We used fluorescence imaging with one nanometer accuracy (FIONA) to analyze organelle movement by conventional kinesin and cytoplasmic dynein in a cell. We located a green fluorescence protein (GFP)-tagged peroxisome in cultured Drosophila S2 cells to within 1.5 nanometers in 1.1 milliseconds, a 400-fold improvement in temporal resolution, sufficient to determine the average step size to be ∼8 nanometers for both dynein and kinesin. Furthermore, we found that dynein and kinesin do not work against each other in vivo during peroxisome transport. Rather, multiple kinesins or multiple dyneins work together, producing up to 10 times the in vitro speed.
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