Figure 5

Correlative observation and interpretation of the structure of CCCP-treated mitochondria in MEFs using live imaging combined with 3D-CLEM. The last frame of live imaging of mitochondria after CCCP treatment showed the globular and ring-shaped mitochondria (red square in a1), which transformed from tube-shaped mitochondria. The whole frame of live imaging is shown in Supplemental Movie 7. Identical mitochondria were reconstructed by FIB-SEM tomography, and the same area is displayed in a2. The volume rendered view of the mitochondrion from the flipped direction shows the cup-shaped feature (a3), and some cristae were observed in cross-sections of the volume (a4). (b,c) Serial slice images of the volume data from different directions. Both discoidal mitochondria and vase-like stomatocyte-shaped mitochondria 10 min after CCCP treatment showed a ring shape in fluorescence microscopy (5d,5e). Two ring-shaped mitochondria (red squares) were selected under fluorescence microscopy, and identical mitochondria were reconstructed using the 3D-CLEM method based on FIB-SEM tomography. One showed a discoidal shape but did not have a through hole or invagination of the cytosol (d). The cross-section through the equator plane showed an erythrocyte-like biconcave shape and a very thin matrix at section 149, almost in contact with the opposite side of the membrane (arrow in section 149). The other (e) shows a large lumen connected to the cytosol through a small orifice (e, arrowhead). The translucent surface view shows the stomatocyte shape, also referred to as the mitochondrial spheroid structure (e), which also had a thin matrix portion (arrow in section 473) in the deepest region of the invagination. This shape was categorised as a “vase” in this work. Scale bar, 2 μm for CLSM images, 1 μm for others.