DepartmentMolecular Cell Biology
Publication date30-Jun-2014

Serial block-face scanning electron microscopy for three-dimensional analysis of morphological changes in mitochondria regulated by Cdc48p/p97 ATPase. Naoyuki Miyazaki, Masatoshi Esaki, Teru Ogura, and Kazuyoshi Murata. J Struct. Biol., in press

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.

This study was supported in part by the program of the Joint Usage/Research Center for Developmental Medicine, IMEG, Kumamoto University.



Figure. Mitochondria are accumulated in a Cdc48-deficient cells
 A-B) Mitochondrial morphology visualized using a mitochondria-targeted fluorescence protein (A, wild type cells; B, Cdc48-deficient cells). Three-dimensional confocal fluorescence microscopy images were projected to 2D.

 C-D) Three-dimensional images constructed from SBF-SEM (C, wild type cells; D, Cdc48-deficient cells) (red, nucleus; blue, vacuoles; green, mitochondria; purple, plasma membranes)