3D Real-Time Orbital Tracking in Zebrafish Embryos: High Spatiotemporal Analysis of Mitochondrial Dynamics in Neurons

Abstract

The main function of mitochondria is to provide cells with adenosintriphosphate (ATP) in regions with high-energy demand. A complex machinery of motor proteins (kinesin, dynein, myosin, etc.) and signaling molecules like calcium ions are responsible for the distribution and recycling of mitochondria in cells. A malfunction in the dynamics of these complexes is one possible reason for neurodegenerative diseases. However, the exact mechanism behind this process is not yet fully understood.To follow the trajectory of individual mitochondria in rohon-beard sensory neurons, we use a home-built, three-dimensional, real-time orbital-tracking microscope with a spatial resolution of a few nm in three dimensions and an acquisition speed of up to 500 Hz. Environmental information is recorded simultaneously with a built-in widefield microscope.Photoactivatable GFP (Tracking microscope) and TAG-RFP (Widefield microscope) are coexpressed in neuronal mitochondria by coinjecting neuron specific driver constructs with separate UAS responder constructs into fertilized single-cell zebrafish eggs. Zebrafish expressing the markers are measured individually three days past fertilization.By photoactivating mitochondria, we are able to obtain single-mitochondria trajectories with a traveled distance of more than 100µm with nm precision. Due to our high spatial and temporal resolution, we can identify several different dynamic populations involved in mitochondrial transport. The environmental information gives insight into the colocalization behavior between stationary and moving mitochondria. By combining the results from the fast and precise tracking microscope with the widefield data, we obtain an in vivo overview over the dynamic processes in rohon-beard sensory neurons which can be used for further disease related experiments.