A possible role of mitochondria in the apoptotic-like programmed nuclear death of Tetrahymena thermophila Takashi Kobayashi and Hiroshi Endoh Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Japan Mitochondria are known to play a major role in apop- tosis or programmed cell death (reviewed in [1,2]). Multiple cell death-associated factors have been identi- fied in mitochondria. These factors may be divided into three categories based on their functions: cyto- chrome c, Smac ⁄ DIABLO, and Omi ⁄ HtrA2, all of which are involved in caspase activation [3–7], while apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are direct effectors of nuclear condensation and DNA degradation [8,9]. The pro- and antiapoptotic members of the Bcl-2 family proteins regulate loss of mitochondrial inner membrane potential, which results in the release of these apoptogenic factors [1,10]. The involvement of mitochondria in apoptosis is common among metazoans and plants [11]. Homologues of the aforementioned mitochondrial apoptosis factors have been identified even in protistans, such as the cellular slime moulds and kinetoplastids [12,13]. Taking these discoveries into consideration, the crucial role played by mitochondria in apoptosis appears to have an early evolutionary origin. The ciliated protozoan Tetrahymena thermophila undergoes a unique process during conjugation, i.e. programmed nuclear degradation. Unicellular Tetra- hymena has two morphologically and functionally dif- ferent nuclei within the same cytoplasm. One is the germinal micronucleus and the other is the somatic macronucleus. These nuclei both originate from a ferti- lized micronucleus (synkaryon) during conjugation [14,15]. As the new macronuclei differentiate from the synkaryon via two postzygotic nuclear divisions, the parental macronucleus begins to degenerate, in a Keywords nuclear apoptosis; autophagosome; endonuclease; mitochondria; Tetrahymena Correspondence T. Kobayashi, Institute for Molecular Science of Medicine, Aichi Medical University, Yazako, Nagakute, Aichi 480-1195, Japan Fax: +81 561 63 3532 Tel: +81 561 62 3311 (ext. 2087) E-mail: 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 endonuclease G was detected in the isolated mitochondria. When the macronuclei were incubated with iso- lated mitochondria in a cell-free system, DNA fragments of 150–400 bp were generated, but no DNA ladder appeared. Taking account of the pre- sent observations and the timing of autophagosome formation, we conclude that mitochondria might be involved in Tetrahymena PND, probably with the process of oligonucleosomal laddering. Abbreviations AIF, apoptosis-inducing factor; DAPI, 4,6-diamino-2-phenylindole; DePsipher, 5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢- tetraethylbenzimidazolylcarbocyanine iodide; EndoG, endonuclease G. 5378 FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS process known as ‘programmed nuclear death’ (PND), because it is controlled by specific gene expression [16]. Programmed nuclear death resembles apoptosis in cer- tain aspects: nuclear condensation, chromatin conden- sation, and DNA laddering are observed during the destruction of the parental macronucleus [16–18], and several studies have demonstrated the involvement of caspase-like enzymes [19,20]. Caspase family proteins are essential for eukaryotic apoptosis, so it seems likely that PND and apoptosis are regulated by similar molecular mechanisms. Previously, we identified caspase-8- and caspase-9- like activities, which appear to be involved in the final resorption of the parental macronucleus during PND in T. thermophila, and suggested the involvement of mitochondria in this process [19]. In mammalian apop- tosis, caspase-8 and caspase-9 are known to be associ- ated with the mitochondrial pathway. Active caspase-8 induces the release of mitochondrial apoptosis factors, in a process that is mediated by tBid (caspase-8- cleaved Bid) [21]. Thus, cytochrome c is released into the cytoplasm where it activates caspase-9 [4]. In addi- tion, mitochondria play a key role in the execution of apoptosis, which is separate from the caspase pathway mentioned above. By analogy, it is reasonable to assume that mitochondria play a key role in PND in Tetrahymena. Unfortunately, the involvement of mito- chondria in PND has not been clarified fully. To eluci- date the role of the mitochondrion as a key effector we studied the localization of mitochondria during the death process and the levels of mitochondrial nuclease activity. Using two different fluorescent dyes, we found that