Image-based techniques to quantify mitochondrial dynamics in neurons.

<p>(A) Mitochondrial motility can be visualized using time-lapse microscopy in neurons that are either transfected with plasmids expressing mitochondrially targeted fluorescent proteins or loaded with mitochondrion-specific fluorescent dyes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001755#pbio.1001755.s001" target="_blank">Video S1</a>). Mitochondria are tracked in the image series either manually or automatically using specific software packages. Various movement parameters (e.g., number and percentage of mobile and stationary mitochondria, the number, length and direction of runs or tracks the mitochondria make, their maximum and average velocity) can be calculated from the data obtained. (B) Mitochondrial fusion and fission can be measured using photoactivatable or photoconvertable mitochondrially targeted fluorescent proteins such as Kikume <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001755#pbio.1001755-Cagalinec1" target="_blank">[20]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001755#pbio.1001755-Habuchi1" target="_blank">[23]</a>. The middle panel shows a fusion event between green- and red-emitting mitochondria, yielding a yellow fusion product after the contents of the mitochondrial matrices are mixed (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001755#pbio.1001755.s002" target="_blank">Video S2</a>). The lower panel demonstrates that the fusion was followed by a fission event. The fate of photoactivated mitochondria can be followed throughout the time-lapse series and the fusion and fission rates calculated. (C) Detection of mitophagy in the cell body of a primary cortical neuron expressing a mitochondrially targeted pH-dependent protein Keima <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001755#pbio.1001755-Katayama1" target="_blank">[24]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001755#pbio.1001755-Klionsky1" target="_blank">[25]</a> whose excitation spectrum shifts from 440 nm to 586 nm (green to red in photo) when mitochondria are delivered to acidic lysosomes, thus enabling easy quantification of mitophagy. An alternative approach is to quantify the co-localization of an autophagosome marker (EGFP-LC3) with a mitochondrial marker.