Bax assembles into large ring‐like structures remodeling the mitochondrial outer membrane in apoptosis

The Bcl‐2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro‐apoptotic proteins. Yet the mechanistic details of the Bax‐induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring‐like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super‐resolution data provide direct evidence in support of large Bax‐delineated pores in the mitochondrial outer membrane as being crucial for Bax‐mediated MOMP in cells.     

Inhibition of Bak Activation by VDAC2 Is Dependent on the Bak Transmembrane Anchor

Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.     

Transmembrane pore formation by the carboxyl terminus of Bax protein

Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal “helix 9” of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide–peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents.     

MIM through MOM: the awakening of Bax and Bak pores

The mechanism whereby mitochondrial DNA (mtDNA) is released into the cytosol and activates the cGAS/STING inflammatory pathway during Bax/Bax‐mediated apoptosis is unknown. In this issue, Riley et al ( 2018 ) report that widening of Bax and Bak pores on the mitochondrial outer membrane (MOM) during apoptosis allows the extrusion of the mitochondrial inner membrane (MIM) into the cytosol and its permeabilization to release mtDNA independently of caspases. In this scenario, Bax and Bak emerge as key modulators of the apoptotic immunogenic response.     

A tale of two mitochondrial channels,MAC and PTP,in apoptosis

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.     

Translocation of a Bak C-terminus mutant from cytosol to mitochondria to mediate cytochrome C release: implications for Bak and Bax apoptotic function

Background

One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear.

Methodology/Principal Findings

To gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C-terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1.

Conclusions/Significance

Residues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax.     

Assembly of the Mitochondrial Apoptosis-induced Channel, MAC

Although Bcl-2 family proteins control intrinsic apoptosis, the mechanisms underlying this regulation are incompletely understood. Patch clamp studies of mitochondria isolated from cells deficient in one or both of the pro-apoptotic proteins Bax and Bak show that at least one of the proteins must be present for formation of the cytochrome c-translocating channel, mitochondrial apoptosis-induced channel (MAC), and that the single channel behaviors of MACs containing exclusively Bax or Bak are similar. Truncated Bid catalyzes MAC formation in isolated mitochondria containing Bax and/or Bak with a time course of minutes and does not require VDAC1 or VDAC3. Mathematical analysis of the stepwise changes in conductance associated with MAC formation is consistent with pore assembly by a barrel-stave model. Assuming the staves are two transmembrane α-helices in Bax and Bak, mature MAC pores would typically contain ∼9 monomers and have diameters of 5.5–6 nm. The mitochondrial permeability data are inconsistent with formation of lipidic pores capable of transporting megadalton-sized macromolecules as observed with recombinant Bax in liposomes.Permeabilization of the mitochondrial outer membrane is the commitment step in intrinsic apoptosis. This process is tightly regulated by Bcl-2 family proteins that control formation of the megachannel mitochondrial apoptosis-induced channel (MAC)² in this membrane. MAC formation correlates with release of pro-apoptotic factors, including cytochrome c from the intermembrane space into the cytosol, and initiates apoptosis (1–7).MAC is absent from normal mitochondria but forms in the outer membrane early in apoptosis, reaching peak conductances of 1.5–5 nS. This channel is formed in the presence of the multidomain pro-apoptotic proteins Bax and/or Bak (8–13), and may be composed of these proteins along with other components (14, 15). Unlike Bax, Bak is normally a resident of the mitochondrial outer membrane and is bound to VDAC2, another outer membrane protein (16). However, Bak is not available for oligomerization until another pro-apoptotic protein, like t-Bid, disrupts the interaction of Bak with VDAC2. In contrast, most Bax is located in the cytoplasm until an apoptotic signal induces the translocation of Bax to the outer membrane of mitochondria and eventual Bax oligomerization in this same membrane (14, 17).Bax and Bak have multiple putative transmembrane domains; the amphipathic helices 5 and 6 of Bax are predicted to form, at least in part, the pore of the cytochrome c release channel (18). Bax lacking helices 5 and 6 does not translocate to mitochondria nor cause cytochrome c release (19, 20). Given the structural similarities between Bax and Bak, the same helices may be important in formation of the MAC pore by both proteins (21). Although Bax and Bak are certainly involved in MAC formation, the exact molecular composition of this channel remains unknown.In this study we report that Bax and Bak are functionally redundant with regard to MAC formation and cytochrome c release in mouse embryonic fibroblasts (MEF). This is true despite the fact that Bak normally resides in the outer membrane, whereas Bax is generally translocated to this membrane to induce MAC formation. Our experimental design bypasses Bax translocation and any underlying autocatalytic mechanism that might be involved (22). Instead, it focuses on formation of the MAC pore. Early MAC-associated conductance increments are relatively small, suggesting that Bax-dependent formation of the cytochrome c-permeable pore does not occur prior to membrane insertion of Bax. Mathematical modeling of the conductance changes indicates that, if MAC is a circular pore assembled by sequential addition of helices 5 and 6 from Bax and/or Bak monomers, the mature, cytochrome c transport-competent pore is likely a 9–10-mer of these proteins.     

