A transient decrease of electrochemical gradient stabilizes DNA structuralchange in single mitochondria of living cells

The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied i n living cells by fluorescence microscopy. Electrochemical gradient perturb ations were induced by the protonophore carbonyl cyanide p-trifluoromethoxy phenylhydrazone and quantified by measuring the mitochondrial membrane pote ntial using tetramethyl rhodamine methyl ester. Under our experimental cond itions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA. Ethidium fluorescence variations ha iie b een used to probe DNA structural changes. This showed that: i) electrochemi cal gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electro chemical gradient; iii) in contrast, a short and weak perturbation of the e lectrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necess ity of studying the effect of energetic perturbations at the level of an in dividual cell. (C) 1999 Editions scientifiques et medicales Elsevier SAS.