Jacobson, Derek William Niall; (2000) Imaging the mitochondrial permeability transition in single cells: Free radicals and calcium as determinants of cell fate. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The textbook interpretation of mitochondrial function has had to be revised in the light of recent discoveries establishing a central role for mitochondria in modulating cell signalling. In particular, the opening of the mitochondrial permeability transition pore (MPTP), a large conductance pore in the tightly impermeable inner mitochondrial membrane, may determine cell fate by inducing either necrotic or apoptotic cell death. I have developed an experimental protocol in which MPTP opening can be reliably induced in living cells. I have then used this as a model to study the relative roles of calcium and reactive oxygen species (ROS) in permeability transition and explored the consequences of these processes for cell fate. The membrane-permeant, fluorescent dye, tetramethylrhodamine ethyl ester (TMRE), accumulates in mitochondria where its fluorescence signals changes in mitochondrial transmembrane potential ( m). Digital imaging of adult rat cortical astrocytes loaded with TMRE revealed spontaneous, transient depolarisations of individual mitochondria leading to a gradual and complete mitochondrial depolarisation in whole cells. Single mitochondria could depolarise and repolarise several times. Treatment of the cells with cyclosporin (an inhibitor of pore opening) significantly reduced the depolarisations suggesting that they were caused by MPTP opening and that TMRE could be used to signal MPTP in intact single cells. This model of MPTP opening was used to study the underlying mechanisms involved in MPTP opening. Illumination of TMRE produces ROS. Attenuation of the excitation light intensity or treatment with free radical scavengers reduced the frequency of the depolarisations, confirming a role for ROS in this model of permeability transition. Mitochondrial calcium loading is thought to be a prerequisite for permeability transition and I found that chelation of intracellular calcium or depletion of endoplasmic reticulum calcium stores inhibited the transient openings. Thus either free radical scavengers or chelation of calcium inhibited pore opening in this model, suggesting an interplay between ROS production and mitochondrial calcium loading in permeability transition, perhaps as a result of ROS-induced calcium release from intracellular stores. The fate of cells that were illuminated with low intensity light, where MPTP was transient, was similar to non-illuminated controls, suggesting that opening of the pore does not inevitably lead to cell death. Those cells in which collapse of [delta][psi]m was complete, however, died by a necrotic pathway.

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