Kilburn et al., 2007; Cole et al., 2008; Gould et al.,2011). Gene expression analysis using RNA seq(Xiong et al., 2012) has also been reported and themicronuclear sequence is also available (http://
www.broadinstitute.org/annotation/genome/

Tetrahymena/MultiHome.html).
Sexual reproduction of ciliates is called“conjugation”. Unlike other eukaryotes, the alteration of generations of ciliates is performed between the two kinds of nuclei through the process
without fusion or disintegration of the cytoplasm.
In Tetrahymena, the progression of conjugation
has been clearly illustrated by Cole and Sugai(2012). Briefly, conjugation is initiated by cell-tocell interaction between different mating types,
followed by meiosis in the micronucleus, exchangeof the haploid meiotic products, and formation of a
diploid zygotic micronucleus, which corresponds
to fertilization in metazoans. The zygotic micronucleus divides mitotically twice (postzygotic divisions), resulting in four micronuclei. Two of theseat the anterior region of the cell differentiate into
new macronuclei, while the other two at the posterior region of the cell remain as micronuclei. The
new macronuclear development involves large
scale genome rearrangement and amplification thatis accomplished by a mechanism involving RNAimediated, heterochromatin formation (Mochizuki
and Gorovsky, 2004; Yao et al., 2007; Noto et al.,2010; Chalker and Yao, 2011). During the process,at least 6,000 distinct sequences, likely remnantsof transposable elements, called Internal Eliminated Sequences (IES), are deleted from the micronuclear genome in a site-specific manner. Concomitant with new macronuclear differentiation, theparental macronucleus starts to degrade and eventually disappears from the cytoplasm. This process
is called Programmed Nuclear Death (PND), in
which only the parental macronucleus is eliminated while other co-existing nuclei, such as new micro- and macronuclei are unaffected (Davis et al.,
1992; Endoh and Kobayashi, 2006).GG
PND plays a critical role in the lifecycle ofciliates because it obligates the parental macronucleus to complete the lifespan and to reset the developmental time of the cell to zero. This is similar
to a death program that almost all eukaryotes are
obligated to for completion of the lifespan of so-

Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

ma. While much has been learned about the mechanisms for new macronuclear development, themechanisms for PND are still largely unexplored.
In this decade, we have been challenged to improve the study of PND and have shown that PND
is a remarkable catabolic process that involves
multiple apoptotic and autophagic markers. In thisreview, we discuss the process of PND in ciliateswith emphasis on Tetrahymena from perspectivesof apoptosis and autophagy. We also discuss its
relation to new macronucler differentiation anddevelopment for a better understanding of the novel lifecycle observed in ciliates.G
INVOLVMENT OF MULTIPLE APOPTOTIC
MARKERS
When PND starts, the parental macronucleus
begins to condense and reduce in size. In this period, nuclear DNA is degraded into high molecularweight fragments of more than 30 kb involving a
Ca2+-independent, Zn2+-insensitive nuclease activity (Mpoke and Wolfe, 1997). Subsequently, anoligonucleosome-sized ladder appears (Kobayashi
and Endoh, 2003). These phenomena are similar to
the apoptotic events in Programmed Cell Death(PCD) in multicellular organisms, so PND issometimes referred to as nuclear apoptosis. Lysosomes cluster around and in close proximity to theparental macronucleus in the later stage of PND,
by which time the nucleus becomes acidic andfinally disappears from the cytoplasm (Lu andWolfe, 2001; Kobayashi and Endoh, 2005). Mpoke
and Wolfe (1996) suggested that acidic enzymes
similar to DNase II, which is involved in apoptosis
in Chinese hamster ovary cells, or DNase ȕ and Ȗ ,both of which are involved in apoptosis, may be
involved in nuclear death in Tetrahymena.
Caspases
When animal cells undergo apoptosis, themorphological features are nuclear condensationand formation of apoptotic bodies without a break-3

down of the plasma membrane (Kerr et al., 1972).
In addition to morphological changes, apoptosis isalso characterized by DNA fragmentation and activation of the caspase enzyme family (Nicholson
and Thornberry, 1997). Caspase is a family of cysteine proteases that play essential roles in apoptosis. Initiator caspases, such as caspase 9, cleave theinactive pro-form of effector caspases (e.g., caspase 3, 6, and 7) to activate them. Sequentially,cleaved (activated) effector caspases then cleavedownstream protein substrates, such as poly(ADPribose) polymerase (PARP), triggering the apoptotic process (Lamkanfi et al., 2007).GG
The involvement of caspase-like activities in
Tetrahymena PND have been identified by in vitro
enzymatic assays showing that competitive inhibitors for caspase-8 (Ac-IETD-CHO) or caspase-9(Ac-LEHD-CHO) inhibited caspase-8 and -9 likeactivities in Tetrahymena extracts. Caspase-3 likeactivity was not detected (Kobayashi and Endoh,2003). Both caspase-8 and -9-like activities significantly increased during the final resorption process. Similarly, a specific inhibitor against caspase-1 (Ac-YVAD-CHO) and a general caspase inhibitor (z-VAD-fmk) blocked PND in vivo, but localization of the activity in the degenerating macronucleus was not detected (Ejercito and Wolfe, 2003),
suggesting its role in PND is indirect. After these
two reports were published in 2003, the complete
macronuclear genome sequence of Tetrahymena
became available (Eisen et al., 2006) and no caspase genes were found. Caspase-dependent pathways
for apoptosis appear to have been established in
independent lineages of animals later in eukaryotic
evolution given that fungi, plants, and protists
commonly lack caspase homologues. Taken together, organisms including ciliates may have independently evolved proteases that may play roles
in each of apoptosis or PCD pathways. In fact,
arginine/lysine-specific proteases called metacaspase are found in plants, fungi, and some protists, but not in slime mold, animals and ciliates. In
an analogous manner to caspases, metacaspases

