Physiol. Rev. 87: 99-163, 2007;
doi:10.1152/physrev.00013.2006
0031-9333/07 $18.00
Mitochondrial Membrane Permeabilization in Cell Death
Guido Kroemer,
Lorenzo Galluzzi and
Catherine Brenner
Institut Gustave Roussy, Institut National de la Santé et de la Recherche Médicale Unit "Apoptosis, Cancer and Immunity," Université de Paris-Sud XI, Villejuif; and Centre National de la Recherche Scientifique UMR 8159, Université de Versailles/Saint-Quentin en Yvelines, Versailles, France
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ABSTRACT
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Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.
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I. INTRODUCTION
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Throughout the second half of the 20th century, mitochondria were exclusively considered as the cell's powerhouse, organelles whose particular architecture and biochemical composition would serve one major purpose, namely, maximization of energy production by oxidative phosphorylation. While many graduate students in chemical, biological, and medical sciences discretely abhorred mitochondria as a site of major metabolic pathways, it became clear around 1995 that mitochondria have a second crucial function, namely, the control of cell death. The discovery that mitochondria control cell death has revitalized the fields of mitochondrial bioenergetics and genetics and has revolutionized the field of cell death research. At first, the idea of mitochondrial cell death control appeared counterintuitive. Specialists in bioenergetics and inveterate biochemists wondered how it is possible that the cell's vital forces concentrated in mitochondria could be perverted to serve a lethal purpose. Similarly, cell death researchers were initially reluctant to accept that an organelle that during apoptosis does not suffer any major alterations in its ultrastructure, for instance, compared with the nucleus, would control the fate of the cell.
Today, the initial resistance against the concept of mitochondrial cell death control has been overcome. Since 2001, more than 13,000 articles that simultaneously mention the keywords apoptosis and mitochondrion have been published in the Medline database. Nowadays, it is difficult to ignore that cell death, in both its physiological and pathological occurrence, is closely linked to mitochondrial structure and (dys)function. The flood of information running through mitochondrial cell death control is so important that it has become an arduous task to understand the complexity of mitochondrial death/life decision making without getting lost in details. The purpose of this review is to provide an ordered vision of mitochondrial cell death control.
In healthy cells, the inner mitochondrial membrane (IM), the frontier between the intermembrane/intercristae space and the matrix, is nearly impermeable to all ions, including protons. This allows complexes I–IV of the respiratory chain to build up, across IM, the proton gradient that is required for oxidative phosphorylation (521, 522). The charge imbalance that results from the generation of an electrochemical gradient across the IM forms the basis of the inner mitochondrial transmembrane potential (
m). Finally, the proton gradient is exploited by complex V of the respiratory chain to drive ATP synthesis. Therefore, the maintenance of the proton gradient is of vital importance for cellular bioenergetics (521, 522), meaning that all constituents of the mitochondrial matrix and all metabolites that cross the IM do so in a tightly regulated fashion, with the help of highly selective channels and transport proteins. Although a transient loss of the m, through the "flickering" of one or several IM pores, may occur in physiological circumstances (404, 896), a long-lasting or permanent m dissipation is often associated with cell death (488, 880), as discussed in this review.
The permeability of the outer mitochondrial membrane (OM), which delimits the outer contour of mitochondria, is also well regulated, both in normal life and during cell death. It has been assumed that OM is freely permeable to small metabolites and solutes up to 
5 kDa, due to the presence of an abundant protein, the voltage-dependent anion channel (VDAC), that would allow for the diffusion of such solutes through the OM. However, this view has been challenged during recent years, because real-time measurements of mitochondrial Ca2+ concentrations coupled to manipulations of the OM protein composition revealed the existence of Ca2+ microdomains in which VDAC and a variety of additional OM proteins control and limit the diffusion of Ca2+ (152, 631). For a comprehensive and detailed analysis on this specific topic, the reader is referred to the excellent review by Rizzuto and Pozzan (648). In cell death, the OM permeability often increases, allowing for the release of soluble proteins that usually are retained within mitochondria, in the intermembrane space (IMS). The death-associated OM permeabilization is not only an accidental process but also a tightly regulated phenomenon, with major consequences for health and disease, as detailed in this review.
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II. AN OVERVIEW OF CELL DEATH PATHWAYS
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A. Apoptosis
In a pioneering study on ischemic liver injury, published in 1972, Kerr et al. (377) observed a novel type of cell death, dubbed "apoptosis," which appeared different from toxin-induced necrotic hepatocyte death. As revealed by electron microscopy, apoptotic cells form small round bodies that are surrounded by membranes and contain intact cytoplasmic organelles or parts of the nucleus. These bodies result from progressive cellular condensation and budding, and eventually are engulfed by resident phagocytic cells (e.g., epithelial cells or fibroblasts). The morphological changes that define apoptosis are nuclear pyknosis (chromatin condensation) and karyorhexis (nuclear fragmentation) (405). The phenomenon of apoptosis has been documented as a prominent player in normal embryonic and postembryonic development, as well as in pathological and therapeutic settings (638, 766). Indeed, apoptosis can be viewed as a process that eliminates superfluous, ectopic, damaged, or mutated cells according to the rule "better death than wrong." Disabled apoptosis is a pathogenic event that contributes to oncogenesis and cancer progression. Unwarranted apoptosis of postmitotic cells (such as neurons or cardiomyocytes) also causes disease. Acute massive apoptosis participates in the pathophysiology of infectious diseases, septic shock, and intoxications (638, 766).
Apoptosis is a genetically predetermined mechanism that may be elicited by several molecular pathways (Fig. 1). The best characterized and the most prominent ones are called the extrinsic and intrinsic pathways. In the extrinsic pathway (also known as "death receptor pathway"), apoptosis is triggered by the ligand-induced activation of death receptors at the cell surface. Such death receptors include the tumor necrosis factor (TNF) receptor-1, CD95/Fas (the receptor of CD95L/FasL), as well as the TNF-related apoptosis inducing ligand (TRAIL) receptors-1 and -2. In the intrinsic pathway (also called "mitochondrial pathway"), apoptosis results from an intracellular cascade of events in which mitochondrial permeabilization plays a crucial role (677).
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FIG. 1. Extrinsic versus intrinsic caspase activation cascades. Left: extrinsic pathway. The ligand-induced activation of death receptors induces the assembly of the death-inducing signaling complex (DISC) on the cytoplasmic side of the plasma membrane. This promotes the activation of caspase-8 (and possibly of caspase-10), which in turn is able to cleave effector caspase-3, -6, and -7. Caspase-8 can also proteolytically activate Bid, which promotes mitochondrial membrane permeabilization (MMP) and represents the main link between the extrinsic and intrinsic apoptotic pathways. The extrinsic pathway includes also the dependency receptors, which deliver a death signal in the absence of their ligands, through yet unidentified mediators. Right: intrinsic pathway. Several intracellular signals, including DNA damage and endoplasmic reticulum (ER) stress, converge on mitochondria to induce MMP, which causes the release of proapoptotic factors from the intermembrane space (IMS). Among these, cytochrome c (Cyt c) induces the apoptosis protease-activating factor 1 (APAF-1) and ATP/dATP to assemble the apoptosome, a molecular platform which promotes the proteolytic maturation of caspase-9. Active caspase-9, in turn, cleaves and activates the effector caspases, which finally lead to the apoptotic phenotype. DNA damage may signal also through the activation of caspase-2, which acts upstream mitochondria to favor MMP. See section IIA for further details.
