Death harmony played by nucleus and mitochondria: nuclear apoptosis during conjugation of tetrahymena

Tetrahymena programmed nuclear death or nuclear apoptosis is a unique process during conjugation in which only the parental macronucleus is eliminated from the progeny cytoplasm, and other nuclei such as new micro- and macronuclei are unaffected. The nuclear death process consists of three successive steps: chromatin cleavage into high-molecular mass DNA, oligonucleosomal laddering concomitant with nuclear condensation, and complete degradation of the nuclear DNA. Following the first step of the death process, the parental macronucleus is engulfed by a large autophagosome in which many mitochondria are incorporated. Those sequestered mitochondria simply break down and release endonuclease similar to mammalian endonuclease G that is responsible for the generation of the DNA ladder, leading to the conclusion that mitochondria play a crucial role in the execution of the death program. Thus, the parental macronucleus is subject to final death by autophagy in collaboration with caspase-like enzymes, resulting in the ultimate outcome of nuclear resorption.     

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

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 encompasses 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 endonuclease with similarities to mammalian endonuclease G was detected in the isolated mitochondria. When the macronuclei were incubated with isolated mitochondria in a cell-free system, DNA fragments of 150-400 bp were generated, but no DNA ladder appeared. Taking account of the present observations and the timing of autophagosome formation, we conclude that mitochondria might be involved in Tetrahymena PND, probably with the process of oligonucleosomal laddering.     

Role of apoptosis-inducing factor (AIF) in programmed nuclear death during conjugation in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Programmed nuclear death (PND), which is also referred to as nuclear apoptosis, is a remarkable process that occurs in ciliates during sexual reproduction (conjugation). In Tetrahymena thermophila, when the new macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation.     

Caspase-like activity in programmed nuclear death during conjugation of Tetrahymena thermophila

Apoptosis, or programmed cell death, is common in a variety of eucaryotes, from unicellular protozoa to vertebrates. The ciliated protozoan Tetrahymena thermophila has a unique apoptosis-like nuclear death during conjugation, called programmed nuclear death. This death program involves nuclear condensation (pyknosis) and oligonucleosomal DNA fragmentation in the parental macronucleus. Subsequently, the condensed nucleus is entirely resorbed in the autophagosome. Here we demonstrate that caspase-8- and -9-like activity was detected, but no caspase-3-like activity, by in vitro assay during the nuclear resorption process, suggesting that caspase-like activity is associated with both programmed cell death and apoptosis-like nuclear death in Tetrahymena. The use of indicator dye to detect the loss of mitochondrial membrane potential suggested the uptake of mitochondria and the degenerating macronucleus by the autophagosome. An involvement of mitochondria in the programmed nuclear death is discussed.     

DNA Digestion and Chromatin Condensation during Nuclear Death inTetrahymena

DNA fragmentation and nuclear condensation are key features in the regulated cell death of higher animal cells. Nuclear death also occurs as part of a developmentally programmed process during the sexual life cycle of the unicellular organismTetrahymena.We examined the regulation of nuclear death and the relationship between DNA fragmentation and chromatin condensation in this model system. Nuclear death is accompanied by DNA digestion to low-molecular-weight oligonucleosomal-length fragments, in agree- ment with a previous study [17], indicating an endonuclease-like activity typical of apoptosis in higher organisms. Actinomycin D and cycloheximide block DNA digestion as well as nuclear condensation suggesting that nuclear death is under genetic regulation. DNA digestion is completely blocked by aurin, a general nuclease inhibitor. In addition, when DNA fragmentation is blocked, nuclear condensation also fails to occur. Moreover, a kinetic analysis of DNA breakdown, using agarose gels, shows that some DNA digestion occurs before nuclear condensation has taken place. Thus the initiation of DNA digestion may provide conditions necessary for nuclear condensation. Temporary inhibition of nuclear death aborts the death program since after removal of inhibitors cells revert to a vegetative pathway without having eliminated the old or developed the new macronucleus. Zn²⁺and EGTA, both of which inhibit apoptosis in some cell types, fail to prevent nuclear condensation or DNA digestion inTetrahymena,suggesting a requirement here for an endonuclease which is Ca²⁺-independent and Zn²⁺-insensitive. With the TUNEL assay, DNA breakdown is detected exclusively in the condensed macronucleus (and occasional micronuclei identified as degenerating haploid products of meiosis), but not in precondensed macronuclei. These studies show that apoptotic-like DNA fragmentation occurs after condensation of the degenerating macronucleus. However, early DNA digestion may be critical for nuclear condensation and subsequent degeneration.     

Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible “attack me” signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.Key words: Tetrahymena, conjugation, nuclear apoptosis, monodansylcadaverine, macroautophagy, phagocytosis marker, glycoconjugates, phosphatidylserine     

Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible "attack me" signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.     

Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation.

A hallmark of apoptosis is the fragmentation of nuclear DNA. Although this activity involves the caspase-3-dependent DNAse CAD (caspase-activated DNAse), evidence exists that DNA fragmentation can occur independently of caspase activity. Here we report on the ability of truncated Bid (tBid) to induce the release of a DNAse activity from mitochondria. This DNAse activity was identified by mass spectrometry as endonuclease G, an abundant 30 kDa protein released from mitochondria under apoptotic conditions. No tBid-induced endonuclease G release could be observed in mitochondria from Bcl-2-transgenic mice. The in vivo occurrence of endonuclease G release from mitochondria during apoptosis was confirmed in the liver from mice injected with agonistic anti-Fas antibody and is completely prevented in Bcl-2 transgenic mice. These data indicate that endonuclease G may be involved in CAD-independent DNA fragmentation during cell death pathways in which truncated Bid is generated.     

Role of class III phosphatidylinositol 3-kinase during programmed nuclear death of Tetrahymena thermophila

Programmed nuclear death (PND) in the ciliate protozoan Tetrahymena thermophila is a novel type of autophagy that occurs during conjugation, in which only the parental somatic macronucleus is destined to die and is then eliminated from the progeny cytoplasm. Other coexisting nuclei, however, such as new micro- and macronuclei are unaffected. PND starts with condensation in the nucleus followed by apoptotic DNA fragmentation, lysosomal acidification, and final resorption. Because of the peculiarity in the process and the absence of some ATG genes in this organism, the mechanism of PND has remained unclear. In this study, we focus on the role of class III phosphatidylinositol 3-kinase (PtdIns3K, corresponding to yeast Vps34) in order to identify central regulators of PND. We identified the sole Tetrahymena thermophila ortholog (TtVPS34) to yeast Vps34 and human PIK3C3 (the catalytic subunit of PtdIns3K), through phylogenetic analysis, and generated the gene knockdown mutant for functional analysis. Loss of TtVPS34 activity prevents autophagosome formation on the parental macronucleus, and this nucleus escapes from the lysosomal pathway. In turn, DNA fragmentation and final resorption of the nucleus are drastically impaired. These phenotypes are similar to the situation in the ATG8Δ mutants of Tetrahymena, implying an inextricable link between TtVPS34 and TtATG8s in controlling PND as well as general macroautophagy. On the other hand, TtVPS34 does not appear responsible for the nuclear condensation and does not affect the progeny nuclear development. These results demonstrate that TtVPS34 is critically involved in the nuclear degradation events of PND in autophagosome formation rather than with an involvement in commitment to the death program.     