4

Programmed
nuclear death in TetrahymenaGinduce PCD in these organisms (Madeo et al.,
2002; Bidle and Falkowski, 2004; Bozhkov et al.,2005).
MitochondriaG
Mitochondria contain multiple cell deathassociated factors, which are known to play a major role in plant and animal PCD processes(Kroemer, 1998). Cytochrome c, Smac/DIABLO,
and Omi/HtrA2 are mitochondrial factors involvedin caspase activation. Furthermore, some caspaseindependent pathways involve either apoptosisinducing factor (AIF) or endonuclease G (EndoG),
both of which are direct effectors of nuclear condensation and DNA fragmentation (Lorenzo et al.,
1999; Low, 2003). These proteins are released
from mitochondria and translocate into the nucleus
owing to breakdown of the mitochondrial membrane potential when the cell responds to apoptosis
signals. Kobayashi and Endoh (2005) confirmed a
relationship between mitochondria and the nucleus
during PND using DePsipher, which stains mitochondria depending upon their membrane potential. During PND, many mitochondria that havelost their membrane potential migrate toward only
the parental macronucleus and seem to cluster in
or in close proximity to the nucleus. The timing of
mitochondrial and macronuclear co-localizationsuggests that some dead or broken mitochondria
are taken into the nucleus, resulting in the execution of nuclear apoptosis.GG
Kobayashi and Endoh (2005) have also identified that mitochondria of Tetrahymena have a
DNase activity. This has an optimal pH at nearly
neutral, requires divalent cations, and is strongly
inhibited by Zn2+, a strong inhibitor of most DNases. These characteristics are reminiscent of thoseof mammalian EndoG, which mediates the caspase-independent pathway in apoptosis (Widlak et al.,2001), suggesting the possibility that the putative
mitochondrial DNase is responsible for DNA fragmentation during PND. This possibility has beenstrengthened by incubation of isolated mitochon-dria with isolated macronuclei in a cell-free system, which generated 150-400 bp DNA fragments,
roughly corresponding in size to the monomericand dimeric units of the DNA ladder (Kobayashi
and Endoh, 2005).G
Apoptosis-inducing factor (AIF)
Akematsu and Endoh (2010) further analyzed PND and its relation to mitochondria, anddemonstrated that PND shared highly conserved
elements with the caspase-independent apoptosis
pathway, an AIF-mediated type of apoptosis pathway in multicellular eukaryotes. AIF is a nuclearencoded mitochondrial flavoprotein that possesses
NADH oxidase activity in its C-terminal region.
The primary sequence of AIF is highly similar to
those of oxidoreductases from animals, fungi,
plants, eubacteria, and archaebacteria (Koonin andAravind, 2002), whereas components for a caspase-cascade have been identified in only animals.
Therefore, AIF-mediated cell death is thought tobe an ancient type of apoptosis. Upon induction of
apoptosis, AIF is released from mitochondrial intermembrane space into the nucleus in a caspaseindependent manner, which causes nuclear condensation and large scale DNA fragmentation(Lorenzo et al., 1999).GG
Observation with a strain expressing Tetrahymena AIF-GFP showed that the signal appeared
to penetrate into the inside of the parental macronucleus, suggesting that AIF is released from mitochondria and differentially translocated into theparental macronucleus (Akematsu and Endoh,2010) (Fig. 1). Somatic knockout of AIF causes
delay of large scale DNA fragmentation and nuclear condensation in the early stage of PND(Akematsu and Endoh, 2010). In Caenorhabditis
elegans, Wah-1/AIF cooperates with Cps-6/EndoG
to promote DNA degradation in vitro (Wang et al.,2002). In addition, Wah-1 (RNAi) strains and Cps6 mutants display similar defects in cell death and
DNA degradation, and both Wah-1 and Cps-6 are
localized to mitochondria (Wang et al., 2002).

Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

Similar to the observation in C. elegans, EndoGlike activity in mitochondria of Tetrahymena isalso affected by AIF. When a plasmid cleavage
assay is performed using mitochondrial proteinsfrom wild-type and AIF deficient strains, absenceof AIF is responsible for a drastic decrease of theactivity in vitro (Akematsu and Endoh, 2010).
These findings suggest that Tetrahymena utilizes
an AIF-mediated apoptosis system, by which thesuicide process of the parental macronucleus may
be facilitated at almost same time with beginningnew macronuclear differentiation. However,knocking out of the AIF gene in the parental macronucleus only slows the early stages of PND by
up to 4 h, including nuclear condensation andkilobase-size DNA fragmentation, and does notcompletely inhibit the progression of PND(Akematsu and Endoh, 2010). Indeed, by the endof conjugation, the AIF-deficient cells are delayed
only approximately 1 h, as compared with the wild
-type controls. Hence, there must be a mechanismthat compensates for the deficiency of AIF, thereby allowing the appropriate execution of the death
program. We have examined several other DNase
activities in mitochondria, which can slightly degrade DNA without AIF (Osada, E., unpublisheddata). These activities may rescue the deficiency in
AIF despite slow progression of the early stage. It
would also be reasonable to assume that DNase IIlike activities found in lysosomes (Mpoke andWolfe, 1996) could assist the digestion of the
DNA into small fragments allowing the cells tocatch up at later stages of the process.G
AUTOPHAGIC/LYSOSOMAL PROCESSIn PND, the parental macronucleus must
trigger signaling pathways to activate the engulfing machinery of a membranous structure to protect other nuclei and cytoplasm from the harm ofdigestive proteins, such as AIF, EndoG-like DNase
and lysosomal enzymes, when commitment ismade to the death program. In the catabolic system

