title: Serial block-face scanning electron microscopy for three-dimensional analysis of morphological changes in mitochondria regulated by Cdc48p/p97 ATPase.
abstract: Cdc48p is a highly conserved cytosolic AAA chaperone that is involved in a wide range of cellular processes. It consists of two ATPase domains (D1 and D2), with regulatory regions at the N- and C-terminals. We have recently shown that Cdc48p regulates mitochondrial morphology, in that a loss of the ATPase activity or positive cooperativity in the D2 domain leads to severe fragmentations and aggregations of mitochondria in the cytoplasm. We have now used serial block-face scanning electron microscopy (SBF-SEM), an advanced three-dimensional (3D) electron microscopic technique to examine the structures and morphological changes of mitochondria in the yeast Saccharomyces cerevisiae. We found that mutants lacking ATPase activity of Cdc48p showed mitochondrial fragmentations and aggregations, without fusion of the outer membrane. This suggests that the ATPase activity of Cdc48p is necessary for fusion of the outer membranes of mitochondria. Our results also show that SBF-SEM has considerable advantages in morphological and quantitative studies on organelles and intracellular structures in entire cells.
authors.affiliation: National Institute for Physiological Sciences, 38 Nishigonaka Myodaiji, Okazaki, Aichi 444-8585, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Miyazaki
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Esaki
authors.affiliation: Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.
authors.forename: Teru
authors.initials: T
authors.lastname: Ogura
authors.affiliation: National Institute for Physiological Sciences, 38 Nishigonaka Myodaiji, Okazaki, Aichi 444-8585, Japan. Electronic address: kazum@nips.ac.jp.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Murata
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D021621:Imaging, Three-Dimensional; D008855:Microscopy, Electron, Scanning; D008928:Mitochondria / Q000201:enzymology / Q000648:ultrastructure*; D051336:Mitochondrial Membranes / Q000378:metabolism / Q000648:ultrastructure; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D009687:Nuclear Proteins; D000251:Adenosine Triphosphatases; C404942:p97 ATPase
keywords: AAA ATPase; Cdc48p/p97; Mitochondrial fusion; Mitochondrial morphology; Protein degradation; Serial block-face SEM