Life and death of lymphocytes: a volume regulation affair

The loss of cell volume, termed apoptotic volume decrease (AVD) has been a hallmark feature of apoptosis. However the role of this characteristic attribute of programmed cell death has always been questioned as to whether it plays an active or passive factor during apoptosis. Here we review studies that suggest that AVD plays an active role during apoptosis and the underlying flux of ions that results in this morphological event regulates the programmed cell death process.     

Cell volume regulation in immune cell apoptosis

The loss of cell volume is an early and fundamental feature of programmed cell death or apoptosis; however, the mechanisms responsible for cell shrinkage during apoptosis are poorly understood. The loss of cell volume is not a passive component of the apoptotic process, and a number of experimental findings from different laboratories highlight the importance of this process as an early and necessary regulatory event in the signaling of the death cascade. Additionally, the loss of intracellular ions, particularly potassium, has been shown to play a primary role in cell shrinkage, caspase activation, and nuclease activity during apoptosis. Thus, an understanding of the role that ion channels and plasma membrane transporters play in cellular signaling during apoptosis may have important physiological implications for immune cells, especially lymphocyte function. Furthermore, this knowledge may also have an impact on the design of therapeutic strategies for a variety of diseases of the immune system in which apoptosis plays a central role, such as oncogenic processes or immune system disorders. The present review summarizes our appreciation of the mechanisms underlying the early loss of cell volume during apoptosis and their association with downstream events in lymphocyte apoptosis.     

Cationic gradient reversal and cytoskeleton-independent volume regulatory pathways define an early stage of apoptosis

Cell shrinkage, or apoptotic volume decrease (AVD), is a ubiquitous characteristic of programmed cell death that is independent of the death stimulus and occurs in all examples of apoptosis. Here we distinguished two specific stages of AVD based on cell size and a unique early reversal of intracellular ions that occurs in response to activation of both intrinsic and extrinsic cell death signal pathways. The primary stage of AVD is characterized by an early exchange of the normal intracellular ion distribution for sodium from 12 to 113.6 mm and potassium from 139.5 to 30 mm. This early ionic reversal is associated with a 20-40% decrease in cell volume, externalization of phosphatidylserine, loss of mitochondrial membrane potential, and caspase activation and activity along with nuclear condensation that occurs independent of actin cytoskeleton disruption. Disruption of the actin cytoskeleton, however, prevents a secondary stage of AVD in apoptotic cells, characterized by a loss of both potassium and sodium that results in an 80-85% loss in cell volume, DNA degradation, and apoptotic body formation. Together these studies demonstrate that AVD occurs in two distinct stages with the earliest stage reflecting a cellular cationic gradient reversal.     

Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis

The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na(+), (86)Rb(+) studies reveal a rapid inhibition of K(+) uptake in response to anti-Fas. These effects on Na(+) and K(+) ions were accounted for by the inactivation of the Na(+)/K(+)-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na(+)/K(+)-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal, i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na(+)/K(+)-ATPase is a likely regulatory component of the death process.     

Osmotic stress resistance imparts acquired anti-apoptotic mechanisms in lymphocytes

Apoptosis is a stochastic, physiological form of cell death that is characterized by unique morphological and biochemical properties. A defining feature of apoptosis in all cells is the apoptotic volume decrease or AVD, which has been considered a passive component of the cell death process. Most cells have inherent volume regulatory increase (RVI) mechanisms to contest an imposed loss in cell size, however T-cells are unique in that they do not have a RVI response. We utilized this property to explore potential regulatory roles of a RVI response in apoptosis. Exposure of immature T-cells to hyperosmotic stress resulted in a rapid, synchronous, and caspase-dependent apoptosis. Multiple rounds of osmotic stress followed by recovery of cells in normal media resulted in the development of a population of cells that were resistant to osmotic stress induced apoptosis. These cells were also resistant to other apoptotic stimuli that activate via the intrinsic cell death pathway, while remaining sensitive to extrinsic apoptotic stimuli. Interestingly, these osmotic stress resistant cells showed no increase in anti-apoptotic proteins, and released cytochrome c from their mitochondria following exposure to intrinsic apoptotic stimuli. The osmotic stress resistant cells developed a RVI response, and inhibition of the RVI restored sensitivity to apoptotic agents. Analysis of apoptotic signaling pathways showed a sustained increase in phospho-AKT, whose inhibition also prevented an RVI response resulting in apoptosis. These results define a critical role of volume regulation mechanisms in apoptotic resistance.     