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Both routes to apoptotic death can be divided at least in three distinct phases: initiation, integration/decision, and execution/degradation (409). The initiation phase is highly heterogeneous and depends on the nature of the death-inducing signal, be it an extrinsic one (the ligation of a death receptor) or an intrinsic one [which may affect any cellular organelle including the nucleus, the endoplasmic reticulum (ER), lysosomes, or mitochondria]. The integration/decision phase involves the near-to-simultaneous activation of caspases and mitochondrial death effectors in a complex molecular interplay that will be dissected in this review. During this phase, the "decision to die" is taken and the "point of no return" is trespassed. The execution/degradation phase, which is essentially a post mortem process, is common to distinct types of apoptosis, meaning that the morphological and biochemical alterations that accompany late-stage apoptosis are independent of the initiating stimulus. Both the extrinsic and the intrinsic routes to apoptosis ultimately lead to cell shrinkage, chromatin condensation, nuclear fragmentation (which is frequently accompanied by internucleosomal DNA fragmentation), blebbing, and phosphatidylserine exposure on the surface of the plasma membrane (Fig. 1) (882).
It appears that the activation of a specific class of proteases, the caspases ("cysteine protease cleaving after Asp"), is required for the rapid and full-blown manifestation of these features of apoptosis. However, not all caspases are required for apoptosis and the process generally results from the activation of a limited subset of caspases, in particular, caspases-3, -6, and -7 (231). These are the "executioner" caspases, and they mediate their effects by the cleavage of specific substrates in the cell. Activation of the executioner caspases-3 and -7 by initiator caspases-8, -9, and -10 define the best understood apoptotic pathways (Fig. 1).
In the extrinsic pathway, ligation of death receptors [a subset of the TNF receptor (TNFR) family, including TNFR1, Fas/CD95, the TRAIL receptors -1 and -2, and probably the death receptor 3, also known as TRAMP, i.e., translocating chain-association membrane protein] causes the recruitment and oligomerization of the adapter molecule FADD (Fas-associating death domain-containing protein) within the death-inducing signaling complex (DISC). Oligomerized FADD binds the initiator caspases-8 and -10, causing their dimerization and activation (Fig. 1) (167). As an alternative, the extrinsic pathway can be activated by the so-called dependency receptors, which are believed to be connected to rapid caspase activation as well. In the absence of ligand, these receptors trigger cell death, thus generating a state of cellular dependence from their ligands. The prototype dependency receptors are the netrin-1 receptors DCC (deleted in colorectal cancer) and UNC5H-1, -2 and -3 (for a review, see Ref. 509).
Most cell death in vertebrates proceeds via the intrinsic or mitochondrial pathway of apoptosis (264). Here, the executioner caspases are cleaved and activated by the initiator caspase-9, which is activated by multimerization on the adapter molecule apoptosis protease activating factor 1 (APAF-1) within a multiprotein complex called "apoptosome." APAF-1 preexists in the cytosol as a monomer, and its activation depends on the presence of cytochrome c (Cyt c) and ATP/dATP (Figs. 1 and 2) (82). The release of Cyt c, which normally resides only in the IMS where it functions as an electron shuttle in the respiratory chain (49), is rate-limiting for the generation of the apoptosome. Hence, mitochondrial membrane permeabilization (MMP) is the critical event responsible for caspase activation in the intrinsic pathway. MMP can even commit a cell to die when caspases are not activated. This "caspase-independent death" (129, 407) can occur because of an irreversible loss of mitochondrial function as well as because of the mitochondrial release of caspase-independent death effectors including apoptosis-inducing factor (AIF) (742), endonuclease G (EndoG) (448), and others (Fig. 2) (129, 407).
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FIG. 2. Release of IMS proteins. Proapoptotic signals resulting in mitochondrial membrane permeabilization (MMP) provide intermembrane space (IMS) proteins with a route for release. Once in the cytosol, IMS proteins follow different fates. 1) Cytochrome c (Cyt c) promotes the formation of the so-called "apoptosome," a molecular platform for the activation of caspase-9 (Casp-9) including also the apoptosis protease activating factor 1 (APAF-1) and ATP/dATP. In turn, active Casp-9 catalyzes the proteolytic activation of the effector caspases, which ultimately contribute to the appearance of the morphological hallmarks of apoptosis (e.g., DNA fragmentation and chromatin condensation). See sections IIA and VIIIA as well as Figure 1 for further information. 2) The caspase-independent death effectors apoptosis-inducing factor (AIF) and endonuclease G (EndoG) translocate from the cytosol to the nuclear compartment where they favor DNA fragmentation and chromatin condensation. Members of the heat shock protein (HSP) family, like HSP70 (i.e., heat shock protein of 70 kDa), antagonize AIF proapoptotic activity by preventing its nuclear import. For additional details, see sections VIII, C and D, as well as Figure 11. 3) Second mitochondria-derived activator of caspase/direct IAP binding protein with a low pI (Smac/DIABLO) and the Omi stress-regulated endoprotease/high temperature requirement protein A2 (Omi/HtrA2), promote apoptosis indirectly, by binding to and antagonizing members of the IAP (inhibitor of apoptosis protein) family. Under normal circumstances, IAPs would exert antiapoptotic effects by preventing the caspase activation. See section VIIIB for more detailed information.
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The cross-talk between the extrinsic and intrinsic pathway has been extensively investigated for death receptors. In the so-called type 1 cells, ligation of death receptors causes the activation of effector caspases without the necessity of MMP. In contrast, in type 2 cells, a complex signaling cascade (caspase-8 activation 
cleavage and activation of Bid Bid-mediated Bax activation MMP Cyt c-dependent caspase-3 activation) critically depends on MMP as a conditio sine qua non for cell death induction (402, 677).
