Disassembly of dying cells in diverse organisms

Abstract

Programmed cell death (PCD) is a conserved phenomenon in multicellular organisms required to maintain homeostasis. Among the regulated cell death pathways, apoptosis is a well-described form of PCD in mammalian cells. One of the characteristic features of apoptosis is the change in cellular morphology, often leading to the fragmentation of the cell into smaller membrane-bound vesicles through a process called apoptotic cell disassembly. Interestingly, some of these morphological changes and cell disassembly are also noted in cells of other organisms including plants, fungi and protists while undergoing ‘apoptosis-like PCD’. This review will describe morphologic features leading to apoptotic cell disassembly, as well as its regulation and function in mammalian cells. The occurrence of cell disassembly during cell death in other organisms namely zebrafish, fly and worm, as well as in other eukaryotic cells will also be discussed.

Cell disassembly in mammalian cells

Morphological changes for cell disassembly

The cell exhibits an active display of morphological changes from the onset of apoptosis to its final removal by phagocytes through efferocytosis. The biochemical pathways controlling the apoptotic process are well described, but the factors driving the physical dismantling of the cell are still being characterized. While cell death was first described in the mid-nineteenth century [1], the term ‘apoptosis’ and disassembly of apoptotic cells forming smaller membrane-bound vesicles called apoptotic bodies (ApoBDs) was first coined by Kerr et al. [2]. While initially thought to be unregulated, we now understand that apoptotic cell disassembly is an ordered and regulated process orchestrated by caspase proteases and rearrangement of the cytoskeletal framework [3]. The progression of apoptotic cell disassembly is divided into three distinct morphological steps namely apoptotic membrane blebbing, thin membrane protrusion formation and ApoBD formation [3–5] (Fig. 1a). Membrane blebbing is observed as the circular bulging and retraction of the plasma membrane. This step initiates with smaller blebs occurring on the plasma membrane, also termed as surface blebs, followed by larger dynamic blebs that involve the entire volume of the cell [5]. The plasma membrane of apoptotic cells can further extend forming thin membrane protrusions [4, 6, 7]. There are three distinct apoptotic membrane protrusions described in literature namely microtubule spikes [7], apoptopodia [6] and beaded apoptopodia [4]. Microtubule spikes were noted by Moss et al. as tubulin-rich rigid spikes in human epithelial cells [7]. Apoptotic cells can also form string-like membrane protrusions termed apoptopodia. Often apoptopodia have a bleb-like structure at the distal end away from the cell body and are observed in cell types including human T cells, primary mouse thymocytes and mouse fibroblasts [6]. Furthermore, beaded apoptopodia were observed in both human monocytic cell lines and CD14⁺ primary monocytes. The term ‘beaded’ was used to describe these protrusions as they resemble ‘beads-on-a-string’ and can extend up to eight times the length of the dying cell [4]. The last step of disassembly is the fragmentation of the apoptotic cell forming discrete ApoBDs. The size of ApoBDs generally ranges from 1 to 5 μm, representing one of the largest types of extracellular vesicles [4]. Numerous types of cells including T cells [6], thymocytes [6, 8] monocytes [4], endothelial cells [9, 10], smooth muscle cells [5] as well as basal epithelial cells [11] can undergo disassembly during apoptosis to generate ApoBDs in vitro and/or in vivo [3]. In particular, recent use of intravital multiphoton microscopy has captured mouse germinal centre B cells undergoing apoptotic cell disassembly, with apoptotic B cells progressing from blebbing to ApoBD formation in vivo [12].

Fig. 1.

Cell death-associated disassembly features noted in cells from different organisms. a Apoptotic mammalian cells undergo a stepwise apoptotic cell disassembly process initiated by membrane blebbing (step 1), followed by thin membrane protrusion formation (step 2) leading to the fragmentation of the cell forming ApoBDs (step 3). Positive and negative regulators of apoptotic cell disassembly are noted for each step. b Developmental apoptosis in embryonic zebrafish neurons exhibit cell disassembly morphologies. c Morphogenesis-driven apoptosis of epithelial cells show cell disassembly during leg development in D. melanogaster. d Apoptotic-like PCD in plant cells show vesicle formation within the cell vacuole and into the periplasmic space. eP. falciparum undergo apoptotic-like PCD-associated fragmentation, forming membrane-bound vesicles called ‘crisis’ forms in red blood cells