Uncoupling cell shrinkage from apoptosis reveals that Na+ influx is required for volume loss during programmed cell death

Cell shrinkage, or the loss of cell volume, is a ubiquitous characteristic of programmed cell death that is observed in all examples of apoptosis, independent of the death stimulus. This decrease in cell volume occurs in synchrony with other classical features of apoptosis. The molecular basis for cell shrinkage during apoptosis involves fluxes of intracellular ions including K+, Na+, and Cl-. Here we show for the first time that these ion fluxes, but not cell shrinkage, are necessary for apoptosis. Using sodium-substituted medium during anti-Fas treatment of Jurkat cells, we observed cellular swelling, a property normally associated with necrosis, in contrast to the typical cell shrinkage. Surprisingly, these swollen cells displayed all of the other classical features of apoptosis, including chromatin condensation, externalization of phosphatidylserine, caspase activity, poly(ADP)-ribose polymerase cleavage, and internucleosomal DNA degradation. These swollen cells had a marked decrease in intracellular potassium, and subsequent inhibition of this potassium loss completely blocked apoptosis. Reintroduction of sodium ions in cell cultures reversed this cellular swelling, resulting in a dramatic loss of cell volume and the characteristic apoptotic morphology. Additionally, inhibition of sodium influx using a sodium channel blocker saxitoxin completely prevented the onset of anti-Fas-induced apoptosis in Jurkat cells. These findings suggest that sodium influx can control not only changes in cell size but also the activation of apoptosis, whereas potassium ion loss controls the progression of the cell death process. Therefore cell shrinkage can be separated from other features of apoptosis.     

Staurosporine-induced apoptotic water loss is cell- and attachment-specific

Apoptotic volume decrease (AVD) is a characteristic cell shrinkage observed during apoptosis. There are at least two known processes that may result in the AVD: exit of intracellular water and splitting of cells into smaller fragments. Although AVD has traditionally been attributed to water loss, direct evidence for that is often lacking. In this study, we quantified intracellular water in staurosporine-treated cells using a previously described optical microscopic technique that combines volume measurements with quantitative phase analysis. Water loss was observed in detached HeLa and in adherent MDCK but not in adherent HeLa cells. At the same time, adherent HeLa and adherent MDCK cells exhibited visually similar apoptotic morphology, including fragmentation and activation of caspase-3. Morphological changes and caspase activation were prevented by chloride channel blockers DIDS and NPPB in both adherent and suspended HeLa cells, while potassium channel blocker TEA was ineffective. We conclude that staurosporine-induced dehydration is not a universal cell response but depends on the cell type and substrate attachment and can only be judged by direct water measurements. The effects of potassium or chloride channel blockers do not always correlate with the AVD.     

