Mitochondrial velocity is similar in WT, myo2(LQ), and myo2-fis1 strains. MYO2 WT, myo2(LQ) mutant, and MYO2 cells expressing Myo2-Fis1 from a multicopy plasmid (myo2-fis1(2µ)) were grown to logarithmic growth phase in synthetic dextrose minimal medium and analyzed by time-resolved 3D fluorescence microscopy. Z stacks of mtGFP-expressing cells were recorded every 2 s and processed by deconvolution and maximum intensity projection. Representative cells are shown in Fig. S3 and Videos 1–3. A total number of 41 tracks of mitochondrial tips consisting of 276 time points were reconstructed in 27 WT cells, 50 tracks consisting of 341 time points in 25 myo2(LQ) mutant cells, and 28 tracks consisting of 123 time points in 16 myo2-fis1(2µ) mutant cells. For each time point, the 2D velocity was determined and expressed as a percentage of total mitochondrial movements for each strain.