Regulation of apoptotic cell disassembly

Apoptotic membrane blebbing is thought to be driven by a number of protein kinases, in particular rho associated kinase 1 (ROCK1), a serine threonine kinase activated by caspase 3 cleavage of its autoinhibitory domain [13, 14]. Upon activation during apoptosis, ROCK1 phosphorylates myosin light chain (MLC), driving actinomyosin contraction and membrane blebbing [13–15]. The role of ROCK1 in this process was demonstrated by pharmacological studies [13, 14, 16] and overexpression of active ROCK1 fragment [14]. Additionally, p21 kinase 2 (PAK2) has also been reported as a positive regulator of apoptotic membrane blebbing downstream of caspase activation [17, 18]. However, expression of the reported active PAK2 domain did not induce or affect blebbing [14]. Caspase 3 activation during apoptosis can further facilitate membrane blebbing by proteolytic processing and deactivating MYPT1, an MLC-binding subunit of myosin phosphatase that functions to dephosphorylate MLC [16]. Furthermore, LIM-kinase 1 (LIMK1) has also been shown to regulate blebbing by blocking the activity of cofilin, an actin depolymeriser [19]. Whether all these molecular factors are required to work in concert (or certain factors are redundant) to mediate apoptotic membrane blebbing remains to be defined. It is interesting to note that anucleate erythrocytes undergoing eryptosis (apoptotic-like cell death) also exhibit membrane blebbing driven by calcium-dependent proteolysis of the cytoskeleton network [20–23]. However, as eryptosis-associated membrane blebbing was shown to proceed with or without activation of caspase 3 [20, 24, 25], it would be of interest to examine if the above kinases driving apoptotic membrane blebbing in nucleated cells are also involved in membrane blebbing during eryptosis.

Thin apoptotic membrane protrusions are formed during or after membrane blebbing and a number of subtypes have been described [4, 6, 7]. First, rigid microtubule spikes were shown to be tubulin-rich structures that could form subsequent to blebbing in apoptotic human epithelial cells [7]. Tubulin filaments were observed to be disassembled early in apoptosis [26], however reorganization of tubulin forming an apoptotic microtubule network was noted at later stages of apoptosis [27, 28], which could aid membrane integrity by limiting caspase access to the cellular cortex [29]. Whether microtubule spikes are an extension of the apoptotic microtubule network or are independently formed structures remains to be defined. The other subtypes of apoptotic membrane protrusions, namely apoptopodia and beaded apoptopodia, were shown to be negatively regulated by pannexin 1 (PANX1) [4, 6]. PANX1 is a membrane channel that could become activated during apoptosis following caspase-mediated cleavage at its C-terminus. Interestingly, PANX1 also functions to release various molecules like ATP, which could act as a ‘find-me’ signal to mediate recruitment of macrophages for cell clearance [30, 31]. Although PANX1 can negatively regulate the generation of apoptopodia during apoptosis in a number of different cell types [4, 6], how PANX1 regulates apoptopodia formation and whether this is linked to the release of certain molecules via this channel is still unclear. Nevertheless, it should be noted that the formation of apoptopodia can be regulated by vesicular trafficking as determined by inhibitor studies [4], yet precisely which molecular components are involved and how this process is regulated requires further determination.

As described above, disassembly of apoptotic cells into ApoBDs involves the formation of plasma membrane blebs and thin membrane protrusions like apoptopodia. Thus, molecular regulators of these apoptotic morphologies, namely ROCK1 and PANX1, are also key regulators of ApoBD formation. In particular, the formation of ApoBD is negatively regulated by PANX1, whereby either inhibition of the channel pharmacologically, expression of a dominant negative mutant, or targeting PANX1 expression genetically, enhanced cell disassembly during apoptosis [4, 6]. Interestingly, gasdermin E (GSDME, also known as DFNA5) was also shown recently to negatively regulate ApoBD formation in human embryonic kidney cells and mouse bone marrow-derived macrophages [32]. During apoptosis, GSDME is activated by caspase 3-mediated cleavage and can subsequently localize to the plasma membrane to initiate secondary necrosis [32, 33]. As a consequence of secondary necrosis, intracellular contents including danger-associated molecular patterns such as double-stranded DNA can be released from cells [34]. Loss of GSDME expression in macrophages promoted the formation of ApoBDs, suggesting the early onset of secondary necrosis driven by GSDME limits the cell to undergo morphological changes necessary for apoptotic cell disassembly [32]. However, these studies on GSDME present an enigmatic process, as it does not align with the non-inflammatory nature of apoptotic cell death. Furthermore, given that ApoBD formation is noted in many in vitro and in vivo settings, what limits the function of GSDME in cells undergoing apoptotic cell disassembly is unclear. Therefore, while some of the regulators of apoptotic cell disassembly have been identified, the molecular mechanism underpinning the generation of ApoBDs, in particular the final separation of ApoBDs from the apoptotic cell body or other ApoBDs remains largely undetermined (Table 1).

