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.     

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.     

Mitochondrial outer membrane permeabilization during apoptosis: The role of mitochondrial fission

Mitochondria continually fuse and divide to yield a dynamic interconnected network throughout the cell. During apoptosis, concomitantly with permeabilization of the mitochondrial outer membrane (MOMP) and cytochrome c release, mitochondria undergo massive fission. This results in the formation of small, round organelles that tend to aggregate around the nucleus. Under some circumstances, preceding their fission, mitochondria tend to elongate and to hyperfuse, a process that is interpreted as a cell defense mechanism. Since many years, there is a controversy surrounding the physiological relevance of mitochondrial fragmentation in apoptosis. In this review, we present recent advances in this field, describe the mechanisms that underlie this process, and discuss how they could cooperate with Bax to trigger MOMP and cytochrome c release. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

PUMA cooperates with direct activator proteins to promote mitochondrial outer membrane permeabilization and apoptosis

The BCL-2 family of proteins regulates apoptosis by controlling mitochondrial outer membrane permeabilization (MOMP). Within the family there are numerous protein?protein interactions that influence MOMP; however, defining the ultimate signal that commits a cell to apoptosis remains controversial. We chose to examine the function of the BH3-only protein, p53 upregulated modulator of apoptosis (PUMA), to define its contribution to MOMP and cooperation with the direct activator proteins. PUMA is a potent regulator of MOMP and our data suggest that this function is attributed to two distinct mechanisms which both rely on PUMA binding to the anti-apoptotic BCL-2 proteins: de-repression and sensitization. Here we will define these interactions and discuss our experiments that suggest PUMA cooperates with direct activator proteins to efficiently induce MOMP and apoptosis.     

Hop cleavage and function in granzyme B-induced apoptosis

Granzyme B (GzmB) is a cytotoxic protease found in the granules of natural killer cells and cytotoxic T lymphocytes. GzmB cleaves multiple intracellular protein substrates, leading to caspase activation, DNA fragmentation, cytoskeletal instability, and rapid induction of target cell apoptosis. However, no known individual substrate is required for GzmB to induce apoptosis. GzmB is therefore thought to initiate multiple cell death pathways simultaneously to ensure the death of target cells. We previously identified Hop (Hsp70/Hsp90-organizing protein) as a GzmB substrate in a proteomic survey (Bredemeyer, A. J., Lewis, R. M., Malone, J. P., Davis, A. E., Gross, J., Townsend, R. R., and Ley, T. J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 11785-11790). Hop is a co-chaperone for Hsp70 and Hsp90, which have been implicated in the negative regulation of apoptosis. We therefore hypothesized that Hop may have an anti-apoptotic function that is abolished upon cleavage, lowering the threshold for GzmB-induced apoptosis. Here, we show that Hop was cleaved directly by GzmB in vitro and in cells undergoing GzmB-induced apoptosis. Expression of the two cleavage fragments of Hop did not induce cell death. Although cleavage of Hop by GzmB destroyed Hop function in vitro, both cells overexpressing GzmB-resistant Hop and cells with a 90-95% reduction in Hop levels exhibited unaltered susceptibility to GzmB-induced death. We conclude that Hop per se does not set the threshold for susceptibility to GzmB-induced apoptosis. Although it is possible that Hop may be cleaved by GzmB as an "innocent bystander" during the induction of apoptosis, it may also act to facilitate apoptosis in concert with other GzmB substrates.     

Dynamics of granzyme B-induced apoptosis: mathematical modeling

Apoptosis is mediated by an intracellular biochemical system that mainly includes proteins (procaspases, caspases, inhibitors, Bcl-2 protein family as well as substances released from mitochondrial intermembrane space). The dynamics of caspase activation and target cleavage in apoptosis induced by granzyme B in a single K562 cell was studied using a mathematical model of the dynamics of granzyme B-induced apoptosis developed in this work. Also the first application of optimization approach to determination of unknown kinetic constants of biochemical apoptotic reactions was presented. The optimization approach involves solving of two problems: direct and inverse. Solving the direct optimization problem, we obtain the initial (baseline) concentrations of procaspases for known kinetic constants through conditional minimization of a cost function based on the principle of minimum protein consumption by the apoptosis system. The inverse optimization problem is aimed at determination of unknown kinetic constants of apoptotic biochemical reactions proceeding from the condition that the optimal concentrations of procaspases resulting from the solution of the direct optimization problem coincide with the observed ones, that is, those determined by biochemical methods. The Multidimensional Index Method was used to perform numerical solution of the inverse optimization problem.     

Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1

We investigated the role of glycogen synthase kinase-3 (GSK-3), which is inactivated by AKT, for its role in the regulation of apoptosis. Upon IL-3 withdrawal, protein levels of MCL-1 decreased but were sustained by pharmacological inhibition of GSK-3, which prevented cytochrome c release and apoptosis. MCL-1 was phosphorylated by GSK-3 at a conserved GSK-3 phosphorylation site (S159). S159 phosphorylation of MCL-1 was induced by IL-3 withdrawal or PI3K inhibition and prevented by AKT or inhibition of GSK-3, and it led to increased ubiquitinylation and degradation of MCL-1. A phosphorylation-site mutant (MCL-1(S159A)), expressed in IL-3-dependent cells, showed enhanced stability upon IL-3 withdrawal and conferred increased protection from apoptosis compared to wild-type MCL-1. The results demonstrate that the control of MCL-1 stability by GSK-3 is an important mechanism for the regulation of apoptosis by growth factors, PI3K, and AKT.     

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Mitochondrial membrane permeabilization (MMP) is considered as the “point-of-no-return” in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential (ΔΨ_m). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several techniques to measure MMP by cytofluorometry and fluorescence microscopy have been developed. Here, we summarize the currently available methods for the detection of MMP, and provide a comparative analysis of these techniques.     

A central role for Bid in granzyme B-induced apoptosis

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Bid-deficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.     

Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario

Mitochondrial outer membrane permeabilization (MOMP) is considered the 'point of no return' as this event is responsible for engaging the apoptotic cascade in numerous cell death pathways. MOMP is directly governed by a subset of the BCL-2 family of proapoptotic proteins, which induce disruptions in the outer mitochondrial membrane (OMM) and subsequent release of death-promoting proteins like cytochrome c. The proposal here is centered on our hypothesis that MOMP is dictated by an interaction between the cytosol and the OMM, and although proteins of the OMM may be important in the process, the 'decision' to undergo apoptosis originates within the cytosol with no participation (in terms of yes, no and when) by mitochondria.     

Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization

Mitochondrial fusion and division play important roles in the regulation of apoptosis. Mitochondrial fusion proteins attenuate apoptosis by inhibiting release of cytochrome c from mitochondria, in part by controlling cristae structures. Mitochondrial division promotes apoptosis by an unknown mechanism. We addressed how division proteins regulate apoptosis using inhibitors of mitochondrial division identified in a chemical screen. The most efficacious inhibitor, mdivi-1 (for mitochondrial division inhibitor) attenuates mitochondrial division in yeast and mammalian cells by selectively inhibiting the mitochondrial division dynamin. In cells, mdivi-1 retards apoptosis by inhibiting mitochondrial outer membrane permeabilization. In vitro, mdivi-1 potently blocks Bid-activated Bax/Bak-dependent cytochrome c release from mitochondria. These data indicate the mitochondrial division dynamin directly regulates mitochondrial outer membrane permeabilization independent of Drp1-mediated division. Our findings raise the interesting possibility that mdivi-1 represents a class of therapeutics for stroke, myocardial infarction, and neurodegenerative diseases.     

Dimerization of Smac is crucial for its mitochondrial retention by XIAP subsequent to mitochondrial outer membrane permeabilization

Following the apoptotic permeabilization of the outer mitochondrial membrane, the inter-membrane space protein second mitochondria-derived activator of caspases (Smac) is released into the cytosol. Smac efficiently promotes apoptosis by antagonizing x-linked inhibitor of apoptosis protein (XIAP), an inhibitor of caspases-9, -3, and -7, via a short NH₂-terminal inhibitor of apoptosis protein (IAP) binding motif (AVPI). Native Smac dimerizes to form a highly stable and inflexible elongated arch, however, a functional role for this outstretched structure so far remained unknown. Using time-lapse single-cell imaging of DLD-1 and HCT-116 colon cancer cells, we here demonstrate that upon mitochondrial outer membrane permeabilization physiological expression levels of XIAP are sufficient to selectively prolong the release of dimeric but not monomeric Smac. Elevating the expression of XIAP further extended the release duration of dimeric Smac and resulted in the mitochondrial retention of a significant proportion of the Smac pool. In contrast, monomeric Smac was always fully released and the release kinetics were not affected by altered XIAP expression. Our findings therefore indicate that the dimerization of Smac is critical for the XIAP-mediated retention of Smac at or inside the mitochondria.     