Ectopic expression of mitochondria endonuclease Pnu1p from Schizosaccharomyces pombe induces cell death of the yeast

Endonuclease G (EndoG) is a mitochondrial non-specific nuclease that is highly conserved among the eukaryotes. Although the precise role of EndoG in mitochondria is not yet known, the enzyme is released from the mitochondria and digests nuclear DNA during apoptosis in mammalian cells. Schizosaccharomyces pombe has an EndoG homolog Pnu1p (previously named SpNuc1) that is produced as a precursor protein with a mitochondrial targeting sequence. During the sorting into mitochondria the signal sequence is cleaved to yield the functionally active endonuclease. From the analogy to EndoG, active extramitochondrial Pnu1p may trigger cell killing by degrading nuclear DNA. Here, we tested this possibility by expressing a truncated Pnu1p lacking the signal sequence in the extramitochondrial region of pnu1-deleted cells. The truncated Pnu1p was localized in the cytosol and nuclei of yeast cells. And ectopic expression of active Pnu1p led to cell death with fragmentation of nuclear DNA. This suggests that the Pnu1p is possibly involved in a certain type of yeast cell death via DNA fragmentation. Although expression of human Bak in S. pombe was lethal, Pnu1p nuclease is not necessary for hBak-induced cell death.     

Mammalian mitochondrial endonuclease activities specific for ultraviolet-irradiated DNA.

Mitochondrial forms of uracil DNA glycosylase and UV endonuclease have been purified and characterized from the mouse plasmacytoma cell line, MPC-11. As in other cell types, the mitochondrial uracil DNA glycosylase has properties very similar to those of the nuclear enzyme, although in this case the mitochondrial activity was also distinguishable by extreme sensitivity to dilution. Three mitochondrial UV endonuclease activities are also similar to nuclear enzymes; however, the relative amounts of these enzyme activities in the mitochondria is significantly different from that in the nucleus. In particular, mitochondria contain a much higher proportion of an activity analogous to UV endonuclease III. Nuclear UV endonuclease III activity is absent from XP group D fibroblasts and XP group D lymphoblasts have reduced, but detectable levels of the mitochondrial form of this enzyme. This residual activity differs in its properties from the normal mitochondrial form of UV endonuclease III, however. The presence of these enzyme activities which function in base excision repair suggests that such DNA repair occurs in mitochondria. Alternatively, these enzymes might act to mark damaged mitochondrial genomes for subsequent degradation.     

Review: nuclear events in apoptosis

Initial apoptosis research characterized this form of cell death based on distinct nuclear morphology that was subsequently shown to be associated with the appearance of oligonucleosomal DNA fragments. More recent evidence has indicated that apoptosis depends upon a tightly regulated cellular program for its successful initiation and execution. Molecular participants in this program are present in different subcellular compartments, including the plasma membrane, cytosol, mitochondria, and nucleus. The interplay among these compartments and the exchange of specific signaling molecules are critical for the systematic progression of apoptosis. While numerous reports have described a key role for caspase activity in the signaling and executive steps of apoptotic cell death, there are some instances where well-established nuclear changes, characteristic of this form of cell death, can occur independently of caspase activity. Moreover, evidence indicates that certain nuclear events, including chromatin condensation and DNA fragmentation, are controlled separately and depend upon a persistent supply of energy in vivo. In this review, we discuss our current understanding of the role and regulation of nuclear events in the apoptotic process with an emphasis on protease and endonuclease activities as well as the ability of certain Bcl-2 family proteins to influence this process.     

Overexpression of Helicard,a CARD-containing helicase cleaved during apoptosis,accelerates DNA degradation

Apoptotic cell death is characterized by several morphological nuclear changes, such as chromatin condensation and extensive fragmentation of chromosomal DNA. These alterations are primarily triggered through the activation of caspases, which subsequently cleave nuclear substrates. Caspase-3 induces processing of Acinus, which leads to chromatin condensation. DNA fragmentation is dependent on the DNase CAD, which is released from its inhibitor, ICAD, upon cleavage by caspase-3. DNA degradation is also induced by AIF and endonuclease G, which are both released from mitochondria upon death stimuli but do not require prior processing by caspases for their DNase activity. Here we report the identification of a widely expressed helicase designated Helicard, which contains two N-terminal CARD domains and a C-terminal helicase domain. Upon apoptotic stimuli, Helicard is cleaved by caspases, thereby separating the CARD domains from the helicase domain. While Helicard localizes in the cytoplasm, the helicase-containing fragment is found in the nucleus. Helicard accelerates Fas ligand-mediated DNA degradation, whereas a noncleavable or a helicase-dead Helicard mutant does not, implicating Helicard in the nuclear remodeling occurring during apoptosis.     