持续性细胞皱缩在人上皮细胞凋亡过程中的必要性

持续性细胞皱缩是凋亡发生的一个主要标志。近期研究发现细胞皱缩在细胞凋亡过程中并不是一个被动的次要事件。在各种细胞中,包括人上皮细胞,凋亡因子（apoptogen）刺激后马上发生全细胞皱缩,又称为凋亡性容积减小（apoptotic　volumede　crease,AVD）,继而发生caspase激活、DNA片段化、细胞破裂死亡。K^＋和Cl^-通道的激活导致KCl外流,诱导AVD发生。抑制AVD发生可以抑制细胞凋亡。AVD与调节性容积增加（regulatory　volume　increase,RVI）异常相伴发生时,人上皮性HeLa细胞发生持续性细胞皱缩。RVI功能受损时,高渗本身就能诱导HeLa细胞持续性细胞皱缩,继而凋亡。即使在正常渗透压、无凋亡因子刺激的情况下,将HeLa细胞置于缺乏Na^＋或Cl。的溶液也会导致细胞持续性皱缩,继而凋亡。因此,AVD诱导和RVI异常所导致的持续性细胞皱缩是人上皮细胞发生凋亡的首要条件。     

Glutathione depletion and disruption of intracellular ionic homeostasis regulate lymphoid cell apoptosis

Intracellular glutathione (GSH) depletion is an important hallmark of apoptosis. We have recently shown that GSH depletion by its extrusion regulates apoptosis independently of excessive reactive oxygen species accumulation. However, the mechanisms by which GSH depletion regulates apoptosis are still unclear. Because disruption of intracellular ionic homeostasis, associated with apoptotic volume decrease (AVD), is necessary for the progression of apoptotic cell death, we sought to evaluate the relationship between GSH transport and ionic homeostasis during Fas ligand (FasL)-induced apoptosis in Jurkat cells. GSH depletion in response to FasL was paralleled by distinct degrees of AVD identified by differences in cellular forward scatter and electronic impedance analysis. Inhibition of GSH efflux prevented AVD, K+ loss, and the activation of two distinct ionic conductances, mediated by Kv1.3 and outward rectifying Cl- channels. Reciprocally, stimulation of GSH loss accelerated the loss of K+, AVD, and consequently the progression of the execution phase of apoptosis. Although high extracellular K+ inhibited FasL-induced apoptosis, GSH depletion was largely independent of K+ loss. These results suggest that deregulation of GSH and ionic homeostasis converge in the regulation of apoptosis in lymphoid cells.     

Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl- conductance

We show that dysregulation of the Cl- homeostasis mediates the staurosporine-induced apoptotic cell death in human ECV304 cells. A pronounced apoptotic volume decrease (AVD), and an increase in plasma membrane Cl- conductance were early (<1 h) events following staurosporine challenge. Both processes were involved in apoptotic death, as demonstrated by the observation that the Cl- channel blocker phloretin inhibited both the staurosporine-evoked Cl- current and AVD, and preserved cell viability. Prolonged incubation (>2 h) with staurosporine caused a decrease in intracellular pH, which, however, was not required for the progression of the apoptotic process, because inhibitors of proton extrusion pathways, which lowered cytoplasmic pH, failed to inhibit both caspase-3 activation and DNA laddering. Moreover, clamping the cytosolic pH to an alkaline value did not prevent the apoptotic cell death. Collectively, these data demonstrate that staurosporine-mediated apoptosis of ECV304 cells is caused by the upregulation of Cl- channel activity and subsequent AVD, but is independent of intracellular acidification.     

Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease

Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation, and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C₆-ceramide. Overexpression of AQP-1 in Chinese hamster ovary (CHO-AQP-1) cells enhanced their rate of apoptosis. The AVD is driven by loss of K⁺ from the cell, and we hypothesize that after the AVD, AQPs become inactive, which halts further water loss and allows K⁺ concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes, and CHO-AQP-1 cells revealed that indeed, the shrunken (apoptotic) subpopulation has very low water permeability compared with the normal-sized (nonapoptotic) subpopulation. In thymocytes, AQP-1 is present and was shown to colocalize with the plasma membrane receptor tumor necrosis factor receptor-1 (TNF-R1) both before and after the AVD, which suggests that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis, and that inactivation after the AVD may help create the low K⁺ concentration that is essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane. water movement; major intrinsic protein; channel; enzyme     

Hypertonicity-induced cation channels rescue cells from staurosporine-elicited apoptosis