5for degenerating disused organelles represented bymacroautophagy (sometimes referred to as justautophagy), formation of a double membranous
structure is crucial for sequestering targeted components from the remaining cytoplasm. This iscalled autophagosome formation (Yang and Klionsky, 2010; Mizushima et al., 2011). Although theorigin of the autohagosome is still controversial,
the autophagosome fuses with lysosomes to form
an autolysosome, and the sequestered components
are degraded by the lysosomal enzymes and thenrecycled. However, no wrapping process of theparental macronucleus with other membranous
structures has been observed during conjugation(Weiske-Benner and Eckert, 1987). Thus, it has
been unclear whether PND involves typical membrane dynamics like mammalian and yeast macroautophagy. Moreover, it has also been unclear why
the PND machinery recognizes the parental macronucleus while the other nuclei are present at thesame time in the same cytoplasm. Such timely andselective elimination of the disused nucleus is essential for ciliates to protect progeny genotype
from the deleterious degradative proteins.G
Formation of an autophagosomal property on theparental macronucleus
To resolve the problem, Akematsu et al.
(2010) tried to observe autophagic/lysosomal processes during PND using a combination of a fluorescent atuophagosome indicator, monodancylcadaverine (MDC), and a lysosome indicator
LysoTracker Red (LTR). MDC is a fluorescentcompound, which can accumulate in the autophagosome but does not accumulate in endoplasmic
reticulum or in the endsome. This has therefore
been used as a useful tracer for the autophagosome(Biederbick et al., 1995). Concomitant with newmacronuclear differentiation and subsequent condensation of the parental macronucleus, a haze of
the MDC fluorescence is exclusively hanging overthe parental macronucleus, and the periphery of
the nucleus becomes uniformly MDC-positive but

Fig. 1. Schematic representation of PND in Tetrahymena thermophila, based on information described in Akematsu and Endoh (2010) and Akematsu et al. (2010). OncePND starts, the parental macronucleus begins to condense and the envelope of the parental macronucleus becomes an autophagosomal membrane together with exposing
its intraluminal sugar and lipid on the surface, and disappearance of the nuclear pore complex from the envelope. Meantime, Atg8 appears around the nucleus, and Twi1scnRNA complex translocates from the parental macronucleus into the developing new macronuclear anlagen. During this time, a methyl mark at histone H3K4 disappears from the parental macronucleus while it turns up in the anlagen. The exposed sugars and lipid on the parental macronuclear surface are responsible for attraction of
mitochondria and lysosomes from the cytoplasm. At the early stage of PND, only AIF and EndoG-like DNase are released from mitochondria into the nucleus, by whichlarge-scale DNA fragmentation and further nuclear condensation is facilitated at neutral pH. EndoG-like DNase is responsible for DNA ladder formation. Lysosomes fusewith the envelope and release their contents including DNase II into the nucleus at the later stage, by which final resorption of the degenerating macronucleus is performed in an acidic environment. Caspase-8 and -9-like activities are involved in this stage, although no caspase homologues have been identified in this organism.G

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LTR-negative without migration or accumulationof other membranous structures or vesicles fromthe cytoplasm (Akematsu et al., 2010). Since specificity of the MDC-staining is derived from interaction with lipid molecules found in the autophagosome (Biederbick et al., 1995), a double membrane autophagosomal structure may be formed
around the degenerating parental macronucleus.
However, such a structure could not be foundaround the nucleus and only the nuclear membrane
was observed throughout PND under electron microscopic observation (Akematsu et al., 2010).
In mammalian and yeast macroautopahgy,
more than 30 Autophagy (Atg)-related proteins
have been characterized, which are required for
autophagosome formation (Mizushima et al.,2011). Among them, Atg8 has been proposed as a
useful tracer for an autophagosome precursor. Recently, a Tetrahymena homologue similar to yeast
ATG8 was identified and its subcellular localization and function were analyzed (Liu and Yao,2012). The signal of both EGFP-Atg8 and mCherry-Atg8 appears in the parental macronucleus at
the beginning of PND, but not in other nuclei.
Similar to the situation with the appearance of the
MDC stainability on the parental macronucleus at
an early stage of PND, it is unlikely generated byrandom aggregations or other causes. Knockingout of the genes causes delay of the final resorption of the parental macronucleus by prevention of
the nuclear acidification (Liu and Yao, 2012).
These observations strongly suggest that membrane lipid composition on the parental macronucleus may be modified at an early stage of PND,
by which the envelope appears to directly change
into an autophagosomal structure unlike the caseof general macroautophagy (Fig. 1). In addition,
under electron microscopy, it has been observedthat the early stage of PND involves loss of nuclear pore complexes, attached ribosomes associatedwith the outer membrane, and continuities with theendoplasmic reticulum (Weiske-Benner and Eckert, 1987). This may be a part of the process to7

ensure the spatial and functional sequestration of
the nucleus from the cytoplasm without othermembrane dynamics like general macroautophagy.G
Degradation of the parental macronucleus in a
stepwise fashion
Akematsu et al. (2010) observed that manysmall MDC-positive vesicles and lysosomes migrate to the nuclear envelope once the autophagosomal property appears on the envelope of theparental macronucleus. The vesicles most likely
contain mitochondria that have undergone loss ofmembrane potential (Fig. 1). This migration causes
acidification of the periphery, whereas the insideof the nucleus remains at neutral pH until the final
stage (Fig. 1). This observation is not contradictedby the observations of Akematsu and Endoh(2010), who demonstrated the collaboration of AIF
and EndoG-like DNase acts under neutral pH, suggesting that only the vesicles containing mitochondria may fuse with the macronuclear envelope inthe early stage of PND, but the lysosomes seem toremain at the periphery and release their contents
into the nucleus in the final stage.GG
Genetic analysis of PND (Davis et al., 1992)
using a Tetrahymena mutant pair nullisomic forchromosome 3 of the micronucleus (NULLI 3)
supports the model that PND appears to occur in a
stepwise fashion. When NULLI 3 cells are mated,the exconjugants failed to establish progeny because the developing macronuclear anlagen lacked
the germinal chromosome 3. In these conjugants,
chromatin condensation and DNA fragmentationin the parental macronucleus occurred normally,
but failed in resorption of the parental macronucleus, because of a lack of zygotic gene expression
from the nullisomic macronucleus. This information suggests that the collaboration of AIF and
EndoG-like DNase functions as a suicide factor in
the parental macronucleus, and that the early stageof suicide and the following final resorptive stage
by lysosomes are genetically distinct.GG
G

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Programmed
nuclear death in TetrahymenaG

