A comparison between nuclear dismantling during plant and animal programmed cell death

1 A comparison between nuclear dismantling during plant and animal programmed cell death Fernando Domínguez and Francisco Javier Cejudo Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC, Avda Américo Vespucio 49, 41092-Sevilla, Spain Correspondence to: Francisco Javier Cejudo; Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC, Avda Américo Vespucio 49, 41092- Sevilla, Spain; E-mail: [email protected] Key words: apoptosis, plant, programmed cell death, nuclease, nucleus dismantling 2 Abstract Programmed cell death (PCD) is a process of organized destruction of cells, essential for the development and maintenance of cellular homeostasis of multicellular organisms. Cells undergoing PCD begin a degenerative process in response to internal or external signals, whereby the nucleus becomes one of the targets. The process of nucleus dismantling includes events affecting the nuclear envelope, such as formation of lobes at the nuclear surface, selective proteolysis of nucleoporins and nuclear pore complex clustering. In addition, chromatin condensation increases in coordination with DNA fragmentation. These processes have been largely studied in animals, but remain poorly understood in plants. The overall process of cell death has different morphological and biochemical features in plants and animals. However, recent advances suggest that nuclear dismantling in plant cells progresses with morphological and biochemical characteristics similar to those in apoptotic animal cells. In this review, we summarize nucleus dismantling in plant PCD, focusing on the similarities and differences with their animal counterparts. 3 Contents 1. Introduction 2. An overview of nuclear dismantling in animal cells 3. Similarities in nuclear dismantling in animal and plant cells undergoing PCD 3.1. Signalling that commits nuclear dismantling in plants 3.2. Sequence of events of plant nuclear dismantling during PCD 3.3. Nuclear dismantling is connected with cytoplasmic events during plant PCD 4. Is nuclear dismantling an autophagic process? 5. Concluding remarks and future perspectives Acknowledgements References 4 1. Introduction Programmed cell death (PCD) is a process of organized destruction of cells that maintains cellular homeostasis and is essential for the successful development of multicellular organisms [1]. PCD is an important process in plants, as in other multicellular organisms, not only for development [2], but also as a mechanism of immunity against pathogen attack [3]. Plant developmental cell death affects to determined cell types at precise stages of development and is characterized by the rupture of the vacuolar tonoplast and subsequent release of hydrolases, which degrade the cellular content and, in some cases, the cell wall. Cell death in biotic stress depends of the type of pathogens, but free radicals seem to exert an important function [4]. The plant cell has peculiar characteristics, most notably the presence of the cell wall and vacuoles, which suggest that the process of PCD takes place with different morphological features from apoptosis of animal cells. Indeed, most of the morphological features of mammalian apoptosis are not found in plant cells undergoing PCD and, thus, it has been proposed that cell death in plants does not take place by the process of apoptosis; however, the degree of conservation of plant and animal cell death programmes is at the moment a matter of debate [4]. The notion that plants seem to have evolved different mechanisms for PCD is reinforced by the biochemical analyses of the components involved in the execution of cell death in different plant systems. Despite intense search, caspases have not been found in plants whereas they are the most characteristic proteases executing apoptosis in animal cells. Still, there are caspase-like proteases involved in plant PCD [5]. Analyses carried out in different plant systems have identified several types of proteases involved in cell death, which include metacaspases [6], subtilisin-like proteases [7], and vacuolar processing enzymes [8]. Therefore, both morphological and biochemical characteristics suggest that plants and animals have developed different strategies that perform cell death. The study of PCD has been a major field of research in different animal model systems not only because it is an essential process to understand development, but also because deregulation of PCD causes alterations of the cellular homeostasis, which have important implications associated with diseases, including cancer or neurodegenerative processes [9]. At the morphological level, animal PCD takes place by two wellestablished