Effects of proton pumping on the structural rigidity of cristae in mitochondria.

Mitochondria change their morphology and inner membrane structure depending on their activity. Since mitochondrial activity also depends on their structure, it is important to elucidate the interrelationship between the activity and structure of mitochondria. However, the mechanism by which mitochondrial activity affects the structure of cristae, the folded structure of the inner membrane, is not well understood. In this study, the effect of the mitochondrial activity on the cristae structure was investigated by examining the structural rigidity of cristae. Taking advantage of the fact that unfolding of cristae induces mitochondrial swelling, we investigated the relationship between mitochondrial activity and the susceptibility to swelling. The swelling of individual isolated mitochondria exposed to a hypotonic solution was observed with an optical microscope. The presence of respiratory substrates (malate and glutamate) increased the percentage of mitochondria that underwent swelling, and the further addition of rotenone or KCN (inhibitors of proton pumps) reversed the increase. In the absence of respiratory substrates, acidification of the buffer surrounding the mitochondria also increased the percentage of swollen mitochondria. These observations suggest that acidification of the outer surface of inner membranes, especially intracristal space, by proton translocation from the matrix to the intracristal space, decreases the structural rigidity of the cristae. This interpretation was verified by the observation that ADP or CCCP, which induces proton re-entry to the matrix, suppressed the mitochondrial swelling in the presence of respiratory substrates. The addition of CCCP to the cells induced a morphological change in mitochondria from an initial elongated structure to a largely curved structure at pH 7.4, but there were no morphological changes when the pH of the cytosol dropped to 6.2. These results suggest that a low pH in the intracristal space may be helpful in maintaining the elongated structure of mitochondria. The present study shows that proton pumping by the electron transfer chain is the mechanism underlying mitochondrial morphology and the flexibility of cristae structure.

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.