Possible “attack-me” signals on the macronuclear membraneG
Early assumptions were that macroautophagy
was a random process. During the last decade,however, many examples of selective macroautophagy, including mitophagy (for mitochondria)(Okamoto et al., 2009), ribophagy (for ribosomes)(Kraft et al., 2008), and pexophagy (for peroxisomes) (Sakai et al., 2006) have been increasing.
In mitophagy, for example, the loss of membranepotential on aged or damaged mitochondria are
specifically targeted by the autophagosome (Kimet al., 2007). By analogy, in mammalian cells, cells
in apoptosis are known to expose some moleculeson the cell surface as “eat-me” signals, toward
activated macrophages. The most common molecules as eat-me signal are phosphatydiylserine(PS) and a variety of glycocalyx (glycoprotein)compounds, both of which are restricted to theinner leaflet of the bilayer plasma membrane byactive transport from the outer to the inner leaflet(Depraetere, 2000; Eda et al., 2004; Savill and
Gregory, 2007). When apoptosis is induced, they
are exposed on the outer surface by randomizingof their asymmetric distributions. Relying on thesignals, receptors on the membrane of macrophages can distinguish the dying cells from normal
cells for phagocytosis. The engulfed cells are finally degraded by phagocytes with activation of thelysosomal enzymes. Considering the information,
we can expect appearance of some signals on themacronuclear membrane. Relying on the signals,
autophagic/lysosomal processes during PND may
selectively recognize the parental macronucleus
destined to die. No such signal molecules have as
yet been identified.GG
In PND, the digestive vesicle complexes
seem to approach the parental macronucleus bytargeting MDC-positive signals of the nuclear envelope, comparable to a pinpoint attack by guided
missiles. We propose to call such a signal an“attack-me” signal. Akematsu et al. (2010) haveobserved specific appearance of PS and glycocalyx

on the parental macronuclear membrane using
FITC-labeled annexin-V and lectin binding specificities as well as being MDC-stainable. Amonglectins, wheat germ agglutinin that recognizes Nacetyl-D-glucosamine and sialic acid has much
more binding specificity than the others. These
marks turn up once the parental macronucleus enters PND, and the stainabilities against FITC are
specifically increased when the nucleus begins tocondense. Similar to the situation with PS exposition in apoptosis, it is most likely that the glycocalyx are not newly synthesized during PND,
usually restricted in the inner leaflet of the macronuclear membrane, and exposed to the outer surface of the envelope with beginning of PND (Fig.
1), because no significant difference is observed
by a lectin western-blot analysis using proteinsextracted from the normal- and degrading parental
macronucleus (Akematsu et al., 2010). These molecules could be candidates for the “attack-me”
signals in Tetrahymena by the autophagic/
lysosomal processes.G
DEATH COMMITMENT AND DEVELOPMENTAL PROGRAMMINGPND appears to occur at almost the sametime as the initiation of new macronuclear differentiation in anlagen. Once this happens, transcriptional activity of the parental macronucleus is replaced by the newly formed macronucleus. For
instance, immunofluorescence against RNApolymerase II, which is a central enzyme for genetranscription, showed that the signal disappears
from the parental macronucleus while it turns up in
the developing new macronuclei (Mochizuki and
Gorovsky, 2004). Immunofluorescence observation of dimethylation of histone H3 at lysine 4(H3K4), which is an epigenetic mark for active
transcription, also demonstrated that the chromatinin the parental macronucleus becomes heterochromatic while in the new macronuclei becomes euchromatic (Akematsu et al., 2010). Indeed, muchJpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

evidence indicates that macronuclear development
is achieved by the transport of small RNAs
(scnRNAs) together with a Tetrahymena argonaute
protein Twi1, both of which are kept in the parental macronucleus until PND is initiated and areessential for removal of Internal Eliminated Sequences (IES) from the new macronuclear genome(Mochizuki, 2010, 2011), suggesting that the two
programs perform in close co-operation.GG
Observations indicating an inextricable link
between the degradation of the parental macronucleus and successful macronuclear differentiation
have been made not only in Tetrahymena but also
in other ciliates including Paramecium. Failure of
the new macronucleus to differentiate can be artificially induced in Tetrahymena by conjugating cellswith different micronuclei (genomic exclusion)(Allen, 1967; Allen et al., 1967) or amicronucleatemutants (Kaney, 1985), allowing cells to mate at
40°C (Scholnick and Bruns, 1982), or UV-B irradiation at meiotic prophase (Kobayashi and Endoh,1998), as well as by mating of NULLI 3 mutants(Davis et al., 1992; Ward et al., 1995). In all ofthese cases, the parental macronucleus is retained
after conjugation, suggesting an inextricable link
between execution of PND and programming of
the new macronucleus, which triggers expressionof the zygotic genes.GG
The behavior of the new macronucleus andthe parental macronuclear fragments of Paramecium caudatum are somewhat different from thoseobserved in Tetrahymena. In P. caudatum, completion of the new macronucleus does not occur
immediately after differentiation but, instead, it
occurs in the fourth cell cycle after conjugation. In
the intervening period, the parental macronucleus
persists in the form of multiple fragments and continues to express its genes (Kimura and Mikami,
2003; Kimura et al., 2004). Once developmental
programming occurs at the fifth cell cycle, justafter completion of the new macronucleus, theparental macronuclear DNA is destined to degrade
and is eliminated within the next few cell cycles

