Apoptosis, or programmed cell death, is an essential physiological process that is required for the normal development and maintenance

of tissue homeostasis1. When misregulated, apoptosis can contribute to various diseases including cancer and autoimmune and neurodegenerative diseases. Central components of the apoptotic death machinery, which have been conserved throughout evolution, include the Bcl-2, Apaf-1 (apoptotic proteaseactivating factor 1) and caspase family members. Caspases (cysteinyl aspartate-specific proteases) are synthesized as dormant proenzymes that, upon proteolytic activation, acquire the ability to cleave key intracellular substrates2, resulting in the morphological and biochemical changes associated with apoptosis (Box 1). Recently, it was found that, in apoptosis triggered by many stimuli, mitochondria play a pivotal role in coordinating caspase activation through the release of cytochrome c. This was first suggested by a pioneering study in which mitochondria were required for cytosolic extracts to induce the morphological changes typical of apoptosis in nuclei isolated from Xenopus eggs3. Further fractionation of cytosolic extracts and subsequent purification revealed that mitochondrial cytochrome c was necessary to activate caspases, along with dATP and Apaf-1 (Ref. 2). The current model in mammalian cells is that, once it is in the cytosol, cytochrome c binds to Apaf-1. In the presence of ATP or dATP, this complex recruits and activates procaspase 9. Activated caspase 9 can, in turn, activate other caspases that are in charge of the cells execution. Similar mechanisms have recently been described in Drosophila4. In addition to cytochrome c, many other proteins normally confined to the intermembrane space of

Mitochondria as the central control point of apoptosis

Solange Desagher and Jean-Claude Martinou

Mitochondria play a major role in apoptosis triggered by many stimuli. They integrate death signals through Bcl-2 family members and coordinate caspase activation through the release of cytochrome c as a result of the outer mitochondrial membrane becoming permeable. The mechanisms that lead to this permeability are not yet completely understood. Here, we attempt to summarize our current view of the mechanisms that lead to the efflux of many proteins from mitochondria during apoptosis and the role played by Bcl-2 family proteins in the control of this event.

BOX 1 APOPTOSIS VERSUS NECROSIS The term apoptosis is currently used as a synonym of programmed cell death. However, it was originally used to describe the morphological characteristics of a certain type of cell death, as opposed to necrosis9. During necrosis, the cell swells, its mitochondria dilate, other organelles dissolve and the plasma membrane ruptures, releasing cytoplasmic material; this often elicits an inflammatory response. By contrast, during apoptosis, the cytoplasm shrinks and the chromatin condenses, but the organelles retain their integrity. The plasma membrane blebbs and exposes phosphatidylserine on its outer surface, which is normally retained in the inner leaflet. However, the plasma membrane does not rupture, preventing the release of cellular compounds into the extracellular medium. In vitro, apoptotic cells ultimately fragment into membrane-enclosed vesicles (apoptotic bodies), whereas, in vivo, they are recognized and removed by phagocytes, thereby avoiding inappropriate inflammation. Biochemical hallmarks of apoptosis also include the activation of endonucleases, DNA degradation into oligonucleosomal fragments and the activation of a family of cysteine proteases called caspases.

mitochondria, such as apoptosis-inducing factor5 (AIF), are released during apoptosis. However, so far, only cytochrome c appears to be essential for apoptosis6. In some systems, such as neurons upon growth-factor deprivation7 or fibroblasts upon c-myc expression8, neutralizing cytochrome c by injecting antibodies into the cytosol is enough to protect the cells from apoptosis. The release of mitochondrial proteins, as we will see, is controlled by the Bcl-2 family members. The main objective of this article is to analyse the mechanisms by which, during apoptosis, the outer mitochondrial membrane becomes permeable to many proteins, including cytochrome c. Other mechanisms through which mitochondria could contribute to cell death, such as the production of reactive oxygen species, are not addressed here. Morphological changes and cellular redistribution of mitochondria According to standard morphological descriptions, mitochondria were long thought to remain unchanged during apoptosis9 but to swell during necrosis (Box 1). However, a review of the past literature on cell death, before apoptosis had even been described, reveals abnormal mitochondria in types of cell death that, retrospectively, can be classified as apoptosis. The most frequent abnormalities are a reduction in mitochondria size and a hyperdensity of their matrix, features often referred to as mitochondrial pyknosis1012. Similar observations (i.e. a transition from an orthodox to a condensed conformation) were recently confirmed as early apoptotic events1315. Experiments performed on sympathetic neurons have shown that,

Solange Desagher is at the UPR CNRS 9023, CCIPE, 141 rue de la Cardonille, F-34094 Montpellier Cedex 5, France; JeanClaude Martinou is at Serono Pharmaceutical Research Institute, chemin des Aulx 14, CH-1228 Plan-les-Ouates, Geneva, Switzerland. E-mail: desagher@ ccipe.montp. inserm.fr, Jean-Claude. Martinou@ Serono.com