mechanisms, apoptosis and necrosis [10]. Apoptosis is characterized by active plasma membrane blebbing and cell fragmentation, forming the so-called 5 apoptotic bodies, which are eventually engulfed by phagocytes. In parallel, the nucleus undergoes dramatic modifications, which include disorganization of the nuclear envelope, chromatin condensation and internucleosomal degradation of DNA in fragments of 180-200 bp and multimers of it, forming a characteristic ladder, which constitutes a hallmark of apoptosis [11]. In contrast, necrosis is characterised by the rupture of the plasma membrane and the consequent degradation of intracellular contents. Necrosis lacks the morphological features of apoptosis, notably the formation of apoptotic bodies. Increasing attention has been devoted to autophagy, a process of cell self-digestion in which cellular components are engulfed in vesicles, called autophagosomes, prior to their degradation by lysosomes. It is well-established that autophagy has pro-death functions, but this process may also have pro-survival functions, and the relationship of autophagy with apoptosis is still the subject of intense debate [12]. The nucleus is the major target of the cell degradation machinery at the onset of PCD. In apoptotic animal cells, the nucleus undergoes a massive reorganization, which includes the condensation of chromatin and internucleosomal fragmentation of DNA [11]. Condensation of chromatin has also been described in different plant systems. This is the case of nuclei from nucellar cells undergoing PCD during wheat grain development, the chromatin of which condenses in a morphologically similar manner as occurs in apoptotic animal cells [13]. Similarly, DNA laddering is also a hallmark of PCD in different plant systems such as pea carpel senescence [14]. However, it should be noted that both chromatin condensation and DNA laddering are not as consistent PCD markers in plants as they are in animal cells. Therefore, despite the clear morphological and biochemical differences of PCD in plants and animals, the phase of nuclear dismantling seems to have similarities in both types of cells. Indeed, factors involved in nuclear dismantling from plant cells are able to induce apoptotic morphology and DNA fragmentation in human cells [15], which suggests the possibility of common mechanisms in PCD of cells from both kingdoms, at least at the stage of nucleus dismantling. In this review, we will summarize the current knowledge of the process of nuclear dismantling during PCD in plant systems, discussing the similarities and differences with the process in animal systems. 2. An overview of nuclear dismantling in animal cells 6 The content of the nucleus, the nucleoplasm, is separated from the cytoplasm by a complex membranous nuclear envelope formed by outer and inner nuclear membranes, which define the perinuclear space. The nuclear envelope is penetrated by nuclear pore complexes that mediate the nucleo-cytoplasmic interchange in both directions [16]. The nuclear matrix at the internal side of the nuclear envelope acts as a skeleton defining nuclear size and shape. Several nuclear membrane proteins localized to the inner side of the nuclear envelope provide binding sites for chromatin and nuclear matrix in animals and plants. In animal cells the nuclear matrix is composed of lamins and lamin-associated proteins [17], nuclei of plant cells lack this structure, with scaffold and structural support exerted by coiled proteins [18]. Morphological and biochemical analyses have led to considerable advances of our knowledge of nuclear dismantling during animal apoptosis [11]. The protein profile of the nucleus in cells undergoing apoptosis is modified by the appearance of transcription factors, protein kinases, proteases and DNases, among other proteins. These proteins are probably translocated from the cytoplasm, as schematically shown in Figure 1. One of the earliest events taking place during nuclear dismantling is the proteolysis of matrix attachment region-binding proteins, which anchor chromatin to the scaffold [19]. This limited cleavage may open nuclease sites on the chromatin structure allowing fragmentation of DNA, which facilitates the subsequent proteolysis of the bulk of nuclear matrix proteins (Figure 1). Several membrane proteins localized to the inner side of the nuclear envelope, such as lamin B receptor, lamin-associated polypeptide 2α and nucleoporin Nup 153, are connected to chromatin. The cleavage of these proteins promotes the detachment of chromatin from the nuclear envelope, which results in nuclear pore clustering. Finally, it has been speculated that cleavage of components of the nuclear pore complex and the nuclear