programmed nuclear death of Tetrahymena thermophila Takashi Kobayashi and Hiroshi Endoh Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University,
Trang 1programmed nuclear death of Tetrahymena thermophila Takashi Kobayashi and Hiroshi Endoh
Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Japan
Mitochondria are known to play a major role in
apop-tosis or programmed cell death (reviewed in [1,2])
Multiple cell death-associated factors have been
identi-fied in mitochondria These factors may be divided into
three categories based on their functions:
cyto-chrome c, Smac⁄ DIABLO, and Omi ⁄ HtrA2, all of
which are involved in caspase activation [3–7], while
apoptosis-inducing factor (AIF) and endonuclease G
(EndoG) are direct effectors of nuclear condensation
and DNA degradation [8,9] The pro- and antiapoptotic
members of the Bcl-2 family proteins regulate loss of
mitochondrial inner membrane potential, which results
in the release of these apoptogenic factors [1,10] The
involvement of mitochondria in apoptosis is common
among metazoans and plants [11] Homologues of the
aforementioned mitochondrial apoptosis factors have
been identified even in protistans, such as the cellular slime moulds and kinetoplastids [12,13] Taking these discoveries into consideration, the crucial role played
by mitochondria in apoptosis appears to have an early evolutionary origin
The ciliated protozoan Tetrahymena thermophila undergoes a unique process during conjugation, i.e programmed nuclear degradation Unicellular Tetra-hymena has two morphologically and functionally dif-ferent nuclei within the same cytoplasm One is the germinal micronucleus and the other is the somatic macronucleus These nuclei both originate from a ferti-lized micronucleus (synkaryon) during conjugation [14,15] As the new macronuclei differentiate from the synkaryon via two postzygotic nuclear divisions, the parental macronucleus begins to degenerate, in a
Keywords
nuclear apoptosis; autophagosome;
endonuclease; mitochondria; Tetrahymena
Correspondence
T Kobayashi, Institute for Molecular
Science of Medicine, Aichi Medical
University, Yazako, Nagakute, Aichi
480-1195, Japan
Fax: +81 561 63 3532
Tel: +81 561 62 3311 (ext 2087)
E-mail: tacobys@aichi-med-u.ac.jp
(Received 13 April 2005, revised 19 July
2005, accepted 24 August 2005)
doi:10.1111/j.1742-4658.2005.04936.x
The ciliated protozoan Tetrahymena has a unique apoptosis-like process, which is called programmed nuclear death (PND) During conjugation, the new germinal micro- and somatic macro-nuclei differentiate from a zygotic fertilized nucleus, whereas the old parental macronucleus degenerates, ensuring that only the new macronucleus is responsible for expression of the progeny genotype As is the case with apoptosis, this process encompas-ses chromatin cleavage into high-molecular mass DNA, oligonucleosomal DNA laddering, and complete degradation of the nuclear DNA, with the ultimate outcome of nuclear resorption Caspase-8- and caspase-9-like activities are involved in the final resorption process of PND In this report,
we show evidence for mitochondrial association with PND Mitochondria and the degenerating macronucleus were colocalized in autophagosome using two dyes for the detection of mitochondria In addition, an endo-nuclease with similarities to mammalian endoendo-nuclease G was detected in the isolated mitochondria When the macronuclei were incubated with iso-lated mitochondria in a cell-free system, DNA fragments of 150–400 bp were generated, but no DNA ladder appeared Taking account of the pre-sent observations and the timing of autophagosome formation, we conclude that mitochondria might be involved in Tetrahymena PND, probably with the process of oligonucleosomal laddering
Abbreviations
AIF, apoptosis-inducing factor; DAPI, 4,6-diamino-2-phenylindole; DePsipher,
5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimidazolylcarbocyanine iodide; EndoG, endonuclease G.
Trang 2process known as ‘programmed nuclear death’ (PND),
because it is controlled by specific gene expression [16]
Programmed nuclear death resembles apoptosis in
cer-tain aspects: nuclear condensation, chromatin
conden-sation, and DNA laddering are observed during the
destruction of the parental macronucleus [16–18], and
several studies have demonstrated the involvement of
caspase-like enzymes [19,20] Caspase family proteins
are essential for eukaryotic apoptosis, so it seems likely
that PND and apoptosis are regulated by similar
molecular mechanisms
Previously, we identified caspase-8- and
caspase-9-like activities, which appear to be involved in the final
resorption of the parental macronucleus during PND
in T thermophila, and suggested the involvement of
mitochondria in this process [19] In mammalian
apop-tosis, caspase-8 and caspase-9 are known to be
associ-ated with the mitochondrial pathway Active caspase-8
induces the release of mitochondrial apoptosis factors,
in a process that is mediated by tBid
(caspase-8-cleaved Bid) [21] Thus, cytochrome c is released into
the cytoplasm where it activates caspase-9 [4] In
addi-tion, mitochondria play a key role in the execution of
apoptosis, which is separate from the caspase pathway
mentioned above By analogy, it is reasonable to
assume that mitochondria play a key role in PND in
Tetrahymena Unfortunately, the involvement of
mito-chondria in PND has not been clarified fully To
eluci-date the role of the mitochondrion as a key effector
we studied the localization of mitochondria during the
death process and the levels of mitochondrial nuclease
activity Using two different fluorescent dyes, we found
that the mitochondria colocalize with the degenerating
macronucleus in autophagosomes In addition, we detected a mitochondrion-derived endonuclease activ-ity, which may be responsible for degrading DNA dur-ing PND A possible role of mitochondria in PND in Tetrahymena is discussed
Fig 1 DePsipher staining of cells during conjugation The cells are
stained with DAPI (left) and the mitochondrial membrane
potential-dependent dye DePsipher (right) (A) A preconjugating cell
Micro-nucleus (mic) and macroMicro-nucleus (Mac) sets are observed by DAPI
staining Most of the mitochondria show red fluorescence, while
green fluorescence is occasionally visible in cells that are stained
with DePsipher (B) Nuclear selection-stage cell (6 h after mating
induction) One of four meiotic products is positioned at the paroral
zone (C) Post-zygotic division I (PZD I)-stage cell (7 h) (D) PZD
II-stage cell (7.5 h) The program for degeneration of the old
paren-tal macronucleus begins at this stage Degenerating meiotic
prod-ucts are observed in the posterior region of the cells (arrowheads
in C and D) Some of these nuclei are stained green by DePsipher
(white arrowheads), while others are not (yellow arrowheads) (E)
Mac IIp-stage cell (12 h) The degenerating old macronucleus
(dOM) is stained green by DePsipher The micronuclei and
macro-nuclear anlagen (MA) do not display this staining pattern (F) Mac
IIe-stage cell (16 h) The dOM also stains green during its
degrada-tion The scale bar indicates 10 lm.
