Chemical Inhibition of the Mitochondrial Division Dynamin Reveals Its Role in Bax/Bak-Dependent Mitochondrial Outer Membrane Permeabilization

SummaryMitochondrial 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

The Pro-Apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane That Is Enhanced by Cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release

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

activate Bax directly and can only act indirectly to reliev

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.publishe

Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

<div><p>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.</p> </div

cBid/Bax–dependent membrane permeabilization: native mitochondrial outer membranes and protein-free liposomes display different permeabilization kinetics, and native membranes respond to lower concentrations of Bax.

<p>Dextran release was measured continuously in rat liver OMVs (A–C) and defined liposomes (D). OMVs were loaded with either 10 kDa dextran-cascade blue (A,C) or 70 kDa dextran-fluorescein (B). Bax concentration was 25 nM (A,B) or as indicated (C,D). Arrows indicate additions of Bax and 0.05% Triton X-100. When present, cBid (40 nM) was added 2–3 min prior to Bax. Bcl-xL (1 µM) was added 2 min before Bax or at later points indicated by arrows (B; blue lines). (E) Respiration-based continuous measurements of Bax-induced MOMP in isolated rat liver mitochondria. Mitochondria were incubated in a KCl-based respiration buffer containing 0.3 µM FCCP, 2 µM myxothiazol (Myxo), 2 mM NADH, and 80 µM cytochrome c (CytC). Under these conditions, the rate of NADH-dependent respiration depends on the rate of cytochrome c permeation through the MOM and therefore reflects the kinetics of MOMP. Arrows indicate additions of myxothiazol, NADH, CytC, and Bax (60 nM) mixed with cBid (40 nM). Control trace (blue line): no cBid/Bax added. Data are plotted as the first derivative of oxygen concentration (i.e., respiration rate) versus time. The insert shows the original oxygen consumption curve, in the presence of cBid and Bax. Note that the kinetics of MOMP in whole mitochondria and OMVs are similarly biphasic. Data shown are representative of at least three independent experiments. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001394#pbio.1001394.s001" target="_blank">Figure S1</a>.</p