transport machinery may stimulate an increase in nuclear pore permeability, facilitating protein translocation from the cytoplasm into the nucleus [19]. Post-translational modifications of nuclear proteins seem to have an important function in nuclear dismantling during apoptosis. Histones H2, H3 and H4, lamins and HMGA1a protein are hyperphosphorylated, whereas histone H1 is dephosphorylated just before DNA fragmentation [19]. It is worth comparing nuclear envelope disassembly during mitosis and apoptosis because disorganization of this structure may share common mechanisms in both processes. Nup98, a peripheral nucleoporin 7 localized on both sides of the nuclear envelope, contains 13 phosphorylation sites that are successively phosphorylated by mitotic kinases, driving nuclear pore complex disassembly and nuclear envelope permeabilization during mitosis [20]. Hyperphosphorylation of Nup98 and other nucleoporins also seems to have a key regulatory role in apoptosis. The post-translational modifications of nuclear proteins are presumably important for three events: chromatin condensation, accessibility of nucleases to DNA and the breakdown of the lamina. 3. Similarities in nuclear dismantling in animal and plant cells undergoing PCD As mentioned above, animal cell nuclei undergo very characteristic morphological changes during apoptosis. The nucleus becomes fragmented and the fragments move to apoptotic bodies, which are subsequently phagocytosed by macrophages, parenchymal or neoplastic cells and degraded by phagolysosomes [21]. Therefore, the nucleus of apoptotic animal cells becomes degraded inside another cell. In contrast, in plant cells undergoing PCD, the nucleus is not degraded in another cell, which is a clear difference of nuclear dismantling between plant and animal cell death. Despite these differences, nuclear extracts of etopoxide-induced apoptotic human cells triggered apoptotic morphology of plant cell nuclei [15]. In the same way nuclear extracts from wheat nucellar cells undergoing PCD induce apoptotic morphology and DNA fragmentation in nuclei from human cells (Figure 2). These results suggest the existence of common mechanisms in the machinery of nuclear dismantling in animal and plant cells. CAD (Caspase Activated DNase) is the first nuclease in apoptotic animal cells responsible for the characteristic internucleosomal degradation of DNA [22]. CAD is maintained in an inactive status by the formation of a complex with its inhibitor [23]. The cleavage of the inhibitor in apoptotic cells releases active CAD, thus allowing the subsequent degradation of DNA [24]. The finding that nuclear extracts from plant cells undergoing PCD can trigger apoptotic morphology and DNA fragmentation in nuclei from human cells prompted us to search for common elements of nuclear dismantling in animal and plant cells. No candidate genes have been identified so far encoding putative CAD or its inhibitor in plants. Apoptosis-inducing factors extracted from nuclei of plant cells undergoing PCD were not inhibited by mammalian CAD inhibitors (Figure 2B). Zn2+ and EDTA, inhibitors of PCD-related Ca2+ and/or Mg2+-dependent endonucleases 8 [15], inhibited the induction of animal apoptosis (Figure 2B). These results suggest that a CAD type nuclease is not responsible for DNA fragmentation in plant cell nuclei undergoing PCD. Therefore nuclear dismantling in plants seems to involve different factors from those in animals. Search for plant homologues of the components of animal cell apoptosis has been carried out by bioinformatic approaches based on sequence similarity, which have not given positive results. The identification of possible shared genes will require searches based on structure and function rather than sequence. 3.1. Signalling that commits nuclear dismantling in plants The process of PCD leading to nuclear dismantling can be schematically subdivided into three major phases: signalling, execution and nuclear dismantling itself. The signalling and execution phases of PCD are beyond the scope of this review. Still, some recent advances in model plant systems, which suggest functional conservation of key events regulating PCD, are here discussed. The rice transcription factor OsNAC4 is translocated into the nucleus in a phosphorylation-dependent manner in pathogentriggered hypersensitive responses. It activates a Ca2+-dependent nuclease, which promotes nuclear DNA fragmentation [25]. The metacaspase mcII-Pa is translocated from the cytoplasm to the nucleus during Norway spruce (Picea abies) embryogenesis and facilitates cell death by cleaving a phylogenetically conserved nuclease [26, 27]. A mitogen-activated protein kinase appears as a signalling factor during early PCD in