Control of mitochondrial outer membrane permeabilization and Bcl-xL levels by thioredoxin 2 in DT40 cells

Mitochondria play a central role in the initiation of apoptosis, which is regulated by various factors such as ATP synthesis, reactive oxygen species, redox status, and outer membrane permeabilization. Disruption of chicken thioredoxin 2 (Trx2), a mitochondrial redox-regulating protein, results in apoptosis in DT40 cells. To investigate the mechanism of this apoptosis, we prepared transfectants expressing control (DT40-TRX2-/-), human thioredoxin 2 (TRX2) (DT40-hTRX2), or redox-inactive TRX2 (DT40-hTRX2CS) in conditional Trx2-deficient DT40 cells containing a tetracycline-repressible Trx2 gene. Production of ATP was not significantly changed by down-regulation of Trx2 expression. The generation of reactive oxygen species was enhanced by the down-regulation of Trx2 expression in DT40-TRX2-/-. Unexpectedly, the change was blocked in both DT40-hTRX2 and DT40-hTRX2CS cells. The down-regulation of Trx2 expression caused the release of cytochrome c and apoptosis-inducing factor on day 3, and apoptosis on day 5. These changes were also suppressed in both DT40-hTRX2 and DT40-hTRX2CS cells, suggesting that TRX2 regulates mitochondrial outer membrane permeabilization and apoptosis by redox-active site cysteine-independent mechanisms. The down-regulation of Trx2 expression caused a decrease in the protein level of Bcl-xL on day 3, whereas the protein level of Bcl-2 did not change until day 4, and the mRNA level of Bcl-xL was unchanged. The decrease in Bcl-xL was not blocked by a caspase 3 inhibitor but blocked in both DT40-hTRX2 and DT40-hTRX2CS. These findings indicate a link between the redox active site cysteine-independent action of TRX2 and the level of Bcl-xL in the regulation of apoptosis.     

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.     

Carbon monoxide prevents hepatic mitochondrial membrane permeabilization

Background  

Low concentrations of carbon monoxide (CO) protect hepatocytes against apoptosis and confers cytoprotection in several models of liver. Mitochondria are key organelles in cell death control via their membrane permeabilization and the release of pro-apoptotic factors.     

Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co²⁺). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co²⁺ (outside of the matrix). Upon induction of apoptosis, such calcein/Co²⁺-labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential ( _m ), the local overproduction of reactive oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co²⁺ (CC) staining can be dissociated from the _m loss, both of which may occur in a caspase-dependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent _m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.     

