Correlative Real-Time and Super Resolution Imaging of Mitochondrial Dynamics

Abstract

Mitochondria are energy producing organelles that play essential functions in all aspects of cell biology. In most mammalian cells mitochondria exist in the form of a highly dynamic interconnected network. Live cell imaging experiments revealed that this network undergoes dramatic shape re-organization as motor proteins actively pull the mitochondrial membrane. In addition, mitochondria that exist as individual organelles can be actively transported from one location to another. Mitochondrial shape changes and their active transport involve microtubules and play key roles in mitochondrial function However, several questions remain unanswered regarding the dynamic behavior of mitochondria: how often do mitochondria change tracks as they are transported along microtubules, how do the underlying microtubule tracks influence the observed shape changes and can mitochondrial membrane be pulled along multiple microtubule tracks.In order to relate the dynamic events of the mitochondrial network to the underlying cytoskeleton, we developed a sequential live-cell and super-resolution imaging approach. Live cell imaging allowed us to track the movement of mitochondria and characterize their dynamic behavior. We could stop the dynamics at a specific time point by in situ fixation on the microscope stage. Subsequent immuno-staining followed by super-resolution imaging using Stochastic Optical Reconstruction Microscopy (STORM) allowed us to obtain a high resolution (∼20 nm) image of the underlying microtubule network. The microtubule network was stabilized through treatment with low concentrations of drugs (nocodazole and taxol) and did not change appreciably during live cell imaging. As a result, we could overlay the dynamic behavior of mitochondria with the high resolution images of microtubule tracks by using fiduciary markers for alignment. This approach allowed us to correlate mitochondrial dynamics and the microtubule tracks at high resolution in order to answer the questions that we pose above.