Papaver rhoeas pollen, stimulating DNA fragmentation [28]. The pathway of PCD culminates in the process of nuclear dismantling in both plant and animal cells. As discussed above, the nuclei of plant and animal cells have very similar structures and, despite the differences, undergo similar modifications, the most relevant being chromatin condensation and DNA fragmentation, in dying cells. Thus the question arises whether the key events of nuclear dismantling are similar in both types of cells. The most relevant events taking place during nuclear dismantling in animal and plant PCD are summarized in Table 1. The first event is the translocation of different factors and enzymes to the nucleus that initiate the process. In apoptotic animal cells the most relevant process that takes place at this stage is the translocation of the CAD complex with its inhibitor [23, 24]. The relevant event in plants at this stage is the translocation of transcription factors, such as OsNAC4 [25], and the metacaspase mcII- 9 Pa [26]. Then, the maturation of apoptogenic factors, or inactivation of their inhibitors, occurs. The relevant activity illustrating this stage is the cleavage of the inhibitor of CAD by caspase-3 in animal cells undergoing apoptosis [24], and the cleavage of the Tudor Staphylococcal Nuclease (TSN) by metacaspase mcII-Pa [27] in plant PCD. The disruption of the nucleo-cytoplasmic barrier, facilitated by limited digestion of some nucleoporins of the nuclear pore complex, which alters the permeability through the pore of an exclusion size of ca. 30 kDa to more than 70 kDa, has been documented in apoptotic animal cells [29] but, to our knowledge, not in plant cells undergoing PCD. Similarly, the protection from cleavage of other nucleoporins until the end of the death process has been documented only in apoptotic animal cells [30], but not in plant cells. A series of events follows with the cleavage of DNA into different conformations (chromatin loop DNA, oligonucleosomal DNA or naked DNA) catalysed by different nucleases. Examples of these nucleases have been reported both in animal and plant PCD (Table 1). Finally, nuclear pore complex clustering, which may facilitate nuclear fragmentation and chromatin release to the cytoplasm, as well as engulfment of nuclear fragments, have also been reported in both types of cells [13, 19, 32, 36]. 3.2. Sequence of events of plant nuclear dismantling during PCD Two types of events can be distinguished in the process of nuclear dismantling; those affecting chromatin structure and DNA fragmentation, and those affecting the nuclear envelope. The most characteristic structural feature in cells undergoing PCD is that chromatin becomes increasingly condensed. This process begins at the inner side of the nuclear envelope, where discrete patches of condensed chromatin can be observed. In animal cell apoptosis the progression of chromatin condensation can be classified in three stages: stage I, or ring condensation, stage II, or necklace condensation, and stage III, or nuclear collapse/disassembly [37, 38], as schematized in Figure 3A. 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Richards, LITTLE NUCLEI genes affecting nuclear morphology in Arabidopsis thaliana. Plant Cell 19 (2007) 2793-2803. 19 Legends of figures Fig. 1. Key events of nuclear dismantling during apoptosis. In animal cells the process of apoptosis culminates in nucleus dismantling. This process requires the translocation of proteins from the cytoplasm to the nucleus, which probably occurs in several waves. Some of the key events of nuclear dismantling are the proteolysis of nuclear matrix and nuclear envelope proteins, allowing the detachment of the nuclear envelope from the chromatin, the subsequent chromatin condensation and the clustering of nuclear pore complex. Cleavage of nuclear pore complex proteins provokes the disruption of nucleocytoplasmic barrier, facilitating the entrance of apoptotic factors. The proteolysis of chromatin-associated proteins close to the detachment of the nuclear envelope may open sites of nuclease hypersensitivity promoting DNA fragmentation by different nucleases. Protein phosphorylation, along with other post-translational modifications, is needed to facilitate nuclear matrix disruption, chromatin condensation and DNA fragmentation. Finally, nuclear fragmentation is produced with the formation of apoptotic bodies and engulfment by macrophages. Fig. 2. Chromatin condensation and DNA fragmentation in human cells can be promoted by plant apoptosis-inducing factors. A, HCT116 human cells were treated with nuclear extracts from wheat nucellar cells undergoing PCD. Clear patches of chromatin condensation can be observed in the nucleus after