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FIGURE 1
Morphological changes and redistribution of mitochondria in HeLa cells overexpressing Bax. (a) Control HeLa cells immunostained with an antibody against mitochondrial Hsp70 in order to reveal mitochondria. (b,c) Mitochondria from HeLa cells labelled with the same antibody, 15 h after transfection with a Bax-encoding cDNA in the presence of the peptide caspase inhibitor z-VAD to prevent apoptosis. Normal cells display worm-like mitochondria (a), but cells overexpressing Bax (assessed by Bax immunostaining, not shown) display either punctiform mitochondria dispersed throughout the cell (b) or mitochondria that have disappeared from the cell periphery to form aggregates around the nucleus (c). These changes are independent of caspase activity. (d) A likely explanation for this is that, following Bax insertion into the outer mitochondrial membrane, mitochondria first condense (pyknotic mitochondria), possibly fragment, and then cluster around the nucleus. The condensed mitochondria have lost their cytochrome c (A. Osen-Sand and J-C. Martinou, unpublished.) Bar, 15 m.

if apoptosis triggered by nerve-growth factor (NGF) deprivation is blocked with caspase inhibitors, the transition from normal to condensed morphology is reversible following readdition of NGF to the neuron culture15. In addition, the cellular distribution of mitochondria is profoundly affected during apoptosis. Mitochondria are normally dispersed throughout the entire cell. However, one of the early events that occurs during apoptosis triggered by tumour necrosis factor (TNF) is a perinuclear clustering of mitochondria that might result from defective kinesinmediated transport of the organelle16. Both mitochondrial condensation and perinuclear clustering can be observed following production of the Bcl-2related proapoptotic protein Bax in many cell types (Fig. 1). These facts leave many questions unanswered about their physiological relevance. For example, does the condensation of mitochondria play a role in the release of cytochrome c? Is the clustering of mitochondria close to the nucleus crucial for generating high ATP levels in a domain rich in energydependent apoptotic events, or is it to facilitate the translocation of mitochondrial proteins (e.g. AIF) to the nucleus? Unravelling the molecular mechanisms underlying these processes could help in understanding the role of mitochondria in the mediation of apoptosis.