9(Kimura et al., 2004). The death commitment of
the cell coincides temporally with completion of
its new macronucleus in the fourth cell cycle after
conjugation. Dimethylation of H3K4 reaches its
maximum level at this stage (Kimura et al., 2006),
as in Tetrahymena. These observations strongly
suggest that gene expression from the parental
macronucleus commits to trigger both its death
program and new macronuclear differentiation,and final resorption of the parental macronucleus
is integrated into the developmental program of the
new generation.GG
This elaborate mechanism for differential
determination of the fates of the two distinct macronuclei may ensure survival of parental clones in
the case of failure to develop the new macronucleus, which could arise from a genetic defect of thegerminal micronucleus. Furthermore, the clones
that succeed in recovering a parental macronucleus
after conjugation will have another chance to produce progeny, by mating with other strains thathave different genetic backgrounds.G
UNANSWERED QUESTIONSMuch remains unknown about commitment
to the death program and what intracellular signaling is activated. If both PND and differentiation of
new macronuclei are commonly regulated, phosphatidylinositol 3-kinase (PI3K) might be a possible candidate for controlling this. The PI3K pathway has been linked to diverse groups of signaltransduction processes in eukaryotic organisms
such as cellular growth, cell survival (antiapoptotic), cell-cycle progression, glucose metabolism, and autophagy (Fruman et al., 1998; Naumann, 2011; Mizushima et al., 2011). Potential
PI3K activities have been detected in the asexual
stage of Tetrahymena with PI3K-inhibitors such aswortmannin and LY294002 (Kovács and Pállinger,
2003; Leondaritis et al., 2005; Deli et al., 2008).
These reports suggest that PI3K is an importantfactor for regulation of proper phagocytotic activi-

10Programmed
nuclear death in TetrahymenaGty and vesicular trafficking in the asexual stage of
Tetrahymena. It can therefore be postulated thatconserved PI3K pathways are utilized in conjugation. Yakisich and Kapler (2004) have provided
data on the effect of PI3K-inhibitors on conjugating Tetrahymena. These authors report that thetreatment blocks final resorption of several typesof nuclei such as meiotic products (pronuclei) andthe parental macronucleus, resulting in the accumulation of extra nuclei in progeny cells. The remaining parental macronuclei during PND showno acidification of the nucleoplasm and actively
synthesize DNA, implying that the treatment disturbs the process of proper alteration of generation
within a single cell. It appears that a potentiallydisturbed pathway during conjugation with the
PI3K-inhibitors is an autophagic pathway involved
in degradation of the pronuclei and in PND. In
mammalian and yeast macroautophagy, it is
known that activation of PI3K complexes is essential for nucleation of the autophagosome (Chenand Klionsky, 2011). Two ubiquitin-like Atg proteins, Atg8 and Atg12, downstream of the nucleation process, are involved in vesicle expansion andcompletion of the autophagosome formation (Chenand Klionsky, 2011). As discussed previously,
knocking-out of Tetrahymena ATG8 causes delayof the final resorption of the parental macronucleus
by prevention of the nuclear acidification (Liu andYao, 2012), suggesting the involvement of PI3K
expression for execution of the autophagic/
lysosomal events during the nuclear degradation.
Moreover, Yakisich and Kapler (2004) also reported that in the presence of PI3K inhibitors, a small
fraction of the remaining pronuclei could undergoa single endo-replication cycle to generate a new
micro- or macronucleus-like form without fertilization. These findings strongly suggest that PI3K
plays a central role that relates to control of both
PND and differentiation of new macronuclei.GG
Another unanswered question is if the parental macronucleus affects the later stage of conjugation even after PND is started. Unlike the case of

Paramecium, the parental macronucleus starts tolose hallmarks for active transcription (H3K4) assoon as the degeneration is initiated, while thedeveloping new macronuclei begin to gain thehallmarks (Mochizuki and Gorovsky, 2004; Mochizuki, 2010, 2011; Akematsu et al., 2010). If
these observations substantially reflect actual transcription, the new macronuclei would serve alltranscription to carry out the later stage of conjugation once the parental macronucleus becomestranscriptionally silent. However, IES eliminationfrom the new macronuclei occurs at the very laststage of conjugation (Mochizuki, 2010, 2011). If
the IES elimination were critical for gene expression, there would be a time lag of transcription
from both the parental- and new macronuclei until
genome re-arrangement was completed in the newmacronuclei. On the other hand, with NULLI 3
mutants (Davis et al., 1992; Ward et al., 1995),
final resorption of the parental macronucleus isstrictly controlled by gene expression from thenew macronuclei. These data suggest that the newmacronuclei could transcribe some genes that participate in the later stages of PND irrespective of
the presence of IES in their genome. Alternatively,
some mRNAs transcribed from the parental macronucleus before PND could be stable until the IES
removal stage, and their translated proteins mightmake up for the limited transcription in the developing new macronuclei with IES. To insure proper
regulation of PND, the amount of transcription
from the parental macronucleus and its timing
might be controlled by upstream signaling of PND.G
CONCLUSION
In unicellular ciliates, the parental cellderived cytoplasm is taken over by the progeny
macronucleus after conjugation. Therefore, evolution of a mechanism for elimination of the parental
macronucleus might have been inevitable when thefirst ciliate ancestors established a spatial differentiation between their soma and germ-line nuclei.G

Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

Ciliate PND is uncoupled from the plasma
membrane events associated with PCD, such as
Fas ligand-Fas receptor binding, but it utilizes thesimilar down stream effectors. In addition, an autophagic/lysosomal system ensures that PND occurs only in a limited area of the cytoplasm. Thus,
ciliates have developed a novel mechanism for
implementing PND that, nonetheless, utilizes someparts of the PCD and autophagy machinery, implying that part of the intrinsic machinery for PCD
and autophagy might have been diverted for use in
PND as a specialized form of apoptosis and autophagy. These features of PND make it an excellent new model system to indentify and highlightthe conserved core mechanisms for timely cell
death and its relation to differentiation(development), and selective catabolism.G
ACKNOWLEDGEMENTS
We acknowledge Rizwan Attiq, Marcella D.
Cervantes, Jyoti Garg, Eileen Hamilton, Hiroshi
Hasegawa, Masaaki Iwamoto, Nahomi Kitada,
Albana Kume, Susanna Marquez, Gavriel Matt,
Kazufumi Mochizuki, Atsushi Mukai, Tomoko
Noto, Eduardo Orias, Judith Orias, Hiroki Sugimoto, Mayumi Sugiura, Toshinobu Suzaki, Yoshiomi
Takagi and Yasuhiro Takenaka for their helpfulcomments and discussions for improvement of thisstudy. This study was supported by Grant-in-Aid
for JSPS Fellows (21-2589) and a Banting Postdoctoral Fellowship through the Natural Sciencesand Engineering Research Council of Canada to
T.A. and grants from the Natural Sciences andEngineering Council of Canada and the Canadian
Institutes for Health Research to R.E.P.G
REFERENCES
Akematsu, T., Endoh, H. (2010) Role of apoptosisinducing factor (AIF) in programmed nuclear
death during conjugation in Tetrahymena
thermophila. BMC Cell Biol., 11, 13.GG