Table 1.

Cell disassembly in different cell types undergoing cell death

Organism	Cell type (tissue)	Cell death stimulus	Disassembly description	References
Human	T cells	Anti-Fas treatment or UV irradiation	Apoptotic cells first underwent cell rounding followed by dynamic membrane blebbing leading to ApoBD formation. Apoptopodia (thin string-like protrusion) were noted with inhibition of PANX1 function	[6]
	Monocytes	UV irradiation or serum starvation	Apoptotic cells displayed dynamic blebbing with small bleb structures, followed by the formation of thin membrane extensions (apoptopodia) that exhibited a ‘beaded’ morphology. Two types of beaded apoptopodia were noted with uniform sized and non-uniform-sized vesicles along the length of the membrane protrusion. Detachment of these vesicles from the cell/protrusions generated ApoBDs	[4]
	Endothelial cells	UV irradiation or serum starvation and tumour necrosis factor-alpha treatment	ApoBDs expressing same surface markers as viable endothelial cells were noted	[9, 10]
	Epithelial cells	UV irradiation	Apoptotic cells displayed dynamic membrane blebbing followed by the formation of rigid microtubule-rich membrane protrusions or ‘spikes’ with a vesicle-like structure at the distal end. Detachment of these vesicles from microtubule spikes to form ApoBDs was observed	[7]
Mouse	Thymocytes	Dexamethasone treatment	Apoptotic cells showed dynamic membrane	[6]
	B cells (lymph nodes)	Immunization with NP15-OVA and HIV envelope antigen	Bleb formation and cell fragmentation was noted in apoptotic cells	[12]
Rat	Thymocytes	X-irradiation (whole body)	Apoptotic cells and apoptotic cell fragments (ApoBDs) with round morphology and smooth surfaces were noted	[8]
Zebrafish	Embryonic cells (cell type and tissue unknown)	Camptothecin treatment	Apoptotic cells showed transient blebbing prior to nuclear fragmentation	[65]
	Endothelial cells	Embryonic development	Apoptotic cells showed membrane blebbing and ApoBD formation	[66]
	Lens retinal cells	Embryonic development	Apoptotic cells showed large membrane blebs and fragmentation	[67]
	Neurons	Embryonic development or UV ablation	Apoptotic cells showed cell body rounding with fragmentation of axon and dendrites Anterograde blebbing followed by axonal fragmentation was noted in apoptotic cells	[67, 68]
D. melanogaster	Embryonic cells (epidermal layer)	Embryonic development or X-irradiation	Apoptotic cells exhibited extensive membrane blebbing and fragmentation into ApoBDs	[80]
	Embryonic cells (cell type and tissue unknown)	DAKT mutation	Ectopic apoptotic cells displayed membrane blebbing	[82]
	Epithelial cells (leg)	Morphogenesis	Apoptotic cells exhibited membrane blebbing and ApoBD formation	[84, 85]
	Squamous cells (imaginal discs)	Morphogenesis	ApoBD generated from apoptotic cells were noted to accumulate in the columnar epithelium	[86]
	Nurse cells (ovaries)	Oogenesis, etoposide or staurosporine treatment	Single membrane-bound vesicles containing fragmented DNA were noted Small vesicles containing chromatin from apoptotic cells were detected in egg chambers	[88]
C. elegans	Neurons	Developmental	Apoptotic cells showed membrane vesicularization and fragmentation	[99]
Tomato	Cells from seedlings (cell type and tissue unknown)	Fumonisin B1 treatment	ApoBD-like vesicles were noted from protoplasts of cells undergoing apoptotic-like PCD	[110]
Tobacco	Suspension cells (cell type and tissue unknown)	Heat (55 °C) treatment	ApoBD-like vesicles were noted 24 h post heat treatment	[112]
Wheat	Parenchymal cells (coleoptile)	Ageing	Cytoplasmic bodies were noted budding into the vacuole region of dying cells containing mitochondria and plastids	[118]
	Cells unknown (leaf apical area)	Ageing	Vesicles containing mitochondria were noted in the vacuole region of cells undergoing apoptotic-like PCD	[118]
Pepper	Mesophyll cells (leaf)	Bacterial race 2 infection	Spherical membranous bodies noted in vacuoles of cells undergoing apoptotic-like PCD	[119]
T. brucei	n/a	Con A treatment	Membrane vesicularization (blebbing) noted 3 h post-cell death stimulus	[140]
T. cruzi	n/a	Treatment with geneticin (G418), staurosporine or sera (containing complement), and nutrient deficiency (non-renewal of growth media) driven differentiation	Dying organisms examined by electron microscopy show extensive membrane blebbing	[135]
P.  berghei	n/a	Life cycle driven	Membrane blebbing and bodies (ApoBD-like) noted in organisms undergoing apoptotic-like PCD	[147]
P. falciparum	n/a	Treated with sera from individuals living in malarial infected regions, chloroquine or etoposide treatment	Fragmentation of the organism, generating ‘crisis forms’ noted Formation of membrane-bound fragments or ‘crisis forms’ can be blocked by pan caspase inhibitor Z-VAD-FMD	[146, 150]