Because apoptosis is involved in the maintenance of tissue homeostasis, it is strictly controlled at multiple, crucial levels (264). In numerous models, mitochondria represent a central checkpoint of apoptosis control by integrating various signals including endogenous factors [e.g., cytosolic and organellar concentrations of protons, Ca2+, Mg2+, K+, and Na+, metabolites such as ATP, ADP, NAD(P), glutathione, lipid second messengers, and multiple proteins including kinases and phosphatases] as well as exogenous factors (e.g., specific viral proteins or xenobiotics). These organelles collect the sum of death-inducing and life-preserving signals at the level of their membranes and, when the lethal signals predominate over the vital ones, mitochondria undergo MMP. MMP is characterized by several hallmarks that include 1) the release of Cyt c, second mitochondria-derived activator of caspase/direct inhibitor of apoptosis binding protein (IAP) with a low pI (Smac/DIABLO), Omi stress-regulated endoprotease/high temperature requirement protein A2 (Omi/HtrA2) through the OM and the subsequent activation of effector caspases (Fig. 2); 2) the release of caspase-independent apoptogenic death effectors, such as AIF and EndoG (Fig. 2); 3) an alteration of the m of the IM; and 4) a bioenergetic catastrophe including the arrest of oxidative phosphorylation, as well as the accumulation of reactive oxygen species (ROS) (Table 1). The multiple relationships existing between apoptosis and bioenergetics are depicted in Figure 3. The kinetic order in which these alterations occur depends on the mechanisms of MMP and in particular whether these mechanisms primarily affect IM or OM, as discussed later in this review.
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FIG. 3. Multiple intersections between apoptosis and bioenergetics. Mitochondria are the cell's powerhouse, the site where the vast majority of ATP is synthesized. ATP synthesis is driven by the electrochemical gradient built across the inner mitochondrial membrane (IM) by the oxidative phosphorylation complexes (OXPHOS). To generate this electrochemical gradient, OXPHOS pump protons from the matrix to the intermembrane space (IMS), thus leading to the formation of a transmembrane potential (m) as well as to the generation of reactive oxygen species (ROS). In healthy cells, ROS are kept at harmless levels by the activity of both nonenzymatic and enzymatic antioxidant systems. Among the former, a prominent role is played by nonoxidized glutathione (GSH), thioredoxin (Trx), and NAD(P)H. On the other hand, glutathione-S-transferase (GST), glutathione peroxidase (GPx), and the manganese-dependent superoxide dismutase (Mn-SOD) represent redox-active enzymes. This delicate equilibrium breaks down when apoptosis is induced, following distinct but sometimes overlapping mechanisms. m dissipation is promoted by proapoptotic stimuli as diverse as members of the Bcl-2 family of proteins (Bax, Bak, tBid), Ca2+ and cytosolic metabolites (all of which promote the opening of the PTPC, i.e., the permeability transition pore complex), and the activation of caspases (that may degrade OXPHOS subunits). The progressive loss of m is often accompanied by an increased generation of ROS, which quickly saturate the antioxidant systems and induce the functional impairment of mitochondria, by arresting oxidative phosphorylation and via feed-forward mechanisms on the PTPC. ROS may accumulate also upon an increase of m, as induced by inhibitors of the ATP synthase (complex V) like oligomycin or by the acidification of the mitochondrial matrix. Finally, decreased ATP production, protein thiol oxidation, lipid peroxidation, and the activation of stress response genes intervene, in the scenario of a bioenergetic crisis that progressively leads the cell to death.
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Defects in initial signal transduction (initiation phase) or in the integration/decision phase of apoptosis can arrest the lethal process. On the contrary, defects in the execution/degradation phase will not affect the fatal outcome, yet change the phenotypic manifestations of cell death, shifting it to a subapoptotic, necrotic, or autophagic appearance. Accordingly, inhibition of caspases has often negligible or little cytoprotective effects, although it drastically affects the morphological appearance of cell death.
B. Autophagic Cell Death
While apoptosis involves the explosive activation of catabolic enzymes leading to the demolition of cellular structures and organelles, autophagy is a slow, circumscript phenomenon in which parts of the cytoplasm are sequestered within double-membraned vacuoles and finally digested by lysosomal hydrolases (406). The functional relationship between apoptosis and autophagy is complex, and autophagy may either contribute to cell death (710) or constitute a cellular defense against acute stress, in particular induced by deprivation of nutrients or obligate growth factors (28, 67). Cells that are deprived from exogenous energy sources catabolize part of their cytoplasm to generate ATP and other intermediate metabolites that allow them to meet their essential energetic demand. Moreover, autophagy allows for the turnover of cytoplasmic regions including protein aggregates and damaged organelles. Thus autophagy prevents the accumulation of misfolded proteins in inclusion bodies, a function that may exert neuroprotective effects. As an example, mice expressing mutant huntingtin protein develop an Huntington-like neurodegenerative disease, which can be prevented by the administration of rapamycin, an inducer of autophagy (634).
Enhanced autophagic vacuolization is observed in some instances of cell death, which has been named "autophagic cell death." It is an ongoing conundrum, however, in which cases autophagic cell death truly occurs through autophagy (meaning that inhibition of autophagy would prevent cell death) and in which cases it occurs with autophagy (meaning that inhibition of autophagy would only affect the morphology of the process, but not the fate of cells). Recently, several loss-of-function studies of autophagy (atg) genes have been performed, and these knock-out (KO) models will clarify the contribution of autophagy to physiological (developmental) and pathological cell death. As it stands, it appears that the essential autophagy gene beclin 1 (also known as atg6) plays an important role in endogenous tumor suppression (444). This tumor suppression may be related to a beclin 1-dependent autophagic control that would avoid the accumulation of cells with damaged organelles, including mitochondria. Beclin 1 was originally identified as a Bcl-2 interacting protein from a mouse brain library (455) and functions in the lysosomal degradation pathway of autophagy (454). Recently, it has been demonstrated that the binding of beclin 1 by Bcl-2 at the ER results in the inhibition of beclin 1-dependent autophagy. According to a rheostat model for the function of the beclin 1-Bcl-2 complex, the relative amounts of the two proteins ensure that autophagy operates at homeostatic levels, sufficient for the cells to cope with starvation or other forms of stress but not enough to promote cell death (602). This scenario, which highlights beclin 1-dependent autophagic cell death as an additional mechanism against tumor progression, raises the possibility that Bcl-2 and the other antiapoptotic members of the Bcl-2 family may function as oncogenes not only by directly blocking apoptosis but also by blocking autophagy (601). It has not yet been demonstrated whether beclin 1 could neutralize the antiapoptotic functions of Bcl-2. Manipulations of other autophagy genes also revealed the importance of autophagy in the elimination of invading microbial pathogens (272).
For the purpose of the present discussion, it is important to note that autophagy is essential for the removal of damaged mitochondria. Thus, when MMP occurs only in a minor subset of mitochondria, in response to a subapoptotic insult, autophagy may constitute the mechanism responsible for the removal of damaged (and permeabilized) organelles. In this scenario, suppression of autophagy will facilitate the induction of apoptosis through the intrinsic pathway (406). The detailed mechanisms through which damaged mitochondria are sequestered in phagosomes for degradation ("mitophagy") remain to be elucidated. According to some studies, mitochondria that undergo permeability transition (PT) with loss of m are preferentially targeted by "mitophagy" (196).
