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The Dynamics of Mitochondrial Transport in Neurons

Robert Francis Niescier
Min, Kyung-Tai
Issued Date
Mitochondria are an essential component of axons, and proper distribution and transport of these organelles is critical for the maintenance of neuronal health. These organelles are responsible for oxidative phosphorylation, the primary energy metabolic pathway in neurons, as well as buffering of calcium micro-spikes that are a consequence of neuronal signaling. While mitochondria are present in the majority of eukaryotic cell types, their positioning is especially important in axons due to the significant length of these extensions, and potential distance from the cell body. Mitochondria in axons are actively transported anterograde and retrograde relative to the cell body, and disruption of this mechanism is known to be responsible for a variety of neurological diseases. The molecular aspects regulating mitochondrial transport have been well studied; however, there has been comparatively little focus on the large-scale aspects of this phenomenon, across the whole axon. Certain assumptions regarding the overall destination and purpose of mitochondrial transport are based upon high-resolution, low field of view microscopy studies, and do not take into account the dynamics of the entire axon.

In this study, we have developed a new method for examining whole-axon movement over significant periods of time. This was done through the use of mitochondria-localized dendra2, a photoconvertible protein that is able to switch from green fluorescence to red fluorescence through a brief exposure to 405 nm laser radiation. By converting mito-dendra2 in either the proximal (soma) or distal areas of the axon, we were able to track individual mitochondria over long periods and distances using low resolution and high field of view microscopy techniques. The resulting data could be used to described total mobility of mitochondria moving in a particular direction, as well as the distribution of stationary mitochondria. We were also able to further characterize the nature of mobile mitochondria by quantifying their movement into a velocity distribution. This distribution could be fitted to a derivation of the Fokker-Planck equation, which examines the nature of movement of particles through a given space. Using this new protocol, we were able to find significant differences between anterograde and retrograde movement. This protocol was also sensitive enough to detect differences in velocity when the mitochondria specific E3 ubiquitin ligase, Parkin, was overexpressed, showing that this method is a useful application for examining elements that perturb mitochondrial movement.

Using this new methodology, we have also examined the role that mitochondrial calcium uniporter (MCU), an inner mitochondrial membrane pore-forming unit, plays in mitochondrial movement. MCU has been previously implicated in mitochondrial mobility regulation through the use of pharmaceutical methods, though a specific biochemical relationship between the mitochondria transport components and MCU has not been established. Here, we have found that reduction in MCU levels in axons significantly reduces both the total number, as well as the velocity, of moving mitochondria. Biochemical analysis reveals that this is due to an interaction with Miro1, the primary regulator of mitochondrial transport. While Miro1 and MCU are outer and inner mitochondrial membrane proteins, respectively, MCU is able to bind to Miro1 through its previously defined mitochondrial targeting sequence (MTS). We found that MCU’s MTS is localized to the mitochondrial outer membrane, and is dispensable for localization of MCU to mitochondria. This interaction is vital for mitochondrial transport to occur, and overexpression of an MCU mutant without the MTS is unable to rescue transport defects caused by MCU knockdown. Altogether, this body of work explores multiple aspects of mitochondrial transport in axons, and reveals levels of complexity not previously described.
Ulsan National Institute of Science and Technology (UNIST)
Departmentof Biological Sciences


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