Function of apoptotic cell disassembly

Apoptotic cells are removed from the tissue via efferocytosis by either neighbouring or immune cells. Defects in the timely clearance of apoptotic cells allow them to proceed to secondary necrosis, which is linked to the development of a number of diseases [3, 34–37]. For example, impaired apoptotic cell clearance in mouse models presented with lupus-like autoimmune features [38]. In humans, sera of a subset of patients with the autoimmune disorder systemic lupus erythematosus contained elevated levels of apoptotic debris and autoantibodies to intracellular moieties [39–41]. Moreover, studies in mouse models linked impaired cell clearance to inflammation associated with colitis [42]. Uncleared apoptotic debris accumulation in atherosclerotic plaques has also been shown to drive inflammation via macrophage and T cell recruitment [43, 44]. Thus, efficient clearance of apoptotic cells is imperative to maintain tissue homeostasis. To ensure rapid removal of apoptotic debris, the dying cell can promote phagocyte recruitment and recognition by the release/exposure of ‘find-me’ and ‘eat-me’ signals, respectively [36, 37]. In addition to these mechanisms, the disassembly of apoptotic cells can also aid cell clearance. For instance, apoptotic membrane blebs are known to harbour endoplasmic reticulum (ER) membranes. The active shuttling of ER was observed in membrane bleb regions [40] and ER membranes were also noted on the outer bleb surfaces that can expose molecules capable of mediating phagocyte recognition [45]. In particular, the exposure of calreticulin (an ER membrane protein) facilitated the binding of C1q and mannose-binding lectin to apoptotic cells and promoted macrophage recognition [46, 47]. Furthermore, pharmacological inhibition of membrane blebbing in apoptotic T cells reduced their uptake by monocyte-derived macrophages [48], supporting the concept that morphological changes during apoptosis could aid the cell clearance process. However, this latter study did not address the mechanism of how apoptotic membrane blebbing is linked to engulfment. Interestingly, ApoBD formation was shown to facilitate efferocytosis, whereby blocking apoptotic neuronal cell fragmentation pharmacologically decreased the engulfment of apoptotic materials by monocyte-derived macrophages [49]. Additionally, the formation of apoptotic membrane protrusions like microtubule spikes in epithelial cells were shown to provide extended surfaces to facilitate their interaction with phagocytes and further aiding cell clearance [7]. Whether the generation of other types of thin apoptotic membrane protrusions like apoptopodia and beaded apoptopodia may also promote phagocyte interaction remains to be defined.