Cell shrinkage is one of the earliest events during apoptosis. Cell shrinkage also occurs upon hypertonic stress, and previous work has shown that hypertonicity-induced cation channels (HICCs) underlie a highly efficient mechanism of recovery from cell shrinkage, called the regulatory volume increase (RVI), in many cell types. Here, the effects of HICC activation on staurosporine-induced apoptotic volume decrease (AVD) and apoptosis were studied in HeLa cells by means of electronic cell sizing and whole-cell patch-clamp recording. It was found that hypertonic stress reduces staurosporine-induced AVD and cell death (associated with caspase-3/7 activation and DNA fragmentation), and that this effect was actually due to activation of the HICC. On the other hand, staurosporine was found to significantly reduce osmotic HICC activation. It is concluded that AVD and RVI reflect two fundamentally distinct functional modes in terms of the activity and role of the HICC, in a shrunken cell. Our results also demonstrate, for the first time, the ability of the HICC to rescue cells from the process of programmed cell death.     

Water and ion balance in rat thymocytes under apoptosis induced with dexamethasone or etoposide. Ion-osmotic model of cell volume decrease

Cell ion and water balance was studied with respect to analysis of the osmotic model of apoptotic volume decrease (AVD) in rat thymocytes under dexamethasone (1 microM, 4-6 h) or etoposide (50 microM, 5 h) treatment. Intracellular water content was determined by measurement of cell buoyant density in continuous Percoll gradient, while intracellular potassium and sodium contents were determined by flame emission analysis. Apoptosis was verified by an increase in cell buoyant density, fluorescence of cells stained with Acridine orange and Ethidium bromide (flow cytometry), by changes in the cell cycle and the appearance of sub-diploid peak in the DNA histogram (flow cytometry), and by a decrease in cell size examined with light microscope. A separate fraction of dense cells with reduced size was found to appear after dexamethasone or etoposide treatment. This fraction was considered as apoptotic. An increase in buoyant density of apoptotic cells corresponded to a decrease in cell water content. In apoptotic cells vs. cells with normal buoyant density, the intracellular potassium content was lower, but sodium content was higher. The sum of potassium and sodium contents was lower in apoptotic cells. Taken into account the loss of anions, associated with the loss of cations, the bulk decrease in ions content has been sufficient to be accounted for cell volume decrease on the basis of the ion-osmotic model.     

Apoptosis, cell volume regulation and volume-regulatory chloride channels

Apoptosis occurs in response to various stimuli under physiological and pathological circumstances. A major hallmark of the programmed cell death is normotonic shrinkage of cells. Induction of the apoptotic volume decrease (AVD) was found to precede cytochrome c release, caspase-3 activation and DNA laddering. A broad-spectrum caspase inhibitor blocked these biochemical apoptotic events but failed to block the AVD. The normotonic AVD induction was coupled to facilitation of the regulatory volume decrease (RVD), which is attained by parallel operation of Cl- and K+ channels, under hypotonic conditions. Both the AVD induction and RVD facilitation were prevented by application of a blocker of volume-regulatory Cl- or K+ channels. Furthermore, apoptotic cell death was rescued by channel blocker-induced prevention of AVD. Thus, it is concluded that the AVD is produced under normotonic conditions by a mechanism similar, though without preceding swelling, to RVD and represents an early prerequisite to apoptotic events leading to cell death. It was previously reported that hypertonic stress triggers apoptosis in cell types that lack the regulatory volume increase (RVI) mechanism. Taken together, it is suggested that 'disordered' or altered cell volume regulation is associated with apoptosis.     