Trang 3Co-localization of mitochondria and the
degenerating macronucleus in the
autophagosome
Previously we proposed an involvement of
mitochon-dria in PND from the results of preliminary
experi-ments with the DePsipher dye, which is useful for
detecting the loss of membrane potential in
mitochon-dria [19] The DePsipher dye accumulates in the
multimeric form in the intermembrane spaces of
chondria, and fluoresces bright red when the
mito-chondria retain membrane potential, whereas the dye
disperses throughout the cytoplasm in monomeric
form, and shows a green fluorescent colour when the
mitochondrial membrane potential is lost, as happens
in apoptotic cells To confirm mitochondrial
involve-ment in PND, more detailed observations were
car-ried out In nonconjugating cells, the vast majority of
mitochondria showed red fluorescence, and only a
small proportion showed green fluorescence in the
cytoplasm (Fig 1A) The fluorescence patterns
remained unchanged in the conjugating cells as long
as the parental macronucleus showed no signs of
degeneration (Fig 1B–D) However, when the
paren-tal macronucleus began to degenerate, the staining
pattern changed drastically, and the nucleus was
stained green (Fig 1E,F) At this stage, the parental
macronucleus has been taken in autophagosome
[17,22,23] In contrast, the precondensed parental
macronucleus, the presumptive micronuclei, and the
developing macronuclear anlagen showed no fluores-cence (Fig 1A–F) These observations suggest that many mitochondria are taken into the autophago-some with the parental macronucleus and have lost membrane potential Thereby, DePsipher changed to the monomeric form (green fluorescence) but would not have diffused into the cytosol through autophago-some membrane, resulting in specific localization to the autophagosome containing degenerating macro-nucleus Small spots of green fluorescence, where some mitochondria are thought to be incorporated into small autophagosomes for turnover, were sporad-ically observed, and some of them correspond to the degenerating meiotic products (Fig 1C and D; white arrowheads)
A macronucleus that is committed to degeneration
is initially surrounded by the autophagosome, and is eventually resorbed [17] Thus, an autophagosome that contains a degenerating macronucleus is called
‘the large autophagosome’ here The large autophago-some fuses with lysoautophago-somes, and becomes acidic in the final step of PND [22,23] DePsipher staining of the macronucleus appeared initially during the stage of autophagosome formation, and persisted until resorp-tion of the parental macronucleus (Fig 1D–F) Based
on these observations, we examined the possibility that the monomeric forms of DePsipher localize to the large autophagosome merely in response to low
pH In order to exclude this possibility, conjugating cells were stained with acridine orange (AO), which is
an indicator dye for acidic organelles [22] Numerous acidic organelles ) stained in orange ) were observed
Fig 2 Distribution of acidic organelles dur-ing degeneration of parental macronucleus The living cells during conjugation were stai-ned with AO, which has different staining characteristics Green and red fluorescence correspond to DNA and acidic organelles, respectively (A) Prezygotic division III (6 h) Many lysosomes are observed Yellow fluor-escence (merged green and red colours) represents the degenerating meiotic prod-ucts (dmic) (B) PZD II (7.5 h) The precon-densed parental macronuclei are still not stained yellow (C) Mac IIp (12 h) (D) Mac IIe (16 h) The condensed parental macro-nucleus displays yellow fluorescence, which indicates the beginning of lysosome fusion Mac, Macronucleus; mic, micronucleus; dmic, degenerating meiotic products; dOM, degenerating old macronucleus The scale bar indicates 10 lm.