In addition to cell clearance, disassembly of apoptotic cells can also facilitate intercellular communication via ApoBDs harbouring biomolecules like DNA and protein [3, 4, 50]. The DNA contained in ApoBDs has been shown to facilitate horizontal gene transfer, whereby Epstein–Barr virus DNA carrying lymphoid cells were able to distribute viral DNA into ApoBDs and their uptake by foetal fibroblasts resulted in the integration and expression of Epstein–Barr viral proteins in the recipient cell [51]. Similarly, oncogenes were also shown to be transferred via ApoBDs from H-ras^V12 and human c-myc transfected rat fibroblasts to recipient mouse embryonic fibroblasts to propagate tumorigenicity in the latter [52]. Endothelial cell-derived ApoBD were also noted to harbour the proinflammatory cytokine interleukin 1 alpha, which when added to viable endothelial cells induced secretion of interleukin 18 and monocyte chemotactic protein-1 [10]. Additionally, ApoBDs generated from epithelial cells carrying alpha-fodrin and Sjögren’s syndrome-related antigen A were shown to induce secretion of proinflammatory cytokines tumour necrosis factor alpha and interleukin 8 from recipient dendritic cells [53]. Together, these examples show that biomolecules carried by ApoBDs can play a number of roles in both physiological and disease settings.

Cell disassembly in zebrafish

The use of zebrafish as a model organisms has numerous advantages especially the ease of genetic manipulation and in vivo imaging in the transparent embryos (reviewed in [54]), providing an unparalleled system to study both physiological and pathological processes [55–58]. The study of apoptosis is of particular interest as the zebrafish has a conserved canonical apoptotic pathway to mammals (reviewed in [59]). Notably, zebrafish pro-apoptotic proteins were functional in mammalian cells whereby expression of zebrafish Bcl-2-associated death promoter (bad) induced DNA fragmentation and chromatin condensation in monkey kidney fibroblast-like cells [60]. Additionally, BH3-only peptides from zebrafish bim, noxa and puma promoted cytochrome c release from mitochondria isolated from mouse embryonic fibroblasts (MEFs) but not from mitochondria isolated from Bax and Bak double-knockout MEFs, indicating zebrafish BH3-only peptides interact with murine pro-apoptotic proteins [61]. More importantly, zebrafish executioner caspases have conserved protein substrates to their mammalian homologs [62, 63] whereby recombinant zebrafish caspase 3a showed high cleavage activity toward the DEVD peptide sequence, similar to caspase 3 and 7 in mammals [64].

The apoptotic cell disassembly process can be observed in a variety of cell types in zebrafish. For example, camptothecin-induced apoptosis in deep cell layer of zebrafish embryos revealed cells undergoing surface membrane blebbing [65]. Furthermore, endothelial cells undergoing apoptosis during pruning of brain blood vessels showed blebbing and cell fragmentation, which were subsequently cleared by microglial cells [66]. Additionally, in vivo imaging of apoptotic cells in the anterior brain of the zebrafish during embryonic development revealed apoptotic lens retina and transganglion cells undergoing blebbing and cell fragmentation forming ApoBD-like vesicles [67]. Zebrafish neurons undergoing apoptosis during embryogenesis also displayed certain cell disassembly morphologies, in which spinal cord interneuron cell body exhibited rounding while the dendrites and axon simultaneously fragmented and readily disappeared (perhaps through clearance), leaving the cell body behind [67] (Fig. 1b). A similar process of apoptotic cell disassembly was observed in UV-ablated neurons in zebrafish embryos at 2–4 days post-fertilization, where the cell showed anterograde (from cell body to axons and dendrites) blebbing and axonal fragmentation. Additionally, the cell body (soma) was cleared by microglia-mediated engulfment over several hours [68].

Similar to mammalian cells, ROCK1 is essential for apoptotic membrane blebbing in zebrafish cells, in which pharmacological inhibition of ROCK1 in whole embryos using Y-27632 reduced blebbing in apoptotic neural tube cells [69]. Notably, zebrafish ROCK1 protein has a conserved caspase 3 cleavage site as mammalian ROCK1, suggesting that zebrafish ROCK1 may also be activated during apoptosis via caspase-mediated cleavage in a similar manner to its mammalian homolog. Interestingly, ROCK1-mediated membrane blebbing in apoptotic neural tube cells during embryonic development promoted directional migration of these cells from the apical region to the central nervous system periphery (basolateral side), which assisted their clearance by microglia [69]. While the mechanism underlying the fragmentation of apoptotic cells in zebrafish is not well defined, a number of cell types in zebrafish also express PANX1 [70], a negative regulator of ApoBD formation in mammalian cells as described above [4, 6]. Furthermore, the caspase cleavage site characterized in mammalian PANX1 [31], is also conserved in zebrafish. Thus, it would be of interest to examine if zebrafish PANX1 functions in a conserved role in apoptotic cell disassembly. Overall, zebrafish cells show many conserved morphological, regulatory and functional aspects of apoptotic cell disassembly to mammalian cells and would be a useful model to further study this process.

