Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis.
Little is known about the temporal relationship between mitochondrial and plasma membrane potential changes and outer mitochondrial membrane permeabilization during apoptosis. Confocal imaging of breast carcinoma and HeLa cells stably transfected with cytochrome-C-GFP demonstrated that mitochondria rapidly depolarized after the release of the fusion protein into the cytosol. Of note, mitochondria did not completely depolarize but established a new steady-state level that could be further dissipated by treatment with the protonophore carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. Treatment with the F(O)F(1)-ATP-synthase inhibitor oligomycin likewise induced a collapse of this steady-state level, suggesting that F(O)F(1)-ATP-synthase reversal maintained mitochondrial potential after outer mitochondrial membrane permeabilization. Treatment with a broad spectrum caspase inhibitor failed to inhibit the partial depolarization of mitochondria during apoptosis, yet potently abolished the activation of effector caspases detected by fluorescence resonance energy transfer analysis in the same experiment. Interestingly, the onset of mitochondrial depolarization was always coupled with a depolarization of the plasma membrane potential. This was associated with the degradation of the regulatory Na(+)/K(+)-ATPase beta-subunit, and both events were blocked by caspase inhibition. Our results demonstrate that outer mitochondrial membrane permeabilization coordinates the depolarization of both membrane potentials during apoptosis.
Düssmann
Heiko
H
Interdisciplinary Center for Clinical Research (IZKF), Westphalian Wilhelms-University, D-48149 Münster, Germany.
Rehm
Markus
M
Kögel
Donat
D
Prehn
Jochen H M
JH
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D005260:Female; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology*; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009369:Neoplasms / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D007476:Ionophores; D011993:Recombinant Fusion Proteins; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D020169:Caspases; D025261:Mitochondrial Proton-Translocating ATPases; D000254:Sodium-Potassium-Exchanging ATPase