Holographic imaging of mitochondrial optical density in living cells reveals that the Mitochondrial Permeability Transition is a two-stage process

Mitochondrial permeability transition is caused by the opening of the Cyclosporin A (CSA) dependent calcium-induced large pore, known as the Permeability Transition Pore (PTP). PTP activation is believed to be a central event in stress-induced cell death. However, the molecular details of PTP opening remain incompletely understood. PTP opening makes mitochondrial inner membrane permeable to the molecules up to 1.5 kDa in size. Solute equilibration with the media in combination with swelling due to the PTP opening make mitochondria optically transparent, a phenomenon that has been widely used as a bona fide "light-scattering" PTP detection method in isolated mitochondria. Here, we utilized holographic microscopy imaging to monitor mitochondrial optical density changes that occur during solute equilibration between matrix and cytoplasm and thus enabled us to assess PTP induction in living cells. This approach relies on label-free, real-time mitochondrial visualization due to refractive index (RI) differences between the mitochondrial matrix and cytoplasm in the intact cells. PTP activation was detected as the decrease in mitochondrial RI. These measurements were done in parallel with measurements of the mitochondrial membrane potential, using the fluorescent probe TMRM. In intact HAP 1 cells, we found that calcium stress caused CSA-sensitive depolarization of the mitochondrial inner membrane. Unexpectedly, high-conductance PTP did not occur until after nearly complete mitochondrial membrane depolarization. In cells lacking c and δ subunits of the ATP synthase, we observed calcium-induced and CSA-sensitive depolarization but not high-conductance PTP. We demonstrate that holographic imaging is a powerful novel tool with unique capabilities that allow measurement of PTP in living cells with high temporal and spatial resolution. We conclude that contrary to the widely accepted view, in living cells, high-conductance PTP is not the cause of calcium-induced membrane depolarization. Further, we provide direct evidence that ATP synthase is essential for high-conductance PTP, but not for calcium-induced CSA-sensitive membrane depolarization. We propose that PTP activation occurs as a two-phase process, where the first phase of the initial membrane depolarization is followed by the second phase of large pore opening that results in high-amplitude membrane permeabilization.