Cell disassembly in D. melanogaster

D. melanogaster is a commonly used model to study the cell death process as apoptosis is important throughout the fly life cycle from embryogenesis to the adult form [71], and the role of caspases in apoptosis of metazoans is conserved [72]. In D. melanogaster, seven caspases orchestrate apoptotic cell death (reviewed in [71, 73]). Among these caspases, drICE is the key executioner caspase and is functionally similar to mammalian caspase 3 [71, 74]. Multiple other apoptotic signalling molecules are also conserved between D. melanogaster and mammals (reviewed in [75]), including Dronc (homolog of caspase 9) [76] and Ark (homolog of Apaf-1) [77]. Moreover, D. melanogaster Bok expression in human embryonic kidney cells induced cytochrome c release and apoptosis similar to mammalian pro-apoptotic Bcl-2 family members [78].

While the disassembly of apoptotic cells is typically not studied using the D. melanogaster model, cellular morphologies associated with cell disassembly were observed in various studies and often used as a hallmark of apoptotic cell death [79]. During embryogenesis, apoptotic cells in the epidermal layer showed fragmentation into ApoBDs as observed by confocal and electron microscopy [80], and these ApoBDs were also observed within phagocytic vesicles of hemocytes and neighbouring cells [80, 81]. Furthermore, embryo expressing mutants of D. melanogaster AKT (protein kinase B, a pro-survival protein) showed increase in apoptosis and dying cells exhibited apoptotic membrane blebbing [82]. Another phase of large turnover of cells in D. melanogaster by various cell death pathways including apoptosis is morphogenesis [71, 83, 84]. Time-lapse imaging of apoptotic epithelial cells in the developing leg during morphogenesis showed membrane blebbing and subsequent cell fragmentation forming distinct ApoBDs [84, 85] (Fig. 1c). Likewise, ApoBDs were also observed from squamous cell apoptosis in imaginal wing disc during morphogenesis [86]. Furthermore, during oogenesis in the adult fly, ex vivo examination of ovaries showed apoptotic nurse cells undergoing blebbing and fragmentation into ApoBDs [87, 88].

The cytoskeletal machinery is important for apoptotic cell disassembly in mammalian cells [13, 14, 89]. In D. melanogaster, cytoskeletal rearrangements forming actin bundling is noted in apoptotic nurse cells during oogenesis, but its function is not defined [87]. Ectopic induction of apoptosis in the wing disc also showed stabilization of filamentous actin in dying cells [84]. It is also interesting to note that one of the key drivers of apoptotic membrane blebbing in mammalian cells, myosin II [13, 14, 89], is also activated downstream of the pro-apoptotic protein reaper in D. melanogaster [84]. While the role of myosin II is described during morphogenesis in D. melanogaster [84, 86, 90], its activation is also noted in non-morphogenetic apoptosis [79] and may potentially drive apoptotic membrane blebbing. Although it is unclear if the disassembly of apoptotic cells aids cell clearance in D. melanogaster, injured and degenerated neurons were fragmented by filamentous actin structures in neighbouring epidermal epithelial cells prior to their engulfment by the latter [91]. Interestingly, this process is also observed in mammalian tissue where adipocytes are fragmented and cleared by multiple macrophages in mice [92], yet the role of phagocytes in assisting the apoptotic cell disassembly process to aid cell clearance is not well understood. Multiple questions remain undefined regarding the disassembly of apoptotic cells in D. melanogaster, in particular the underpinning molecular mechanism and the function of this process throughout the life cycle.