Mitochondria as a major target for Bcl-2 family proteins Currently, 15 Bcl-2 family proteins have been identified in mammals17. All contain at least one of the four conserved regions called Bcl-2 homology domains (BH1BH4). These motifs are formed by helices and enable the different members of the family to form either homo- or heterodimers and to regulate each other18,19. The Bcl-2-related proteins display either antiapoptotic or proapoptotic function. The members that inhibit apoptosis, such as Bcl-2 and Bcl-xL, harbour at least three BH domains. Among the death promoters, some proteins (e.g. Bax, Bak) contain BH1, BH2 and BH3, and closely resemble Bcl-2. Others (e.g. Bid, Bad) possess only the BH3 domain, which is essential for both their interaction with other family members and their death-promoting activity18; these are called BH3-only proteins. Most Bcl-2 family proteins also contain a C-terminal ~20-residue hydrophobic domain that targets them to intracellular membranes, predominantly to the outer membrane of mitochondria20. The principal mechanism by which Bcl-2 family proteins regulate apoptosis is probably by controlling cytochrome-c release. Overexpression of the antiapoptotic proteins Bcl2 or Bcl-xL in many cell types subjected to various cytotoxic treatments prevents cytochrome-c release from mitochondria, caspase activation and cell
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Death receptors Survival factors Fas ligand death2123. Moreover, the use of cellIndependent stimuli free systems indicates that exogenous Fas Bcl-2 must be associated with mitochondria to prevent cytochrome-c DISC release21. Conversely, ectopic expression 14-3-3 P P P P of the death promoter Bax triggers FADD cytochrome-c release from mitochondria Bax in the absence of any death signal2428. This loss of cytochrome c is associated Bid Calcineurine Kinases with caspase activation and the inducPP1a (Akt, MAPK, Caspase 8 tion of apoptosis. The direct addition PKA, Rsks, of Bax to isolated mitochondria also tBid Bcl-x Bad PAK) L induces cytochrome-c release25,26,2831. These effects can be blocked by overexpression of Bcl-xL in intact cells or addition of recombinant Bcl-xL to isolated Caspase 9 mitochondria25,28,30. However, caspase inhibitors do not affect Bax-induced cytoApaf-1 Cyt c chrome-c leakage but they do effecdATP tively block caspase activation and delay apoptosis2426,28. These data thus Downstream suggest that Bax can directly induce the caspases loss of cytochrome c and indicate that trends in Cell Biology caspases do not participate in this event. Although Bcl-2 appears to be exclu- FIGURE 2 sively membrane bound, particularly Many death signals converge onto mitochondria and are mediated through members of the Bcl-2 in mitochondria32, other related pro- protein family called BH3-only proteins, such as Bid and Bad. These proteins are recruited to specific teins (e.g. Bid, Bad and Bim) are cyto- pathways. In some cells, binding of Fas ligand to its receptor Fas leads to the trimerization of Fas and solic but translocate to mitochondria to the formation of the death-inducing signalling complex (DISC). This complex is formed by during apoptosis3335. These proteins association of the cytoplasmic region of Fas, the adaptor protein FADD (Fas-associating protein with play a major role in transducing signals death domain) and procaspase 8, which is proteolytically cleaved to generate the active enzyme2. from the cytosol to mitochondria, Caspase 8 then cleaves Bid, whose C-terminal fragment (tBid) translocates to mitochondria, where it where they bind to and regulate the activates Bax or Bax-like proteins and results in cytochrome-c (cyt c) release. tBid might also act on its activity of the Bcl-2 family members own to trigger cytochrome-c release. Once in the cytosol, cytochrome c activates caspase 9 by of this pathway has recently been shown by that control the release of cytochrome binding to Apaf-1 and dATP. The physiological relevance69 c (Fig. 2). Translocation of these pro- the resistance of Bid-deficient mice to Fas hepatotoxicity . Caspase 8 can also initiate a direct signalling pathway that is independent of mitochondria by cleaving and activating downstream teins is triggered by specific posttranscaspases. Death-receptor-independent stimuli and growth-factor deprivation can trigger apoptosis by lational modifications such as dephosinducing translocation of Bax or Bad to mitochondria. In healthy cells, Bad can be phosphorylated in 35 phorylation (in the case of Bad ) and response to survival factors by several kinases, including Akt, mitogen-activated protein kinase (MAPK), cleavage by caspase 8 (in the case of Erk, protein kinase A (PKA), Rsks (MAPK-activated kinases) and p21-activated kinase 1 (PAK). The two Bid2). Similarly, under normal condi- serine residues that are phosphorylated are embedded in a 1433 consensus site. Phosphorylation of tions, Bax appears to be predominantly either residue or both results in the sequestration of Bad in the cytosol through its binding to 1433. cytosolic and, in some cells, in periph- During apoptosis, Bad is dephosphorylated by the Ca2-sensitive phosphatase calcineurine or the eral association with mitochondria. protein phosphatase 1 (PP1) and translocates to mitochondria, where it binds to Bcl-xL. This displaces Following a death signal, several Bcl-xL from Bcl-xLBax heterodimers, thereby inhibiting the death-repressor activity of Bcl-xL. changes affect Bax that lead to its activation (Fig. 3). First, Bax translocates from cytosol to mitochondria32,36 unless Bax is almitochondria by treatment with recombinant fulllength or truncated Bid33,37. Both Bcl-xL and Bid ready loosely attached to the organelle20,33. Then, the conformation of Bax changes, unmasking its N seem to exert their influence by interacting directly terminus20,33. This is accompanied by an apparent with Bax33,37. Taken together, these data suggest that oligomerization, which was detected by crosslinking Bax or a Bax-like protein act as a kind of onoff experiments36,37. Finally, Bax inserts into the outer switch regulating cytochrome-c release, and that Bax or a Bax-like protein is the focus of many apopmembrane of mitochondria and becomes an intetosis-regulating pathways. gral membrane protein20,37. This is rapidly followed Finally, most effects of the Bcl-2 family proteins by cytochrome-c release from mitochondria. that have been described seem to be conditional on Interestingly, it has been shown that enforced their mitochondrial localization. This does not exdimerization of Bax is sufficient to induce Bax clude the possibility that Bcl-2-related proteins could translocation, mitochondrial dysfunction and influence apoptosis independently of mitochonapoptosis, which indicates that the mitochondrial dria. Consistent with this thesis, some Bcl-2 mutants localization of Bax and its oligomerization play a that are not addressed to the mitochondrial memcrucial role in triggering apoptosis36. brane retain significant ability to inhibit apoptosis19. These changes in Bax can be prevented by Bcl-2 and Bcl-xL33,36, and they can be reproduced in isolated Similarly, apoptosis in different cell lines induced by
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Bax
Cytosol expansion of the inner membrane upon matrix swelling can break the outer membrane, and such rupture has been observed in different apoptotic systems23. This would be expected to trigger the release and irreversible dilution in the cytosol of the whole content of the intermembrane space, because it is no longer constrained by the outer membrane. However, the content of the matrix is retained in mitochondria because the inner membrane remains intact even though it expands. ? Two models can account for matrix swelling. The first model implicates the Cytochrome c initial hyperpolarization of the inner Matrix membrane that precedes cytochrome-c release in some systems23,42 (Fig. 4a). trends in Cell Biology According to Vander Heiden et al.43, FIGURE 3 this hyperpolarization might result from the inability to exchange mitoAfter an apoptotic signal, Bax moves from the cytosol to the mitochondria. In some cell types, Bax is chondrial ATP for cytosolic ADP during already loosely attached to the organelles and this translocation cannot be detected. After this, Bax apoptosis. This antiport is normally undergoes a conformational change, oligomerizes and inserts into the outer mitochondrial mediated by the voltage-dependent membrane. This is rapidly followed by cytochrome-c release. It is possible that Bax inserts into anion channel (VDAC, or mitochonthe membrane before it oligomerizes. All these events can be induced by either full-length Bid or drial porin) located in the outer memcaspase-8-cleaved Bid (tBid) and prevented by Bcl-2 or Bcl-xL, probably by direct interaction brane and the adenylate translocator with Bax33,37. Another Bax-like protein, Bak, is activated through similar mechanisms33,70. (ANT), which resides in the inner membrane. Impairment of ATPADP exchange by VDAC closure should inhibit F1F0-ATPase microinjection of cytochrome c is strongly reduced by Bcl-2 overexpression38. In some circumstances, activity, resulting in an inhibition of H re-entry to Bcl-2 has also been shown to inhibit Baxthe matrix, and should thereby contribute to the induced caspase activation and cell death without hyperpolarization of the inner mitochondrial memblocking cytochrome-c release24. Taken together, brane. Such an increase of the mitochondrial transmembrane potential (m) is predicted to promote these observations suggest that antiapoptotic proteins can also act downstream of cytochrome-c an osmotic matrix swelling44. However, these hyporelease. It has been proposed that Bcl-xL might inthetical mechanisms conflict with another model that implicates a mitochondrial megachannel called teract with Apaf-1 and prevent it from activating the permeability transition pore (PTP; Fig. 4b). caspase 9 (Refs 39 and 40). However, this assumption The PTP is a high-conductance unselective chanis very controversial and requires further investigation. nel that can be formed by the apposition of transRecently, Goldstein et al.41 studied the kinetics membrane proteins from the inner and outer mitoof cytochrome-c release from mitochondria using chondrial membranes at contact sites between the cytochrome c tagged with green fluorescent protein two membranes45. Although the exact composition (GFP) and found that, during apoptosis induced by a variety of stimuli, all of the cytochrome-cGFP was of the PTP is still elusive, accumulating data suggest released from all mitochondria of the cell within that its basic unit might be formed by the associ5 min. They also reported that the release was temation of ANT, VDAC and cyclophilin D, a waterperature independent. Although these observations soluble matrix protein (Fig. 5). Opening of the PTP were made using an artificial protein, they argue can be triggered by several physiological effectors strongly against the involvement of enzymatic such as Ca2, reduced concentrations of adenine transport systems. However, what exactly are the nucleotides, inorganic phosphate, reactive oxygen mechanisms that underlie cytochrome-c release? species, changes in pH or low m (Ref. 45) and posTwo prevailing theories have been postulated: the sibly Bax46. PTP opening causes a sudden increase in nonspecific rupture of the outer mitochondrial memthe permeability of the inner mitochondrial membrane and the formation of cytochrome-c-conducting brane to molecules of mass 1.5 kDa. This event, channels (Fig. 4). the permeability transition, results in the immediate dissipation of the proton-dependent m and chemical equilibration between the cytoplasm and The theory of the outer-mitochondrialmitochondrial matrix (Ref. 45). This causes a promembrane rupture gressive osmotic swelling of the matrix owing to its According to the first theory, water and solutes high solute concentration and can ultimately lead enter the matrix during apoptosis, causing swelling to disruption of the outer membrane. of the mitochondria (Fig. 4a,b). Because the inner The first argument supporting the PTP model is membrane, with its numerous cristae, has a considthe observation of a m collapse preceding nuclear erably larger surface area than the outer membrane, Apoptotic signal