C. Necrosis
The cell's decision to die from necrosis or apoptosis is dictated at least in part by the abundance of intracellular energy stores. Indeed, whereas apoptosis requires a minimal amount of intracellular ATP, necrosis is generally accompanied by its total depletion (550). Thus necrosis may be viewed as an accidental type of cell death. Necrosis is not genetically predetermined and normally occurs within a short period following the triggering insult (2–4 h). The final phenotypic appearance of necrotic cells is highly dependent on the severity of the injury. The main features of necrosis include a gain in cell volume (oncosis) that finally leads to rupture of the plasma membrane and the unorganized dismantling of swollen organelles. Hence, necrosis lacks a specific biochemical marker, apart from the presence of plasma membrane permeabilization, and can be detected only by electron microscopy. Necrosis is considered to be harmful because it is often associated with pathological cell loss and because of the ability of necrotic cells to promote local inflammation that may support tumor growth (782). Importantly, cell death that usually occurs with an apoptotic morphology can be shifted to a more necrotic phenotype when caspase activation is inhibited by pharmacological inhibitors or by the elimination of essential caspase activators such as APAF-1 (257, 407).
Recently, the molecular events occurring during TNF-induced cell death have been investigated in more detail, revealing a contribution of caspase activation and protein synthesis to necrosis (407, 664). These observations as well as recent KO studies (540) argue against the concept of necrosis as a merely accidental cell death and rather suggest that the susceptibility to undergo necrosis is partially determined by the cell (and not only by the stimulus) and that the necrotic process involves an active contribution of cellular enzymes, implying that it is subjected to regulation. A recent study has demonstrated that the kinase RIP (receptor interacting protein), which is essential for TNF-induced necrosis, can inhibit ATP/ADP exchange on mitochondrial membranes by a direct interaction with the adenine nucleotide translocase (ANT), thereby causing mitochondrial dysfunction and cell death (760). Thus mitochondrial alterations may constitute a rate-limiting step of necrotic cell death, at least in some instances.
In accord with this idea, in several paradigms, overexpression of Bcl-2, an antiapoptotic protein that stabilizes mitochondrial membranes, can prevent or retard necrotic cell death. This applies, for instance, to necrosis induced by cyanide or by the simultaneous treatment with chemotherapeutic agents and the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk) (305, 404). Similarly, the KO of cyclophilin D (CypD), a protein that is required for some modes of MMP (29, 36), has been shown to prevent necrotic cell death of hepatocytes responding to the Ca2+ ionophore A23187 or to H2O2 (540). These results demonstrate that MMP can be rate-limiting for necrotic cell death.
D. Mitotic Catastrophe
Mitotic catastrophe represents a type of cell death that occurs during mitosis. Thus the morphological aspect of cells dying during mitosis is different from that of cells dying from classical apoptosis (that mostly occurs in the interphase). Mitotic catastrophe often involves micronucleation and multinucleation events that occur before cell death. Mitotic catastrophe results from a combination of deficient cell cycle checkpoints (in particular the DNA structure and the spindle assembly checkpoints) and cellular damage (for a review, see Ref. 96). Failure to arrest the cell cycle before or at mitosis triggers an attempt of aberrant chromosome segregation, which culminates in the activation of the apoptotic pathway and ultimately leads to the cellular demise. Cell death occurring during the metaphase/anaphase transition is often characterized by the activation of caspase-2 (which can occur in response to DNA damage) (96) and/or MMP with release of cell death effectors such as AIF and the caspase-9 and -3 activator Cyt c (553).
When cell death resulting from mitotic catastrophe is inhibited (for instance by overexpression of Bcl-2), the abortion of irregular mitoses is avoided and cells divide asymmetrically resulting in the generation of aneuploid daughter cells (397). Thus mitotic catastrophe may be viewed as a mechanism that protects against unwarranted (and possibly oncogenic) aneuploidization (96, 397). As it stands, mitotic catastrophe is a complex process that is controlled by numerous molecular players including kinases involved in cell cycle control (e.g., the cyclin-dependent kinase Cdk1, polo-like kinases, and Aurora kinases), cell cycle checkpoint proteins, survivin, p53, caspases, and members of the Bcl-2 family. Nonetheless, as the other cell death modalities, the fatal outcome of mitotic catastrophe depends, at least in part, on mitochondrial permeabilization.
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III. DETECTION OF MITOCHONDRIAL MEMBRANE PERMEABILIZATION
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MMP is a universal feature of cell death and is often considered as the "point of no return" in the cascade of events leading to apoptosis (263, 264). In general, it represents a defining stigma of death affecting cells as diverse as lymphocytes, cardiomyocytes, hepatocytes, neurons, osteoblasts, keratinocytes, or kidney epithelial cells. The mechanisms underlying MMP are complex and probably result from the coordinate execution of several interdependent steps. Before we discuss the intricate mechanisms of MMP, we recapitulate here the technologies that can be used to detect permeabilization events affecting the IM or OM of mitochondria. These technologies are very different. OM is normally permeable to metabolites but not to proteins, meaning that OM permeabilization is mostly assessed by determining the translocation of proteins through OM, from the IMS to the extramitochondrial compartment. In contrast, IM is usually impermeable to ions and water, so its permeabilization is measured by physicochemical methods assessing the capacity of IM to maintain an electrochemical gradient or to separate low-molecular-weight solutes from each other.
A. Signs of Outer Mitochondrial Permeabilization
OM permeabilization is generally detected by determining the subcellular localization of proteins that are normally retained within the IMS by the protein-impermeable OM. Immunoblot detection of such proteins [including Cyt c, AIF, or adenylate kinase (ADK)], in an extramitochondrial compartment (cytosol, nuclei) purified according to subcellular fractionation procedures is interpreted as a reliable sign of OM permeabilization. Similarly, two-color immunofluorescence experiments can be performed to detect the presence of AIF, Cyt c, or ADK outside of mitochondria, by visualizing them in a separate localization from sessile mitochondrial markers such as heat shock protein 60 (HSP60) (394, 450, 741). Based on microinjection experiments with recombinant proteins, Cyt c and AIF are considered the prototypes of apoptogenic proteins released upon MMP, since each of them suffice to trigger nuclear apoptosis (466, 742, 891). However, proteomic analysis of the supernatants of mitochondria with permeabilized OM revealed that Cyt c and AIF are released together with numerous other key proteins, including but not limited to procaspases, Smac/DIABLO, Omi/HtrA2, and EndoG (5, 394, 600, 665, 741). While all these proteins are released to the cytosol, some additionally translocate to the nucleus (e.g., AIF, EndoG) or interact with receptors on the ER (e.g., Cyt c).