the mitochondria colocalize with the degenerating macronucleus in autophagosomes. In addition, we detected a mitochondrion-derived endonuclease activ- ity, which may be responsible for degrading DNA dur- ing PND. A possible role of mitochondria in PND in Tetrahymena is discussed. A A’ B B’ C C’ D D’ E E’ F F’ Fig. 1. DePsipher staining of cells during conjugation. The cells are stained with DAPI (left) and the mitochondrial membrane potential- dependent dye DePsipher (right). (A) A preconjugating cell. Micro- nucleus (mic) and macronucleus (Mac) sets are observed by DAPI staining. Most of the mitochondria show red fluorescence, while green fluorescence is occasionally visible in cells that are stained with DePsipher. (B) Nuclear selection-stage cell (6 h after mating induction). One of four meiotic products is positioned at the paroral zone. (C) Post-zygotic division I (PZD I)-stage cell (7 h). (D) PZD II-stage cell (7.5 h). The program for degeneration of the old paren- tal macronucleus begins at this stage. Degenerating meiotic prod- ucts are observed in the posterior region of the cells (arrowheads in C and D). Some of these nuclei are stained green by DePsipher (white arrowheads), while others are not (yellow arrowheads). (E) Mac IIp-stage cell (12 h). The degenerating old macronucleus (dOM) is stained green by DePsipher. The micronuclei and macro- nuclear anlagen (MA) do not display this staining pattern. (F) Mac IIe-stage cell (16 h). The dOM also stains green during its degrada- tion. The scale bar indicates 10 lm. T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS 5379 Results Co-localization of mitochondria and the degenerating macronucleus in the autophagosome Previously we proposed an involvement of mitochon- dria in PND from the results of preliminary experi- ments with the DePsipher dye, which is useful for detecting the loss of membrane potential in mitochon- dria [19]. The DePsipher dye accumulates in the multimeric form in the intermembrane spaces of mito- chondria, and fluoresces bright red when the mito- chondria retain membrane potential, whereas the dye disperses throughout the cytoplasm in monomeric form, and shows a green fluorescent colour when the mitochondrial membrane potential is lost, as happens in apoptotic cells. To confirm mitochondrial involve- ment in PND, more detailed observations were car- ried out. In nonconjugating cells, the vast majority of mitochondria showed red fluorescence, and only a small proportion showed green fluorescence in the cytoplasm (Fig. 1A). The fluorescence patterns remained unchanged in the conjugating cells as long as the parental macronucleus showed no signs of degeneration (Fig. 1B–D). However, when the paren- tal macronucleus began to degenerate, the staining pattern changed drastically, and the nucleus was stained green (Fig. 1E,F). At this stage, the parental macronucleus has been taken in autophagosome [17,22,23]. In contrast, the precondensed parental macronucleus, the presumptive micronuclei, and the developing macronuclear anlagen showed no fluores- cence (Fig. 1A–F). These observations suggest that many mitochondria are taken into the autophago- some with the parental macronucleus and have lost membrane potential. Thereby, DePsipher changed to the monomeric form (green fluorescence) but would not have diffused into the cytosol through autophago- some membrane, resulting in specific localization to the autophagosome containing degenerating macro- nucleus. Small spots of green fluorescence, where some mitochondria are thought to be incorporated into small autophagosomes for turnover, were sporad- ically observed, and some of them correspond to the degenerating meiotic products (Fig. 1C and D; white arrowheads). A macronucleus that is committed to degeneration is initially surrounded by the autophagosome, and is eventually resorbed [17]. Thus, an autophagosome that contains a degenerating macronucleus is called ‘the large autophagosome’ here. The large autophago- some fuses with lysosomes, and becomes acidic in the final step of PND [22,23]. DePsipher staining of the macronucleus appeared initially during the stage of autophagosome formation, and persisted until resorp- tion of the parental macronucleus (Fig. 1D–F). Based on these observations, we examined the possibility that the monomeric forms of DePsipher localize to the large autophagosome merely in response to low pH. In order to exclude this possibility, conjugating cells were stained with acridine orange (AO), which is an indicator dye for acidic organelles [22]. Numerous acidic