transduced iPSC-derived neurons 16 hours prior to imaging with the BacMam CellLight® Mitochondria-GFP construct, which utilizes the leader sequence of E1 alpha-pyruvate dehydrogenase (3.1 kDa) to label mitochondria. By adjusting the relative number of viral particles, it was possible to efficiently label the mitochondria in mature iPSC-derived neurons and carry out continuous observation of mitochondrial movement with no cytotoxicity. We followed mitochondrial trafficking in individual neurons at day 40, day 70 and day 100 of differentiation and analyzed retrograde (towards the cell body) and anterograde (away from the cell body) mitochondrial movements (where a movement required at least a 10μm displacement in one direction) as well as stationary mitochondria as percentage of mitochondrial motility (Figure 3D). The corresponding kymograph (Figure 3D, bottom) illustrates mitochondrial movement (x axis) over time (y axis), in which the stationary mitochondria are displayed as straight lines and the moving mitochondria as diagonal lines in either direction. Overall, individual mitochondria exhibited a variety of dynamic behaviors as shown in Supplemental Movie 1. For example, some mitochondria were largley static while others moved at high speed, some stopped abruptly before continuing to move, or reversed direction entirely. Overall, the micropatterned neurons cultured for 100 days showed a