Cell disassembly in C. elegans

Pioneering work using C. elegans as a model organism uncovered many molecular mechanisms and regulation of apoptotic cell death [93], including genes that control the execution of apoptosis (ced-3, ced-4, ced-9, and egl-1) as well as recognition and removal of apoptotic cells (ced-1, ced-2, ced-5, ced-6, ced-7, ced-10 and ced-12) (reviewed in [94]). During development in C. elegans, apoptosis is pre-programmed where invariably 131 cells out of a total 1090 cells will die by this process [95–97]. Morphologically, these apoptotic cells became refractive as determined by Nomarski (differential interference confocal) microscopy [98]. Detailed examination of an apoptotic nerve cell in the ventral nerve cord showed whirling of internal and external membranes, followed by loss of cell shape and formation of membrane-bound remains [99]. Fragmentation of late apoptotic cells were also noted when genes namely ced-5, ced-6, ced-7 and ced-10 involved in cell clearance were mutated [100]. Thus, while fragmentation of dying cells is evident in C. elegans, the morphological steps of apoptotic cell disassembly including membrane blebbing and thin protrusion formation were not observed. Interestingly, the lack of clear morphological features during apoptosis may be linked to how apoptotic cells are cleared in C. elegans. Cell engulfment in C. elegans has been shown to actively promote apoptosis, whereby worms harbouring mutations in any engulfment gene reduced the number of cells proceeding to apoptosis [101, 102]. Furthermore, apoptotic cells can be cleared prior to any observable morphological features [101]. Thus, it is likely that apoptotic cell disassembly is not needed in C. elegans as the cell clearance process is efficient and/or early engulfment of the apoptotic cell does not leave sufficient time for most cells to disassemble.

Cell disassembly in other organisms

While apoptotic pathways are well described in metazoans, similar pathways termed apoptosis-like PCD are also characterized in plants, fungi and Protista. This section will discuss the cellular morphology associated with apoptosis-like PCD and drawing parallels to the mammalian apoptotic cell disassembly process.

Cell disassembly in plants?

Programmed cell death in plants is characterized into three categories, namely apoptotic-like PCD, senescence and, vacuole-mediated cell death [103]. In metazoans, apoptosis is a caspase-dependent process, whereas no caspases have been identified in plants. However, other caspase-like proteases and metacaspases are shown to drive apoptotic-like PCD in plants [103]. The terminology ‘apoptotic-like’ to describe this process including the functional similarity of metacaspases to caspases is still a topic of debate [103–105]. Interestingly, expression of anti-apoptotic proteins such as human Bcl-2, chicken Bcl-xL and C. elegans CED-9 in tobacco plants conferred resistance to herbicide-induced cell death [106], and expression of pro-apoptotic murine Bax in tobacco plants was able to induce hypersensitive response (apoptosis-like PCD surrounding viral-infected cells) [107]. While these suggest some conservation, however, as the molecular machinery driving apoptosis-like PCD in plants is not fully defined [103, 108, 109], it is uncertain how these overexpression studies relate to apoptosis described in metazoans.

Interestingly, plants including tomato (root, stem, cotyledon tissues and suspension cell cultures) [110], carrot (embryonic cells from hypocotyl explants) [111], tobacco (leaf disc and suspension cell culture) [110, 112], mustard (suspension cell culture) [113] and sunflower (anther) [114] showed cells undergoing apoptotic-like PCD exhibited features similar to apoptosis when induced in culture, but also during development and in response to pathogens (reviewed in [103, 115]). These features include shrinking of the cell away from the cell wall and membrane blebbing [111], DNA condensation and laddering [110–112], cytochrome c release [113, 114] and, loss of membrane asymmetry and exposure of phosphatidylserine [116, 117]. The fragmentation of the dying plant cell into membrane-bound vesicles akin to ApoBDs have been observed in tomato seedlings treated with mycotoxin and in heat (55 °C) treated tobacco cell cultures [110, 112]. However, these observations have not been reported in whole plant tissue [103]. Additionally, vesicles containing organelles including the mitochondria are seen budding into the vacuole space of wheat leaf and coleoptile cells undergoing ageing-driven apoptotic-like PCD when examined by electron microscopy [118] (Fig. 1d). Similar vesicles were also observed in pepper leaf cells infected with bacterial race 2 to cause hypersensitive cell death [119]. As to how and why these cells undergo fragmentation in plants is uncertain given that the cell clearance process is not evident and probably not necessary in plant tissue. Moreover, the presence of the cell wall hinders interaction with neighbouring cells, thus limiting the possibility of these plant-derived vesicles mediating intercellular communication. However, plant cells have been reported to secrete and deliver exosome-like extracellular vesicles containing small RNA to invading fungal pathogen cells [120], thus the cell wall may not be completely impermeable to the trafficking of extracellular vesicles. A number of questions including the regulation of apoptotic-like PCD still need to be characterized before we begin to unfold the role of these associated morphological features in plants.