Trang 4in the cytoplasm of the conjugating cells, while intact
macro- and micronuclei were stained green with AO
(Fig 2) The localization of the acidic organelles
(Fig 2) is clearly different in distribution and in
num-ber from that of the green fluorescent signals of the
DePsipher dye seen in Fig 1C and D, indicating that there is no interaction between DePsipher monomers and acidic organelles When the extrameiotic products (Fig 2A) and the parental macronucleus (Fig 2C and D) began to degenerate, they were stained in yellow (merged colour of green and orange), resulting from the fusion of the nuclei and lysosomes, as reported previously [22]
Green fluorescence of DePsipher did not directly show the localization of mitochondria in the autophag-osome, as the red fluorescence corresponding to intact mitochondria was not observed in the area Therefore,
to confirm further the localization, the MitoTracker Green ) a dye that accumulates in the lipid environ-ment of mitochondria ) was used With this dye, mito-chondria can be easily localized, irrespective of membrane potential In the nonconjugating cells, the mitochondria were arranged mainly along ciliary lows (Fig 3A) Similar staining patterns were observed for conjugating cells (Fig 3B–E) MitoTracker stained the degenerating parental macronucleus, but not the other nuclei (Fig 3C–E) Moreover, the density of staining was high around the degenerating macronucleus, pre-sumably corresponding to the space between the autophagosomal membrane and nuclear envelope (Fig 3C–E) In a previous study, mitochondria were not observed in or outside the large autophagosome using the electron microscope [17] Considering this report and our observations of the monomeric form of DePsipher in the autophagosome together, the mito-chondria taken in the autophagosome might be broken, once they were incorporated into the autophagosomes These observations led us to an idea that the appar-ently dead mitochondria (or broken membrane frag-ments) that have lost membrane potential, together with the degenerating parental macronucleus, are taken
up preferentially by the autophagosome This, in turn, suggests that some molecules released from the incor-porated broken mitochondria may play a role in the execution of the death program
Mitochondrion-derived nuclease activities The uptake of mitochondria coincides with nuclear condensation and oligonucleosomal DNA laddering [19] The hypothesis that mitochondria are associated with nuclear condensation and⁄ or DNA degradation
in PND is linked with the notion of mitochondrial nuclease activities In order to examine whether the mitochondria in Tetrahymena have any nuclease activ-ity, the mitochondria were purified from vegetatively growing cells and incubated with a circular plasmid as the substrate DNA The substrate plasmid DNA was
Fig 3 Mitochondrial staining by a membrane potential-independent
dye The cells were stained with DAPI (left) or MitoTracker Green
(right) (A) A preconjugating cell (B) Conjugant during meiotic
divi-sion II (6 h after mating induction) (C and D) Mac IIp-stage (12 h).
The MitoTracker fluorescence is localized around the degenerating
old parental macronucleus (dOM) (E) Mac IIe-stage cell (14 h).
Scale bar ¼ 10 lm.
Trang 5coincubated with the isolated mitochondria at neutral
pH, and an experimental condition was surveyed (Fig 4A) All of the following experiments were car-ried out in the following conditions: 200 lL reaction containing 20 lg protein, incubated for 120 min at
30C The putative DNase had an optimum pH of 6.0–6.5 for the digestion of circular DNA (Fig 4B, lane 3 and 4) The divalent cation requirement for the mitochondrial DNase activity was investigated (Fig 4C) As shown by inhibition with EDTA (Fig 4C, lanes 6–8), the mitochondrial nuclease activ-ity required divalent cations However, higher concen-trations (5 and 10 mm) of Mg2+ inhibited the DNA cleavage activity (Fig 4C, lane 4 and 5) and weak inhi-bition was observed even in 1 mm of Mg2+ (compare lane 2 and 3 in Fig 4C), indicating a different nature from most other DNases On the other hand, nicking activity was unaffected by Mg2+, as shown by the increased amounts of open circular DNA (Fig 4C, lanes 4 and 5) The addition of Mn2+and Ca2+ gave similar inhibition results (data not shown) In the pre-sent experiment, which involved mixing mitochondria with plasmid DNA, the low levels of endogenous diva-lent cations carried across with the mitochondria may have been sufficient to support nuclease activity Zinc (Zn2+) ions, which are strong inhibitors of DNases, inhibited completely the nuclease activity (Fig 4C, lanes 9–11) The presence of the DNase activity in mitochondria is reminiscent of mammalian mitochond-rial EndoG, which mediates the caspase-independent pathway of apoptosis
Fig 4 Mitochondrial nuclease activity Purified mitochondria were incubated with plasmid DNA under various conditions (A) Basic assay for mitochondrial nuclease activity The assay was performed under various conditions Lanes 1–5: isolated mitochondria (approximately 0–20 lg protein) and 2 lg substrate DNA were coincubated for 120 min at 30 C in 200 lL reaction buffer (50 m M Hepes ⁄ NaOH pH 7.0, 10 m M KCl,
1 m M MgCl2) The DNA was then purified and electrophoresed Lanes 6–10, mitochondria (20 lg protein) and substrate DNA were coincubated
in reaction buffer at 30 C for 0–120 min Lanes 11–16, the assay was carried out for 120 min at 0–50 C PH (preheated sample) denotes the mixtures that were preincubated at 90 C for 5 min before the reaction The substrate DNA appears in the nicked open circular (OC), linear (L), and supercoiled (SC) forms (B) Optimal pH of the nuclease activity The assay was performed at various pH values The reaction mixtures con-tained 50 m M sodium citrate (pH 5.0 or 5.5), Mops (pH 6.0 or 6.5) or Hepes (pH 7.0, 7.5, 8.0), and 20 m M KCl (C) Divalent cation requirement
of the mitochondrial nuclease activity Reaction mixtures that contained 50 m M Mops (pH 6.5) and 10 m M KCl, together with 1, 5, and 10 m M MgCl2(lanes 3, 4, and 5, respectively), 1, 5, and 10 m M EDTA (lanes 6, 7, and 8, respectively), and 0.1, 1, and 5 m M ZnCl2(lanes 9, 10, and 11, respectively) were assayed at 30 C for 120 min A standard reaction (S) was performed with 50 m M Mops (pH 6.5) and 10 m M KCl (lane 2) The undigested sample (U) was similar to the standard reaction, but contained no test sample (lane 1).