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FIGURE 4
Five models for the release of cytochrome c from mitochondria during apoptosis. (a,b) The outer mitochondrial membrane ruptures as a result of swelling of the mitochondrial matrix, allowing cytochrome c and other proteins to escape from the intermembrane space. Model (a) involves closure of the voltage-dependent anion channel (VDAC) and impairment of ATPADP exchange43. Under normal conditions (left), the protons that accumulate in the intermembrane space as a result of electron transport re-enter the mitochondrial matrix through F1F0ATPase (complex V of the respiratory chain). By inhibiting F1F0ATPase, impairment of ATPADP exchange results in the accumulation of protons in the intermembrane space and an increase in the mitochondrial transmembrane potential (m), which is predicted to promote osmotic swelling (right). Model (b) suggests that the permeability-transition pore (PTP) opens and that the inner membrane permeability increases, causing mitochondrial-matrix swelling47. Bax might induce this pore to open by binding to the adenine-nucleotide translocator (ANT) at the contact sites between the inner and outer membranes or after translocation to the inner membrane46. (ce) A large channel forms in the outer mitochondrial membrane, allowing cytochrome-c release, but mitochondria are not damaged. This channel could be formed by Bax only64 (c), Bax in association with VDAC51 (d) or could be a lipidic channel or proteinlipid complex (e) formed after Bax insertion into the outer mitochondrial membrane67.

apoptosis in many cell types in response to various toxic stimuli47. Consistently, specific inhibitors of the PTP such as bongkrekic acid (an antagonizing ligand of ANT) or cyclosporin A (a ligand of cyclophilin D) can prevent apoptosis in different models, while opening of the PTP by different pharmacological agents like atractyloside (an agonistic ligand of ANT) can induce matrix swelling and apoptosis47. In addition, members of the Bcl-2 family can regulate opening of the PTP48. Bcl-2 can prevent the PTPmediated mitochondrial depolarization in intact cells and isolated mitochondria47,49, but Bax can provoke m loss, matrix swelling and cytochromec release in some cell types and isolated mitochondria in a cyclosporin-A-inhibitable fashion27,29,30,46. Bax could trigger PTP opening through binding to ANT, as suggested by coimmunoprecipitation and yeast two-hybrid studies46. Taken together, these results suggest that opening of the PTP might be retrends in CELL BIOLOGY (Vol. 10) September 2000

sponsible for cytochrome-c release during cell death and might be a target for the regulatory effects of Bcl-2 family. However, conflicting data question whether PTP opening is the cause or the consequence of cytochrome-c leakage from mitochondria. Indeed, many studies indicate that cytochrome-c release can occur in the absence of or before m disruption21,22,41,50. Accordingly, in some experimental conditions, treatment of isolated mitochondria with recombinant Bax or Bid can induce cytochrome-c release without triggering any fall in m, mitochondrial swelling or physical disruption of the outer membrane15,25,28,31. Moreover, Eskes et al.26 have reported that Bax-induced cytochrome-c redistribution cannot be inhibited by cyclosporin A or by bongkrekic acid either in intact cells or in isolated mitochondria. Finally, the interaction between ANT and Bax is controversial51. These data thus suggest that, at