organelles ) stained in orange ) were observed Fig. 2. Distribution of acidic organelles dur- ing degeneration of parental macronucleus. The living cells during conjugation were stai- ned with AO, which has different staining characteristics. Green and red fluorescence correspond to DNA and acidic organelles, respectively. (A) Prezygotic division III (6 h). Many lysosomes are observed. Yellow fluor- escence (merged green and red colours) represents the degenerating meiotic prod- ucts (dmic). (B) PZD II (7.5 h). The precon- densed parental macronuclei are still not stained yellow. (C) Mac IIp (12 h). (D) Mac IIe (16 h). The condensed parental macro- nucleus displays yellow fluorescence, which indicates the beginning of lysosome fusion. Mac, Macronucleus; mic, micronucleus; dmic, degenerating meiotic products; dOM, degenerating old macronucleus. The scale bar indicates 10 lm. Mitochondria in nuclear death of Tetrahymena T. Kobayashi and H. Endoh 5380 FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS in the cytoplasm of the conjugating cells, while intact macro- and micronuclei were stained green with AO (Fig. 2). The localization of the acidic organelles (Fig. 2) is clearly different in distribution and in num- ber from that of the green fluorescent signals of the DePsipher dye seen in Fig. 1C and D, indicating that there is no interaction between DePsipher monomers and acidic organelles. When the extrameiotic products (Fig. 2A) and the parental macronucleus (Fig. 2C and D) began to degenerate, they were stained in yellow (merged colour of green and orange), resulting from the fusion of the nuclei and lysosomes, as reported previously [22]. Green fluorescence of DePsipher did not directly show the localization of mitochondria in the autophag- osome, as the red fluorescence corresponding to intact mitochondria was not observed in the area. Therefore, to confirm further the localization, the MitoTracker Green ) a dye that accumulates in the lipid environ- ment of mitochondria ) was used. With this dye, mito- chondria can be easily localized, irrespective of membrane potential. In the nonconjugating cells, the mitochondria were arranged mainly along ciliary lows (Fig. 3A). Similar staining patterns were observed for conjugating cells (Fig. 3B–E). MitoTracker stained the degenerating parental macronucleus, but not the other nuclei (Fig. 3C–E). Moreover, the density of staining was high around the degenerating macronucleus, pre- sumably corresponding to the space between the autophagosomal membrane and nuclear envelope (Fig. 3C–E). In a previous study, mitochondria were not observed in or outside the large autophagosome using the electron microscope [17]. Considering this report and our observations of the monomeric form of DePsipher in the autophagosome together, the mito- chondria taken in the autophagosome might be broken, once they were incorporated into the autophagosomes. These observations led us to an idea that the appar- ently dead mitochondria (or broken membrane frag- ments) that have lost membrane potential, together with the degenerating parental macronucleus, are taken up preferentially by the autophagosome. This, in turn, suggests that some molecules released from the incor- porated broken mitochondria may play a role in the execution of the death program. Mitochondrion-derived nuclease activities The uptake of mitochondria coincides with nuclear condensation and oligonucleosomal DNA laddering [19]. The hypothesis that mitochondria are associated with nuclear condensation and ⁄ or DNA degradation in PND is linked with the notion of mitochondrial nuclease activities. In order to examine whether the mitochondria in Tetrahymena have any nuclease activ- ity, the mitochondria were purified from vegetatively growing cells and incubated with a circular plasmid as the substrate DNA. The substrate plasmid DNA was AA’ BB’ C C’ D D’ E E’ Fig. 3. Mitochondrial staining by a membrane potential-independent dye. The cells were stained with DAPI (left) or MitoTracker Green (right). (A) A preconjugating cell. (B) Conjugant during meiotic divi- sion II (6 h after mating induction). (C and D) Mac IIp-stage (12 h). The MitoTracker fluorescence is localized around the degenerating old parental macronucleus (dOM). (E) Mac IIe-stage cell (14 h). Scale bar ¼ 10 lm. T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS 5381 coincubated with the isolated mitochondria at neutral pH, and an experimental condition was surveyed (Fig. 4A). All of the following experiments were car- ried out in the following conditions: 200 