title: Isolation of mitochondria by gentle cell membrane disruption, and their subsequent characterization.
abstract: 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.
authors.affiliation: Division of Biotechnology and Life Sciences, Institute of Engineering, Tokyo University of Agriculture and Technology, Nakacho 2-24-16, Koganei, Tokyo 184-8588, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Shibata
authors.affiliation: Division of Biotechnology and Life Sciences, Institute of Engineering, Tokyo University of Agriculture and Technology, Nakacho 2-24-16, Koganei, Tokyo 184-8588, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Division of Biotechnology and Life Sciences, Institute of Engineering, Tokyo University of Agriculture and Technology, Nakacho 2-24-16, Koganei, Tokyo 184-8588, Japan.
authors.forename: Kotoe
authors.initials: K
authors.lastname: Hirusaki
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Katoh
authors.affiliation: Division of Biotechnology and Life Sciences, Institute of Engineering, Tokyo University of Agriculture and Technology, Nakacho 2-24-16, Koganei, Tokyo 184-8588, Japan. Electronic address: ohta@cc.tuat.ac.jp.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohta
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate
keywords: FRET; Fluorescence microscopy; Isolation; Mitochondria; Streptolysin-O; Super-resolution microscopy