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The permeability transition pore signals apoptosis by directing Bax translocation and multimerization¹

FRANCESCA DE GIORGI, LYDIA LARTIGUE, MANUEL K. A. BAUER^*, ALEXIS SCHUBERT^*, STEFAN GRIMM^*, GEORGE T. HANSON, S. JAMES REMINGTON, RICHARD J. YOULE and FRANÇOIS ICHAS²

European Institute of Chemistry and Biology, and INSERM E.9929, Victor Segalen-Bordeaux 2 University, 33076 Bordeaux cedex, France;
^* Max-Planck-Institute for Biochemistry, 82152 Martinsried, Germany;
Institute of Molecular Biology and Departments of Chemistry and Physics, University of Oregon, Eugene, Oregon, USA; and
Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA

²Correspondence: European Institute of Chemistry and Biology, and INSERM E.9929, Victor Segalen-Bordeaux 2 University, 33076 Bordeaux cedex, France. E-mail: ichas{at}u-bordeaux2.fr

SPECIFIC AIMS

The permeability transition pore (PTP) is a channel spanning the inner and outer mitochondrial membranes that has been proposed to play a key role during programmed cell death; based on in vitro observations, the model prevailing in the literature proposes that PTP gating would cause osmotic swelling of the mitochondrial matrix compartment and subsequent outer membrane rupture responsible for an aspecific leakage of cytochrome c.

To elucidate whether PTP operation in the live cell is sufficient to trigger execution of the apoptotic program and cause cytochrome c release, we developed a cellular model where PTP activity can be experimentally provoked and manipulated in situ and in which subsequent apoptotic events can be kinetically and spatially resolved at a submicron scale by high-resolution imaging.

PRINCIPAL FINDINGS

1. TMRM is a mitochondrially targeted, photoactivable trigger of the PTP in the live cell
To elicit PTP gating in situ, we relied on tetramethylrhodamine methyl ester (TMRM), which is a fluorescent lipophilic cation that selectively accumulates in mitochondria down the electrical potential of the inner mitochondrial membrane (). Photoexcitation of TMRM results in fluorescence emission that provides readout and locally releases free radicals that are potent agonists of the PTP. During imaging, mitochondria initially appear to be steadily polarized, but soon a phase of stochastic flickering (i.e., transient redistribution of TMRM) concerning increasing fractions of the mitochondrial population is observed. Eventually, collapses in the entire mitochondrial population (Fig. 1 ). By expressing an artificial pH-sensitive GFP mutant, we found that TMRM photoactivation operates a mitochondrial transmembrane proton-dissipative pathway that is highly sensitive to Bongkrekic acid, a selective inhibitor of the PTP, and modulated by overexpression of the main endogenous PTP regulator proteins: Bcl-2, Bax, and cyclophilin D (CypD). Indeed, Bcl-l2 overexpression resulted in a dramatic inhibition of flickering and delayed terminal depolarization compared with the control cells; in contrast, Bax and CypD behaved as strong coagonists of TMRM photoactivation, increasing the rate of flickering and accelerating the occurrence of the terminal depolarization. This series of results demonstrates that in the live cell, TMRM can be used as a mitochondrially targeted photoactivable trigger of the PTP (Fig. 1) .

View larger version (37K): [in this window] [in a new window]   Figure 1. Directed PTP gating causes delayed all-or-nothing cytochrome c release and phenotypic apoptosis. , EGFP-Cyt.c, and cellular permeability to propidium iodide were imaged simultaneously in a 2H18 cell for 9 h. a) Expanded spatiotemporal stack showing PTP gating during the first 20 min of the recording. b) Entire (red)/EGFP-Cyt.c (green)/PI (red) spatiotemporal stack overlay showing the succession: PTP gating, 2D images (c), bar=10 µm; EGFP-Cyt.c release, 2D images (d); cellular and nuclear shrinkage, 2D images (e); plasma membrane blebbing with PI entry, 2D images (f). g) Kinetics of PTP gating at the single organelle level (yellow) and in the mitochondrial population (red) of this 2H18 cell. h) Delayed cytochrome c release and its kinetics in the same cell (green symbols) vs. control

2. Directed gating of the PTP triggers a delayed ‘all-or-nothing’ cytochrome c release not associated with mitochondrial swelling or caused by a mechanical rupture of the outer mitochondrial membrane
Using this model of directed gating, we directly studied morphological changes affecting the mitochondrial matrix after PTP operation in cells overexpressing mtGFP as a morphological marker of the mitochondrial matrix compartment. We found no evidence to support the idea that PTP gating in the live cell could result in significant matrix swelling, indicating that operation of the PTP in a cellular context is not likely to cause per se any mechanical rupture of the outer membrane.