A
B
Fig 5 (A) Fractionation PCR A partial fragment of the
mitochond-rial large subunit ribosomal RNA (23S rRNA) was amplified by PCR,
using fraction samples that contained equal amounts of protein.
Lane, 1 pre-mitochondrial fraction; lane 2, mitochondrial fraction;
lane 3, post-mitochondrial fraction 1; lane 4, post-mitochondrial
frac-tion 2; lane 5, cytosolic fracfrac-tion PCR products were observed in
fractions 1–3 (lanes 1–3) (B) The nuclease activities of the fractions
under two different pH conditions The reaction mixtures (200 lL)
contained 50 m M sodium acetate (pH 5.0) or Mops (pH 6.5), 10 m M
KCl, 20 lg plasmid DNA as substrate, and 20 lg protein from each
fraction The isolation of each fraction is described in Experimental
procedures Lanes 1 and 6, pre-mitochondrial fraction; lanes 2 and
7, mitochondrial fraction; lanes 3 and 8, post-mitochondrial fraction
1; lanes 4 and 9, post-mitochondrial fraction 2; lanes 5 and 10,
cyto-solic fraction.
Trang 6Lysosomal contamination of the mitochondrial
frac-tion used in this study was unavoidable To confirm
that the nuclease activity was derived from
mitochon-dria, we prepared pre- and postmitochondrial fractions
for testing in the DNase assay (see Experimental
pro-cedures) The relative ratios of mitochondria and
lyso-somes in each fraction were compared by using PCR
analysis for the mitochondria and acid phosphatase
assays for the lysosomes (Fig 5A, Table 1) Fraction 2
was used as the mitochondrial fraction in the above
experiments (Fig 5A, lane 2) Although mitochondria
were also detected in fractions 1 (the premitochondrial
fraction) and 3 (postmitochondrial fraction 1) by PCR
amplification, they were not detected in fractions 4 and
5, and mitochondria were most abundant in fraction 2
(Fig 5A) On the other hand, acid phosphatase
activ-ity was higher in fractions 3 and 4 than in fraction 2
(Table 1) These results indicate that fraction 2
con-tains a significant number of mitochondria, and that
fraction 3 is the main lysosomal fraction The
DNA-cleavage activities in each fraction were compared at
pH 5.0 and pH 6.5 (Fig 5B) Under somewhat acidic
conditions (pH 5.0) the nuclease activity was
consider-ably inhibited and there was no significant difference
between the fractions (Fig 5B lane 1–5), suggesting
that the lysosomal nuclease might be activated only
under more acidic conditions As expected, fraction 2 had the highest DNA-cleavage activity at pH 6.5 (Fig 5B, lane 7), although fraction 1 (premitochon-drial faction) and the two postmitochon(premitochon-drial fractions (3 and 4) also showed nuclease activities, probably due
to low-level contamination with mitochondria and⁄ or the lysosomal enzyme itself (Fig 5B, lanes 6, 8, 9) Taking these results into consideration, it can be judged that the DNase activity was derived mainly from mitochondria rather than lysosomes
To determine whether chromatin-associated DNAs,
as opposed to naked DNAs, are degraded by this DNase the mitochondria were incubated with isolated macronuclei as the substrate (Fig 6) Under the pre-sent experimental conditions of low osmotic pressure and⁄ or freeze–thawing of the mitochondrial fraction, mitochondria are usually burst, resulting in the release
of the putative DNase as well as divalent cations Pro-longed incubation enhanced DNA cleavage, thereby generating fragments of approximately 150–400 bp (Fig 6 lanes 3–5) Although the chromatin-sized lad-ders were not identified, their sizes corresponded roughly to the monomeric and dimeric forms of the DNA ladder, as demonstrated previously for Tetra-hymena[16,19]
Discussion
In the ciliated protozoan Tetrahymena, apoptosis-like cell death is known to occur following treatment with staurosporine [24], C2 ceramide [25], or Fas-ligand [26] On the other hand, PND is a process in which only the parental macronucleus is removed from the cytoplasm of the next generation This degradative process occurs in a restricted area of the cytoplasm and does not affect other nuclei that are located within the same cytoplasm Since they are unicellular, this process must have been developed in a ciliate ancestor that evolved spatial differentiation of the germline and soma Factors that resemble those operating in apop-tosis also participate in nuclear death, which suggests that PND is a modified form of apoptosis In this study, a possible involvement of mitochondria in PND was suggested, as shown by the simultaneous uptake
of mitochondria and the parental macronucleus in autophagosomes This finding leads us to hypothesis that some of the mitochondria are taken into the large autophagosome, and the incorporated mitochondria subsequently lose membrane potential or break down,
as indicated by the staining with two different dyes, which leads to the release of mitochondrial factors into
a limited space, without affecting other organelles within the same cytoplasm Alternatively,
mitochond-Table 1 Acid phosphatase activities of Tetrahymena cell fractions.