Granzyme B-induced mitochondrial ROS are required for apoptosis

Caspases and the cytotoxic lymphocyte protease granzyme B (GB) induce reactive oxygen species (ROS) formation, loss of transmembrane potential and mitochondrial outer membrane permeabilization (MOMP). Whether ROS are required for GB-mediated apoptosis and how GB induces ROS is unclear. Here, we found that GB induces cell death in an ROS-dependent manner, independently of caspases and MOMP. GB triggers ROS increase in target cell by directly attacking the mitochondria to cleave NDUFV1, NDUFS1 and NDUFS2 subunits of the NADH: ubiquinone oxidoreductase complex I inside mitochondria. This leads to mitocentric ROS production, loss of complex I and III activity, disorganization of the respiratory chain, impaired mitochondrial respiration and loss of the mitochondrial cristae junctions. Furthermore, we have also found that GB-induced mitocentric ROS are necessary for optimal apoptogenic factor release, rapid DNA fragmentation and lysosomal rupture. Interestingly, scavenging the ROS delays and reduces many of the features of GB-induced death. Consequently, GB-induced ROS significantly promote apoptosis.To induce cell death, human granzyme B (GB) activates effector caspase-3 or acts directly on key caspase substrates, such as the proapoptotic BH3 only Bcl-2 family member Bid, inhibitor of caspase-activated DNase (ICAD), poly-(ADP-ribose) polymerase-1 (PARP-1), lamin B, nuclear mitotic apparatus protein 1 (NUMA1), catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and tubulin.^{1, 2, 3} Consequently, caspase inhibitors have little effect on human GB-mediated cell death and DNA fragmentation.² GB causes reactive oxygen species (ROS) production, dissipation of the mitochondrial transmembrane potential (ΔΨm) and MOMP, which leads to the release of apoptogenic factors such as cytochrome c (Cyt c), HtrA2/Omi, endonuclease G (Endo G), Smac/Diablo and apoptosis-inducing factor, from the mitochondrial intermembrane space to the cytosol.^{4, 5, 6, 7, 8, 9, 10, 11} Interestingly, cells deficient for Bid, Bax and Bak are still sensitive to human GB-induced cell death,^{5, 11, 12, 13} suggesting that human GB targets the mitochondria in another way that needs to be characterized. Altogether, much attention has been focused on the importance of MOMP in the execution of GB-mediated cell death, leaving unclear whether ROS production is a bystander effect or essential to the execution of GB-induced apoptosis. The mitochondrial NADH: ubiquinone oxidoreductase complex I is a key determinant in steady-state ROS production. This 1 MDa complex, composed of 44 subunits,¹⁴ couples the transfer of two electrons from NADH to ubiquinone with the translocation of four protons to generate the ΔΨm. The importance of ROS has been previously demonstrated for caspase-3 and granzyme A (GA) pathways through the cleavage of NDUFS1 and NDUFS3, respectively.^{15, 16, 17, 18} GA induces cell death in a Bcl-2-insensitive and caspase- and MOMP-independent manner that has all the morphological features of apoptosis.^{1, 16, 17, 18, 19, 20} As GA and GB cell death pathways are significantly different, whether ROS are also critical for GB still need to be tested. Here, we show that GB induces ROS-dependent apoptosis by directly attacking the mitochondria in a caspase-independent manner to cleave NDUFS1, NDUFS2 and NDUFV1 in complex I. Consequently, GB inhibits electron transport chain (ETC) complex I and III activities, mitochondrial ROS production is triggered and mitochondrial respiration is compromised. Interestingly, MOMP is not required for GB to cleave the mitochondrial complex I subunits and ROS production. Moreover, GB action on complex I disrupts the organization of the respiratory chain and triggers the loss of the mitochondrial cristae junctions. We also show that GB-mediated mitocentric ROS are necessary for proper apoptogenic factor release from the mitochondria to the cytosol and for the rapid DNA fragmentation, both hallmarks of apoptosis. Moreover, GB-induced ROS are necessary for lysosomal membrane rupture. Thus, our work brings a new light to the GB pathway, showing that GB-mediated mitochondrial ROS are not adventitious waste of cell death, but essential mediators of apoptosis.     

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, ‘worm-like'' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death ‘athetosis''. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.     

Mitochondrial membrane permeabilization upon interaction with lysozyme fibrillation products: Role of mitochondrial heterogeneity

Mitochondrial dysfunction is a common feature of many neurodegenerative disorders, although the relative degree of this functional impairment and the mechanism of selective vulnerability in different regions of the brain related to various neurological diseases are not completely understood. In a recent study, we reported on brain mitochondrial membrane permeabilization upon interaction with hen egg white lysozyme (HEWL) protofibrils, and came to the conclusion that mitochondrial heterogeneity could offer an explanation for some of our observations. Accordingly, the first part of the present investigation was devoted on studies involving interaction of HEWL fibrillation products with mitochondria isolated from various areas of the brain, known to be affected in a number of well-characterized neurodegenerative conditions. This was followed by looking at heart and liver mitochondria. Membrane permeabilization was investigated by monitoring release of mitochondrial enzymes. Mitochondria isolated from cortex and hippocampus showed greater sensitivities than those prepared from substantia nigra. Results clearly demonstrate heterogeneity in brain mitochondria together with a higher resistance to permeabilization in these organelles in comparison with those isolated from liver and heart. Calcium and spermine were found to be more effective in preventing permeabilization in brain mitochondria, as compared to liver and heart. The structure-function aspects and physiological significance of the observations in relation to differences in composition, biophysical nature and morphological properties of mitochondria are discussed. It is argued that studies on heterogeneity in cellular membranes, the primary targets of toxic protofibrils, may provide important insights into mechanism of toxicity, with clinical and pathological manifestations.