lL reaction containing 20 lg protein, incubated for 120 min at 30 °C. The putative DNase had an optimum pH of 6.0–6.5 for the digestion of circular DNA (Fig. 4B, lane 3 and 4). The divalent cation requirement for the mitochondrial DNase activity was investigated (Fig. 4C). As shown by inhibition with EDTA (Fig. 4C, lanes 6–8), the mitochondrial nuclease activ- ity required divalent cations. However, higher concen- trations (5 and 10 mm)ofMg 2+ inhibited the DNA cleavage activity (Fig. 4C, lane 4 and 5) and weak inhi- bition was observed even in 1 mm of Mg 2+ (compare lane 2 and 3 in Fig. 4C), indicating a different nature from most other DNases. On the other hand, nicking activity was unaffected by Mg 2+ , as shown by the increased amounts of open circular DNA (Fig. 4C, lanes 4 and 5). The addition of Mn 2+ and Ca 2+ gave similar inhibition results (data not shown). In the pre- sent experiment, which involved mixing mitochondria with plasmid DNA, the low levels of endogenous diva- lent cations carried across with the mitochondria may have been sufficient to support nuclease activity. Zinc (Zn 2+ ) ions, which are strong inhibitors of DNases, inhibited completely the nuclease activity (Fig. 4C, lanes 9–11). The presence of the DNase activity in mitochondria is reminiscent of mammalian mitochond- rial EndoG, which mediates the caspase-independent pathway of apoptosis. A B C Fig. 4. Mitochondrial nuclease activity. Purified mitochondria were incubated with plasmid DNA under various conditions. (A) Basic assay for mitochondrial nuclease activity. The assay was performed under various conditions. Lanes 1–5: isolated mitochondria (approximately 0–20 lg protein) and 2 lg substrate DNA were coincubated for 120 min at 30 °C in 200 lL reaction buffer (50 m M Hepes ⁄ NaOH pH 7.0, 10 mM KCl, 1m M MgCl 2 ). The DNA was then purified and electrophoresed. Lanes 6–10, mitochondria (20 lg protein) and substrate DNA were coincubated in reaction buffer at 30 °C for 0–120 min. Lanes 11–16, the assay was carried out for 120 min at 0–50 °C. PH (preheated sample) denotes the mixtures that were preincubated at 90 °C for 5 min before the reaction. The substrate DNA appears in the nicked open circular (OC), linear (L), and supercoiled (SC) forms. (B) Optimal pH of the nuclease activity. The assay was performed at various pH values. The reaction mixtures con- tained 50 m M sodium citrate (pH 5.0 or 5.5), Mops (pH 6.0 or 6.5) or Hepes (pH 7.0, 7.5, 8.0), and 20 mM KCl. (C) Divalent cation requirement of the mitochondrial nuclease activity. Reaction mixtures that contained 50 m M Mops (pH 6.5) and 10 mM KCl, together with 1, 5, and 10 mM MgCl 2 (lanes 3, 4, and 5, respectively), 1, 5, and 10 mM EDTA (lanes 6, 7, and 8, respectively), and 0.1, 1, and 5 mM ZnCl 2 (lanes 9, 10, and 11, respectively) were assayed at 30 °C for 120 min. A standard reaction (S) was performed with 50 m M Mops (pH 6.5) and 10 mM KCl (lane 2). The undigested sample (U) was similar to the standard reaction, but contained no test sample (lane 1). A B Fig. 5. (A) Fractionation PCR. A partial fragment of the mitochond- rial large subunit ribosomal RNA (23S rRNA) was amplified by PCR, using fraction samples that contained equal amounts of protein. Lane, 1 pre-mitochondrial fraction; lane 2, mitochondrial fraction; lane 3, post-mitochondrial fraction 1; lane 4, post-mitochondrial frac- tion 2; lane 5, cytosolic fraction. PCR products were observed in fractions 1–3 (lanes 1–3). (B) The nuclease activities of the fractions under two different pH conditions. The reaction mixtures (200 lL) contained 50 m M sodium acetate (pH 5.0) or Mops (pH 6.5), 10 mM KCl, 20 l g plasmid DNA as substrate, and 20 lg protein from each fraction. The isolation of each fraction is described in Experimental procedures. Lanes 1 and 6, pre-mitochondrial fraction; lanes 2 and 7, mitochondrial fraction; lanes 3 and 8, post-mitochondrial fraction 1; lanes 4 and 9, post-mitochondrial fraction 2; lanes 5 and 10, cyto- solic fraction. Mitochondria in nuclear death of Tetrahymena T. Kobayashi and H. Endoh 5382 FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS Lysosomal contamination of the mitochondrial frac- tion used in this study was unavoidable. To confirm that the nuclease activity was derived from mitochon- dria, we prepared pre- and postmitochondrial fractions for testing in the DNase assay (see Experimental pro- cedures). The relative ratios of mitochondria and lyso- somes in each fraction were compared by using PCR analysis for the mitochondria and acid phosphatase assays for the lysosomes (Fig. 5A, Table 1). Fraction 2 was used as the mitochondrial fraction in the above experiments (Fig. 5A, lane 2). Although mitochondria were also detected in fractions 1 (the premitochondrial fraction) and 3 (postmitochondrial fraction 1) by PCR amplification, they were not detected in fractions 4 and 5, and mitochondria were most abundant in fraction 2 (Fig. 5A). On the other hand, acid phosphatase activ- ity was higher in fractions 3 and 4 than in fraction 2 (Table 1). These results indicate that fraction 2 con- tains a significant number of mitochondria, and that fraction 3 is the main lysosomal fraction. The DNA- cleavage activities in each fraction were compared at pH 5.0 and pH 6.5 (Fig. 5B). Under somewhat acidic conditions (pH 5.0) the nuclease activity was consider- ably inhibited and there was no significant difference between the fractions (Fig. 5B lane 1–5), suggesting that the lysosomal nuclease might be activated only under more acidic conditions. As expected, fraction 2 had the highest DNA-cleavage activity at pH 6.5 (Fig. 5B, lane 7), although fraction 1 (premitochon- drial faction) and the two postmitochondrial fractions (3 and 4) also showed nuclease activities, probably due to low-level contamination with mitochondria and ⁄ or the lysosomal enzyme itself (Fig. 5B, lanes 6, 8, 9). Taking these results into consideration, it can be judged that the DNase activity was derived mainly from mitochondria rather than lysosomes. To determine whether chromatin-associated DNAs, as opposed to naked DNAs, are degraded by this DNase the mitochondria were incubated with isolated macronuclei as the substrate (Fig. 6). Under the pre- sent experimental conditions of low osmotic pressure and ⁄ or freeze–thawing of the mitochondrial fraction, mitochondria are usually burst, resulting in the release of the putative DNase as well as divalent cations. Pro- longed incubation enhanced DNA cleavage, thereby generating fragments of approximately 150–400 bp (Fig. 6 lanes 3–5). Although the chromatin-sized lad- ders were not identified, their sizes corresponded roughly to the monomeric and dimeric forms of the DNA ladder, as demonstrated previously for Tetra- hymena [16,19]. Discussion In the ciliated protozoan Tetrahymena, apoptosis-like cell death is known to occur following treatment with staurosporine [24], C 2 ceramide [25], or Fas-ligand [26]. On the other hand, PND is a process in which only the parental macronucleus is removed from the cytoplasm of the next generation. This degradative process occurs in a restricted area of the cytoplasm and does not affect other nuclei that are located within the same cytoplasm. Since they are unicellular, this process must have been developed in a ciliate ancestor that evolved spatial differentiation of the germline and soma. Factors that resemble those operating in apop- tosis also participate in nuclear death, which suggests that PND is a modified form of apoptosis. In this study, a possible involvement of mitochondria in PND was suggested, as shown by the simultaneous uptake of mitochondria and the parental macronucleus in autophagosomes. This finding leads us to hypothesis that some of the mitochondria are taken into the large autophagosome, and the incorporated mitochondria subsequently lose membrane potential or break down, as indicated by the staining with two different dyes, which leads to the release of mitochondrial factors into a limited space, without affecting other organelles within the same cytoplasm. Alternatively, mitochond- Table 1. Acid phosphatase activities of Tetrahymena cell fractions. Fractions AP activity (mAÆmin )1 Ælg )1 protein) Relative value 1 Pre-mitochondrial 0.8958 ± 0.1802 1.24 2 Mitochondrial 0.7196 ± 0.0435 1.00 3 Post-mitochondrial 1 3.1093 ± 0.1531 4.32 4 Post-mitochondrial 2 2.0750 ± 0.2412 2.88 5 Cytoplasmic 0.4356 ± 0.2008 0.61 Fig. 6. Nuclear DNA degradation by mitochondrial nucleases. The isolated nuclei were incubated with mitochondria. The reaction was carried out for 0 min (lane 1), 30 min (lane 2), 60 min (lane 3), 90 min (lane 4), and 120 min (lane 5). M represents the 100-bp DNA ladder. T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS 5383 rial degeneration may play a crucial role in autophago- some formation, as the scattered small autophago- somes shown by green fluorescence are probably formed prior to the formation of the large autophago- some (Fig. 1C and D). In either case, the autophago- some can acquire some key molecule from the sequestered mitochondria. This notion is supported by the presence of a nuclease