The protonophore CCCP induces mitochondrial permeability transition without cytochrome <i>c</i> release in human osteosarcoma cells

Lim, Maria L. R.; Minamikawa, Tetsuhiro; Nagley, Phillip

doi:10.1016/s0014-5793(01)02693-x

Cited by 110 publications

(90 citation statements)

References 45 publications

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“…None of these treatments provoked the apoptotic morphology observed upon treatment with 1 M staurosporine ( Figure 3B). The relatively low toxicity of mitochondria-specific drugs was confirmed by the observation that cccp-treated cells remained viable for up to 24 h and that their mitochondria retained cytochrome c (our unpublished data), as described previously (Lim et al, 2001).…”

Section: Inner Membrane Potential Is Required For Mitochondrial Fusionsupporting

confidence: 79%

Mitochondrial Fusion in Human Cells Is Efficient, Requires the Inner Membrane Potential, and Is Mediated by Mitofusins

Legros

Lombès

Frachon

et al. 2002

MBoC

586

399

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Mitochondrial fusion remains a largely unknown process despite its observation by live microscopy and the identification of few implicated proteins. Using green and red fluorescent proteins targeted to the mitochondrial matrix, we show that mitochondrial fusion in human cells is efficient and achieves complete mixing of matrix contents within 12 h. This process is maintained in the absence of a functional respiratory chain, despite disruption of microtubules or after significant reduction of cellular ATP levels. In contrast, mitochondrial fusion is completely inhibited by protonophores that dissipate the inner membrane potential. This inhibition, which results in rapid fragmentation of mitochondrial filaments, is reversible: small and punctate mitochondria fuse to reform elongated and interconnected ones upon withdrawal of protonophores. Expression of wild-type or dominant-negative dynamin-related protein 1 showed that fragmentation is due to dynamin-related protein 1-mediated mitochondrial division. On the other hand, expression of mitofusin 1 (Mfn1), one of the human Fzo homologues, increased mitochondrial length and interconnectivity. This process, but not Mfn1 targeting, was dependent on the inner membrane potential, indicating that overexpressed Mfn1 stimulates fusion. These results show that human mitochondria represent a single cellular compartment whose exchanges and interconnectivity are dynamically regulated by the balance between continuous fusion and fission reactions. INTRODUCTIONThe morphology and distribution of mitochondria differ significantly between the cells of different species and tissues. In addition, mitochondrial volume and morphology vary in function of cellular metabolism, are modulated during cell cycle and development, and during apoptosis (Stevens, 1981;Tzagoloff, 1982;Bereiter-Hahn and Voth, 1994;Church and Poyton, 1998;Diaz et al., 1999;Frank et al., 2001). Live microscopy has revealed that mitochondrial morphology is continuously remodeled by fission and fusion (Nunnari et al., 1997;Rizzuto et al., 1998). In yeast, selective inhibition of either process significantly modifies mitochondrial size and interconnectivity (Bleazard et al., 1999;Sesaki and Jensen, 1999). Among the best known proteins involved in mitochondrial dynamics are Fzo/ mitofusin, a transmembrane GTPase involved in fusion (Hales and Fuller, 1997;Hermann et al., 1998;Santel and Fuller, 2001;Rojo et al., 2002), and Dnm1p/dynamin-related protein 1 (Drp1), a dynamin-related protein involved in fission (Bleazard et al., 1999;Pitts et al., 1999;Smirnova et al., 2001).In yeast, mitochondrial fusion has been demonstrated by the diffusion and/or mixing of different matrix proteins during mating of haploid cells (Azpiroz and Butow, 1993;Nunnari et al., 1997). In contrast, mitochondrial fusion has not been studied with similar assays in human cells, and it is not known to what extent the apparent fusion events observed by live microscopy correspond to the formation of intermitochondrial junctions (Bakeeva et al., 1978;Amche...