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Water, solutes Caspase-dependent opening of the PTP could form an amplification loop through which the initial cytochrome-c release would induce matrix swelling, rupture of the outer membrane and loss Outer BPR VDAC of the remaining cytochrome c and membrane other intermembrane proteins. This model would reconcile the observation CK that cytochrome-c release often occurs before m dissipation with the strong ANT correlation between opening of the PTP and cell death. Alternatively, caspases Intermembrane could target proteins involved in the space Cph. D control of mitochondrial homeostasis, such as Bcl-2, thereby contributing to the late mitochondrial dysfunction that Atractyloside often follows apoptosis in vitro56,57. Ca2+, Bax, Whether such a late event also occurs ROS Cyclosporin A in vivo, before plasma-membrane Inner modifications and phagocytosis (Box 1), membrane Bongkrekic acid, ATP remains to be determined. In summary, rupture of the outer mitochondrial membrane is, of course, Matrix an attractive hypothesis because it trends in Cell Biology could explain the release of many proteins from the intermembrane space in FIGURE 5 addition to cytochrome c, such as some The possible components of mitochondrial permeability transition pore (PTP). In the open configuration, caspases, AIF (57 kDa), Hsp60, Hsp10, water and solutes enter the matrix, causing matrix swelling and rupture of the outer mitochondrial adenylate kinase (25.2 kDa) and sulfite membrane. Abbreviations: ANT, adenine nucleotide translocator; BPR, benzodiazepine peripheral oxidase (104 kDa)5,31,42. However, such receptor; CK, creatine kinase; HK, hexokinase; VDAC, voltage-dependent anion channel; Cph. D, cyclophilin a physical rupture has rarely been deD. The different inducers [atractyloside, Ca2, Bax, reactive oxygen species (ROS)] and inhibitors scribed and many data suggest that it is (cyclosporin A, bongkrekic acid, ATP) of PTP opening are represented according to their site of action. more likely to be a consequence than a cause of cytochrome-c release. This argues for the existence of other mechanisms responleast in some circumstances, cytochrome-c release sible for the initial cytochrome-c leakage. is independent of PTP opening and mitochondrial swelling. In an attempt to explain these apparent discrepThe hypothesis of cytochrome-c-conducting ancies, it has been proposed that the PTP might channels flicker between open and closed states, resulting in According to the second theory, channels are transient and asynchronous mitochondrial depolarformed that are large enough for the passage of solization and swelling. This could affect the permeuble proteins (Fig. 4ce). One clue to how Bcl-2 famability of the outer membrane while allowing the ily proteins exert their mitochondrial activity has inner membrane to restore the m and the normal come from the three-dimensional structure of Bcl-xL homeostasis of the matrix29. Transient opening of (Ref. 58). Bcl-xL consists of two central, predomithe PTP appears to be its normal mode of behaviour, nantly hydrophobic, helices (5 and 6) suras demonstrated at the single-cell and single-mitorounded by five amphipathic helices58. A similar chondrion levels by imaging techniques52,53. structure can be predicted for other Bcl-2 family members such as Bcl-2 and Bax, which have a high However, such reversible openings have not yet degree of sequence homology with Bcl-xL (Ref. 59). been found to induce either cytochrome-c release or cell death. Surprisingly, however, the structure of Bid is also Alternatively, PTP opening could occur downsimilar to that of Bcl-xL, even though the amino acid stream of cytochrome c release as a consequence of sequence identity between Bid and Bcl-xL is limited electron-transport inhibition (by reducing m and to the 16-residue BH3 domain60,61. ATP levels) or of caspase activation. In support of this Importantly the structure of Bcl-xL and Bid resemlatter hypothesis, caspases have been shown to inbles that of the pore-forming domains of diphtheria duce opening of the PTP48,54. Caspase inhibitors can toxin and some bacterial colicins58, which can form consistently prevent dissipation of m and cell channels for ions and proteins. Bcl-xL, Bcl-2, Bax59 death under conditions where they are unable to and the truncated form of Bid62 have been shown to affect cytochrome-c release28,50. Furthermore, in form functional ion channels in synthetic lipid vesicles and planar lipid bilayers. In a similar way to thymocytes from Apaf-1-knockout mice, the strong bacterial toxin pores, these channels have multiple reduction in caspase activation correlates with mainconductance states, are pH and voltage sensitive, tenance of m in response to various apoptotic and show a low ion selectivity. The intrinsic channel stimuli, but cytochrome-c release occurs normally55. HK

