Imaging the Permeability Pore Transition in Single Mitochondria

Abstract

In mitochondria the opening of a large proteinaceous pore, the "mitochondrial permeability transition pore" (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (delta psi(m)) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of delta psi(m) due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of delta psi(m). Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (approximately 620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized delta psi(m) in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.

Cyclosporin A corrects mitochondrial dysfunction and muscle apoptosis in patients with collagen VI myopathies

“…As long as the energy demand can be matched, the fibers would behave normally, but dysfunction would be precipitated by increased workload and/or by increased PTP flickering, events that will eventually lead to individual muscle fiber death observed in vivo (2). This sequence of events is consistent with the occurrence of transient PTP openings in isolated mitochondria (8,9) and intact cells (10,11) and with the therapeutic effect of short-term treatment of Col6a1 Ϫ/Ϫ mice with cyclosporin A (CsA) at 10 mg⅐kg Ϫ1 ⅐day Ϫ1 (2), which desensitizes the PTP in vivo (12). Myoblasts from patients affected by Ullrich congenital muscular dystrophy (UCMD; Mendelian Inheritance in Man no.…”

Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death

“…We investigated ROS-induced PT using high-power laser illumination of TMRM. This generates superoxide ROS production and consequently triggers PT (12,13). Illumination of the cells induced PT in both control (n ϭ 26) and MGP-transfected (n ϭ 22) HepG2 cells, within 5-6 min (SI Fig.…”

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

“…2) may be employed to measure transient IM permeabilization events. According to this technique, cells are loaded with the fluorescent probe calcein, as well as with its quencher, cobalt (Co 2+ ) [91]. In its acetoxymethyl ester form, calcein diffuses to all subcellular compartments, including mitochondria, whereas Co 2+ ions are excluded from the mitochondrial matrix by the Co 2+ -impermeable IM.…”