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.     

Activation of Bax by joint action of tBid and mitochondrial outer membrane: Monte Carlo simulations

The mitochondrial pathway of apoptosis proceeds when molecules, such as cytochrome c, sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane (MOM) permeabilization. Bax, a member of the Bcl-2 protein family, plays a pivotal role in mitochondrion-mediated apoptosis. In response to apoptotic stimuli, Bax integrates into the MOM, where it mediates the release of cytochrome c from the intermembrane space into the cytosol, leading to caspase activation and cell death. The pro-death action of Bax is regulated by interactions with both other prosurvival proteins, such as tBid, and the MOM, but the exact mechanisms remain largely unclear. Here, the mechanisms of integration of Bax into a model membrane mimicking the MOM were studied by Monte Carlo simulations preceded by a computer prediction of the docking of tBid with Bax. A novel model of Bax activation by tBid was predicted by the simulations. In this model, tBid binds to Bax at an interaction site formed by Bax helices α1, α2, α3 and α5 leading, due to interaction of the positively charged N-terminal fragment of tBid with anionic lipid headgroups, to Bax reorientation such that a hydrogen-bonded pair of residues, Asp98 and Ser184, is brought into close proximity with negatively charged lipid headgroups. The interaction with these headgroups destabilizes the hydrogen bond which results in the release of helix α9 from the Bax-binding groove, its insertion into the membrane, followed by insertion into the membrane of the α5–α6 helical hairpin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

The tumor suppressor DAP-kinase links cell adhesion and cytoskeleton reorganization to cell death regulation

Summary Death-associated protein (DAP)-kinase, an actin-cytoskeleton localized serine/threonine kinase, functions as a novel tumor suppressor and participates in a wide variety of cell death systems. Recent studies indicate that DAP-kinase elicits a potent cytoskeletal reorganization effect and is capable of modulating integrin inside-out signaling. Using this understanding of DAP-kinase protein function as a framework, we discuss the functional mechanisms of this kinase in regulating death-associated morphological and signaling events. Furthermore, a potential role of DAP-kinase to be a drug target is also discussed.     

Yersinia YopP-induced apoptotic cell death in murine dendritic cells is partially independent from action of caspases and exhibits necrosis-like features

Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of κB kinase (IKK)-β resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells (DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential ΔΨ_m and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs independently from apoptotic events or as an apoptotic epiphenomenon.     

Vanilloid receptor agonists and antagonists are mitochondrial inhibitors: how vanilloids cause non-vanilloid receptor mediated cell death

Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.     

The cell death machinery controlled by Bax and Bcl-XL is evolutionarily conserved in <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

Bax and Bcl-XL are key regulators of apoptosis in mammals. Here we report the functional characterization of two Bcl-2 homologues, ciBax and ciBcl-XL, in a basal invertebrate-chordate ascidian Ciona intestinalis. CiBax is a Ciona homologue of the BH1-3 pro-apoptotic protein Bax, whereas ciBcl-XL is a Bcl-XL-like anti-apoptotic protein. Molecular modeling analysis showed that ciBax and ciBcl-XL share both sequence and structural similarities to human Bax and Bcl-XL, respectively. Like their human counterparts, ciBax could form a homodimer or oligomers as well as heterodimerize with ciBcl-XL, and overexpression of ciBax caused apoptosis that could be attenuated by ciBcl-XL. Mutagenesis studies showed that the BH3 domain of ciBax is critical for its cell death-inducing function and also for its interaction with ciBcl-XL. In Ciona embryos, ectopic expression of ciBax but not its BH3 deletion mutant resulted in cell dissociation and apoptosis after late gastrula stage of embryonic development. Moreover, not only wild type ciBcl-XL but also a mutant ciBcl-XL(F101V), which is unable to interact with ciBax, could block cell dissociation and developmental deficit in Ciona embryos induced by overexpression of ciBax. Taken together, these findings suggest that functional homologues of both the BH1-3 death effector Bax and the pro-survival Bcl-XL exist in sea squirt Ciona intestinalis, and they control the cell death machinery independent of their heterodimerization.     

The victorin-induced mitochondrial permeability transition precedes cell shrinkage and biochemical markers of cell death, and shrinkage occurs without loss of membrane integrity

Summary In this study, we determined the timing of events associated with cell death induced by the host-selective toxin, victorin. We show that the victorin-induced collapse in mitochondrial transmembrane potential (Deltapsi(m)), indicative of a mitochondrial permeability transition (MPT), on a per cell basis, did not occur simultaneously in the entire mitochondrial population. The loss of Deltapsi(m) in a predominant population of mitochondria preceded cell shrinkage by 20-35 min. Rubisco cleavage, DNA laddering, and victorin binding to the P protein occurred concomitantly with cell shrinkage. During and following cell shrinkage, tonoplast rupture did not occur, and membranes, including the plasma membrane and tonoplast, retained integrity. Ethylene signaling was implicated upstream of a victorin-induced loss in mitochondrial motility and the collapse in Deltapsi(m). Results suggest that the victorin-induced collapse in Deltapsi(m) is a consequence of an MPT and that the timing of the victorin-induced MPT is poised to influence the cell death response. The retention of plasma membrane and tonoplast integrity during cell shrinkage supports the interpretation that victorin induces an apoptotic-like cell death response.