Necroptosis Interfaces with MOMP and the MPTP in Mediating Cell Death

During apoptosis the pro-death Bcl-2 family members Bax and Bak induce mitochondrial outer membrane permeabilization (MOMP) to mediate cell death. Recently, it was shown that Bax and Bak are also required for mitochondrial permeability transition pore (MPTP)-dependent necrosis, where, in their non-oligomeric state, they enhance permeability characteristics of the outer mitochondrial membrane. Necroptosis is another form of regulated necrosis involving the death receptors and receptor interacting protein kinases (RIP proteins, by Ripk genes). Here, we show cells or mice deficient for Bax/Bak or cyclophilin D, a protein that regulates MPTP opening, are resistant to cell death induced by necroptotic mediators. We show that Bax/Bak oligomerization is required for necroptotic cell death and that this oligomerization reinforces MPTP opening. Mechanistically, we observe mixed lineage kinase domain-like (MLKL) protein and cofilin-1 translocation to the mitochondria following necroptosis induction, while expression of the mitochondrial matrix isoform of the antiapoptotic Bcl-2 family member, myeloid cell leukemia 1 (Mcl-1), is significantly reduced. Some of these effects are lost with necroptosis inhibition in Bax/Bak1 double null, Ppif^-/-, or Ripk3^-/- fibroblasts. Hence, downstream mechanisms of cell death induced by necroptotic stimuli utilize both Bax/Bak to generate apoptotic pores in the outer mitochondrial membrane as well as MPTP opening in association with known mitochondrial death modifying proteins.     

Apoptosis-associated mitochondrial outer membrane permeabilization assays

Following most cell death signals, pro-apoptotic Bcl-2 members as Bax and Bak are activated and oligomerize into the mitochondria outer membrane, triggering its permeabilization and release into the cytosol of soluble apoptogenic factors such as cytochrome c involved in caspase activation. Thus, in many studies focused on apoptosis, cytochrome c release within cells is frequently examined to assess Bax/Bak activation and mitochondrial outer membrane permeabilization. In addition, cytochrome c release can also be investigated in vitro in functional mitochondria that have been isolated from cultured cells, offering a number of advantages. Here, protocols for measuring cytochrome c release from intact cells as well as from isolated mitochondria is detailed. Finally, assays to investigate Bax/Bak activation and olimerization are also presented.     

Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores

Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual‐color single‐molecule localization‐based super‐resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc‐shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.     

Where Killers Meet—Permeabilization of the Outer Mitochondrial Membrane during Apoptosis

