| Opening of high conductance permeability transition (PT) pores in the mitochondrial inner membrane causes onset of the mitochondrial permeability transition (MPT) and is a major cause of mitochondrial dysfunction in cells exposed to stresses causing necrotic and apoptotic cell death. Autophagy is the major mechanism for turnover of mitochondria, but functional changes to mitochondria preceding autophagy are unknown, since conventional methods cannot discern dysfunction of single, individual mitochondria within living cells. A novel technique utilizing fluorescence resonance energy transfer (FRET) permitted selective visualization of single depolarized mitochondria. Cultured rat hepatocytes were loaded with green-fluorescing MitoTracker Green-FM (MTG) and red-fluorescing tetramethylrhodamine methylester (TMRM). As both fluorophores accumulated into polarized mitochondria, the green fluorescence of MTG was quenched by TMRM via FRET. After depolarization, mitochondria released TMRM, but MTG remained bound by virtue of covalent bonding to protein sulfhydryls. As a consequence, green MTG fluorescence recovered as mitochondria depolarized, whereas red TMRM fluorescence disappeared. After autophagic stimulation of hepatocytes by glucagon (1 muM) and nutrient deprivation, spontaneous depolarization of individual mitochondria increased 5-fold, as revealed by unquenching of green MTG fluorescence and loss of red TMRM fluorescence. Autophagic stimulation also increased the number of LysoTracker Red-labeled acidic vesicles by 5-fold. Co-labeling experiments revealed that depolarizing mitochondria moved into these autophagosomes. Cyclosporin A (CsA) and tacrolimus bind calcineurin in their roles as immunosuppressants, but of the two, only CsA blocks conductance of PT pores. CsA (5 muM) prevented mitochondrial depolarization after autophagic stimulation, and CsA but not tacrolimus blocked autophagosomal proliferation. Because the MPT is also a critical step in tumor necrosis factor-alpha (TNFalpha)-induced apoptosis, the ability of autophagic stimulation by glucagon to enhance TNFalpha-induced autophagy was assessed. Autophagic stimulation doubled cell killing in hepatocytes exposed to low-dose TNFalpha, but did not increase killing of hepatocytes exposed to higher dose TNFalpha. In conclusion, the initial step in mitochondrial autophagy is onset of the MPT causing mitochondrial depolarization and subsequent sequestration of depolarized mitochondria into autophagosomes. Signaling of autophagy by the MPT provides a means of ridding the cell of dysfunctional mitochondria, but may also potentiate cell killing in the presence of apoptotic stimuli. |
