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Department of Botany, University of British Columbia, 6270 University Blvd., Vancouver, V6T 1Z4 Canada
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ABSTRACT |
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In N. crassa and N. intermedia, the kalilo plasmid triggers senescence by insertion into mitochondrial DNA. To investigate the cell death pathway induced by this plasmid, juvenile and senescent subcultures of several senescent strains were examined by light and transmission electron microscopy, and at the DNA level. There were no signs of apoptotic events, such as shrinkage of the cytoplasm away from the cell wall, apoptotic bodies, internucleosomal DNA fragmentation or condensation of the cytoplasm while retaining mitochondria and endomembrane structure. Instead, swollen mitochondria lacking cristae and containing amorphous inclusions, and disruption of nuclear and mitochondrial membranes indicated a necrotic mode of cell death.
Key words: apoptosis, kalilo, mitochondria, necrosis, Neurospora, plasmid, senescence
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The necrotic mode of cell death (Mastrangelo and Betenbaugh 1998) generally is caused externally, by severe physical or chemical damage to the cell, by pathogen attack or environmental stress. For example, in animal cells, necrosis involves the disruption of endomembrane integrity by swelling and osmotic lysing. In mitochondria, there is vesiculation, distortion and fragmentation of membranes, loss of cristae and the appearance of large, characteristic aggregations of dense material or vacuoles in the mitochondrial matrix (Buja et al 1976, Ali et al 1997). These signs of degeneration are like those found in mitochondria during in vitro cell lysis (Trump et al 1965).
In contrast to this passive death mechanism, apoptosis represents an internally programmed type of cell death that is important in the development and homeostasis of multicellular organisms. It is found in most cell types that undergo cellular suicide in response to nonlethal stimuli. The characteristics of apoptosis are cell shrinkage, membrane blebbing and fragmentation of the cell into apoptotic bodies, chromatin condensation and fragmentation of nuclear DNA cleaved by a specific Ca++/Mg++-dependent endonuclease at internucleosomal sites (Wyllie 1980). Apoptosis is a cascade of events controlled by endogenous cellular genes, enzymes and signaling systems (Mastrangelo and Betenbaugh 1998). Parts of the apoptotic pathway have been conserved among worms, insects, vertebrates (Steller 1995), plants and fungi (Wang et al 1996, Havel and Durzan 1996, Jacobson et al 1998, Umar and Griensven 1997). In fungi, other indicators of apoptosis are plugged septal pores (Beckett et al 1974, Battu et al 1998, Avallone et al 2002, Kimura et al 1997) and shells of walls containing small condensed and disorganized masses (Jacobson et al 1998).
The kalilo mitochondrial plasmid, isolated from Hawaiian strains of the fungus Neurospora intermedia, was originally of interest because kalilo-bearing strains routinely senesce and die (reviewed by Griffiths 1992). One of the characteristic and unique properties of the kalilo plasmid DNA is its ability to insert into the mitochondrial DNA (mtDNA). From the time at which insertions can be detected, there is a progressive loss of normal mitochondrial functions associated with oxidative phosphorylation (OXPHOS) up to the death of the host. However, the precise pathway leading to senescence is not known, be it necrosis or apoptosis. Kalilo-induced senescence is a complicated process that involves interaction between nuclear DNA, mtDNA and plasmid DNA (Griffiths 1992). Although, as parasitic elements, plasmids might be considered prime candidates as agents of necrosis, it is known that damage to the mitochondria of mammals can release apoptosis-inducing proteins, so in theory plasmids could work through the apoptotic pathway (Wallace 1999).
In this study, ultrastructural changes and internucleosomal DNA fragmentation patterns of senescing kalilo strains of Neurospora were used to deduce the senescence pathway. Our results suggest that kalilo plasmid-induced senescence acts through necrosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and Strain 773 (a synthetic histidine-requiring strain of Oak Ridge background, bearing the prototypic kalilo plasmid, Chan et al 1991). Two kalilo-bearing natural strains of N. intermedia from Hawaii were used, P561 and P790 (Griffiths and Bertrand 1984). All culturing and manipulation of strains used standard techniques devised for Neurospora (Davis and deSerres 1970).
Isolation of plasmid DNA –
Plasmid DNA was co-purified with mtDNA using the small scale mtDNA isolation method described by Myers (1988). Proteinase K digestion was necessary before phenol/chloroform extraction of proteins.
Southern analysis –
The Southern blotting and hybridization methodology was similar to that described by Maniatis et al (1982). An 8.2 kb Kpn I restriction fragment, which encompasses most of the kalilo plasmid, was cloned in a pUC18 vector and used as a probe to detect kalilo-homologous sequences. The DNA probe was labeled with 32P-dCTP, using an oligolabelling kit (Pharmacia).
Light microscopy – Mycelia grown on cellophane over Vogel's minimal media were examined after incubation for 24 h at 30 C. Photographs were processed to give a final magnification of 120 x with Zeiss Axioskop (12V 50W light bulb) microscopy.
