mtDNA dynamics in living cells with YFP-tagged mitochondria. Cells were grown (30 min) in 0.1 μg/ml ethidium, the ethidium washed away, the cells regrown for 10 min, and single confocal sections collected every 10 sec for ≤500 sec; mitochondria and mtDNA are marked by YFP and ethidium respectively. (A,B) Two views of mitochondria in one cell at one time. Ethidium is locally concentrated in (mtDNA) foci, against a background (bound to RNA). S1: distance between two foci. S2: distance between one focus and mitochondrial tip. (C,D) Frames showing ethidium fluorescence (marking mtDNA) from two movies. In each case, the first (0 sec; left) and last (500 sec; middle) frames show the two mtDNA foci move little relative to each other; this is confirmed by superimposing all frames in each movie (right). Bar: 2 μm. (E) Changes in separations S1 and S2 seen in (B) over time; movement is erratic. (F) Mean square displacement (MSD; n = 20) of one mt DNA focus relative to another (DNA), or one tip relative to another (tip); movement of foci is more restrained than that of tips. The MSD of one terminal focus relative to the tip is similar to that of one focus relative to another and is not shown. (G) Autocorrelation analysis. The autocorrelation (ac) between the displacement distances (Δd) for time points separated by different time intervals (Δt) of 10 sec (dashed lines show limits of 5% significance). The pattern is typical of a random walk; there is no periodicity, and the first value (the only significant one) is negative. (H) The distribution of step lengths (Δd) is typical of a random walk. Mean = 0.01 μm (SD ± 0.4). The maximum value was 2.4 μm, and is not shown.