Disruption of mitochondrial quality control genes promotes caspase-resistant cell survival following apoptotic stimuli.

In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases, a subpopulation of treated cells can exhibit a sublethal response, termed "minority MOMP." In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease caspase-activated DNase (DNA fragmentation factor subunit beta). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.

Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.

Phenotypic selection with an intrabody library reveals an anti-apoptotic function of PKM2 requiring Mitofusin-1.

Bcl-2 family proteins control a decisive apoptotic event: mitochondrial outer membrane permeabilization (MOMP). To discover MOMP-regulating proteins, we expressed a library of intracellular single-chain variable fragments (scFvs) ("intrabodies") and selected for those rescuing cells from apoptosis induced by BimS (the short isoform of Bim). One anti-apoptotic intrabody, intrabody 5 (IB5), recognized pyruvate kinase M2 (PKM2), which is expressed in cancer cells. PKM2 deletion ablated this clonogenic rescue; thus, IB5 activated a latent cytoprotective function of PKM2. This resulted not from pyruvate kinase activity per se but rather from the formation of an active tetrameric conformation of PKM2. A stably tetrameric PKM2 mutant, K422R, promoted cell survival even in the absence of IB5, and IB5 further increased survival. Mitochondria isolated from IB5-expressing cells were relatively resistant to MOMP in vitro. In cells, IB5 expression up-regulated Mitofusin-1 (Mfn1) and increased mitochondrial length. Importantly, Mfn1 deficiency abrogated IB5's cytoprotective effect. PKM2's anti-apoptotic function could help explain its preferential expression in human cancer.

Mitochondrial residence of the apoptosis inducer BAX is more important than BAX oligomerization in promoting membrane permeabilization.

Permeabilization of the mitochondrial outer membrane is a key step in the intrinsic apoptosis pathway, triggered by the release of mitochondrial intermembrane space proteins into the cytoplasm. The BCL-2-associated X apoptosis regulator (BAX) protein critically contributes to this process by forming pores in the mitochondrial outer membrane. However, the relative roles of the mitochondrial residence of BAX and its oligomerization in promoting membrane permeabilization are unclear. To this end, using both cell-free and cellular experimental systems, including membrane permeabilization, size-exclusion chromatography-based oligomer, and retrotranslocation assays, along with confocal microscopy analysis, here we studied two BAX C-terminal variants, T182I and G179P. Neither variant formed large oligomers when activated in liposomes. Nevertheless, the G179P variant could permeabilize liposome membranes, suggesting that large BAX oligomers are not essential for the permeabilization. However, when G179P was transduced into BAX/BCL2 agonist killer (BAK) double-knockout mouse embryonic fibroblasts, its location was solely cytoplasmic, and it then failed to mediate cell death. In contrast, T182I was inefficient in both liposome insertion and permeabilization. Yet, when transduced into cells, BAXT182I resided predominantly on mitochondria, because of its slow retrotranslocation and mediated apoptosis as efficiently as WT BAX. We conclude that BAX's mitochondrial residence in vivo, regulated by both targeting and retrotranslocation, is more significant for its pro-apoptotic activity than its ability to insert and to form higher-order oligomers in model membranes. We propose that this finding should be taken into account when developing drugs that modulate BAX activity.

Opa1-mediated cristae opening is Bax/Bak and BH3 dependent, required for apoptosis, and independent of Bak oligomerization.

Controversy surrounds the role and mechanism of mitochondrial cristae remodeling in apoptosis. Here we show that the proapoptotic BH3-only proteins Bid and Bim induced full cytochrome c release but only a subtle alteration of crista junctions, which involved the disassembly of Opa1 complexes. Both mitochondrial outer membrane permeabilization (MOMP) and crista junction opening (CJO) were caspase independent and required a functional BH3 domain and Bax/Bak. However, MOMP and CJO were experimentally separable. Pharmacological blockade of MOMP did not prevent Opa1 disassembly and CJO; moreover, expression of a disassembly-resistant mutant Opa1 (Q297V) blocked cytochrome c release and apoptosis but not Bax activation. Thus, apoptosis requires a subtle form of Opa1-dependent crista remodeling that is induced by BH3-only proteins and Bax/Bak but independent of MOMP.