Transmission electron microscopy –
Strains were inoculated onto Vogel's medium. After two days of incubation at 25 C, the agar in the region of the mycelial tips was chopped into 1 mm blocks. The blocks were fixed in 2.0% glutaraldehyde in 0.05 M sodium cacodylate (pH 7.1) for 8 h at room temperature, then rinsed and washed overnight in the sodium cacodylate buffer. They were post-fixed in 1% osmium tetroxide for 1 h at room temperature (Adams et al 1995). They were dehydrated in a graded series of ethanol and embedded in Spurr's resin, which was polymerized 16 h at 60 C (Spurr 1969). Ultra-thin sections were prepared on a LKB ultratome V, using a diamond knife. Sections were stained with uranyl acetate and Reynolds' lead (Reynolds 1963). Stained sections were examined in a Zeiss transmission electron microscope (ZEISS TEM 10C). We fixed more than 20 blocks/each sample of subculture, cut five blocks from 20 blocks and stained several sections/each sample to determine best three sections/each sample. If necessary, we repeated sampling to create better pictures.
Analysis of DNA fragmentation – Mycelia grown in liquid Vogel's medium were harvested by filtration and ground in the presence of DNA extraction buffer (LETS buffer—0.1 M LiCl, 10 mM EDTA, 10 mM Tris-HCl, 0.5% SDS, pH 8). Phenol and chloroform-isoamyl alcohol (25:24:1) extraction was performed twice to extract nucleic acid. The aqueous phase after centrifugation was precipitated with 2.5 volumes of ethanol/0.2 M ammonium acetate and stored overnight at -20 C. After centrifugation (10 000 g for 20 min), the DNA pellet was air-dried then resuspended in Tris EDTA buffer (10 mM Tris-HCl, 1 mM EDTA) with RNase. After incubation at 55 C for one hour, loading buffer [0.25% bromophenol blue, 0.25% (v/v) xylenecyanol, 30% (v/v) glycerol] was added and the samples were loaded onto a 2% agarose gel and subjected to electrophoresis at 5 v/cm in TAE buffer (40 mM Tris, 1.14% (v/v) acetic acid, 10 mM EDTA, pH 8). DNA was visualized by ethidium bromide staining.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, and our unpublished data).
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). Plasma membranes, ribosomes and small vacuoles were visible.
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Organelle degradation is evident in the senescent samples. Most were irregularly shaped and could not be identified with certainty. Amorphous inclusions were found within the matrix of grossly swollen mitochondria (Figs. 5–8). Such amorphous inclusions probably represent precipitates of denatured matrical proteins formed during the evolution of irreversible cellular damage (Buja et al 1976) and are a characteristic sign of necrotic cell death (Ali et al 1997). Other signs of necrosis were observed, including loss of mitochondrial cristae (Figs. 5–8) and the appearance of intramitochondrial vacuoles (Figs. 6, 7). Nuclei were distinguishable but became irregularly shaped, swollen and disrupted (Figs. 5–7).
Regular features of apoptosis, such as cell shrinkage, chromatin condensation, membrane blebbing and fragmentation of cells into apoptotic bodies, were not found. Plugged septal pores also are common in apoptosis in fungi (Beckett et al 1974, Jacobson et al 1998) but could not be found in the set of senescent samples. Figure 9 shows an unplugged pore with a nearby mitochondrion.
Analysis of DNA fragmentation –
During apoptosis, chromatin is cleaved at regularly spaced sites leading to a typical oligonucleosomal DNA ladder that serves as biochemical indicator for this type of death (Mastrangelo and Betenbaugh 1998). However, in our samples, the DNA did not show much difference in smearing compared to the juvenile culture and OR control on the gel, and no signs of fragmentation were found (Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Wang et al 1996). Neither could we find shells of wall, containing small, condensed masses of disorganized material (Jacobson et al 1998). In addition, the lack of detectable nuclear DNA ladders argues against apoptosis. Other groups have reported various types of cell death in fungi that could be thought of as apoptosis (Umar and Van Griensven 1997, Worrall 1997, Jacobson et al 1998). Those studies were not of plasmid-induced senescence. The results did not parallel ours.
The key to the necrotic process presumably is insertion of the plasmid into host mtDNA, acting as an insertional mutagen, and severely disrupting OXPHOS (Rieck et al 1982, Bertrand et al 1985). Such disruption is known to produce reactive oxygen species (ROS; Wallace, 1999). Large amounts of ROS damage the mitochondria and ultimately the cell. Trump et al (1981) reported that such necrogenic agents interfere with the homeostatic regulation of Ca++ ions and calmodulin by direct assault on the cell membrane and/or depletion of mitochondrial ATP. Mitochondria become porous, consume substrates at maximal rate and, instead of synthesizing ATP, hydrolyze ATP by reversal of the H+-ATP-synthetase reaction under de-energized conditions. Kalilo-induced senescence shows loss of cytochrome c oxidase and cytochrome b (Rieck et al 1983, Bertrand et al 1985). Papa and Skulachev (1997) reported that cytochrome c oxidase can lower the level of ROS. The content of cytochrome b and the activity of the cytochrome bc1 complex exhibit a marked reduction with age in human females (Boffoli et al 1996).
Damage in the mtDNA could lead to damage in the nuclear DNA. It seems quite probable that even small age-linked ROS-induced injuries of mtDNA, which are not sufficient to cause measurable decline of the mitochondrial ATP production, result in increased ROS formation due to slight inhibition of the electron flow in rotenone- or antimycin-sensitive sites of the respiratory chain. Such a small effect might trigger a vicious cycle (Wallace 1999). As a result, the level of ROS becomes so high that even nuclear DNA is oxidized. However, we have no direct proof of nuclear DNA damage.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication September 8, 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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