Time-lapse imaging of Ca²⁺-induced swelling and permeability transition: Single mitochondrion study

Mitochondrial functions are closely related to the membrane structure. Mitochondrial swelling, which is accompanied with dissipation of the crista structure and rupture of the outer membrane, have been observed as mitochondrial damage when mitochondria are under Ca²⁺-overload or oxidative stress. Although these phenomena have been well studied, the detailed behaviors of individual mitochondria upon swelling remain unknown. The aim of this study was to investigate the detailed behavior of mitochondrial volume upon addition of Ca²⁺. Here, we report for the first time, time-lapse measurements of single mitochondrion swelling and permeability transition induced by Ca²⁺ by optical microscopy. We added 220 μM Ca²⁺ to mitochondria, and found that 1) the swelling rate depended on the mitochondrion, 2) a small number of mitochondria showed step-like swelling, 3) cyclosporin A decreased the percentage of mitochondria that underwent swelling induced by Ca²⁺, but did not affect the amplitude of swelling, 4) permeability transition is necessary but not sufficient for Ca²⁺-induced swelling, 5) permeability transition is more sensitive to Ca²⁺ than swelling, 6) Ca²⁺ stimulated mitochondrial swelling after permeability transition. These results suggest that single mitochondrion measurement of swelling is a powerful tool for examining the regulation of mitochondrial structure.