Fractions
AP activity (mAÆmin)1Ælg)1protein)
Relative value
3 Post-mitochondrial 1 3.1093 ± 0.1531 4.32
4 Post-mitochondrial 2 2.0750 ± 0.2412 2.88
Fig 6 Nuclear DNA degradation by mitochondrial nucleases The
isolated nuclei were incubated with mitochondria The reaction was
carried out for 0 min (lane 1), 30 min (lane 2), 60 min (lane 3),
90 min (lane 4), and 120 min (lane 5) M represents the 100-bp
DNA ladder.
Trang 7rial degeneration may play a crucial role in
some formation, as the scattered small
autophago-somes shown by green fluorescence are probably
formed prior to the formation of the large
some (Fig 1C and D) In either case, the
autophago-some can acquire autophago-some key molecule from the
sequestered mitochondria This notion is supported by
the presence of a nuclease activity in the mitochondria
of Tetrahymena
DNase activities of isolated mitochondria
In general, mitochondria have signalling pathways that
involve either AIF or EndoG, in which these molecules
execute apoptosis in a caspase-independent manner [2]
To identify mitochondrial factors in Tetrahymena, we
focused on EndoG-like enzyme activities, as EndoG is
a nuclease and AIF is not In this study, we detected
strong nuclease activities in isolated mitochondria
(Fig 4) This activity required divalent cations and
was strongly inhibited by the addition of Zn2+ In
addition, the optimal pH of this activity was pH 6.5,
while the activity was inhibited at lower pH (5.0;
Fig 4B), suggesting that the DNase and lysosomal
enzymes function in different steps of PND These
characteristics suggest similarities with the mammalian
EndoG Indeed, the mammalian EndoG also requires
divalent cations, such as Mg2+ and Mn2+, exhibits
biphasic pH optima of 7.0 and 9.0, and is potently
inhibited by Zn2+ [27] Digestion using the cell-free
system, in which isolated macronuclei and
mitochon-dria were mixed, generated nucleosome-sized DNA
fragments, although a laddering pattern was not
observed (Fig 6) In Arabidopsis, the mitochondria
alone can induce large-sized DNA fragments (30 kb)
and chromatin condensation, whereas an additional
cellular factor is required for DNA laddering in the
cell-free system [28] An additional factor would be
insufficient for ladder formation in the present study
However, our findings imply that the nuclease activity
is involved in the process of DNA laddering (as is the
case with EndoG) rather than in the production of
large-sized DNA fragments, considering the timing of
uptake of the mitochondria in the autophagosome, as
discussed below
Mitochondria as a possible executor of PND
The process of DNA degradation during PND can be
divided into three different steps, based on the sizes of
the DNA fragment generated [16–19]: (a) initial
gen-eration of high-molecular-weight (30-kb) DNA
frag-ments, followed by (b) oligonucleosome-sized ladder
formation, and (c) eventual complete degradation of the DNA The initial higher-order DNA fragmentation precedes nuclear condensation [18] Moreover, this DNA fragmentation is a prerequisite for nuclear con-densation An as yet unidentified enzyme has been sug-gested to act as a Ca2+-independent, Zn2+-insensitive nuclease [18] In mammalian apoptosis, AIF is known
to act as a caspase-independent death effector that localizes to the mitochondrial intermembrane space and translocates to the nucleus after its release from mitochondria Apoptosis-inducing factor causes chro-matin condensation and degrades DNA into fragments
of sizes > 50 kb To date, there has been no evidence
of an association between mitochondria and Tetrahym-ena cell death, and mitochondrial homologues of mam-malian apoptosis factors, such as AIF, have not been identified in the Tetrahymena genome, despite the ongoing Tetrahymena genome sequencing project Therefore, it seems likely that the putative mitochond-rial apoptosis factor is not involved in the initial DNA fragmentation step Following the initial stage des-cribed above, the DNA is degraded to oligonucleo-some-sized ( 180-bp) fragments The uptake of mitochondria into the large autophagosome is observed at this stage (Figs 1 and 3) According to the observation made by Lu and Wolfe [23], who used a combination strategy of 4,6-diamino-2-phenylindole (DAPI) staining for the detection of DNA and Azo dye staining for the identification of acid phosphatase activity, lysosomal bodies approach the condensed macronucleus prior to the formation of the large autophagosome It seems likely that the lysosomal bodies incorporate some mitochondria, as indicated by the dispersed small green fluorescence (Fig 1) As the nucleus becomes more condensed, many lysosomal bodies fuse with each other, thereby forming lamellar vesicles Eventually, the macronucleus is completely enveloped by a lamellar vesicle, which then corres-ponds to the large autophagosome Despite the enclo-sure of the nucleus within the lamellar vesicle, acid phosphatase activity is restricted to the lamellar vesicle