Thus multiple IMS proteins can be used as markers of OM permeabilization. It should be noted, however, that not all IMS proteins are released from mitochondria simultaneously. Depending on the apoptotic model that is studied, it has been found that the release of Cyt c can occur before or after that of AIF (526). Similarly, Smac/DIABLO may be released from mitochondria before Cyt c and AIF (22). This has been interpreted as an indication of differential release mechanisms (for instance distinct OM pores) (666), yet may also be explained by distinct mechanisms of mitochondrial retention. For instance, Cyt c must be desorbed from its interaction with the IM lipid cardiolipin (see also Fig. 10), presumably through cardiolipin oxidation (573), while AIF must be cleaved by a non-caspase protease to remove its anchorage in the IM (see also Fig. 11) (615). Thus it may be an advantage to monitor the subcellular localization of several IMS proteins rather than a single one to detect OM permeabilization.
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FIG. 4. Detection of OM and IM permeabilization. Mitochondrial outer membrane (OM) permeabilization (A, B, G, H): under physiological conditions (A, G), cytochrome c-green fluorescent protein (Cyt c-GFP) fusions are retained in mitochondria, thanks to the diffusional barrier provided by the OM. This is visualized as a tubular pattern of green fluorescence by fluorescence microscopy (A). Upon OM permeabilization (B, H), intermembrane space (IMS) proteins, including Cyt c-GFP chimeras, redistribute to the cytoplasm, resulting in a diffuse green fluorescence of lower intensity. In A and B, blue fluorescence identifies the nucleus (Hoechst DNA staining). Mitochondrial inner membrane (IM) permeabilization (C–F, J, L): under physiological conditions (C, E, J), calcein loaded into cells as its acetoxymethyl ester freely diffused to all subcellular compartments, whereas its quencher (Co2+) is excluded from the mitochondrial matrix due to the fact that IM is impermeable to this ion. As a consequence, fluorescence microscopy may be employed to identify functional mitochondria, which appear as brightly fluorescent spots (C). This corresponds to an intense cellular fluorescence, as assessed by monoparametric fluorescence-activated cell sorter (FACS) analysis (E). When cells are treated with calcimycin (a Ca2+ ionophore which promotes IM permeabilization) (D, F, L), Co2+ gain access to the mitochondrial matrix, where they quench the calcein signal, as assessed by fluorescence microscopy (C), as well as by monoparametric FACS analysis (D). As schematized in K, the calcein-Co2+ technique does not identify OM permeabilization. Similarly, whereas IM permeabilization results in osmotic swelling of the mitochondrial matrix, IMS proteins are not released so far as the OM remains intact (I). See section III for further details.
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FIG. 5. Mechanisms for OM permeabilization. Under physiological conditions, mitochondria exhibit a high mitochondrial transmembrane potential (m), intermembrane space (IMS) proteins are retained in IMS, proapoptotic members of the Bcl-2 family are in their inactive state (either soluble in the cytoplasm, as Bax and Bid, or anchored to the mitochondrial OM, as Bak) and the permeability transition pore complex (PTPC) ensures the exchange of metabolites between the cytosol and the matrix, in virtue of its "flickering" activity. In these circumstances, interactions of hexokinase (HK) and cyclophilin D (CypD) with the scaffold structure of the permeability transition pore complex (PTPC) are likely to inhibit OM permeabilization. OM permeabilization, which leads to the release in the cytosol of the IMS proteins and eventually to cell death, may occur through several mechanisms. 1) Proapoptotic signals may directly promote the destabilization of mitochondrial lipids, thus favoring the formation of pores which allow for the release of IMS proteins. 2) Long-lasting opening of the PTPC, associated with the loss of antiapoptotic interactions with HK and CypD, may lead to the dissipation of m, followed by an osmotic imbalance that induces the swelling of the mitochondrial matrix. Due to the surface area of the mitochondrial IM, largely exceeding that of the OM, swelling may culminate in the physical rupture of the OM. See sections IIIA and IV as well as Figure 6 for additional information. 3) Upon activation, proapoptotic members of the Bcl-2 family may translocate from the cytosol to OM (e.g., Bax and Bid) or undergo conformational changes (e.g., Bak) to bind to components of the PTPC. The resulting heterooligomers may provide IMS proteins with a route for release. See section IVA for further details. 4) Alternatively, activated proapoptotic proteins of the Bcl-2 family may assemble into large multimers, allowing for the release of IMS proteins. See section IVA for further details.
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FIG. 6. Role of the voltage-dependent anion channel (VDAC) in OM permeabilization. Two models have been put forward to explain the role of VDAC in the mitochondrial OM permeabilization. According to Tsujimoto and co-workers (a) (713, 773), in physiological conditions VDAC would exist prominently in a low conductance state, within a "flickering" permeability transition pore complex (PTPC), to ensure the exchange of metabolites between mitochondria and cytosol. Upon apoptosis induction, VDAC would exhibit an increased conductance associated with long-lasting PTPC opening, thus leading to dissipation of mitochondrial transmembrane potential (m), efflux of IMS proteins, and eventually cell death. On the contrary, Thompson and colleagues (b) (784–786) proposed that the physiological role of VDAC would be exerted by its high conductance state and that under proapoptotic conditions the closed state of VDAC would be favored. This would bring about a transient mitochondrial hyperpolarization, followed by osmotic imbalance, OM rupture, release of the IMS proteins, and ultimately cell death. See section IVB for more detailed information.
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FIG. 7. PTPC architecture. Although the exact molecular composition of the permeability transition pore complex (PTPC) has not been clearly established yet, a consensus has started to emerge about the proteins involved in its scaffold structure, which builds up at the contact sites between the mitochondrial outer and inner membranes (OM and IM, respectively). In addition to the VDAC and the adenine nucleotide translocase (ANT), which represent the main PTPC components, these include hexokinase (HK, interacting with VDAC from the cytosol), creatine kinase (CK, interacting with PTPC from the intermembrane space, IMS), peripheral-type benzodiazepine receptor (PBR, interacting with PTPC from OM), and cyclophilin D (CypD, interacting with ANT from the mitochondrial matrix). HK and CK seemingly associate with the PTPC scaffold in a mutually exclusive fashion. Both anti- and proapoptotic members of the Bcl-2 family modulate the activity of PTPC, through direct interactions with ANT or VDAC. The table reports some inhibitors and facilitators of mitochondrial membrane permeabilization (MMP) for which the target within PTPC has been identified. PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide.