Bax activation initiates the assembly of a multimeric catalyst that facilitates Bax pore formation in mitochondrial outer membranes.

Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for "intrinsic" apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the "hit-and-run" hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.

Targeting Ca2+ and Mitochondrial Homeostasis by Antipsychotic Thioridazine in Leukemia Cells.

Mitochondria have pivotal roles in cellular physiology including energy metabolism, reactive oxygen species production, Ca2+ homeostasis, and apoptosis. Altered mitochondrial morphology and function is a common feature of cancer cells and the regulation of mitochondrial homeostasis has been identified as a key to the response to chemotherapeutic agents in human leukemias. Here, we explore the mechanistic aspects of cytotoxicity produced by thioridazine (TR), an antipsychotic drug that has been investigated for its anticancer potential in human leukemia cellular models. TR exerts selective cytotoxicity against human leukemia cells in vitro. A PCR array provided a general view of the expression of genes involved in cell death pathways. TR immediately produced a pulse of cytosolic Ca2+, followed by mitochondrial uptake, resulting in mitochondrial permeabilization, caspase 9/3 activation, endoplasmic reticulum stress, and apoptosis. Ca2+ chelators, thiol reducer dithiothreitol, or CHOP knockdown prevented TR-induced cell death. TR also exhibited potent cytotoxicity against BCL-2/BCL-xL-overexpressing leukemia cells. Additionally, previous studies have shown that TR exhibits potent antitumor activity in vivo in different solid tumor models. These findings show that TR induces a Ca2+-mediated apoptosis with involvement of mitochondrial permeabilization and ER stress in leukemia and it emphasizes the pharmacological potential of TR as an adjuvant in antitumor chemotherapy.

Bcl-2-family proteins and the role of mitochondria in apoptosis.

Mitochondria are central to many forms of cell death, usually via the release of pro-apoptotic proteins from the mitochondrial intermembrane space. Some intermembrane space proteins, including cytochrome c, Smac/DIABLO, and Omi/Htra2, can induce or enhance caspase activation, whereas others, such as AIF and endonuclease G, might act in a caspase-independent manner. Intermembrane space protein release is often regulated by Bcl-2-family proteins. Recent evidence suggests that pro-apoptotic members of this family, by themselves, can permeabilize the outer mitochondrial membrane without otherwise damaging mitochondria. Mitochondria can contribute to cell death in other ways. For example, they can respond to calcium release from the endoplasmic reticulum by undergoing the mitochondrial permeability transition, which in turn causes outer membrane rupture and the release of intermembrane space proteins. Bcl-2-family proteins can influence the levels of releasable Ca(2+) in the endoplasmic reticulum, and thus determine whether the released Ca(2+) is sufficient to overload mitochondria and induce cell death.

Caspase-2-induced apoptosis requires bid cleavage: a physiological role for bid in heat shock-induced death.

The mechanisms through which Caspase-2 leads to cell death are controversial. Here we show, using a combination of cell-free and cell culture-based approaches, that cleavage of the Bcl-2-family protein Bid is required for the induction of apoptosis by Caspase-2. Caspase-2 promoted cytochrome c release from mitochondria in the presence of cytosol from wild-type, but not Bid-deficient, mouse embryonic fibroblasts (MEFs). Recombinant wild-type Bid, but not a noncleavable mutant (D59E), restored cytochrome c release. Similarly, Bid-null MEFs were relatively resistant to apoptosis triggered by active Caspase-2, and apoptosis was restored in Bid-null cells by the expression of wild-type, but not D59E, Bid. Finally, Bid-null MEFs were substantially more resistant to apoptosis induced by heat shock, which has been shown to be dependent on apical activation of Caspase-2. The data are consistent with a model in which Caspase-2 induces apoptosis via cleavage of Bid at D59 and the subsequent engagement of the mitochondrial (intrinsic) pathway.