Although mitochondria are usually considered as supporters of life, they are also involved in cellular death. Mitochondrial outer membrane permeabilization (MOMP) is a crucial event during apoptosis because it causes the release of proapoptotic factors from the mitochondrial intermembrane space to the cytosol. MOMP is mainly controlled by the Bcl-2 family of proteins, which consists of both proapoptotic and antiapoptotic members. We discuss the current understanding of how activating and inhibitory interactions within this family lead to the activation and oligomerization of MOMP effectors Bax and Bak, which result in membrane permeabilization. The order of events leading to MOMP is then highlighted step by step, emphasizing recent discoveries regarding the formation of Bax/Bak pores on the outer mitochondrial membrane. Besides the Bcl-2 proteins, the mitochondrial organelle contributes to and possibly regulates MOMP, because mitochondrial resident proteins and membrane lipids are prominently involved in the process.Mitochondria are essential for the life of the cell. They produce most of the ATP via oxidative phosphorylation thanks to the respiratory chain that is embedded in the inner mitochondrial membrane. Consequently, mitochondrial dysfunction is implicated in the development of many human diseases, in particular, neurodegenerative disorders (Lin and Beal 2006). Mitochondria are also prominently involved in cell death, because they play a crucial role in many apoptotic responses. Apoptosis is a self-destruction program that is essential during the development of multicellular organisms. Its dysregulation has also been recognized as a main feature of many pathological conditions, especially cancer (Llambi and Green 2011).The executioners of apoptosis are a family of cysteine proteases termed caspases that cleave a variety of cellular targets, resulting in morphological changes, degradation of genomic DNA, and, ultimately, phagocytic removal of the apoptotic cell (Taylor et al. 2008). Caspases are synthesized as inactive zymogens that become activated after regulated limited proteolysis. Two different pathways of apoptotic signaling that result in the activation of executioner caspases 3 and 7 can be distinguished. In the extrinsic pathway, binding of ligands such as FasL or TNFα to a death receptor on the plasma membrane leads to the activation of initiator caspase 8. Active caspase 8 propagates the signal by directly cleaving and thereby activating caspases 3 and 7, which continue a proteolytic cascade ultimately leading to the removal of the cell.The intrinsic pathway, on the other hand, is initiated upon exposure to a number of stress situations, including DNA damage. A subclass of the Bcl-2 protein family termed BH3-only proteins (see below) becomes activated after an internal stress stimulus and translocates to the outer mitochondrial membrane (OMM), where they orchestrate a process called mitochondrial outer membrane permeabilization (MOMP). As an outcome of this process, pores are formed in the OMM, membrane integrity is lost, and contents of the intermembrane space gain access to the cytosol. One of the molecules that is rapidly released to the cytosol is cytochrome c, which is normally a soluble electron carrier between respiratory complexes III and IV. Together with the proapoptotic cytosolic factor APAF1, cytochrome c assembles into a caspase-activating complex termed the “apoptosome.” This complex subsequently activates caspase 9, which is able to cleave caspases 3 and 7, proceeding with the same downstream cascade as in the extrinsic pathway. Other intermembrane space proteins also contribute to cell death after being released into the cytosol (e.g., SMAC/Diablo, which blocks the caspase inhibitor protein XIAP).Remarkably, the two pathways are not completely independent. Cross talk between the extrinsic and intrinsic pathways exists because of caspase 8-dependent cleavage of the BH3-only protein Bid. Upon cleavage, Bid becomes activated, and the truncated version, tBid, translocates to the surface of mitochondria to induce MOMP. In so-called type II cells, this mitochondrial feedback loop is needed to induce apoptosis through the extrinsic pathway, because of the requirement of XIAP antagonism by SMAC.The loss of OMM integrity caused by MOMP is usually considered the point of no return in the whole process, because cells are committed to die once MOMP is initiated. Therefore, this process represents a major checkpoint of apoptosis and must be tightly controlled to ensure that it is initiated at the right time and place. The main molecular players of MOMP belong to the Bcl-2 protein family. Integration of proapoptotic and antiapoptotic signals by the network of Bcl-2 proteins determines whether or not the OMM is permeabilized. In the following sections, we describe in detail the stimulatory and inhibitory protein–protein interactions within this family, discussing various models of how the MOMP effectors, Bax and Bak, become activated. Furthermore, we focus on the actual event of membrane permeabilization, summarizing the current understanding of how pores are formed in the OMM by Bax and Bak oligomers.     

Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC₅₀ = ～80 μM), along with apoptotic sub-G₁ cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δψ_m) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G₁ cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δψ_m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c, causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.     

Bax/Bak-dependent,Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.     

Use of Human Cancer Cell Lines Mitochondria to Explore the Mechanisms of BH3 Peptides and ABT-737-Induced Mitochondrial Membrane Permeabilization

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.     

Bak and Bax are non-redundant during infection- and DNA damage-induced apoptosis

Mitochondrial outer membrane permeabilization (MOMP) and release of mitochondrial intermembrane proteins like cytochrome c are critical steps in the control of apoptosis. Previous work has shown that MOMP depends on the functionally redundant multidomain proapoptotic proteins, Bak and Bax. Here we demonstrate that Bak and Bax are functionally non-redundant during Neisseria gonorrhoeae (Ngo)- and cisplatin-induced apoptosis. While the activation of Bak is caspase independent Bax activation needs Bak and active caspases. Silencing of either Bak or Bax resists both Ngo- and cisplatin- but not TNFalpha-induced apoptosis. Activation of Bak is required to release cytochrome c from the mitochondria; however, Bax is still required to activate effector caspases. Thus, both Bak and Bax are necessary to accomplish DNA damage and Ngo-induced apoptosis.     