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FIG. 8. Cristae remodeling. Under physiological conditions, mitochondrial membranes define the boundaries of at least three submitochondrial compartments: the mitochondrial matrix is enclosed within the mitochondrial IM, the intermembrane space (IMS) is located between the IM and the mitochondrial OM, the intracristae space (ICS) is delimited by the convoluted folds of the IM, namely, cristae. Most cytochrome c (Cyt c) resides in the ICS, which communicates with IMS via tight bottleneck-like junctions, forming a diffusional barrier. It appears that ICS undergoes deep structural rearrangements during apoptosis to promote the complete release of Cyt c and other IMS proteins. In healthy cells, cristae structure is maintained with the help of Opa1 (optic atrophy 1) oligomers, which are constituted by both the IM integral form of Opa1 and by its IMS soluble counterpart. The serine protease PARL (presenilin-associated rhomboid-like) is responsible for the production of the soluble form of Opa1 that is required for the assembly of Opa1 oligomers. Upon apoptosis induction, Opa1 oligomers are disrupted (for instance following the translocation of truncated Bid, i.e., tBid). Then profound rearrangements of the submitochondrial structure take place, resulting also in the loss of the diffusion barrier between IMS and ICS. Taken together, these rearrangements have been called "cristae remodeling" and promote the mobilization of the pool of IMS proteins, including Cyt c, previously sequestered in ICS. Finally, mitochondrial membrane permeabilization (MMP) allows for the release of the mobilized IMS proteins. For further details see section VI.
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FIG. 9. Signals converging on mitochondria to induce MMP. Mitochondria represent crucial checkpoints of apoptosis control, where lethal and vital signals emanating from different intracellular compartments, as well as from the extracellular microenvironment, converge and are integrated to decide the cell's fate. 1) Pro- and antiapoptotic members of the Bcl-2 family exert their activities not only at mitochondria but also at the endoplasmic reticulum (ER), where they have been shown to regulate the size of the intra-ER Ca2+ pool. The amount of Ca2+ available for release upon activation of the inositol 1,4,5-trisphosphate receptor (IP3R) determines the mitochondrial response to this ion, which may range from metabolic activation to mitochondrial membrane permeabilization (MMP), release of intermembrane space (IMS) proteins, and apoptosis. For further details, see section VIIB. 2) p53 controls part of the response of mammalian cells to DNA damage, by means of both transcriptional (for instance by upregulating the expression of proapoptotic members of the Bcl-2 family like Bax) and transcription-independent mechanisms. Among the latter, p53 may favor MMP by direct interactions with Bak, Bax, Bcl-2, and Bcl-XL at the outer mitochondrial membrane (OM). p53 transcriptional and transcription-independent activities may be promoted by the binding of glycogen synthase kinase 3 (GSK-3). For further details, see section VIIA. 3) Death receptors transduce proapoptotic signals from the extracellular environment, along the extrinsic pathway, into the activation of the caspase cascade. The extrinsic pathway is linked to mitochondria by caspase-8, which mediates the proteolytic maturation of Bid. tBid favors MMP by promoting the pore-forming activity of Bak and Bax, the dismantling of Opa1 (optic atrophy 1) oligomers (a process that results in cristae remodeling), and mitochondrial dysfunctions through interactions with cardiolipin (CL) in the IM. For further details on the extrinsic pathway of apoptosis, see section IIA and Figure 1. For additional information on cristae remodeling, see section VI and Figure 8. 4) When lysosomal membranes break down, cathepsins and other hydrolases are released into the cytoplasm, where they promote apoptosis and/or necrosis. Some cathepsins are able to activate Bid, as well as to induce mitochondrial dysfunctions by cleaving specific subunits of the oxidative phosphorylation complexes (OXPHOS), thus enhancing reactive oxygen species (ROS) generation. The same has been reported for caspase-3, which may enter IMS upon limited OM permeabilization and participate in amplificatory loops for MMP. For further details on the cross-talk between lysosomes and mitochondria, see section VIIC. For additional information about the involvement of caspases in amplification loops MMP, see Figure 10 and Ref. 242. 5) Multiple signals coming from the cytosol lead to MMP. These include but are not limited to the following: ROS, metabolites (e.g., glucose-6-phosphate, palmitate) and the activation of specific kinases [e.g., GSK-3; PKC , i.e., protein kinase C, isoform; members of the c-Jun NH2-terminal kinase (JNK) family]. 6) On the other side, numerous endogenous modulators inhibit the permeability transition pore complex (PTPC) and protect mitochondria from MMP. These include metabolites [e.g., NAD(P)H and UTP], the antiapoptotic members of Bcl-2 family, antioxidant enzymes (e.g., glutathione-S-transferase) and several prosurvival kinases, like Akt. Akt inhibits apoptosis via multiple distinct pathways, such as the activation of NF B, the inhibition of caspases and of GSK-3, and through hexokinase II (HKII)-dependent mechanisms (activated also by glucose). See section VIID for more detailed information.
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FIG. 10. Amplification loops for Cyt c release. 1) In healthy cells, a major fraction of cytochrome c (Cyt c) is associated with the mitochondrial IM lipid cardiolipin (CL). This pool of Cyt c may be mobilized by the disruption of electrostatic interactions with CL or by the oxidation of CL mediated by reactive oxygen species (ROS) and by the CL-oxygenase activity exhibited by Cyt c itself. A limited release of Cyt c enhances the generation of ROS at the levels of the oxidative phosphorylation complexes (OXPHOS) I and III, thus favoring CL peroxidation and further Cyt c release. For additional details, see section VIIIA and Ref. 242. 2) Once in the cytosol, low amounts of Cyt c released following a limited mitochondrial membrane permeabilization (MMP) are sufficient to activate part of the caspase-3 (Casp-3) pool. Activated Casp-3, then, can enter the IMS through the partially permeabilized mitochondrial OM and cleave a 75-kDa component of the respiratory complex I. In turn, this provokes the disruption of the respiratory chain followed by an intense generation of ROS, which favor MMP by interacting with the permeability transition pore complex (PTPC) and/or support further Cyt c release by oxidizing CL. For more detailed information, see Ref. 242. 3) At the ER, the second messenger IP3 binds to its receptor (IP3R) to modulate Ca2+ release. While physiological concentrations of Ca2+ enhance the IP3R channel activity, higher concentrations inhibit the receptor, therefore establishing a negative-feedback regulatory loop. Low amounts of cytosolic Cyt c are able to bind to type I IP3R and remove such Ca2+-dependent inhibition, thus promoting unrestrained release of Ca2+ from the ER. In turn, Ca2+ favors MMP by direct effects on the PTPC. See sections VII, B and D, and VIIIA as well as Ref. 242 for further information. 4) The mobilization of ICS proteins occurring along with cristae remodeling represents an additional mechanism to account for the biphasic release of Cyt c during apoptotis. For additional details about cristae remodeling, see Figure 8 and section VI.
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To visualize OM permeabilization in living cells, they can be transiently or stably transfected with chimeric cDNA constructs that contain a mitochondrial IMS localization sequence (MLS) linked to a green fluorescent protein (GFP) moiety (Fig. 4). With the use of this system, it is possible to generate cells that express a fluorescent fusion protein (such as Cyt c-GFP or AIF-GFP chimeras) in the IMS until they receive an apoptotic stimulus (Fig. 4). Videomicroscopy revealed that the mitochondrial release of AIF-GFP or Cyt c-GFP chimeric proteins occurs in a rapid, coordinate fashion affecting all mitochondria of a cell in usually <5 min (256, 466).