Lysosomal enzymes in the macronucleus of Tetrahymena during its apoptosis-like degradation

A key characteristic of apoptosis is its regulated nuclear degradation. Apoptosis-like nuclear degradation also occurs in the ciliated unicellular organism, Tetrahymena thermophila. Chromatin of the macronucleus undergoes massive condensation, a process that can be blocked by caspase inhibitors. The nucleus becomes TUNEL-positive, and its DNA is cleaved into nucleosome-sized fragments. In a matter of hours the macronucleus is completely degraded, and disappears. The condensed nucleus sequesters acridine orange, which means that it might become an acidic compartment. We therefore asked whether lysosomal bodies fuse with the condensed macronucleus to form an autophagosome. We monitored acid phosphatase (AP) activity, which is associated with lysosomal bodies but is not found in normal nuclei. We find that after the macronucleus condenses AP activity is localized in cap-like structures at its cortex. Later, after the degrading macronucleus loses much of its DNA, acid phosphatase deposits appear deeper within the nucleus. We conclude that although macronuclear elimination is initiated by an apoptosis-like mechanism, its final degradation may be achieved through autophagosomy.     

Dynamic nuclear reorganization during genome remodeling of Tetrahymena

The single-celled ciliate Tetrahymena thermophila possesses two versions of its genome, one germline, one somatic, contained within functionally distinct nuclei (called the micronucleus and macronucleus, respectively). These two genomes differentiate from identical zygotic copies. The development of the somatic nucleus involves large-scale DNA rearrangements that eliminate 15 to 20 Mbp of their germline-derived DNA. The genomic regions excised are dispersed throughout the genome and are largely composed of repetitive sequences. These germline-limited sequences are targeted for removal from the genome by a RNA interference (RNAi)-related machinery that directs histone H3 lysine 9 and 27 methylation to their associated chromatin. The targeting small RNAs are generated in the micronucleus during meiosis and then compared against the parental macronucleus to further enrich for germline-limited sequences and ensure that only non-genic DNA segments are eliminated. Once the small RNAs direct these chromatin modifications, the DNA rearrangement machinery, including the chromodomain proteins Pdd1p and Pdd3p, assembles on these dispersed chromosomal sequences, which are then partitioned into nuclear foci where the excision events occur. This DNA rearrangement mechanism is Tetrahymena's equivalent to the silencing of repetitive sequences by the formation of heterochromatin. The dynamic nuclear reorganization that occurs offers an intriguing glimpse into mechanisms that shape nuclear architecture during eukaryotic development.     

Cell nucleus and DNA fragmentation are not required for apoptosis

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.     

Small RNAs in genome rearrangement in Tetrahymena

Small RNAs produced by an RNAi-related mechanism are involved in DNA elimination during development of the somatic macronucleus from the germline micronucleus in Tetrahymena. The properties of these small RNAs can explain how the primary sequence of the parental macronucleus epigenetically controls genome rearrangement in the new macronucleus and provide the first demonstration of an RNAi-mediated process that directly alters DNA sequence organization. Methylation of histone H3 on lysine 9 and accumulation of chromodomain proteins, hallmarks of heterochromatin, also occur specifically on sequences undergoing elimination and are dependent on the small RNAs. These findings contribute to a new paradigm of chromatin biology: regulation of heterochromatin formation by RNAi-related mechanisms in eukaryotes.     

Cover Picture

Programmed nuclear death in conjugating Tetrahymena: The parental macronucleus (pale‐green) is attacked by many lysosomes and autophagosomes (pale‐yellow), while new micro‐ and macronuclei (blue) remain intact.