11Akematsu, T., Pearlman, R. E. and Endoh, H.
(2010) Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila. Autophagy, 6, 901–911.GG
Allen, S. L. (1967) Cytogenetics of genomic exclusion in Tetrahymena. Genetics, 55, 797–
822.GG
Allen, S. L., File, S. K. and Koch, S. L. (1967)
Genomic exclusion in Tetrahymena. Genetics, 55, 823–837.
Bidle, K. D. and Falkowski, P. G. (2004) Cell
death in planktonic, photosynthetic microorganisms. Nat. Rev. Microbiol., 2, 643–655.GG
Biederbick, A., Kern, H. F. and Elsässer, H. P.
(1995) Monodansylcadaverine (MDC) is a
specific in vivo marker for autophagic vacuoles. Eur. J. Cell Biol., 66, 3–14.
Bozhkov, P. V., Suarez, M. F., Filonova, L. H.,
Daniel, G., Zamyatnin, A. A. Jr, RodriguezNieto. S., Zhivotovsky. B. and Smertenko,
A. (2005) Cysteine protease mcII-Pa executes programmed cell death during plant
embryogenesis. Proc. Natl. Acad. Sci. U S A,
102, 14463–14468.
Cech, T. R. (1990) Nobel lecture. Self-splicing andenzymatic activity of an intervening sequence RNA from Tetrahymena. Biosci.
Rep., 10, 239–261.
Chalker, D. L. and Yao, M. C. (2011) DNA elimination in ciliates: transposon domesticationand genome surveillance. Annu. Rev. Genet.,
45, 227–246.
Chen, Y. and Klionsky, D. J. (2011) The regulation of autophagy - unanswered questions. J.
Cell. Sci., 15, 161–170.
Cole, E. S., Anderson, P. C., Fulton, R. B., Majerus, M. E., Rooney, M. G., Savage, J. M.,
Chalker, D., Honts, J., Welch, M. E., Wentland, A. L., Zweifel, E. and Beussman, D. J.
(2008) A proteomics approach to cloning
fenestrin from the nuclear exchange junctionof Tetrahymena. J. Eukaryot. Microbiol., 55,245–256.

12

Programmed
nuclear death in TetrahymenaG
Cole, E. and Sugai, T. (2012) Developmental progression of Tetrahymena through the cell
cycle and conjugation. Methods Cell Biol.,
109, 177–236.
Coyne, R. S., Thiagarajan, M., Jones, K.
M.,Wortman, J. R., Tallon, L. J., Haas, B. J.,
Cassidy-Hanley, D. M., Wiley, E. A., Smith,
J. J., Collins, K., Lee, S. R., Couvillion, M.
T., Liu, Y., Garg, J., Pearlman, R. E., Hamilton, E. P., Orias, E., Eisen, J. A. and Methe,
B. A. (2008) Refined annotation and assembly of the Tetrahymena thermophila genome
sequence through EST analysis, comparative
genomic hybridization, and targeted gap closure. BMC Genomics, 9, 562.
Davis, M. C., Ward, J. G., Herrick, G. and Allis,
C. D. (1992) Programmed nuclear death:
apoptotic-like degradation of specific nuclei
in conjugating Tetrahymena. Dev. Biol., 154,419–432.
Deli, D., Leondaritis, G., Tiedtke, A. and Galanopoulou, D. (2008) Deficiency in lysosomal
enzyme secretion is associated with upregulation of phosphatidylinositol 4-phosphate in
Tetrahymena. J. Eukaryot. Microbiol., 55,343–350.
Depraetere, V. (2000) "Eat me" signals of apoptotic bodies. Nat. Cell. Biol., 2, E104.Eda, S.,
Yamanaka, M. and Beppu, M. (2004) Carbohydratemediated phagocytic recognition ofearly apoptotic cells undergoing transient
capping of CD43 glycoprotein. J. Biol.
Chem., 279, 5967–5974.
Eisen, J. A., Coyne, R. S., Wu, M., Wu, D., Thiagarajan, M., Wortman, J. R., Badger, J. H.,
Ren, Q., Amedeo, P., Jones, K. M., Tallon,
L. J., Delcher, A. L., Salzberg, S. L., Silva, J.
C., Haas, B. J., Majoros, W. H., Farzad, M.,
Carlton, J. M., Smith, R. K. Jr., Garg, J.,
Pearlman, R. E., Karrer, K. M., Sun, L.,
Manning, G., Elde, N. C., Turkewitz, A. P.,
Asai, D. J., Wilkes, D. E., Wang, Y., Cai, H.,
Collins, K., Stewart, B. A., Lee, S. R., Wila-