at this stage, which indicates that the lysosomal enzyme is not localized inside the nucleus In this instance, the intranuclear pH should still be close to neutral As mentioned above, the putative mitochond-rial nuclease presented here has an optimal pH of 6.5 During the second period of PND, the nuclease that is released from mitochondria is transported selectively into the enclosed nucleus, where the second step of DNA degradation occurs, resulting in DNA laddering Evidence for this stage is provided by the observation showing the localization of mitochondria at the circumference of the nucleus (Fig 3.C–E) This
Trang 8hypo-thesis is consistent with our previous finding that the
initial degradation of DNA into the chromatin-sized
ladder is suspended once for a few hours, after which
period final DNA loss occurs rapidly [19] In the final
stage, during which the macronucleus is resorbed, acid
phosphatase activity becomes localized deeper inside
the nucleus, as supported by acridine orange staining,
which reveals that the most highly condensed
macro-nuclei are acidic [22] In addition, the caspase-8- and
caspase-9-like activities increase dramatically just
before this stage [19]
These three steps of DNA degradation are similar to
those seen in the apoptotic nucleus [29,30] The
large-fragment-size DNA fragmentation and DNA laddering
are characteristics of the apoptotic nucleus, and the
final DNA degradation step in the autophagosome may
correspond to the phagocytosis of apoptotic bodies by
macrophages The machinery for apoptosis may have
originated in the era of unicellular protistans, whereas
the apoptotic function of mitochondria is thought to
have evolved relatively recently For instance, the
nematode Caenorhabditis elegans seems to have no
pathway for caspase activation by cytochrome c In
contrast, homologues of mitochondrial
caspase-inde-pendent apoptosis effectors, as well as caspase
homo-logues (paracaspases and metacaspases), have been
identified in certain plants, fungi, and protistans, such
as Dictyostelium and Leishmania [11–13] Indeed, the
role of AIF in apoptosis is widely conserved in
phylo-genetically distant eukaryotes, such as the cellular slime
mould [12] and nematode [31] More advanced
mecha-nisms may have evolved independently in each
eukary-otic lineage In this context, it is likely that PND in
Tetrahymenais the simplest and most primitive form of
apoptosis
Experimental procedures
Stock strains, culturing methods, and induction
of conjugation
Tetrahymena thermophila strains CU813 and CU428.2,
which were kindly supplied by P Bruns (Cornell
Univer-sity, Ithaca, NY), were used for all experiments Conditions
for cell culture and mating induction have been described
previously [32]
Cytological analysis
The DePsipher Kit (Trevigen Inc., Gaithersburg, MD) was
used to detect changes in mitochondrial membrane
poten-tial Conjugated cells were transferred to 5 lgÆmL)1
DePsipher
(5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzimi-dazolylcarbocyanine iodide) in 10 mm Tris⁄ HCl pH 7.5 along with stabilizer solution, and incubated for 1.5–2 h at
26C The cells were then transferred to 10 mm Tris ⁄ HCl
pH 7.5 with stabilizer solution Cells were observed imme-diately under a fluorescence microscope with fluorescein isothiocyanate (FITC) and green filters For photography, the cells were fixed with formalin (final concentration 0.5%) and stained with DAPI (4,6-diamino-2-phenylindole)
to visualize the nucleus Acridine orange staining was per-formed as described in Mpoke and Wolfe (1997) [22] Mito-Tracker Green (Molecular Probes Inc., Eugene, OR) stain-ing has been described previously [33]
Subcellular fractionation The late log phase cells were harvested by centrifugation at
1000 g for 5 min and washed with cold 10 mm Tris⁄ HCl
pH 7.5 The washed cells were resuspended in a cold solu-tion of 0.35 m sucrose, 10 mm Tris⁄ HCl pH 7.5, 2 mm EDTA (MIB; mitochondria isolation buffer), and homo-genized using a Polytron homogenizer To remove nuclei and unbroken cells, the homogenate was centrifuged twice
at 1000 g for 5 min, and the precipitate was used as frac-tion 1 To sediment the mitochondria, the supernatant (fraction 1; premitochondrial fraction) was centrifuged at
8700 g for 10 min To increase the purity, the crude mito-chondria were resuspended in MIB that contained 10% Percoll (Amersham Pharmacia Biotech AB, Uppsala, Swe-den) and centrifuged at 5300 g for 5 min The purified mitochondria were washed once to remove Percoll and re-suspended in MIB (fraction 2; mitochondrial fraction) The supernatant of the crude mitochondrial fraction was centri-fuged at 10 700 g for 10 min, and then the obtained super-natant was further centrifuged at 18 100 g for 10 min Both precipitates were resuspended in MIB (fraction 3 designated
as postmitochondrial fraction 1, and fraction 4 as designa-ted postmitochondrial fraction 2, respectively) The final supernatant was used as the cytosolic fraction (fraction 5) Each fraction was stored at)80 C until use
PCR
To assess the amount of mitochondria in each fraction, we used a modified whole-cell PCR method [34] Aliquots of each fraction (4 lg protein in 5 lL) were added to 5 lL 1% Nonidet P-40 (NP-40) The mixture was incubated at 65C for 10 min, followed by 92C for 3 min, and 10 lL of