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells placed into hyperosmotic medium

Time course of changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium with addition of 200 mM sucrose was studied. Ouabain-sensitive and ouabain-resistant Rb+ (K+) influxes were measured during regulatory cell volume increase (RVI) and apoptotic volume decrease (AVD). Microscopy of cells stained by Acrydine orange, Ethydium bromide, APOPercenrage Dye and polycaspase marker FLICA was performed. We found that initial osmotic cell shrinkage induced both RVI and AVD responses. RVI dominated at the early stage whereas AVD prevailed at the later stage. In view of the data obtained in U937 cells the current opinion that RVI "dysfunction" is a prerequisite for apoptosis and AVD (Subramanyam et al., 2010) should be revised. U937 cells are capable to trigger of apoptosis and AVD in spite of the unimpaired RVI response. It is concluded that AVD plays a significant role in preventing osmotic lysis of apoptotic cells rather than in the initiation of apoptosis.     

The potential role of caveolin-1 in inhibition of aquaporins during the AVD

BACKGROUND INFORMATION: During apoptosis, the first morphological change is a distinct cell shrinkage known as the AVD (apoptotic volume decrease). This event is driven by a loss of intracellular K(+), which creates an osmotic gradient, drawing water out of the cell through AQPs (aquaporins). Loss of water in balance with K(+) would create a shrunken cell with an equivalent intracellular concentration of K(+) ([K(+)](i) = 140 mM). However, we have previously shown that the [K(+)](i) of the shrunken apoptotic cell is 35 mM, and this level is absolutely essential for the activation of apoptotic enzymes. We have recently found that AQPs are inactivated following the AVD, so that continued loss of K(+) will reduce the intracellular concentration to this critical level. Using thymocytes, we have investigated the expression profile and regulation of the AQP family members. RESULTS: In the present study, we have found that AQP1, AQP8 and AQP9 are present in non-apoptotic thymocytes and localized primarily to the plasma membrane. Expression and localization did not change when these cells were induced to undergo apoptosis by growth factor withdrawal for 24 h. To explore other possible mechanisms by which these water channels are inactivated, we investigated their association with CAV-1 (caveolin-1), binding to which is known to inactivate a variety of proteins. We found that CAV-1 is present in thymocytes and that this protein co-localizes with a portion of AQP1 in normal (non-apoptotic) thymocytes. However, thymocytes induced to undergo apoptosis greatly increase their AQP1/CAV-1 association. CONCLUSIONS: Taken together, these results indicate that AQPs are localized to the plasma membrane of shrunken apoptotic thymocytes where increased binding to CAV-1 potentially inactivates them. AQP inactivation, coupled with continued K(+) efflux, then allows the [K(+)](i) to decrease to levels conducive for the activation of downstream apoptotic enzymes and the completion of the apoptotic cascade.     

Identification of potassium-dependent and -independent components of the apoptotic machinery in mouse ovarian germ cells and granulosa cells

Recent studies with thymocytes have suggested a critical role for intracellular potassium in the regulation of apoptosis. In this study, we examined the pathways of K(+) regulation during ovarian cell death. In initial studies, fluorographic analysis demonstrated a significant loss of K(+) during apoptosis stimulated by doxorubicin in oocytes and trophic hormone deprivation in granulosa cells. In oocytes, suppression of potassium efflux by potassium-enriched medium prevented condensation, budding, and fragmentation, although it did not block DNA degradation, suggesting the existence of potassium-independent nucleases in oocytes. Culture of granulosa cells in potassium-enriched medium inhibited internucleosomal DNA cleavage, although high-molecular weight DNA cleavage was apparent, suggesting that the nuclease or nucleases responsible for generating 50-kilobase (kb) fragments in these cells is potassium independent. To address this directly, isolated granulosa cell nuclei were stimulated to autodigest their DNA, and internucleosomal, but not large-fragment, cleavage was completely blocked by 150 mM potassium. We next examined whether the proapoptotic caspases are targets for potassium regulation. In cell-free assays, processing of pro-interleukin-1beta and proteolysis of cellular actin by recombinant caspase-1 and caspase-3, respectively, were suppressed by the presence of 150 mM potassium. Other monovalent ions (NaCl, LiCl) exerted a similar effect in these cell-free assays. Thus, in oocytes and granulosa cells, potassium efflux appears to occur early in the cell death program and may regulate a number of apoptotic events including caspase activity and internucleosomal DNA cleavage. However, there also exist novel potassium-independent pathways in both ovarian germ cells and somatic cells that signal certain apoptotic events, such as large-fragment DNA cleavage.     