activity in the mitochondria of Tetrahymena. DNase activities of isolated mitochondria In general, mitochondria have signalling pathways that involve either AIF or EndoG, in which these molecules execute apoptosis in a caspase-independent manner [2]. To identify mitochondrial factors in Tetrahymena,we focused on EndoG-like enzyme activities, as EndoG is a nuclease and AIF is not. In this study, we detected strong nuclease activities in isolated mitochondria (Fig. 4). This activity required divalent cations and was strongly inhibited by the addition of Zn 2+ .In addition, the optimal pH of this activity was pH 6.5, while the activity was inhibited at lower pH (5.0; Fig. 4B), suggesting that the DNase and lysosomal enzymes function in different steps of PND. These characteristics suggest similarities with the mammalian EndoG. Indeed, the mammalian EndoG also requires divalent cations, such as Mg 2+ and Mn 2+ , exhibits biphasic pH optima of 7.0 and 9.0, and is potently inhibited by Zn 2+ [27]. Digestion using the cell-free system, in which isolated macronuclei and mitochon- dria were mixed, generated nucleosome-sized DNA fragments, although a laddering pattern was not observed (Fig. 6). In Arabidopsis, the mitochondria alone can induce large-sized DNA fragments (30 kb) and chromatin condensation, whereas an additional cellular factor is required for DNA laddering in the cell-free system [28]. An additional factor would be insufficient for ladder formation in the present study. However, our findings imply that the nuclease activity is involved in the process of DNA laddering (as is the case with EndoG) rather than in the production of large-sized DNA fragments, considering the timing of uptake of the mitochondria in the autophagosome, as discussed below. Mitochondria as a possible executor of PND The process of DNA degradation during PND can be divided into three different steps, based on the sizes of the DNA fragment generated [16–19]: (a) initial gen- eration of high-molecular-weight (30-kb) DNA frag- ments, followed by (b) oligonucleosome-sized ladder formation, and (c) eventual complete degradation of the DNA. The initial higher-order DNA fragmentation precedes nuclear condensation [18]. Moreover, this DNA fragmentation is a prerequisite for nuclear con- densation. An as yet unidentified enzyme has been sug- gested to act as a Ca 2+ -independent, Zn 2+ -insensitive nuclease [18]. In mammalian apoptosis, AIF is known to act as a caspase-independent death effector that localizes to the mitochondrial intermembrane space and translocates to the nucleus after its release from mitochondria. Apoptosis-inducing factor causes chro- matin condensation and degrades DNA into fragments of sizes > 50 kb. To date, there has been no evidence of an association between mitochondria and Tetrahym- ena cell death, and mitochondrial homologues of mam- malian apoptosis factors, such as AIF, have not been identified in the Tetrahymena genome, despite the ongoing Tetrahymena genome sequencing project. Therefore, it seems likely that the putative mitochond- rial apoptosis factor is not involved in the initial DNA fragmentation step. Following the initial stage des- cribed above, the DNA is degraded to oligonucleo- some-sized ( 180-bp) fragments. The uptake of mitochondria into the large autophagosome is observed at this stage (Figs 1 and 3). According to the observation made by Lu and Wolfe [23], who used a combination strategy of 4,6-diamino-2-phenylindole (DAPI) staining for the detection of DNA and Azo dye staining for the identification of acid phosphatase activity, lysosomal bodies approach the condensed macronucleus prior to the formation of the large autophagosome. It seems likely that the lysosomal bodies incorporate some mitochondria, as indicated by the dispersed small green fluorescence (Fig. 1). As the nucleus becomes more condensed, many lysosomal bodies fuse with each other, thereby forming lamellar vesicles. Eventually, the macronucleus is completely enveloped by a lamellar vesicle, which then corres- ponds to the large autophagosome. Despite the enclo- sure of the nucleus within the lamellar vesicle, acid phosphatase activity is restricted to the lamellar vesicle at this stage, which indicates that the lysosomal enzyme is not localized inside the nucleus. In this instance, the intranuclear pH should still be close to neutral. As mentioned above, the putative mitochond- rial nuclease presented here has an optimal pH of 6.5. During the second period of PND, the nuclease that is released from mitochondria is transported