(A, B) Analysis of TOM-LOV-labeled mitochondrial movement in COS cells expressing TOM-LOV and 560-PDZ together with the control iRFP or MAP7-FL-iRFP. High magnification TOM-LOV images of the boxed areas in (A) are shown at time 0’ and at time 1’ in (B). Arrows indicate the mitochondria analyzed below in (C, D). (C) Sequential images at 1-s intervals are projected with temporal color-coding to show the trajectories of motile mitochondria. Arrows indicate the beginning and the end of each trajectory. The two ends of the color bar represent 0’ and 1’ respectively. Asterisks (*) indicate pause followed by sharp turns and hashes (#) indicate pauses followed by straightruns. (D) Kymographs (left panel) of TOM-LOV-labeled mitochondria are generated based on the trajectories in (C) andeach run track is represented by line drawing (right panel) to display regions of movement (yellow) and regions of pausing (red). Asterisks (*) and hashes (#) indicate corresponding pauses in (C). (E–G) Comparisons of mitochondria run speed (E), pause frequency (F), and sharp turn frequency (G) between cells expressing TOM-LOV, 560-PDZ, and the iRFP control (ctrl), MAP7-FL-iRFP (FL), or MAP7-ΔC-iRFP (ΔC). n = 8 cells (>4 tracks per cell). T-test (Mean ±SEM) for (E): Ctrl-FL, p=0.04; Ctrl-ΔC, p=0.52; FL-ΔC, p=0.10; for (F): Ctrl-FL, p=0.04; Ctrl-ΔC, p=0.32; FL- ΔC, p=0.67; and for (G): Ctrl-FL, p=0.007; Ctrl-ΔC, p=0.10; FL-ΔC, p=0.0009. (H–J) Comparisons of peroxisome run speeds (H), pause frequency (I), and sharp turn frequency (J) between cells expressing PEX-LOV, 560-PDZ, and the iRFP control (ctrl), MAP7-FL-iRFP (FL), or MAP7-ΔC-iRFP (ΔC). n = 8 cells (>4 tracks per cell). T-test (Mean ±SEM) for (H): Ctrl-FL, p=0.31; Ctrl-ΔC, p=0.37; FL-ΔC, p=0.01; for (I): Ctrl-FL, p=0.008; Ctrl-ΔC, p=0.13; FL-ΔC, p=0.10; and for (J): Ctrl-FL, p=0.001; Ctrl-ΔC, p=0.38; FL- ΔC, p=0.006. *p<0.05, **p<0.005, ***p<0.001 and ns: not significant. Scale bars: 5 µm.