In selected cases, OM permeabilization has been detected by electron microscopy, by the visualization of gaps in OM, through which IM herniation may occur (762, 785). Osmotic matrix swelling due to the influx of water may culminate in OM rupture. This is possible because the surface area of the IM with its folded cristae largely exceeds that of the OM. Obviously, this mode of OM permeabilization is irreversible and so are the associated mitochondrial dysfunction and release of apoptogenic factors. Indeed, according to immunoelectron microscopy determinations, upon physical rupture of the OM following PT induction, VDAC and the subunit F1 of ATPase remain associated with mitochondrial membranes, while Cyt c staining is lost (762). Nonetheless, there is no consensus on the contribution of OM ruptures to OM permeabilization. Confocal fluorescence microscopy has been used to visualize large Bax/Bak pore-forming oligomers assembled within or in the vicinity of OM (417, 546), and such complexes have been suggested to explain OM permeabilization without permanent membrane rupture.
Also, biochemical tests have been used to measure the OM integrity. For instance, the diffusion of metabolites generated by IMS enzymes (e.g., phosphocreatine produced by creatine kinase) can be used to determine the permeability of OM (and that of the metabolite channel in OM, VDAC) (659). Similarly, the accessibility of the enzymes of the respiratory chain (e.g., NADH oxidase, Cyt c oxidase) to exogenously administered substrates (e.g., NADH or Cyt c) can be quantified to determine the opening state of VDAC (which is responsible for the diffusion of NADH). Moreover, there have been attempts to measure the permeability of OM to Cyt c in cells after the permeabilization of their plasma membranes (528). These methods, however, have not yet been established for the routine monitoring of OM permeabilization in cell death research.
B. Signs of Inner Mitochondrial Permeabilization
IM permeabilization implies the formation of pores or channels that cause the dissipation of the m built across IM. Lipophilic cations accumulate in the mitochondrial matrix, driven by the m according to the Nernst equation, which states that (at +37°C) a hyperpolarization by 61.5 mV corresponds to a 10-fold increase in the intramitochondrial concentration of monovalent cations. Since in physiological conditions the m ranges from 120 to 180 mV (the intramitochondrial side being electronegative), the concentration of such cations is normally 2 to 3 logs higher in the mitochondrial matrix than in the cytosol. As a result, several different cationic fluorochromes can be employed to measure the m (95, 513). These markers include 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] (which emits in green, 552 nm), chloromethyl-X-rosamine (CMXRos, also known as MitoTracker Red) (emitting in red, at 599 nm), tetramethylrhodaminemethylester (TMRM) (emitting in orange, with a peak at 580 nm), and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) (which emits in red or green, according to its oligomerization status). Compared with rhodamine-123, which we do not recommend for cytofluorometric analyses, TMRM and DiOC6(3) offer the important advantage that they can be used at relatively low concentrations that do not cause major quenching effects (513). JC-1 incorporates into mitochondria where it either forms monomers (emitting in green, at 527 nm) or, at higher dye concentrations (implicating a high m), aggregates (emitting in red, at 590 nm). Thus the ratio between green and red JC-1 fluorescence provides an estimate of m that is (relatively) independent of mitochondrial mass. These m-sensitive fluorochromes may be used to assess the apoptosis-associated m loss. For a critical evaluation of the methods currently available to measure m, the reader may refer to Ref. 189.
As a caveat, it has to be taken into account that m dissipation may result from inhibited respiration not followed by IM permeabilization. Moreover, IM pore opening may be transient, leading to a transient m loss. To measure such a transient IM permeabilization event ("flickering pores"), one may use the calcein quenching method. This method relies on the loading of cells with the fluorescent probe calcein (molecular mass 620 Da) and its quencher, cobalt (Co2+) (321). When loaded into cells in its acetoxymethyl ester form, calcein is trapped in all subcellular compartments including mitochondria, whereas Co2+ is excluded from the mitochondrial matrix due to the IM impermeability to this ion. As a consequence, when the barrier provided by IM is functional, a distinct punctuate fluorescence signal from calcein clearly identifies mitochondria (Fig. 4C). Conversely, upon IM permeabilization induced by Ca2+ overload or oxidative stress (551, 607), Co2+ enters the mitochondria matrix, and quenches the calcein signal (Fig. 4D). In most models of apoptosis induced by DNA damage and p53 activation, IM permeabilization, as measured by either of these methods, occurs at the same time or shortly after mitochondrial translocation of Bax (599, 691, 822).
Following IM permeabilization, an increase in mitochondrial matrix volume occurs as a consequence of the massive entry of solutes and water (320). This is the result of the colloid osmotic pressure of the matrix, which is tightly packed with metabolically relevant enzyme complexes. This swelling gives rise to a distension and disorganization of the cristae as well as to a reduction of the electron density of the matrix. In vivo, such an alteration has been observed for the first time in hepatocytes undergoing apoptosis induced by Fas or glutathione depletion and later confirmed in several models (209, 286). Thus electron microscopy constitutes an additional means to investigate the contribution of mitochondria to cell death.
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IV. MECHANISMS OF MITOCHONDRIAL OUTER MEMBRANE PERMEABILIZATION
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A. Bax/Bak-Mediated Permeabilization
Bcl-2 is the prototype member of a family of proteins containing at least one Bcl-2 homology (BH) region. For classification purposes, the family may be divided into antiapoptotic multidomain proteins (prototypes: Bcl-2, Bcl-XL), which contain four BH domains (BH1234); proapoptotic multidomain proteins (prototypes: Bax, Bak), which contain three BH domains (BH123); and proapoptotic BH3-only proteins (prototypes: Bid, Bad) (442). Some members of all the subgroups share an additional COOH-terminal transmembrane domain, which mediates their insertion into the OM and other intracellular membranes (e.g., the ER membrane). The main site of action of Bcl-2-like proteins is probably the mitochondrial membrane (408). As a rule, BH1234 proteins mainly reside in OM, where they protect mitochondria against MMP, presumably by binding to and neutralizing other proapoptotic proteins from the Bcl-2 family, which on the contrary induce MMP. However, some BH1234 proteins act also at the ER membrane. The same has been reported also for some BH123 proteins, and the effect of Bcl-2-like proteins on the ER is discussed in section VIIB, when the impact of lethal signals emanating from the ER is mentioned.