selectively into the enclosed nucleus, where the second step of DNA degradation occurs, resulting in DNA laddering. Evidence for this stage is provided by the observation showing the localization of mitochondria at the circumference of the nucleus (Fig 3.C–E). This hypo- Mitochondria in nuclear death of Tetrahymena T. Kobayashi and H. Endoh 5384 FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS thesis is consistent with our previous finding that the initial degradation of DNA into the chromatin-sized ladder is suspended once for a few hours, after which period final DNA loss occurs rapidly [19]. In the final stage, during which the macronucleus is resorbed, acid phosphatase activity becomes localized deeper inside the nucleus, as supported by acridine orange staining, which reveals that the most highly condensed macro- nuclei are acidic [22]. In addition, the caspase-8- and caspase-9-like activities increase dramatically just before this stage [19]. These three steps of DNA degradation are similar to those seen in the apoptotic nucleus [29,30]. The large- fragment-size DNA fragmentation and DNA laddering are characteristics of the apoptotic nucleus, and the final DNA degradation step in the autophagosome may correspond to the phagocytosis of apoptotic bodies by macrophages. The machinery for apoptosis may have originated in the era of unicellular protistans, whereas the apoptotic function of mitochondria is thought to have evolved relatively recently. For instance, the nematode Caenorhabditis elegans seems to have no pathway for caspase activation by cytochrome c.In contrast, homologues of mitochondrial caspase-inde- pendent apoptosis effectors, as well as caspase homo- logues (paracaspases and metacaspases), have been identified in certain plants, fungi, and protistans, such as Dictyostelium and Leishmania [11–13]. Indeed, the role of AIF in apoptosis is widely conserved in phylo- genetically distant eukaryotes, such as the cellular slime mould [12] and nematode [31]. More advanced mecha- nisms may have evolved independently in each eukary- otic lineage. In this context, it is likely that PND in Tetrahymena is the simplest and most primitive form of apoptosis. Experimental procedures Stock strains, culturing methods, and induction of conjugation Tetrahymena thermophila strains CU813 and CU428.2, which were kindly supplied by P. Bruns (Cornell Univer- sity, Ithaca, NY), were used for all experiments. Conditions for cell culture and mating induction have been described previously [32]. Cytological analysis The DePsipher Kit (Trevigen Inc., Gaithersburg, MD) was used to detect changes in mitochondrial membrane poten- tial. Conjugated cells were transferred to 5 l gÆmL )1 DePsipher (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢ -tetraethylbenzimi- dazolylcarbocyanine iodide) in 10 mm Tris ⁄ HCl pH 7.5 along with stabilizer solution, and incubated for 1.5–2 h at 26 °C. The cells were then transferred to 10 mm Tris ⁄ HCl pH 7.5 with stabilizer solution. Cells were observed imme- diately under a fluorescence microscope with fluorescein isothiocyanate (FITC) and green filters. For photography, the cells were fixed with formalin (final concentration 0.5%) and stained with DAPI (4,6-diamino-2-phenylindole) to visualize the nucleus. Acridine orange staining was per- formed as described in Mpoke and Wolfe (1997) [22]. Mito- Tracker Green (Molecular Probes Inc., Eugene, OR) stain- ing has been described previously [33]. Subcellular fractionation The late log phase cells were harvested by centrifugation at 1000 g for 5 min and washed with cold 10 mm Tris ⁄ HCl pH 7.5. The washed cells were resuspended in a cold solu- tion of 0.35 m sucrose, 10 mm Tris ⁄ HCl pH 7.5, 2 mm EDTA (MIB; mitochondria isolation buffer), and homo- genized using a Polytron homogenizer. To remove nuclei and unbroken cells, the homogenate was centrifuged twice at 1000 g for 5 min, and the precipitate was used as frac- tion 1. To sediment the mitochondria, the supernatant (fraction 1; premitochondrial fraction) was centrifuged at 8700 g for 10 min. To increase the purity, the crude mito- chondria were resuspended in MIB that contained 10% Percoll (Amersham Pharmacia Biotech AB, Uppsala, Swe- den) and centrifuged at 5300 g for 5 min. The purified mitochondria were washed once to remove Percoll and re- suspended in MIB (fraction 2; mitochondrial fraction). The supernatant of the crude mitochondrial fraction was centri- fuged at 10 700 g for 10 min, and then the obtained super- natant was further centrifuged at 18 100 g for 10 min. Both