Mitochondria: pharmacological manipulation of cell death.

Cell death by apoptosis or necrosis is often important in the etiology and treatment of disease. Since mitochondria play important roles in cell death pathways, these organelles are potentially prime targets for therapeutic intervention. Here we discuss the mechanisms through which mitochondria participate in the cell death process and also survey some of the pharmacological approaches that target mitochondria in various ways.

Pro-apoptotic Bax molecules densely populate the edges of membrane pores.

How the pro-apoptotic Bax protein permeabilizes the mitochondrial outer membrane is not fully understood. Previously, using cryo-electron microscopy (cryo-EM), we showed that activated Bax forms large, growing pores. Whether formed in liposomes or in mitochondrial outer membranes, Bax-induced pores exhibit the same morphology, with negative curvature flanking the edges and with no visible protein structure protruding from the membranes. Here we used cryo-EM to show that gold-labeled Bax molecules, after activation by Bid, became localized strictly at pore edges. This argues that Bax acts at short range to deform the membrane. Also, Bax molecules populated the walls of both small and large pores at the same density, implying that Bax is continuously recruited to the pores as they widen. Moreover, because all Bax molecules became oligomerized after membrane insertion, we infer that Bax oligomers are present at pore edges. We suggest that oligomerization may promote pore enlargement.

Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes.

Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼ 100-160 nm in diameter after 60-90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10-500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.

Bioenergetics and cell death.

Mitochondrial bioenergetic function is a key to cell life and death. Cells need energy not only to support their vital functions but also to die gracefully. Execution of an apoptotic program includes energy-dependent steps, including kinase signaling, formation of the apoptosome, and effector caspase activation. Under conditions of bioenergetic collapse, cells are diverted toward necrotic demise. Mitochondrial outer membrane permeabilization (MOMP) is a decisive event in the execution of apoptosis. It is also causally linked to a decline in bioenergetic function via different mechanisms, not merely due to cytochrome c dispersion. MOMP-induced bioenergetic deficiency is usually irreversible and commits cells to die, even when caspases are inactive. Here, we discuss the mechanisms by which MOMP impacts bioenergetics in different cell death paradigms.

Caspase-independent mitochondrial cell death results from loss of respiration, not cytotoxic protein release.

In apoptosis, mitochondrial outer membrane permeabilization (MOMP) triggers caspase-dependent death. However, cells undergo clonogenic death even if caspases are blocked. One proposed mechanism involved the release of cytotoxic proteins (e.g., AIF and endoG) from mitochondria. To initiate MOMP directly without side effects, we created a tamoxifen-switchable BimS fusion protein. Surprisingly, even after MOMP, caspase-inhibited cells replicated DNA and divided for approximately 48 h before undergoing proliferation arrest. AIF and endoG remained in mitochondria. However, cells gradually lost mitochondrial membrane potential and ATP content, and DNA synthesis slowed to a halt by 72 h. These defects resulted from a partial loss of respiratory function, occurring 4-8 h after MOMP, that was not merely due to dispersion of cytochrome c. In particular, Complex I activity was completely lost, and Complex IV activity was reduced by approximately 70%, whereas Complex II was unaffected. Later, cells exhibited a more profound loss of mitochondrial protein constituents. Thus, under caspase inhibition, MOMP-induced clonogenic death results from a progressive loss of mitochondrial function, rather than the release of cytotoxic proteins from mitochondria.

GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation.

In cells undergoing apoptosis, mitochondrial outer-membrane permeabilization (MOMP) is followed by caspase activation promoted by released cytochrome c. Although caspases mediate the apoptotic phenotype, caspase inhibition is generally not sufficient for survival following MOMP; instead cells undergo a "caspase-independent cell death" (CICD). Thus, MOMP may represent a point of commitment to cell death. Here, we identify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a critical regulator of CICD. GAPDH-expressing cells preserved their clonogenic potential following MOMP, provided that caspase activation was blocked. GAPDH-mediated protection of cells from CICD involved an elevation in glycolysis and a nuclear function that correlated with and was replaced by an increase in Atg12 expression. Consistent with this, protection from CICD reflected an increase in and a dependence upon autophagy, associated with a transient decrease in mitochondrial mass. Therefore, GAPDH mediates an elevation in glycolysis and enhanced autophagy that cooperate to protect cells from CICD.

Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration.

The release of mitochondrial intermembrane space proteins to the cytosol is a key event during apoptosis. We used in situ fluorescent labeling of proteins tagged with a short tetracysteine-containing sequence to follow the release of Smac, Omi, adenylate kinase-2, cytochrome c, and apoptosis-inducing factor (AIF) during apoptosis and compared the release with that of cytochrome c tagged with GFP in individual cells observed over time. We observed a caspase-independent, simultaneous release of cytochrome c, Smac, Omi, and adenylate kinase-2. Although AIF release also was caspase-independent and commenced with that of the other proteins, it proceeded much more slowly and incompletely from mitochondria, perhaps because of a requirement for a secondary event. These results suggest that these proteins are released through the same mitochondrial pore and that apoptosis may not be regulated through a selective release of individual mitochondrial proteins. The timing and extent of AIF release makes it unlikely that it is involved in the induction of apoptosis, either upstream or downstream of mitochondrial outer membrane permeabilization.

PUMA couples the nuclear and cytoplasmic proapoptotic function of p53.

The Trp53 tumor suppressor gene product (p53) functions in the nucleus to regulate proapoptotic genes, whereas cytoplasmic p53 directly activates proapoptotic Bcl-2 proteins to permeabilize mitochondria and initiate apoptosis. Here, we demonstrate that a tripartite nexus between Bcl-xL, cytoplasmic p53, and PUMA coordinates these distinct p53 functions. After genotoxic stress, Bcl-xL sequestered cytoplasmic p53. Nuclear p53 caused expression of PUMA, which then displaced p53 from Bcl-xL, allowing p53 to induce mitochondrial permeabilization. Mutant Bcl-xL that bound p53, but not PUMA, rendered cells resistant to p53-induced apoptosis irrespective of PUMA expression. Thus, PUMA couples the nuclear and cytoplasmic proapoptotic functions of p53.

Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes.

A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mm KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes.

The multidomain proapoptotic molecules Bax and Bak are directly activated by heat.

During apoptosis, engagement of the mitochondrial pathway involves a decisive event characterized by the release of mitochondrial intermembrane space proteins, such as cytochrome c. This permeabilization of the mitochondrial outer membrane depends on activation and oligomerization of multidomain Bcl-2-family proteins Bax or Bak. Although specific members of the Bcl-2 family can activate these proapoptotic proteins, we found that heat directly activated Bax or Bak to induce cytochrome c release. A preparation of mitochondria heated at 43 degrees C released cytochrome c in association with Bak oligomerization, and Bcl-xL prevented these events. Similarly, heat induced the oligomerization of recombinant Bax, conferring an ability to permeabilize mitochondria. Compared with wild-type cells, bax(-/-)bak(-/-) mouse embryonic fibroblasts and mitochondria isolated from these cells were resistant to heat-induced cytochrome c release. Cytosol from untreated cells inhibited heat-activated Bax or Bak; however, depletion of cytosolic Bcl-xL ablated this protection. Although mitochondria heated in the presence of cytosol did not release cytochrome c, they displayed a dramatic increase in sensitivity to permeabilization by the BH3-only protein Bid. Additionally, a peptide corresponding to the BH3 domain of Puma counteracted the inhibitory effect of cytosol and permitted heat-activated Bak to permeabilize the mitochondria. Therefore, heat represents a condition under which multidomain proapoptotic proteins are activated, and this activation is regulated by both antiapoptotic and BH3-only members of the Bcl-2 family. Our results support an emerging paradigm, wherein the activation of Bax or Bak and the blockade of antiapoptotic Bcl-2 proteins are pivotal steps in the mitochondrial pathway of apoptosis.

BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly.

Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.