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properties of pro- and antiapoptotic proteins differ significantly, which might account for their opposite influences on cytochrome-c release63. More interestingly, Bcl-2 is able to prevent Bax-channel formation in liposomes64. These data, taken together with the fact that the addition of Bax or Bak directly to mitochondria triggers cytochrome-c release, suggest that these proapoptotic members of the Bcl-2 family could form mitochondrial channels. Could these channels be responsible for the outer mitochondrial membrane permeabilization that occurs during apoptosis? Bcl-2-related proteins have only two helices long enough to span a membrane bilayer, which is insufficient to form an aqueous channel. However, Bax can oligomerize under some conditions, allowing it to form very-high-conductance channels in liposomes and to trigger cytochrome-c release from mitochondria65. Oligomerization, a process so far described only for Bax, might represent the mechanism by which proapoptotic proteins form megachannels (Fig. 4c). Alternatively, it has been found that Bax and the truncated form of Bid (tBid), but not Bcl-xL, can decrease the stability of planar phospholipid bilayers, suggesting that proapoptotic proteins might act directly by destabilizing the outer mitochondrial membrane66,67. By reducing the linear tension of the membrane, Bax and tBid could promote the formation of a lipidic pore or a proteinlipid complex large enough to allow intermembrane proteins to diffuse into the cytosol (Fig. 4e). A third possibility is that Bax cooperates with VDAC to form a large cytochrome-c-conducting channel (Fig. 4d). When VDAC is reconstituted in liposomes, Bak and Bax promote opening of the channel, whereas Bcl-xL facilitates its closure. Furthermore, monitoring the movement of fluorescein-labelled cytochrome c indicates that Bax and Bak allow cytochrome c to pass through VDAC, possibly by inducing a conformational change or by cooperating with VDAC to form a megapore. However, none of Bax, Bak or VDAC is able by itself to form channels permeable to cytochrome c in liposomes51. In summary, the channel theory offers the advantage of maintaining functional mitochondria during the time necessary for the activation of caspases, because the latter process requires ATP. Nevertheless, we have thus far no evidence that Bcl-2 family proteins do form channels in vivo. Whether the lumen diameter of these pores would be sufficient to allow the passage of cytochrome c and other proteins is even less certain. Regulation of mitochondrial homeostasis by antiapoptotic Bcl-2 family proteins Mitochondria play a vital role in the cell, providing most of the cells energy and participating in the Ca2, redox and pH homeostasis. This means that a major mitochondrial dysfunction is likely to cause cell death. For instance, disruption of electron transport might be responsible for the increased production of reactive oxygen species (ROS) and the cytoplasmic acidification that are observed early during
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apoptosis. In such circumstances, the electrons that escape in large amounts from the respiratory chain can reduce molecular oxygen to produce superoxide anions, and the reduced ATP synthesis promotes the accumulation of lactate by stimulating glycolysis. Consistent with the idea that mitochondrial status can influence the lifedeath decision, antiapoptotic Bcl-2 proteins have been implicated in the protection of mitochondrial integrity. One of the first antiapoptotic functions attributed to Bcl-2 was the reduction of ROS levels44. Since then, Bcl-2 has been shown to stabilize m, to affect mitochondrial proton flux and to modulate mitochondrial Ca2 homeostasis47,49,68; similarly, Bcl-xL has been implicated in the regulation of mitochondrial metabolism and matrix volume23,43. Some of these effects can be ascribed to the blockade of cytochrome-c release by Bcl-2 and Bcl-xL because the depletion of cytochrome c from mitochondria can impair electron transport and be responsible for some types of mitochondrial dysfunction. Alternatively, the control of cytochrome c release by antiapoptotic proteins might be the indirect result of their effects on mitochondrial homeostasis. For example, Bcl-2 can maintain m in response to various agents such as Ca2 and ROS by enhancing proton efflux49. By offsetting ion imbalances that would otherwise result in a lowered m and an increased probability of PTP opening, Bcl-2 could prevent cytochrome-c redistribution. Similarly, Bcl-xL appears to inhibit cytochrome-c release by maintaining mitochondrial ADPATP exchange and by preventing mitochondrial swelling23,43. Therefore, Bcl-2 and Bcl-xL seem to regulate an aspect of mitochondrial physiology more general than the simple redistribution of cytochrome c. Concluding remarks Progress over the past few years has led to the recognition that, in addition to their established role in generating energy for the cell, mitochondria play a key role into controlling life and death by releasing cytochrome c into the cytosol, thereby activating caspases. With a few exceptions (e.g. Fas or TNF-triggered apoptosis in certain cells), mitochondria represent an essential component of many apoptotic pathways. The physiological importance of cytochrome-c release and the subsequent activation of caspase 9 has been shown by Apaf-1- or caspase-9-knockout mice, which die perinatally with brain overgrowth and reduced apoptosis in the central nervous system1. More recently, these observations have been confirmed by the generation of cytochrome-c-deficient mice6. These mice die earlier during embryonic development, but this is presumably due to defective mitochondrial oxidative phosphorylation. Nevertheless, cells derived from the cytochrome-c-knockout embryos (as well as cells lacking Apaf-1 or caspase 9) are resistant to a wide range of apoptotic stimuli, even though they undergo apoptosis normally after Fas or TNF-receptor activation1,6. Although the molecular mechanisms underlying cytochrome-c redistribution during apoptosis are