mowska, K., Weinberg, Z., Ruzzo, W. L.,
Wloga, D., Gaertig, J., Frankel, J., Tsao, C.
C., Gorovsky, M. A., Keeling, P. J., Waller,
R. F., Patron, N. J., Cherry, J. M., Stover, N.
A., Krieger, C. J., del Toro, C., Ryder, H. F.,
Williamson, S. C., Barbeau, R. A., Hamilton,
E. P. and Orias, E. (2006) Macronuclear genome sequence of the ciliate Tetrahymena
thermophila, a model eukaryote. PLoS Biol.,4, e286.
Ejercito, M. and Wolfe, J. (2003) Caspase-like
activity is required for programmed nuclear
elimination during conjugation in Tetrahymena. J. Eukaryot. Microbiol., 50, 427–429.
Endoh, H. and Kobayashi, T. (2006) Death harmony played by nucleus and mitochondria: nuclear apoptosis during conjugation of Tetrahymena. Autophagy, 2, 129–131.
Fruman, D. A., Meyers, R. E. and Cantley, L. C.
(1998) Phosphoinositide kinases. Annu. Rev.
Biochem., 67, 481–507.
Gould, S. B., Kraft, L. G., van Dooren, G. G.,
Goodman, C. D., Ford, K. L., Cassin, A. M.,
Bacic, A., McFadden, G. I. and Waller, R. F.
(2011) Ciliate pellicular proteome identifies
novel protein families with characteristic
repeat motifs that are common to alveolates.
Mol. Biol. Evol., 28, 1319–1331.
Greider, C. W. and Blackburn, E. H. (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell,
43, 405–413.
Iwamoto, M., Mori, C., Kojidani, T., Bunai, F.,
Hori, T., Fukagawa, T., Hiraoka, Y. andHaraguchi, T. (2009) Two distinct repeat
sequences of Nup98 nucleoporins characterize dual nuclei in the binucleated ciliate Tetrahymena. Curr. Biol., 19, 843–847.
Iwamoto, M. (2011) Nuclear pore complex andnuclear transport in the ciliate Tetrahymena
thermophila. Jpn. J. Protozool., 44, 103–113.
Jacobs, M. E., DeSouza, L. V., Samaranayake, H.,
Pearlman, R. E., Siu, K. W. and Klobutcher,Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

L. A. (2006) The Tetrahymena thermophila
phagosome proteome. Eukaryot. Cell, 5,1990–2000.
Kaney, A. R. (1985) A transmissible developmental block in Tetrahymena thermophila. Exp.
Cell Res., 157, 315–321.
Kerr, J. F., Wyllie, A. H. and Currie, A. R. (1972)
Apoptosis: a basic biological phenomenon
with wide-ranging implications in tissue kinetics. Br. J. Cancer, 26, 239–257.
Kilburn, C. L., Pearson, C. G., Romijn, E. P.,
Meehl, J. B., Giddings Jr., T. H., Culver, B.
P., Yates 3rd, J. R. and Winey, M. (2007)
New Tetrahymena basal body protein components identify basal body domain structure. J. Cell Biol., 178, 905–912.
Kim, I., Rodriguez-Enriquez, S. and Lemasters, J.
J. (2007) Selective degradation of mitochondria by mitophagy. Arch. Biochem. Biophys., 15, 245–253.
Kimura, N. and Mikami, K. (2003) Interactions
between newly developed macronuclei andmaternal macronuclei in sexually immature
multinucleate exconjugants of Paramecium
caudatum. Differentiation, 71, 337–345.
Kimura, N., Mikami, K. and Endoh, H. (2004)
Delayed degradation of parental macronuclear DNA in programmed nuclear death of
Paramecium caudatum. Genesis, 40, 15–21.
Kimura, N., Mikami, K., Nakamura, N. and Endoh, H. (2006) Alteration of developmental
program in Paramecium by treatment withtrichostatin a: a possible involvement of histone modification. Protist, 157, 303–314.
Kobayashi, T. and Endoh, H. (1998) Abortive conjugation induced by UV-B irradiation at meiotic prophase in Tetrahymena thermophila.
Dev. Genet., 23, 151–157.
Kobayashi, T. and Endoh, H. (2003) Caspase-like
activity in programmed nuclear death during
conjugation of Tetrahymena thermophila.
Cell Death Differ., 10, 634–640.
Kobayashi, T. and Endoh, H. (2005) A possible

13role of mitochondria in the apoptotic-like
programmed nuclear death of Tetrahymena
thermophila. FEBS J., 272, 5378–5387.
Koonin, E. V. and Aravind, L. (2002) Origin andevolution of eukaryotic apoptosis: the bacterial connection. Cell Death Differ., 9, 394–
404.
Kovács, P. and Pállinger, E. (2003) Phosphatidylinositol 3-kinase-like activity in Tetrahymena. Effects of wortmannin and LY
294002. Acta Protozool., 42, 277–285.
Kraft, C., Deplazes, A., Sohrmann, M. and Peter,M. (2008) Mature ribosomes are selectively
degraded upon starvation by an autophagy
pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat. Cell Biol., 10, 602–610.
Kroemer, G. (1998) The mitochondrion as an integrator/coordinator of cell death pathways.
Cell Death Differ., 5, 547.
Lamkanfi, M., Festjens, N., Declercq, W., Vanden
Berghe, T. and Vandenabeele, P. (2007)
Caspases in cell survival, proliferation anddifferentiation. Cell Death Differ., 14, 44–55.
Leondaritis, G., Tiedtke, A. and Galanopoulou, D.
(2005) D-3 phosphoinositides of the ciliate
Tetrahymena: characterization and study of
their regulatory role in lysosomal enzyme
secretion. Biochim. Biophys. Acta, 1745,
330–341.
Liu, M. L. and Yao, M. C. (2012) Role of ATG8
and autophagy in programmed nuclear degradation in Tetrahymena thermophila. Eukaryot. Cell, 11, 494–506.
Lorenzo, H. K., Susin, S. A., Penninger, J. andKroemer, G. (1999) Apoptosis inducing factor (AIF): a phylogenetically old, caspaseindependent effector of cell death. Cell
Death Differ., 6, 516–524.
Low, R. L. (2003) Mitochondrial Endonuclease G
function in apoptosis and mtDNA metabolism: a historical perspective. Mitochondrion,
2, 225–236.
Lu, E. and Wolfe, J. (2001) Lysosomal enzymes in14Programmed
nuclear death in TetrahymenaGthe macronucleus of Tetrahymena during its
apoptosis-like degradation. Cell Death Differ., 8, 289–297.
Madeo, F., Herker, E., Maldener, C., Wissing, S.,
Lächelt, S., Herlan, M., Fehr, M., Lauber, K.,
Sigrist, S. J., Wesselborg, S. and Fröhlich, K.
U. (2002) A caspase-related protease regulates apoptosis in yeast. Mol. Cell, 9, 911–
917.
Miao, W., Xiong, J., Bowen, J., Wang, W., Liu,Y., Braguinets, O., Grigull, J., Pearlman, R.
E., Orias, E. and Gorovsky, M. A. (2009)
Microarray analyses of gene expression during the Tetrahymena thermophila life cycle.
PLoS ONE, 4, e4429.
Mizushima, N., Yoshimori, T. and Ohsumi, Y.
(2011) The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev.
Biol., 27, 107–132.
Mochizuki, K. and Gorovsky, M. A. (2004) Small
RNAs in genome rearrangement in Tetrahymena. Curr. Opin. Genet. Dev., 14, 181–187.
Mochizuki, K. (2010) DNA rearrangements directed by non-coding RNAs in ciliates.
Wiley Interdiscip. Rev. RNA, 1, 376–387.
Mochizuki, K. (2011) Developmentally programmed, RNA-directed genome rearrangement in Tetrahymena. Dev. Growth Differ.,
54, 108–119.
Mpoke, S. and Wolfe, J. (1996) DNA digestionand chromatin condensation during nuclear
death in Tetrahymena. Exp. Cell Res., 225,357–365.
Mpoke, S. and Wolfe, J. (1997) Differential staining of apoptotic nuclei in living cells: application to macronuclear elimination in Tetrahymena. J. Histochem. Cytochem., 45, 675–
683.
Naumann, R. W. (2011) The role of the phosphatidylinositol 3-kinase (PI3K) pathway in thedevelopment and treatment of uterine cancer.
Gynecol. Oncol., 123, 411–420.
Nicholson, D. W. and Thornberry, N. A. (1997)