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Section: Inner Membrane Potential Is Required For Mitochondrial Fusionsupporting

confidence: 79%

Mitochondrial Fusion in Human Cells Is Efficient, Requires the Inner Membrane Potential, and Is Mediated by Mitofusins

Legros

Lombès

Frachon

et al. 2002

MBoC

586

399

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“…onophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), has been shown to induce mitochondrial permeability transition and mitochondrial swelling without cytochrome c release or subsequent cell death (45,46). There is little doubt that release of intramitochondrial cytochrome c into the cytoplasm leads to apoptotic cell death by binding to APAF and procaspase-9 to form the so-called apoptosome, which in turn leads to activation of the caspase cascade (25).…”

Section: Discussionmentioning

confidence: 99%

Pseudoapoptosis Induced by Brief Activation of ATP-gated P2X7 Receptors

Mackenzie

Young

Adinolfi

et al. 2005

Journal of Biological Chemistry

155

160

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P2X 7 receptors are ATP-gated ion channels primarily expressed on antigen-presenting immune cells where they play a role in the acute inflammatory response. These ion channels couple not only to influx of cations, including calcium, but also to rapid alterations in cell morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding). These features resemble the extranuclear events associated with end stages of apoptosis but cell death does not occur if receptor activation is brief. Here we delineate two signaling pathways underlying these apoptotic-like processes. Loss of membrane asymmetry occurs within seconds, which directly triggers cytoskeletal disruption and zeiotic membrane blebbing; this is readily reversible and requires both calcium influx through P2X 7 channels and mitochondrial calcium increase but is not associated with cytochrome c release. A slower, calcium-independent, ROCK-1-dependent cascade that does not involve rapid loss of membrane asymmetry but is associated with cytochrome c release is secondarily activated. The ROCK-1 pathway appears largely responsible for cell death, which occurs after prolonged stimulation of P2X 7 receptors. We suggest that the former mechanism underlies the reversible pseudoapoptotic events induced by brief activation of P2X 7 receptors.

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“…The release was also observed with other anti-tubulin agents, either microtubule-stabilizing agents like docetaxel or microtubule-depolymerizing agents like nocodazole, CI 980, and vinorelbine (10). Anti-mitochondrial agents such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and arsenic trioxide induce the release of cytochrome c from isolated mitochondria (11,12) and, interestingly, also act like anti-tubulin agents by depolymerizing microtubules in intact cells and in vitro experiments (12-14.). In con-* The costs of publication of this article were defrayed in part by the payment of page charges.…”

mentioning

confidence: 99%

Tubulin Is an Inherent Component of Mitochondrial Membranes That Interacts with the Voltage-dependent Anion Channel

Carré

André²,

Carles³

et al. 2002

Journal of Biological Chemistry

253

202

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We have previously reported that anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. In this study, we show that tubulin is present in mitochondria isolated from different human cancerous and non-cancerous cell lines. The absence of polymerized microtubules and cytosolic proteins was checked to ensure that this tubulin is an inherent component of the mitochondria. In addition, a salt wash did not release the tubulin from the mitochondria. By using electron microscopy, we then showed that tubulin is localized in the mitochondrial membranes. As compared with cellular tubulin, mitochondrial tubulin is enriched in acetylated and tyrosinated ␣-tubulin and is also enriched in the class III ␤-tubulin isotype but contains very little of the class IV ␤-tubulin isotype. The mitochondrial tubulin is likely to be organized in ␣/␤ dimers and represents 2.2 ؎ 0.5% of total cellular tubulin. Lastly, we showed by immunoprecipitation experiments that the mitochondrial tubulin is specifically associated with the voltage-dependent anion channel, the main component of the permeability transition pore. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with the permeability transition pore.

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The protonophore CCCP induces mitochondrial permeability transition without cytochrome c release in human osteosarcoma cells

Cited by 110 publications

References 45 publications

Mitochondrial Fusion in Human Cells Is Efficient, Requires the Inner Membrane Potential, and Is Mediated by Mitofusins

Mitochondrial Fusion in Human Cells Is Efficient, Requires the Inner Membrane Potential, and Is Mediated by Mitofusins

Pseudoapoptosis Induced by Brief Activation of ATP-gated P2X7 Receptors

Tubulin Is an Inherent Component of Mitochondrial Membranes That Interacts with the Voltage-dependent Anion Channel

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