Induction of apoptosis by nitric oxide in macrophages is independent of apoptotic volume decrease

Apoptosis occurs through a sequence of specific biochemical and morphological alterations that define the progress of cell death. The changes of the mitochondrial inner membrane potential (DeltaPsi(m)), the release of cytochrome c to the cytosol, the apoptotic volume decrease (AVD) and the activation of caspases have been measured in RAW 264.7, HeLa and Jurkat T cells incubated with molecules that induce apoptosis through the mitochondrial pathway. Our data show that NO, staurosporine, etoposide and camptothecin increased DeltaPsi(m) in macrophages but not in HeLa and Jurkat cells, that exhibited a DeltaPsi(m) decrease. Moreover, the apoptosis induced by NO in macrophages, but not that promoted by staurosporine, might occur in the absence of AVD. Analysis of the sequence of apoptotic manifestations shows that DeltaPsi(m) precedes AVD and caspase activation in RAW 264.7 cells. Inhibition of AVD abrogates apoptosis in HeLa and Jurkat T cells regardless of the stimuli used. These data suggest that the changes of DeltaPsi(m) are cell-type dependent and that AVD is dispensable for apoptosis in macrophages.     

Role of potassium channels in chlorogenic acid-induced apoptotic volume decrease and cell cycle arrest in Candida albicans

BackgroundChlorogenic acid (CRA) is an abundant phenolic compound in the human diet. CRA has a potent antifungal effect, inducing cell death in Candida albicans. However, there are no further studies to investigate the antifungal mechanism of CRA, associated with ion channels.MethodsTo evaluate the inhibitory effects on CRA-induced cell death, C. albicans cells were pretreated with potassium and chloride channel blockers, separately. Flow cytometry was carried out to detect several hallmarks of apoptosis, such as cell cycle arrest, caspase activation, and DNA fragmentation, after staining of the cells with SYTOX green, FITC-VAD-FMK, and TUNEL.ResultsCRA caused excessive potassium efflux, and an apoptotic volume decrease (AVD) was observed. This change, in turn, induced cytosolic calcium uptake and cell cycle arrest in C. albicans. Moreover, CRA induced caspase activation and DNA fragmentation, which are considered apoptotic markers. In contrast, the potassium efflux and proapoptotic changes were inhibited when potassium channels were blocked, whereas there was no inhibitory effect when chloride channels were blocked.ConclusionsCRA induces potassium efflux, leading to AVD and G2/M cell cycle arrest in C. albicans. Therefore, potassium efflux via potassium channels regulates the CRA-induced apoptosis, stimulating several apoptotic processes.General significanceThis study improves the understanding of the antifungal mechanism of CRA and its association with ion homeostasis, thereby pointing to a role of potassium channels in CRA-induced apoptosis.     

The morphology of apoptosis

The concept of apoptotic cell death as an essential part of the development and life of complex organisms has been devised in different situations and tested from various angles. This review article discusses the morphological changes during death by apoptosis. In cells undergoing apoptosis, an intracellular signalling pathway operates cell autonomously to implement the death and disposal of the cell. The similarity of the biochemical events during apoptosis in different situations is reflected by a high uniformity of morphological changes in many situations of naturally occurring or experimentally induced cell death. The unifying concept of apoptosis has been derived from the observation of this morphological consistency of dying cells almost 30 years ago. Since then, we have learned much about the intracellular signalling in the apoptotic process and the molecular background has been delineated which guides the initiation of the morphological changes. Here, an attempt is made to present the current knowledge about the molecular events in the development of these morphological alterations and to place these changes in the context of apoptotic cell death.