In healthy cells, the BH123 protein Bak is associated with the OM, whereas the other BH123 protein Bax resides in the cytosol, under normal circumstances. The expression of at least one of the two BH123 proteins (Bax or Bak) is required for MMP, in a series of different models of apoptosis induction (823). Accordingly, fibroblasts from mice that lack both Bax and Bak (but not cells from animals deficient solely for Bax or Bak) are highly resistant against MMP induction and against the activation of cell death by the intrinsic pathway (823). The genetic invalidation of Bax and Bak proved their determining role in the disruption of mitochondrial function promoted by a plethora of stimuli including, but not limited to, staurosporine, ultraviolet irradiation, growth factor deprivation, etoposide, and the ER stress inducers thapsigargin and tunicamycin (823). Although it has been assumed that mitochondria from Bax/Bak double KO fibroblasts would be completely resistant to MMP, it appears that such mitochondria can be permeabilized by alternative mechanisms such as high Ca2+ concentrations, which can induce the phenomenon known as "permeability transition" (PT) (174), or by a hexokinase/VDAC-dependent mechanism (477), which is discussed below.
As just mentioned, in physiological conditions Bax is a cytosolic protein. However, upon apoptosis induction, Bax inserts into the OM (836), where it is thought to form supramolecular openings, alone or in association with other proapoptotic members such as Bak or tBid (truncated Bid) (417). Such openings might result from the formation of homooligomeric Bax-containing pores or from the destabilization of the lipid bilayer, resulting in transient discontinuities within OM (Fig. 5). Relocalization of Bax is required for its proapoptotic function. Indeed, if Bax is retained in the cytosol by interaction with the Ku autoantigen of 70 kDa (Ku70), as well as with Ku70-derived peptides, mitochondrial damage and apoptosis are efficiently prevented (674). Although the precise mechanisms of MMP are debated (878), MMP can result from a conformational change of Bax or Bak (with exposure of their NH2 terminus), their full insertion into mitochondrial membranes as homooligomerized multimers, and formation of giant protein-permeable pores (Fig. 5) (417). It is an ongoing conundrum, however, how Bax (and Bak) "find their way" to mitochondria and whether they are attracted through specific properties of the lipid or protein composition of OM. Reportedly, large Bax oligomers organize in clusters near the mitochondria shortly after its translocation to mitochondria (546). Bak colocalizes in these apoptotic clusters, in contrast to other Bcl-2 family members, including Bid and Bad. Formation of these complexes has been reported to occur in a caspase-independent fashion and to be inhibited completely and specifically by Bcl-XL (546).
BH3-only proteins can exert their proapoptotic action by two different mechanisms. Some BH3-only proteins (the "facilitators," prototype: Bad) preferentially interact with BH1234 proteins, dissociating them from other BH3-only or from BH123 proteins, which in turn promote MMP. Others (the "activators," prototype: tBid) directly activate BH123 proteins to initiate MMP, either by stimulating the translocation of Bax to mitochondrial membranes or by local effects on Bak (442). As a result, it is possible to generate two different types of "BH3 mimetics," a class of pharmacological agents that bind to multidomain Bcl-2 family proteins or so-called "BH3 receptors." One type of BH3 mimetics (prototype: ABT-737) only binds to BH1234 proteins (and hence facilitates apoptosis induction by neutralizing the antiapoptotic proteins of the Bcl-2 family) (570), while a second type of BH3 mimetics also binds to BH123 proteins and directly induces apoptosis in a Bax/Bak-dependent fashion (805).
How the molecular openings induced by Bax/Bak and/or Bax/tBid mediate Cyt c release is still a highly controversial issue. Indeed, some discrepancies between in vitro models based on purified cellular components (e.g., reconstituted proteoliposomes, black lipid membranes) and cell cultures have been reported. Evidence supporting the importance of lipid-protein interactions has started to accumulate (259, 469, 878), and it remains formally possible that Bax or Bak simply destabilize lipid bilayers instead of forming specific "pores" (35). Nonetheless, further studies are needed to completely elucidate the Bax/Bak- and/or Bax/tBid-mediated OM permeabilization.
According to some reports, Bax engages in a close molecular cooperation with proteins from the permeability transition pore complex (PTPC), such as ANT and/or VDAC, to induce MMP (Fig. 5). This has been demonstrated, for instance, by electrophysiological experiments involving purified recombinant Bax and purified ANT or VDAC (for a review, see Ref. 264). However, experiments on isolated mitochondria and liposomes suggest that Bax can permeabilize OM and release Cyt c in a fashion that does not involve any of the critical components of the PTPC, including VDAC, ANT, or CypD (201, 417). The two modes of Bax-mediated MMP (PTPC dependent versus PTPC independent) are not mutually exclusive, and they may coexist in specific proapoptotic settings. Indeed, the contribution of the PTPC to Bax-mediated MMP may be dictated by the concentration of Bax and its oligomerization status (599). Accordingly, when added to isolated mitochondria, Bax promotes cyclosporin A (CsA)-sensitive PT, depolarization, swelling, and release of Cyt c and of matrix-entrapped calcein only when used above a certain concentration threshold. Conversely, when lower amounts of Bax are used, mitochondrial swelling and depolarization are not detected (599).
The way the antiapoptotic members of the Bcl-2 family inhibit MMP is also a matter of debate. According to some authors, antiapoptotic members of the Bcl-2 family would simply act as inhibitors of their proapoptotic counterparts, without any independent effects on other mitochondrial proteins. This suppression of MMP could be either achieved by direct interaction with the pore-forming members of the Bcl-2 family, or indirectly by neutralizing BH3-only proteins (442). However, some data indicate that Bcl-2 and Bcl-XL can interact with sessile mitochondrial proteins including ANT (495) and VDAC (711). In vitro, the overexpression of Bcl-2 in cells or the addition of Bcl-2 to isolated mitochondria reduces the PT probability (495, 708). Moreover, recombinant Bcl-2 can inhibit the formation of pores by purified ANT or VDAC reconstituted into artificial membranes (71, 708), while enhancing the ADP/ATP antiporter activity of ANT (71). Accordingly, chemical inhibitors of Bcl-2 that have been designed to block a hydrophobic pocket within its BH3-binding domain can sensitize isolated mitochondria to permeability transition in vitro (518). One possible interpretation of these data would require the assumption that Bcl-2-like proteins have two functions on mitochondria, namely, the inhibition of pore formation by Bax and Bak as well as the inhibition of pores formed by proteins from the PTPC, such as VDAC and ANT.
In summary, the proapoptotic members of Bcl-2 family like Bax and Bak can induce MMP either independently or in coordination with PTPC proteins, by means of interactions occurring before or after PTPC opening (171, 621, 822, 830). Therefore, as depicted in Figure 5, two MMP mechanisms may coexist: a Bax-mediated OM permeabilization that occurs independently of any early and direct effect on the IM (214, 215) and a PTPC-mediated permeabilization, which on the contrary involves IM. The possible cooperation between these pathways and their relative weight in different cell death settings are still debated. Nonetheless, both routes eventually lead to the permeabilization of both mitochondrial membranes, release of proapoptotic proteins from IMS, and functional collapse of the organelle and apoptosis, irrespective of the initial trigger. It appears plausible that either of the two mechanisms may prevail
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