Cell disassembly in fungi?

Apoptosis has been described in a number of fungal species, with the majority of studies focussing on yeast models including S. cerevisiae and the pathogenic C. albicans [121, 122]. Apoptosis in yeast is dependent on a number of regulators including metacaspase yCa1p, an ortholog of mammalian caspases [123, 124]. Moreover, study of apoptosis in yeast has served as a model to study human diseases including neurodegeneration, cardiovascular disease and cancer [124–127].

Apoptosis in yeast shows a number of features that are similar to mammalian cells undergoing apoptosis. These include the condensation of chromatin, DNA fragmentation, externalization of phosphatidylserine, and depolarization of the mitochondrial matrix [128, 129]. However, morphological changes associated with mammalian apoptotic cell disassembly are absent. Notably, apoptotic yeast cells displayed enlargement rather than cell shrinkage as observed in mammalian apoptosis [128]. Interestingly, tiny buds containing chromatin have been noted on the apoptotic cell plasma membrane, which were suggested to be precursors of vesicles like ApoBDs [129]. However, it should be noted that these tiny buds on the plasma membrane did not separate from the dying cell [129]. It is also worth noting that yeast undergoing cell death can release factors such as nutrients that can promote survival of other cells in the colony [130, 131], but unlike multicellular organisms there may be no need for these factors to be packaged into membrane-bound vesicles.

Cell disassembly in Protista (Protozoa)

A number of protozoans have been reported to exhibit apoptosis-like PCD [132–134] and much of our understanding of this process comes from the studies of parasitic protozoans including Trypanosoma [135–140], Leishmania [141–144], Plasmodium [145–147] and Toxoplasma [148, 149]. Factors that could promote apoptotic-like PCD in these organisms including density control, immune silencing, differentiation and stress response [133]. The molecular mechanism underpinning apoptosis-like PCD in protozoans involves metacaspases and has been suggested to have divergently evolved to apoptosis in mammals [132]. Interestingly, apoptotic features such as cell shrinkage, chromatin condensation, DNA fragmentation, loss of mitochondrial membrane potential and externalization of phosphatidylserine are noted in protozoans undergoing apoptotic-like PCD [132–134].

In addition to apoptotic features mentioned above, morphological changes similar to certain stages of mammalian apoptotic cell disassembly are also observed in protozoans. T. brucei treated with Con A to induce apoptotic-like PCD showed surface membrane vesicularization (resembling blebbing) [140]. T. cruzi undergoing cell death in vitro during differentiation and induction by treatments including complement containing sera also showed membrane blebbing when examined by electron microscopy [135]. Additionally, electron micrographs of P. berghei ookinetes undergoing apoptosis-like PCD in vivo (midgut of A. stephensi) or in vitro cultures showed membrane blebs and the presence of ApoBD-like vesicles [147]. Furthermore, when examining immune sera-induced apoptosis-like PCD in P. falciparum within erythrocytes (ex vivo), ‘crisis form’ characterized by disintegration of the parasite were noted [150]. These disintegrated fragments or ‘crisis forms’ were also observed with etoposide- and chloroquine-induced P. falciparum cell death, and were suggested to be related to ApoBDs generated from mammalian cells [146, 151] (Fig. 1e). Notably, the generation of these ‘crisis forms’ by P. falciparum undergoing apoptotic-like PCD could be blocked by the pan caspase inhibitor Z-VAD-FMK [146]. Although the function of this cell disassembly process in protozoans is not well defined, it is suggested—that packaging of pathogen cell contents by vesicle formation may prevent direct presentation of antigen to the host cell [151]. Thus, understanding how and why these parasitic protozoans disassemble during apoptosis-like PCD may provide novel therapeutic targets to treat parasitic infections.

Concluding remarks

It is evident that features of cell disassembly are noted in cells from various organisms undergoing apoptosis or apoptotic-like PCD. These observations however raise several intriguing questions including how the disassembly of dying cells is regulated and whether this is a conserved phenomenon or if this process is independently developed through convergent evolution. Moreover, the formation of ApoBDs and similar vesicles has been shown to play physiologically relevant roles in mammals. Thus, it would be of interest to further study the role of dying cell disassembly in processes such as cell clearance and intercellular communication in other organisms.