10· PCR buffer (Promega Inc., Madison WI), 10 lL of
25 mm MgCl2, 2 lL of 10 mm dNTPs, 2 lL of each primer (100 pmol), 1 U Taq polymerase (Promega), and 60 lL H2O were added, to give a total reaction volume of 100 lL PCR was performed as follows: 25 cycles of 92C for 30 s, 50 C for 45 s, and 72C for 20 s The following oligonucleotides were used to amplify the partial sequence of the
Trang 9mitochond-rial large subunit rRNA (mtLSUrRNA) gene: mtLSU-3,
5¢-TACAACAGATAGGGACCAA-3¢; and mtLSU-4,
5¢-CCTCCTAAAAAGTAACGG-3¢ The PCR products were
cloned into the pBluescript II SK– vector (Stratagene Inc.,
La Jolla, CA) and sequenced using the SQ-5500 DNA
sequencer (Hitachi, Tokyo, Japan)
Acid phosphatase assay
Acid phosphatase activities were assayed using
p-nitrophe-nol phosphate [35,36] Each fraction sample (10 lL) was
mixed with 190 lL 5 mm p-nitrophenol phosphate dissolved
in 50 mm sodium acetate buffer (pH 5.0), and the mixture
was incubated at 30C for 60 min To stop the reaction,
1 mL 0.4 m NaOH was added The amount of liberated
p-nitrophenol was determined spectrophotometrically
at 410 nm
Agarose gel assay for mitochondrial nuclease
activity
The standard nuclease reaction (200 lL) contained 20 lg of
the protein in the subcellular fraction, 2 lg substrate DNA
[pT7Blue (R) vector; Novagen Inc., San Diego, CA],
50 mm Hepes⁄ NaOH pH 7.0, 10 mm KCl The reaction
was incubated at 30C for 120 min To stop the reaction,
300 lL of stop solution (100 mm Tris⁄ HCl pH 7.5, 50 mm
EDTA, 2% SDS, 0.2 mgÆmL)1 proteinase K) was added to
the reaction, and the mixture was incubated at 50C for
60 min The stopped reaction was deproteinized with
phe-nol⁄ chloroform (1 : 1), and the DNA was precipitated with
an equal volume of isopropanol The precipitated DNA
was washed with 70% ethanol and diluted with 50 lL of
TE buffer (pH 8.0) The DNA samples (10 lL) were loaded
onto a 1% agarose gel, electrophoresed, and visualized by
staining with ethidium bromide
In vitro nuclear apoptosis
Tetrahymena nuclei were isolated by the modified method
of Mita et al [37] Late log phase cells were harvested, and
washed with cold solution 1 (0.25 m sucrose, 10 mm
Tris⁄ HCl pH 7.5, 10 mm MgCl2, 3 mm CaCl2, 25 mm
KCl) The packed cells were resuspended in 9 vols solution
1 To lyse the cells, 1⁄ 5 volumes of 1% NP-40 in solution 1
were added, and the mixture was homogenized using a
magnetic stirrer The cell lysate was placed on 2 vols
solu-tion 2 (0.33 m sucrose, 10 mm Tris⁄ HCl pH 7.5, 10 mm
MgCl2, 3 mm CaCl2, 25 mm KCl), and centrifuged at
1200 g for 5 min The pellet was resuspended in solution 1,
and washed three times using the sucrose superposition
method described above The nuclear pellet was washed
three times in solution 1 with centrifugation at 400· g for
10 min Finally, the nuclear pellet was washed with solution
3 (0.25 m sucrose, 10 mm Tris⁄ HCl pH 7.5, 1 mm MgCl2) and resuspended in solution 3 to a concentration of 0.5· 106
macronucleiÆmL)1 The isolated nuclei (approximately 10 000 macronuclei) were incubated with mitochondrial fractions (20 lg pro-teins) in 200 lL of reaction buffer (50 mm Mops pH 6.5,
10 mm KCl) at 30C To stop the reaction, 300 lL of stop solution (100 mm Tris⁄ HCl pH 7.5, 50 mm EDTA, 2% SDS, 0.2 mgÆmL)1 proteinase K, 100 lgÆmL)1 RNase A) was added to the reaction, and the mixture was incu-bated at 50C for 60 min The stopped reaction was de-proteinized with phenol⁄ chloroform (1 : 1), and the DNA was precipitated with an equal volume of isopropanol The precipitated DNA was washed with 70% ethanol and diluted in TE buffer (pH 8.0) The DNA samples were loaded onto a 2% agarose gel, electrophoresed, and visualized by staining with ethidium bromide
References
1 Kroemer G (1999) Mitochondrial control of apoptosis:
an overview Biochem Soc Symp 66, 1–15
2 Gurp M, Festjens N, van Loo G, Saelens X & Vandena-beele P (2003) Mitochondrial intermembrane proteins
in cell death Biochem Biophys Res Commun 304, 487– 497
3 Liu X, Kim CN, Yang J, Jemmerson R & Wang X (1996) Induction of apoptotic program in cell free extracts: requirement for dATP and cytochrome c Cell 86, 147–157
4 Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahamd M, Alnemri ES & Wang X (1997) Cytochrome
c and dATP-dependent formation of Apaf-1⁄ caspase-9 complex initiates an apoptotic protease cascade Cell 91, 479–489
5 Du C, Fang M, Li Y, Li L & Wang X (2000) Smac,
a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition Cell 102, 33–34
6 Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly
LM, Reid GE, Moritz RL, Simpson RJ & Vaux DL (2000) Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins Cell 102, 43–53
7 Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K
& Takahashi R (2001) A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death Mol Cell 8, 613–621
8 Susin SA, Lorenzo HK, ZamZami N, Marzo I, Snow
BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM & Kroemer G (1999) Molecular characterization of mitochondrial apoptosis-inducing factor Nature 397, 441–446