Assessing mitochondrial outer membrane permeabilization during apoptosis

Mitochondria play a pivotal role in the regulation of apoptosis. An imbalance in apoptosis can lead to disease. Unscheduled apoptosis has been linked to neurodegeneration while inhibition of apoptosis can cause cancer. An early and key event during apoptosis is the release of factors from mitochondria. In apoptosis the mitochondrial outer membrane becomes permeable, leading to release of apoptogenic factors into the cytosol. One such factor, cytochrome c, is an electron carrier of the respiratory chain normally trapped within the mitochondrial intermembrane space. Many apoptotic studies investigate mitochondrial outer membrane permeabilization (MOMP) by monitoring the release of cytochrome c. Here, we describe three reliable techniques that detect cytochrome c release from mitochondria, through subcellular fractionation or immunocytochemistry and fluorescence microscopy, or isolated mitochondria and recombinant Bax and t-Bid proteins in vitro. These techniques will help to identify mechanisms and characterize factors regulating MOMP.     

Modulation of Bax mitochondrial insertion and induced cell death in yeast by mammalian protein kinase Cα

Protein kinase Cα (PKCα) is a classical PKC isoform whose involvement in cell death is not completely understood. Bax, a major member of the Bcl-2 family, is required for apoptotic cell death and regulation of Bax translocation and insertion into the outer mitochondrial membrane is crucial for regulation of the apoptotic process. Here we show that PKCα increases the translocation and insertion of Bax c-myc (an active form of Bax) into the outer membrane of yeast mitochondria. This is associated with an increase in cytochrome c (cyt c) release, reactive oxygen species production (ROS), mitochondrial network fragmentation and cell death. This cell death process is regulated, since it correlates with an increase in autophagy but not with plasma membrane permeabilization. The observed increase in Bax c-myc translocation and insertion by PKCα is not due to Bax c-myc phosphorylation, and the higher cell death observed is independent of the PKCα kinase activity. PKCα may therefore have functions other than its kinase activity that aid in Bax c-myc translocation and insertion into mitochondria. Together, these results give a mechanistic insight on apoptosis regulation by PKCα through regulation of Bax insertion into mitochondria.     

MAC and Bcl-2 family proteins conspire in a deadly plot

Apoptosis is an elemental form of programmed cell death; it is fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Apoptosis is also involved in many pathologies including cancer, neurodegenerative diseases, aging, and infarcts. This cell death program is tightly regulated by Bcl-2 family proteins by controlling the formation of the mitochondrial apoptosis-induced channel or MAC. Assembly of MAC corresponds to permeabilization of the mitochondrial outer membrane, which is the so called commitment step of apoptosis. MAC provides the pathway through the mitochondrial outer membrane for the release of cytochrome c and other pro-apoptotic factors from the intermembrane space. While overexpression of anti-apoptotic Bcl-2 eliminates MAC activity, oligomers of the pro-apoptotic members Bax and/or Bak are essential structural component(s) of MAC. Assembly of MAC from Bax or Bak was monitored in real time by directly patch-clamping mitochondria with micropipettes containing the sentinel tBid, a direct activator of Bax and Bak. Herein, a variety of high affinity inhibitors of MAC (iMAC) that may prove to be crucial tools in mechanistic studies have recently been identified. This review focuses on characterization of MAC activity, its regulation by Bcl-2 family proteins, and a discussion of how MAC can be pharmacologically turned on or off depending on the pathology to be treated.     

“Licensed to kill”: Tyrosine dephosphorylation and Bak activation

The genomes of multicellular organisms are under constant assault from a host of environmental agents. The efficient elimination of cells harboring damage is essential to avoid the accumulation of deleterious changes that may promote tumorigenesis. Consequently, a complex and elaborate series of damage responses have evolved to either ensure that correct repair of the DNA has been carried out, or alternatively, to initiate programmes that result in the ablation of the damaged cell. Apoptosis is recognized as both a fast an efficient way of disposing of damaged or unwanted cells before they accumulate changes that may result in the acquisition of neoplastic autonomy. The mitochondrial apoptotic pathway relies upon two effector proteins of the Bcl2 family, Bax and Bak, that when activated form pores in the outer mitochondrial membrane that release cytochrome c and other apoptogenic factors. We have recently shown that the initiation of Bak activation is controlled by dephosphorylation. In particular, we found that a specific tyrosine dephosphorylation was required for Bak activation to proceed and that tyrosine phosphatases may serve to integrate apoptotic signals that culminate in Bak dephosphorylation. Here, we discuss these findings and present additional data underlining the importance of dephosphorylation in the Bak activation process and how the modulation of Bak phosphorylation status may be modified to enhance cell killing.Key words: apoptosis, mitochondria, phosphatase, BH3, p53, Bak, signaling, phosphorylation