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not yet completely understood, it is now accepted that this process must not cause a great mitochondrial dysfunction because this would lead to necrosis because of the energy-production catastrophe. Instead, ATP supply should be sustained to allow caspase activation and apoptosis to proceed normally. It is also clear that, depending on the cell type and the nature of the cellular damage, various molecular signals converge onto mitochondria, where they are integrated by the Bcl-2 family proteins. Unravelling the precise mechanism of action of these proteins will undoubtedly help our understanding of how the outer mitochondrial membrane becomes permeable during apoptosis. References
1 2 3 Vaux, D.L. et al. (1999) Cell death in development. Cell 96, 245254 Budihardjo, I. et al. (1999) Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15, 269290 Newmeyer, D.D. et al. (1994) Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell 79, 353364 Abrams, J.M. (1999) An emerging blueprint for apoptosis in Drosophila. Trends Cell Biol. 9, 435440 Susin, S.A. et al. (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441446 Li, K. et al. (2000) Cytochrome c deficiency causes embryonic lethality and attenuates stress-induced apoptosis. Cell 101, 389399 Neame, S.J. et al. (1998) Blocking cytochrome c activity within intact neurons inhibits apoptosis. J. Cell Biol. 142, 15831593 Juin, P. et al. (1999) c-myc-induced sensitization to apoptosis is mediated through cytochrome c release. Genes Dev. 13, 13671381 Kerr, J.F.R. et al. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239257 Mannweiler, K. et al. (1957) Recherches ultrastructurales sur une tumeur rnale experimentale du hamster. J. Ultrastruct. Res. 1, 158169 Rouiller, C. (1957) Contribution de la microscopie lectronique a ltude du foie normal et pathologique. Ann. Anat. Pathol. 2, 548562 Hackenbrock, C.R. (1968) Chemical and physical fixation of isolated mitochondria in low-energy and high-energy states. Proc. Natl. Acad. Sci. U. S. A. 61, 598605 Mancini, M. et al. (1997) Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line. J. Cell Biol. 138, 449469 Zhuang, J. et al. (1998) Apoptosis, in human monocytic THP.1 cells, results in the release of cytochrome c from mitochondria prior to their ultracondensation, formation of outer membrane discontinuities and reduction in inner membrane potential. Cell Death Differ. 5, 953962 Martinou, I. et al. (1999) The release of cytochrome c from mitochondria during apoptosis of NGF-deprived sympathetic neurons is a reversible event. J. Cell Biol. 144, 883889 De Vos, K. et al. (1998) The 55-kDa tumor necrosis factor receptor induces clustering of mitochondria through its membrane-proximal region. J. Biol. Chem. 273, 96739680 Gross, A. et al. (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13, 18991911 Kelekar, A. et al. (1998) Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol. 8, 324330 Oltvai, Z.N. et al. (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74, 609619 Goping, I.S. et al. (1998) Regulated targeting of BAX to mitochondria. J. Cell Biol. 143, 207215 Kluck, R.M. et al. (1997) The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 11321136 Yang, J. et al. (1997) Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 11291132 Vander Heiden, M.G. et al. (1997) Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91, 627637 Ross, T. et al. (1998) Bcl-2 prolongs cell survival after Bax-induced release of cytochrome c. Nature 391, 496499 Jrgensmeier, J.M. et al. (1998) Bax directly induces release of cytochrome c from isolated mitochondria. Proc. Natl. Acad. Sci. U. S. A. 95, 49975002 Eskes, R. et al. (1998) Bax-induced cytochrome c release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2 ions. J. Cell Biol. 143, 217224 27 Pastorino, J.G. et al. (1998) The overexpression of Bax produces cell death upon induction of the mitochondrial permeability transition. J. Biol. Chem. 273, 77707775 28 Finucane, D.M. et al. (1999) Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J. Biol. Chem. 274, 22252233 29 Pastorino, J.G. et al. (1999) Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore. J. Biol. Chem. 274, 3173431739 30 Narita, M. et al. (1998) Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc. Natl. Acad. Sci. U. S. A. 95, 1468114686 31 Kluck, R.M. et al. (1999) The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J. Cell Biol. 147, 809822 32 Hsu, Y-T. et al. (1997) Cytosol-to-membrane redistribution of Bax and Bcl-xL during apoptosis. Proc. Natl. Acad. Sci. U. S. A. 94, 36683672 33 Desagher, S. et al. (1999) Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144, 891901 34 Puthalakath, H. et al. (1999) The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex. Mol. Cell 3, 287296 35 Downward, J. (1999) How BAD phosphorylation is good for survival. Nat. Cell Biol. 1, E33E35 36 Gross, A. et al. (1998) Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17, 38783885 37 Eskes, R. et al. (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell. Biol. 20, 929935 38 Zhivotovsky, B. et al. (1998) Injected cytochrome c induces apoptosis. Nature 391, 449450 39 Hu, Y. et al. (1998) Bcl-xL interacts with Apaf-1 and inhibits Apaf-1dependent caspase-9 activation. Proc. Natl. Acad. Sci. U. S. A. 95, 43864391 40 Pan, G. et al. (1998) Caspase 9, Bcl-XL, and Apaf-1 form a ternary complex. J. Biol. Chem. 273, 58415845 41 Goldstein, J.C. et al. (2000) The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat. Cell Biol. 2, 156162 42 Samali, A. et al. (1999) Presence of a pre-apoptotic complex of procaspase-3, Hsp60 and Hsp-10 in the mitochondrial fraction of Jurkat cells. EMBO J. 18, 20402048 43 Vander Heiden, M.G. et al. (1999) Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Mol. Cell 3, 159167 44 Vander Heiden, M.G. et al. (1999) Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis? Nat. Cell Biol. 1, E209E216 45 Crompton, M. (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem. J. 341, 233249 46 Marzo, I. et al. (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281, 20272031 47 Kroemer, G. et al. (1997) Mitochondrial control of apoptosis. Immunol. Today 18, 4451 48 Marzo, I. et al. (1998) The permeability transition pore complex: a target for apoptosis regulation by caspases and Bcl-2-related proteins. J. Exp. Med. 187, 12611271 49 Shimizu, S. et al. (1998) Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. Proc. Natl. Acad. Sci. U. S. A. 95, 14551459 50 Bossy-Wetzel, E. et al. (1998) Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17, 3749 51 Shimizu, S. et al. (1999) Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399, 483487 52 Petronilli, V. et al. (1999) Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys. J. 76, 725734 53 Huser, J. et al. (1999) Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore. Biochem. J. 343, 311317 54 Susin, S.A. et al. (1997) The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO1/CD95- and ceramide-induced apoptosis. J. Exp. Med. 186, 2537