staining with propidium iodide. B, DNA extracted from these cells was analysed in agarose gels and stained with ethidium bromide. C, control untreated cells; +N, samples treated with nuclear extracts from wheat nucellar cells undergoing PCD, in the absence (-) or the presence of purified Inhibitor of CAD (ICAD), Zn2+ or EDTA. This result has not previously been reported, details of experimental conditions as previously described [19]. Fig. 3. The proposed sequence of events of nuclear dismantling during plant PCD involves chromatin condensation, DNA fragmentation and nuclear envelope disruption. A, Features of chromatin condensation distinguishing ring, necklace and collapse/disassembly morphologies [39]. B, Progressive fragmentation of DNA: high molecular weight DNA cleavage, low molecular weight DNA cleavage and naked DNA unspecific cleavage. C, Nuclear envelope disassembly. Sequential cleavage of plant nucleoporins Nup 93, Nup 96, Nup 214, Nup 50, Nup 62 and Tpr/NUA was proposed 20 based on apoptotic animal cells [30]. Proteolysis of plant lamin-like proteins and Nup 136 nucleoporin was proposed based on the similar role of animal lamins and Nup 155, although there is no sequence homology. 21 Table 1 Relevant events in nuclear dismantling Event Examples and References Animals Plants Translocation to the nucleus CAD-Inhibitor complex [11, 23, 24] Transcription factor OsNAC4 [25] Metacaspase mcII-Pa [26] Processing of apoptogenics factors or cleavage of their inhibitors Cleavage of CAD inhibitor by caspase-3 [24] Cleavage of tudor staphylococcal nuclease (TSN) by a metacaspase mcII-Pa [27] Disruption of nucleocytoplasmic barrier Degradation of cytoplasmic filaments and nuclear basket from nuclear pore complex [29] n.d. Protection of cleavage sites of some nucleoporins Nucleoporin Nup62 [30] n.d. Accessibility to the conformation of DNA - Chromatin loops Chromatin loop nuclease [31] Chromatin loop nuclease [32] - Internucleosomal DNA CAD nuclease [22, 23] D-mannose-induced nuclease [33] PCD-related nuclease [15, 34] - Naked DNA Different nucleases [11] ZEN1 nuclease [35] Nuclear Pore Complex clustering Nuclear Pore Complex clustering in apoptosis [19] Nuclear Pore Complex clustering in plant PCD [32] Nuclear fragment engulfment Phagocytosis of apoptotic bodies by macrophages [36] Autophagic process to remove nuclear fragments [13, 32] n.d.: not determined 22 Table 2 Sequence of cytoplasmic events preparing for plant nuclear dismantling Organelle Event References ER-derived compartments Accumulation and release of cysteine proteases and nucleases in the onset of PCD [41, 55] ER-Golgi Chaperoning and inhibition of cysteine proteases by protein disulfide isomerase-5 during trafficking to vacuole before PCD [56] Vacuole (PSV and Lytic Vacuole) -Self-processing of vacuolar processing enzyme at acid pH -Maturation of precursors of PR proteins and hydrolases by vacuolar processing enzyme -Accumulation of aspartate proteases, cysteine proteases and nucleases until their release in PCD -Tonoplast disruption [4, 57] Mitochondria Release of endonucleases, cytochrome c and other apoptogenic factors [58] Choroplast Release of ROS [59] Cytoskeleton Depolymerization of microtubule and reorganization of actin network providing a skeleton to trigger autophagosome formation and promoting autophagic movements for cell content engulfment [60] Autophagosomes and autophagic vacuoles Digestion of remnants of membrane-less nuclear fragments [13, 32, 40] 23 Early proteolysis of MAR-binding proteins Detachment of the nuclear envelope from chromatin Breakdown of nuclear matrix and entire nucleus Proteases Transcription Factors Protein kinases DNases Increase/Modification in nuclear pore permeability TRANSLOCATION Nuclear Pore Complex clustering Proteases Transcription Factors Protein kinases Dnases Proteolysis of nuclear membrane and pore proteins Chromatin condensation Opened sites of nuclease hypersensitivity DNA Fragmentation P P P CYTOPLASM NUCLEUS Nuclear Envelope Nuclear Matrix Nuclear Pore Complex Proteolytic event PPhosphorylationChromatin Figure 1 24 2.0 1.0 0.6 0.4 0.2 kbp C +N -ICAD Zn EDTA 8.00 μm A B Figure 2 25 Nuclear basket ONM INM Nuclear Matrix Cytoplasmic filaments Nup 93 Nup 96 Nup 62? Nup 214 Nup 50 Lamin-like proteins Tpr/NUA Nup 136 Stage I Necklace condensation Ring peripheral condensation Collapse condensation Stage II Stage III 50 kpb Loop DNases 30 nm DNA filament “Beads on a string” DNA filament Internucleosomal DNases Caspase-like proteases Other proteases 180 bp oligomers DNA filaments DNA smear “Naked” DNA filaments Unspecific DNases 50 kbp oligomers DNA filament A Chromatin Condensation B DNA Fragmentation C Nuclear Envelope / Nuclear Pore Disassembly Figure 3