precipitates were resuspended in MIB (fraction 3 designated as postmitochondrial fraction 1, and fraction 4 as designa- ted postmitochondrial fraction 2, respectively). The final supernatant was used as the cytosolic fraction (fraction 5). Each fraction was stored at )80 °C until use. PCR To assess the amount of mitochondria in each fraction, we used a modified whole-cell PCR method [34]. Aliquots of each fraction (4 lg protein in 5 lL) were added to 5 lL1% Nonidet P-40 (NP-40). The mixture was incubated at 65 °C for 10 min, followed by 92 °C for 3 min, and 10 lLof 10 · PCR buffer (Promega Inc., Madison WI), 10 lLof 25 mm MgCl 2 ,2lLof10mm dNTPs, 2 lL of each primer (100 pmol), 1 U Taq polymerase (Promega), and 60 lLH 2 O were added, to give a total reaction volume of 100 lL. PCR was performed as follows: 25 cycles of 92 °C for 30 s, 50 °C for 45 s, and 72 °C for 20 s. The following oligonucleotides were used to amplify the partial sequence of the mitochond- T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena FEBS Journal 272 (2005) 5378–5387 ª 2005 FEBS 5385 rial large subunit rRNA (mtLSUrRNA) gene: mtLSU-3, 5¢- TACAACAGATAGGGACCAA-3¢; and mtLSU-4, 5¢- CCTCCTAAAAAGTAACGG-3¢. The PCR products were cloned into the pBluescript II SK– vector (Stratagene Inc., La Jolla, CA) and sequenced using the SQ-5500 DNA sequencer (Hitachi, Tokyo, Japan). Acid phosphatase assay Acid phosphatase activities were assayed using p-nitrophe- nol phosphate [35,36]. Each fraction sample (10 lL) was mixed with 190 lL5mm p-nitrophenol phosphate dissolved in 50 mm sodium acetate buffer (pH 5.0), and the mixture was incubated at 30 °C for 60 min. To stop the reaction, 1 mL 0.4 m NaOH was added. The amount of liberated p-nitrophenol was determined spectrophotometrically at 410 nm. Agarose gel assay for mitochondrial nuclease activity The standard nuclease reaction (200 lL) contained 20 lgof the protein in the subcellular fraction, 2 lg substrate DNA [pT7Blue (R) vector; Novagen Inc., San Diego, CA], 50 mm Hepes ⁄ NaOH pH 7.0, 10 mm KCl. The reaction was incubated at 30 °C for 120 min. To stop the reaction, 300 lL of stop solution (100 mm Tris ⁄ HCl pH 7.5, 50 mm EDTA, 2% SDS, 0.2 mgÆmL )1 proteinase K) was added to the reaction, and the mixture was incubated at 50 °C for 60 min. The stopped reaction was deproteinized with phe- nol ⁄ chloroform (1 : 1), and the DNA was precipitated with an equal volume of isopropanol. The precipitated DNA was washed with 70% ethanol and diluted with 50 lLof TE buffer (pH 8.0). The DNA samples (10 lL) were loaded onto a 1% agarose gel, electrophoresed, and visualized by staining with ethidium bromide. In vitro nuclear apoptosis Tetrahymena nuclei were isolated by the modified method of Mita et al. [37]. Late log phase cells were harvested, and washed with cold solution 1 (0.25 m sucrose, 10 mm Tris ⁄ HCl pH 7.5, 10 mm MgCl 2 ,3mm CaCl 2 ,25mm KCl). The packed cells were resuspended in 9 vols solution 1. To lyse the cells, 1 ⁄ 5 volumes of 1% NP-40 in solution 1 were added, and the mixture was homogenized using a magnetic stirrer. The cell lysate was placed on 2 vols solu- tion 2 (0.33 m sucrose, 10 mm Tris ⁄ HCl pH 7.5, 10 mm MgCl 2 ,3mm CaCl 2 ,25mm KCl), and centrifuged at 1200 g for 5 min. The pellet was resuspended in solution 1, and washed three times using the sucrose superposition method described above. The nuclear pellet was washed three times in solution 1 with centrifugation at 400 · g for 10 min. Finally, the nuclear pellet was washed with solution 3 (0.25 m sucrose, 10 mm Tris ⁄ HCl pH 7.5, 1 mm MgCl 2 ) and resuspended in solution 3 to a concentration of 0.5 · 10 6 macronucleiÆmL )1 . The isolated nuclei (approximately 10 000 macronuclei) were incubated with mitochondrial fractions (20 lg pro- teins) in 200 lL of reaction buffer (50 mm Mops pH 6.5, 10 mm KCl) at 30 °C. To stop the reaction, 300 lLof stop solution (100 mm Tris ⁄ HCl pH 7.5, 50 mm EDTA, 2% SDS, 0.2 mgÆmL )1 proteinase K, 100 lg ÆmL )1 RNase A) was added to the reaction, and the mixture was incu- bated at 50 °C for 60 min. The stopped reaction was de- proteinized with phenol ⁄ chloroform (1 : 1), and the DNA was precipitated with an equal volume of isopropanol. 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A possible role of mitochondria in the apoptotic-like programmed nuclear death of Tetrahymena thermophila Takashi Kobayashi and Hiroshi Endoh Division. h). The dOM also stains green during its degrada- tion. The scale bar indicates 10 lm. T. Kobayashi and H. Endoh Mitochondria in nuclear death of Tetrahymena FEBS