Abstract
Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named “MitoQuant”. This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0268-3) contains supplementary material, which is available to authorized users.
Highlights
Mitochondria act as both critical powerhouse and important signaling station(s) in eukaryotic cells
Our microfluidic system made it possible to track most, if not all, mitochondria in the field of interest because most axonal mitochondrial signals were contained inside parallel axonal microgrooves, allowing efficient identification of mitochondria moving in anterograde or retrograde directions
Our data show that rotenone treatment significantly increased the percentage of mitochondria in ST state, but reduced the proportion of mitochondria in either dynamic pause (DP) or running states (AR or retrograde running (RR); Fig. 3B, 3E and 3F)
Summary
Mitochondria act as both critical powerhouse and important signaling station(s) in eukaryotic cells (for recent reviews see Wang and Schwarz, 2009a; Wallace 2013). Active cargo transport between the cell body and neuronal processes (neurites) is essential for proper distribution of critical materials and energy to their respective subcellular locations (Radad et al, 2006; Singh et al, 2014). The assignment progress was achieved by matching the measurements (the mitochondrial position and morphology in current frame) with their prediction made by the IMM filter. More details about the MiTracker are described in the Supplementary program package and its Tutorial
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