Temporal depolarization of mitochondria during M phase.

Mitochondrial activity in cells must be tightly controlled in response to changes in intracellular circumstances. Despite drastic changes in intracellular conditions and mitochondrial morphology, it is not clear how mitochondrial activity is controlled during M phase of the cell cycle. Here, we show that mitochondrial activity is drastically changed during M phase. Mitochondrial membrane potential changed during M phase progression. Mitochondria were polarized until metaphase to the same extent as mitochondria in interphase cells, but were depolarized at around telophase and cytokinesis. After cytokinesis, mitochondrial membrane potential was recovered. In addition, the generation of superoxide anions in mitochondria was significantly reduced at metaphase even in the presence of antimycin A, an inhibitor of complex III. These results suggest that the electron supply to the mitochondrial electron transfer chain is suppressed during M phase. This suppression might decrease the reactive oxygen species generated by the fragmentation of mitochondria during M phase.

Isolation of mitochondria by gentle cell membrane disruption, and their subsequent characterization.

Mitochondria play a key role in several physiological processes as in integrating signals in the cell. However, understanding of the mechanism by which mitochondria sense and respond to signals has been limited due to the lack of an appropriate model system. In this study, we developed a method to isolate and characterize mitochondria without cell homogenization. By gently pipetting cells treated with streptolysin-O, a pore-forming membrane protein, we disrupted the cell membrane and were able to isolate both elongated and spherical mitochondria. Fluorescence imaging combined with super resolution microscopy showed that both the outer and inner membranes of the elongated mitochondria isolated using the newly developed method were intact. In addition, a FRET-based ATP sensor expressed in the mitochondrial matrix demonstrated that ATP generation by FoF1-ATPase in the isolated elongated mitochondria was as high as that in intracellular mitochondria. On the other hand, some of the spherical mitochondria isolated with this method had the outer membrane that no longer encapsulated the inner membrane. In addition, all mitochondria isolated using conventional procedures involving homogenization were spherical, many of them had damaged membranes, and low levels of ATP generation. Our results suggest that elongated mitochondria isolated from cells through gentle cell membrane disruption using a pore-forming protein tend to be more similar to intracellular mitochondria, having an intact membrane system and higher activity than spherical mitochondria.