ETC-inhibited mitochondria are arrested by the actin cytoskeleton. (A) Cartoon of the rapalog system used to drive a constitutively active motor onto mitochondria. (B) Representative kymographs demonstrating the increased anterograde movement of mitochondria caused by the addition of rapalog. Hippocampal neurons expressing a mitochondria-targeted FKBP in green (mito-meGFP-FKBP) and a constitutive active kinesin (HA-Kif5b-MD-FRB) were co-expressed together with an axon initial segment marker TRIM46-mCherry in magenta (to identify axons) and iRFP706. Cells were treated with 1 µM rapalog or vector for 3–6 min and then imaged. (C) Representative kymographs demonstrating mitochondrial movement in neurons exposed for 1 h to 4 nM AntA or vehicle and then treated with rapalog as in B. (D) Quantification of mitochondrial movement with or without adding rapalog as described in B. n = 7–9 axons per treatment from four independent animals. (E) Quantification of rapalog-induced mitochondrial movement in hippocampal neurons treated with AntA or vehicle as described in C. n = 6–11 axons per treatment from four independent animals. (F) Quantification of mitochondrial motility in cultured hippocampal neurons treated for 3 h with 5 µM LatA or vehicle. After 3 h, 4 nM AntA or vehicle was added for one more hour. n = 7–10 axons per treatment from 6 independent animals. (G) Representative kymographs of mitochondrial movement from the experiment described in F. Mitochondria are magenta, and axons are green. For all graphs, bars on boxplots show the 10–90th percentile. P values for D and E were calculated by two-tailed, unpaired t tests with Welch’s correction. For all images, scale bars = 20 μm. For F, P values were calculated by performing a blocked one-way ANOVA with Tukey’s multiple comparison correction. Only P values ≤0.05 are shown.