Using a cell line stably expressing an EGFP-cytochrome c (EGFP-Cyt.c) chimera, we investigated the possible diffusion of cytochrome c out of the mitochondrial intermembrane space subsequent to PTP operation. Definitive PTP opening was followed after a delay of 4.35 ± 2.73 h (n=5) by a complete all-or-nothing coordinated release of cytochrome c taking place over 7.54 ± 2.02 min (n=5). The release was rapidly followed by a sequence of cellular shrinkage, nuclear condensation, plasma membrane blebbing, and, finally, cytolysis (Fig. 1) . In parallel experiments, we found that subsequent to and distant from PTP gating, caspase 3 activation occurred over 10.34 ± 4.87 min (n=3), with an activation profile kinetically superposable to the cytochrome c release process.

3. PTP gating causes delayed cytochrome c release by signaling relocation of cytosolic Bax to the outer mitochondrial membrane
Using EGFP-Bax-overexpressing cells and TMRM, we investigated the possibility that Bax may be the key element responsible for the delayed cytochrome c release observed after PTP operation. We found that subsequent to and at a distance from PTP gating, EGFP-Bax does indeed undergo a redistribution to the outer mitochondrial membrane, where the chimera eventually forms visible multimeric aggregates 100 nm in diameter. A similar process was observed in cells where mitochondria where depolarized in situ using the protonophore FCCP in the presence of oligomycin, indicating that the signal causing Bax redistribution subsequent to PTP gating is distal to collapse. Note that the process of Bax redistribution and clustering is kinetically slow and reaches a maximum only after several hours (Fig. 2 ).

View larger version (76K): [in this window] [in a new window]   Figure 2. a) Directed gating of PTP signals Bax redistribution and clustering. The PTP was gated using TMRM in a COS-7 cell expressing EGFP-Bax while and EGFP-Bax distribution were imaged simultaneously in a cellular subregion for 7 h (bar=2 µm). The 3 image pairs show progressive Bax redistribution and formation of Bax clusters at the mitochondrial periphery. b) Left panel: kinetics of EGFP-Bax redistribution observed after directed PTP gating (green symbols) vs. a control trace recorded in an EGFP-Bax-expressing cell imaged in the absence of TMRM (dotted trace). c) Right panels: FCCP (150 nM) treatment in the presence of oligomycin (500 nM) mimics the effects of PTP gating in a COS-7 cell expressing EGFP-Bax and imaged in the absence of TMRM (bar=10 µM).

4. Clustering and multimerization of Bax at the level of mitochondria is responsible for the release of cytochrome c observed after PTP gating
To possibly correlate the processes of cytochrome c release and Bax redistribution occurring subsequent to PTP gating, we operated the PTP in cells coexpressing EGFP-Bax and a blue mutant of GFP targeted to the mitochondrial matrix compartment. Once the PTP gated, we imaged EGFP-Bax redistribution, fixed the cells at various reference times during the redistribution process, and treated them to reveal cytochrome c by immunofluorescence using a red-emitting secondary antibody. Triple wavelength imaging was performed to image the mitochondrial matrix compartment (blue) and the distribution of Bax (green) and cytochrome c (red). After PTP gating, but before any visible sign of Bax redistribution, no cytochrome c release was detected. Strikingly, cytochrome c still showed a mitochondrial distribution after Bax had redistributed to mitochondria and homogeneously delineated the organelles. However, during the subsequent process of Bax clustering, cytochrome c eventually diffused out of the intermembrane space into the cytosol. Using fluorescence resonance energy transfer imaging and ECFP-Bax plus EYFP-Bax chimeras, we observed that macroscopic Bax clustering in the live cell was associated with the formation of Bax multimers.

These results indicate that docking of Bax to the outer mitochondrial membrane and its subsequent in situ clustering/multimerization are responsible for the release of cytochrome c observed after PTP gating (Fig. 3 ).

View larger version (73K): [in this window] [in a new window]   Figure 3. Schematic diagram of the proapoptotic mechanism of PTP. A) In resting conditions PTP is closed, cytochrome c is in the intermembrane space, and inactive Bax is soluble in the cytosolic compartment. B) PTP operation opens a proton dissipative pathway that induces mitochondrial collapse. C) Mitochondrial depolarization signals a conformational change of cytosolic Bax, which is progressively recruited on the mitochondrial outer membrane. D) Bax oligomerization on the outer membrane forms channels for cytochrome c release.

CONCLUSIONS

PTP operation is proapoptotic and able to direct coordinated redistribution of the cytosolic protein Bax to the outer mitochondrial membrane, where, after a silent phase of docking, Bax progressively forms multimers and clusters in situ that coincide with the event of cytochrome c release. Our results reveal that the PTP is not an intrinsic component of the cytochrome c release machinery, but an upstream trigger able to signal Bax-dependent outer membrane permeabilization (Fig. 3) . The signal(s) emitted by the PTP and triggering the Bax cascade are being investigated.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0269fje; to cite this article, use FASEB J. (February 25, 2002) 10.1096/fj.01-0269fje

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