​Mitochondria visualized via Tom20

Z-stack

​High magnification timelapse (cell division)

Figure. Live cell movies of Tom20 in Mitochondria. Left: Z-stack of live hiPS cells expressing mEGFP-tagged Tom20 imaged on a spinning-disk confocal microscope. Images start from the bottom of the cells and end at the top. Right: Timelapse movie of hiPS cells expressing mEGFP-tagged Tom20 imaged on a Zeiss LSM880 Airyscan FAST in super-resolution mode. Images were collected in 3D every 30 seconds for 30 minutes. Images show a single slice near the bottom of the cell; playback speed is 150x real time.
​
Observations

• Tom20 is a member of the TOM (translocase of the outer membrane) complex, which permits movement of proteins through the outer mitochondrial membrane and into the intermembrane space of mitochondria.
• In hiPS cells, mitochondria are localized primarily to the top of cells in the nucleus-free ‘cytoplasmic pocket.’ In the center of cells, they localize perpendicular to the substrate and appear like hollow tubes in cross section, as expected for an outer mitochondrial membrane protein. There are fewer mitochondria at the bottom of cells. This observation is consistent with mitochondrial positioning in the cell being primarily dependent on microtubule positioning during interphase.
• Mitochondria form long, interconnected tubules as well as smaller separated structures. The longest tubules are most visible at the bottom of cells where mitochondria are less crowded. Mitochondria are dynamic, jiggling due to Brownian motion, moving (presumably along microtubules) and exhibiting fission and fusion dynamics.
• In mitotic cells mitochondria are more evenly distributed throughout the cell and tend to cluster towards the cell periphery, outside of the mitotic spindle.