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Acknowledgements We thank Bruno Antonsson, Peter Clarke and Kinsey Maundrell for critical reading of the manuscript, Astrid Osen for the immunofluorescence experiment, and Christopher Hebert for artwork. Owing to space limitations, many studies could not be cited and we apologize to those researchers for their omission.
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55 Yoshida, H. et al. (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739750 56 Chen, Q. et al. (2000) Distinct stages of cytochrome c release from mitochondria: evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death Differ. 7, 227233 57 Cheng, E.H-Y. et al. (1997) Conversion of Bcl-2 to a Bax-like death effector by caspases. Science 278, 19661968 58 Muchmore, S.W. et al. (1996) X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381, 335341 59 Schendel, S.L. et al. (1998) Bcl-2 family proteins as ion-channels. Cell Death Differ. 5, 372380 60 Chou, J.J. et al. (1999) Solution structure of Bid, an intracellular amplifier of apoptotic signaling. Cell 96, 615624 61 McDonnell, J.M. et al. (1999) Solution structure of the proapoptotic molecule Bid: a structural basis for apoptotic agonists and antagonists. Cell 96, 625634 62 Schendel, S.L. et al. (1999) Ion channel activity of the BH3 only Bcl-2 family member, BID. J. Biol. Chem. 274, 2193221936 63 Schlesinger, P.H. et al. (1997) Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc. Natl. Acad. Sci. U. S. A. 94, 1135711362 Antonsson, B. et al. (1997) Inhibition of Bax channel-forming activity by Bcl-2. Science 277, 370372 Antonsson, B. et al. (2000) Bax oligomerization is required for channelforming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochem. J. 345, 271278 Kudla, G. et al. The destabilization of lipid membranes induced by the C-terminal fragment of caspase 8-cleaved Bid is inhibited by the N-terminal fragment. J. Biol. Chem. (in press) Basaez, G. et al. (1999) Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. U. S. A. 96, 54925497 Zhu, L. et al. (1999) Modulation of mitochondrial Ca2 homeostasis by Bcl-2. J. Biol. Chem. 274, 3326733273 Yin, X-M. et al. (1999) Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400, 886891 Griffiths, G.J. et al. (1999) Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis. J. Cell Biol. 144, 903914

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Genes encoding semaphorins have been highly conserved in evolution, from invertebrates to humans (Fig. 1). At least 20 semaphorins are known, recently divided into seven subclasses and renamed according to structural similarities1. An additional group contains viral-encoded semaphorins, found in the genome of non-neurotropic DNA viruses2. Semaphorins can also be distinguished by their biochemical structure as being secreted, membrane glycosylphosphatidylinositol (GPI)-anchored or transmembrane molecules. A distinctive protein module of 500 amino acids, called the sema domain, characterizes all semaphorins2. Interestingly, sema domains are also present in the extracellular moiety of other cellsurface proteins, namely the plexins, shown recently to be functional semaphorin receptors, and the scatter factor receptors of the MET proto-oncogene family, controlling similar biological functions3,4. The sema domain is known to mediate receptorbinding specificity of semaphorins5. Additional protein motifs and structural modules conserved in the extracellular domain of semaphorins, plexins and scatter factor receptors further underline the phylogenetic relationships between the genes encoding these three protein families3,4 (see Fig. 1). There are two plexin genes in the fly Drosophila melanogaster3. In humans, there are at least nine different genes, grouped into four subfamilies according to sequence similarity6 (plexins A to D). The intracellular domain of plexins6,7, the sex-plexin (SP) domain, is unrelated to other known proteins. However, its primary sequence is highly conserved among family members and across evolution, suggesting that plexins share common biochemical functions and signal-transduction pathways. Neuropilins, the other component of semaphorin receptors, are unrelated phylogenetically to plexins or semaphorins. Two neuropilin genes are present in the genome of birds and mammalians (NPN-1 and NPN-2, reviewed in Ref. 8), but none has been identified in invertebrates; this might be consistent with their evolution in vertebrates as co-receptors
trends in CELL BIOLOGY (Vol. 10) September 2000

Signalling by semaphorin receptors: cell guidance and beyond

Luca Tamagnone and Paolo M. Comoglio
Semaphorins are a large family of secreted or cell-bound signals, known to guide axons in developing nervous tissue. They are expressed in a variety of adult and embryonic tissues and are thought to have a broader spectrum of functions. Recent evidence suggests that semaphorins and their receptors play a key role in the control of cellular interactions, most likely in cellcell repulsion. A subset of semaphorins interacts with neuropilins cell-surface molecules lacking a signalling-competent cytoplasmic domain. Another large family of transmembrane molecules, namely plexins, bind specifically to semaphorins. Thus plexins, alone, or in association with neuropilins, behave as fully functional semaphorin receptors. The intracellular responses elicited by plexins are unknown, but their large cytoplasmic moiety, containing the strikingly conserved sex-plexin (SP) domain, is likely to trigger novel signal-transduction pathways.

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