Trang 109 Li LY, Luo X & Wang X (2001) Endonuclease G is an
apoptotic DNase when released from mitochondria
Nature 412, 95–99
10 Zamzami N & Kroemer G (2001) The mitochondrion in
apoptosis: How Pandora’s box opens Nat Rev Mol Cell
Biol 2, 67–71
11 Ameisen JC (2002) On the origin, evolution, and nature
of programmed cell death: a timeline of four billion
years Cell Death Differ 9, 367–393
12 Arnoult D, Tatischeff I, Estaquier J, Girard M, Sureau
F, Tissier JP, Grodet A, Dellinger M, Traincard F,
Kahn A, Ameisen JC & Petit PX (2001) On the
evolu-tionary conservation of the cell death pathway:
mito-chondrial release of an apoptosis-inducing factor during
Dictyostelium discoideumcell death Mol Biol Cell 12,
3016–3030
13 Arnoult D, Akarid DA, Grodet A, Petit PX, Estaquier
J & Ameisen JC (2002) On the evolution of
pro-grammed cell death: apoptosis of the unicellular
eukar-yote Leishmania major involves cysteine proteinase
activation and mitochondrion permeabilization Cell
Death Differ 9, 65–81
14 Martindale DW, Allis CD & Bruns PJ (1982)
Conjuga-tion in Tetrahymena thermophila A temporal analysis of
cytological stages Exp Cell Res 140, 227–236
15 Orias E (1986) Ciliate conjugation In The Molecular
Biology of Ciliated Protozoa(Gall, JG, ed.), pp 45–84
Academic Press, San Diego
16 Davis MC, Ward JG, Herrick G & Allis CD (1992)
Programmed nuclear death: apoptotic-like degradation
of specific nuclei in conjugating Tetrahymena Dev Biol
154, 419–432
17 Weiske-Benner A & Eckert WA (1987) Differentiation
of nuclear structure during the sexual cycle in
Tetra-hymena thermophila; II Degeneration and autolysis of
macro- and micronuclei Differentiation 34, 1–12
18 Mpoke S & Wolfe J (1996) DNA digestion and
chroma-tin condensation during nuclear death in Tetrahymena
Exp Cell Res 225, 357–365
19 Kobayashi T & Endoh H (2003) Caspase-like activity in
programmed nuclear death during conjugation of
Tetra-hymena thermophila Cell Death Differ 10, 634–640
20 Ejercito R & Wolfe J (2003) Caspase-like activity is
required for programmed nuclear elimination during
conjugation in Tetrahymena J Euk Microbiol 50,
427–429
21 Esposti MD (2002) The role of Bid Apoptosis 7,
433–440
22 Mpoke S & Wolfe J (1997) Differential staining of
apoptotic nuclei in living cell: Application to
macro-nuclear elimination in Tetrahymena J Histochem
Cyto-chem 45, 675–683
23 Lu E & Wolfe J (2001) Lysosomal enzymes in the
macronucleus of Tetrahymena during its apoptosis-like
degradation Cell Death Differ 8, 289–297
24 Christensen ST, Chemnitz J, Strarup EM, Kristiansen
K, Wheatley DN & Rasmussen L (1998) Staurosporine-induced cell death Tetrahymena thermophila has mixed characteristics of both apoptotic and autophagic degen-eration Cell Biol Int 2, 591–598
25 Kova´cs P, Hegyesi H, Ko¨hidai L, Nemes P & Csaba G (1999) Effect of C2ceramide on the inositol phospholipid metabolism (uptake of32P,3H-serine and3H-palmitic acid) and apoptosis-related morphological changes in Tetrahymena Comp Biochem Physiol C 122, 215–224
26 Jaso-Friedmann L, Leary JH III & Evans DL (2000) Role of nonspecific cytotoxic cells in the induction of programmed cell death of pathogenic protozoans: Parti-cipation of the Fas ligand-Fas receptor system Exp Parasitol 96, 75–88
27 Widlak P, Li LY, Wang X & Garrard WT (2001) Action of recombinant human apoptotic endonuclease
G on naked DNA and chromatin substrates: coopera-tion with exonuclease and DNase I J Biol Chem 276, 48404–48409
28 Balk J, Chew SK, Leaver CJ & McCabe PF (2003) The intermembrane space of plant mitochondria contains a DNase activity that may be involved in programmed cell death Plant J 34, 573–583
29 Robertson JD, Orrenius S & Zhivotovsky B (2000) Nuclear events in apoptosis J Struct Biol 129, 346–358
30 Nagata S, Nagase H, Kawane K, Mukae N &
Fukuyama H (2003) Degradation of chromosomal DNA during apoptosis Cell Death Differ 10, 108–116
31 Wang X, Yang C, Chai J, Shi Y & Xue D (2002) Mechanisms of AIF-mediated apoptotic DNA degra-dation in Caenorhabditis elegans Science 298, 1587– 1592
32 Kobayashi T & Endoh H (1998) Abortive conjugation induced by UV-B irradiation at meiotic prophase in Tetrahymena thermophila Dev Genet 23, 151–157
33 Kobayashi T & Endoh H (2004) Unusual distribution
of mitochondrial large subunit rRNA in the cytosol during conjugation in Tetrahymena thermophila Genes Genet Syst 79, 255–262
34 Mayer KM, Mikami K & Forney JD (1998) A mutation
in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements Genetics 148, 139–149
35 Lau K, Farley J & Baylink D (1985) Phosphotyrosyl-specific protein phosphatase activity of a bovine skeletal acid phosphatase isoenzyme Comparison with the phos-photyrosyl protein phosphatase activity of skeletal alka-line phosphatase J Biol Chem 260, 4653–4660
36 Kiy T, Vosskuhler C, Rasmussen L & Tiedtke A (1993) Three pools of lysosomal enzymes in Tetrahymena thermophila Exp Cell Res 205, 286–292
37 Mita T, Shiomi H & Iwai K (1966) Isolation of nuclei from exponentially growing Tetrahymena pyriformis Exp Cell Res 43, 696–699