Caspases: killer proteases. Trends Biochem.
Sci., 22, 299–306.
Noto, T., Kurth, H. M., Kataoka, K., Aronica, L.,
DeSouza, L.V., Siu, K. W., Pearlman, R. E.,
Gorovsky, M. A. and Mochizuki, K. (2010)
The Tetrahymena argonaute-binding protein
Giw1p directs a mature argonaute-siRNAcomplex to the nucleus. Cell, 140, 692–703.
Okamoto, K., Kondo-Okamoto, N. and Ohsumi, Y.
(2009) Mitochondria-anchored receptor
Atg32 mediates degradation of mitochondria
via selective autophagy. Dev. Cell, 17, 87–
97.
Orias, E., Cervantes, M. D. and Hamilton, E. P.
(2011) Tetrahymena thermophila, a unicellular eukaryote with separate germline andsomatic genomes. Res. Microbiol., 162, 578–
586.
Sakai, Y., Oku, M., van der Klei, I. J. and Kiel, J.
A. (2006) Pexophagy: autophagic degradation of peroxisomes. Biochim. Biophys. Acta., 1763, 1767–1775.
Savill, J. and Gregory, C. (2007) Apoptotic PS tophagocyte TIM-4: eat me. Immunity, 27, 830
–832.
Scholnick, S. B. and Bruns, P. J. (1982) A genetic
analysis of Tetrahymena that have aborted
normal development. Genetics, 102, 29–38.
Smith, J. C., Northey, J. G., Garg, J., Pearlman, R.
E. and Siu, K. W. (2005) Robust method forproteome analysis by MS/MS using an entire
translated genome: demonstration on the
ciliome of Tetrahymena thermophila. J. Proteome Res., 4, 909–919.
Smith, D. G., Gawryluk, R. M., Spencer, D. F.,
Pearlman, R. E., Siu, K.W. and Gray, M.W.
(2007) Exploring the mitochondrial proteome of the ciliate protozoon Tetrahymena
thermophila: direct analysis by tandem mass
spectrometry. J. Mol. Biol., 30, 837–867.
Wang, X., Yang, C., Chai, J., Shi, Y. and Xue, D.
(2002) Mechanisms of AIF-mediated apoptotic DNA degradation in Caenorhabditis

Jpn. J. Protozool. Vol. 45, No. 1, 2. (2012)

elegans. Science, 298, 1587–1592.
Ward, J. G., Davis, M. C., Allis, C. D. and Herrick, G. (1995) Effects of nullisomic chromosome deficiencies on conjugation events in
Tetrahymena thermophila: insufficiency ofthe parental macronucleus to direct
postzygotic development. Genetics, 140, 989
–1005.
Weiske-Benner, A. and Eckert, W. A. (1987) Differentiation of nuclear structure during thesexual cycle in Tetrahymena thermphila; II.
Degeneration and autolysis of macro-andmicronuclei. Differentiation, 34, 1–12.
Widlak, P., Li, L. Y., Wang, X. and Garrard, W.,
T. (2001) Action of recombinant human
apoptotic endonuclease G on naked DNA
and chromatin substrates: cooperation withexonuclease and DNase I. J. Biol. Chem.,
276, 48404–48409.
Xiong, J., Lu, X., Zhou, Z., Chang, Y., Yuan, D.,
Tian, M., Zhou, Z., Wang, L., Fu, C., Orias,
E. and Miao, W. (2012) Transcriptome analysis of the model protozoan, Tetrahymena

15

thermophila, using Deep RNA sequencing.
PLoS ONE, 7, e30630.
Xiong, J., Yuan, D., Fillingham, J. S., Garg, J., Lu,
X., Chang, Y., Liu, Y., Fu, C., Pearlman, R.
E. and Miao, W. (2011) Gene network landscape of the ciliate Tetrahymena thermophila. PLoS ONE, 6, e20124.
Yakisich, J. S. and Kapler, G. M. (2004) The effectof phosphoinositide 3-kinase inhibitors on
programmed nuclear degradation in Tetrahymena and fate of surviving nuclei. Cell Death
Differ., 11, 1146–1149.
Yang, Z. and Klionsky, D. J. (2010) Mammalian
autophagy: core molecular machinery andsignaling regulation. Curr. Opin. Cell Biol.,
22, 124–131.
Yao, M. C., Yao, C. H., Halasz, L. M., Fuller, P.,
Rexer, C. H., Wang, S. H., Jain, R., Coyne,
R. S. and Chalker, D. L. (2007) Identificationof novel chromatin-associated proteins involved in programmed genome rearrangements in Tetrahymena. J. Cell Sci., 120,1978–1989.