Hand-over-hand stepping of Myo4p-She3p bound to She2p. (A) Diagram of the experimental setup for the TIRF processivity assay. Steps are tracked by Qdots bound to the N terminus of the motor domain. For the trace shown in B, one head is labeled with a 565-nm Qdot (green star) and the other with a 655-nm Qdot (red star). TM, tropomyosin. (B) Myo4p processive run with heads labeled with different colored Qdots showing a hand-over-hand stepping pattern, with lead and trailing heads exchanging position with each step. The displacement versus time for the 565- and 655-nm Qdots is shown by green and red open circles, respectively. Qdot positions were determined by the ImageJ plugin SpotTracker (Sage et al., 2005). Steps were identified using the Kerssemakers step-finding algorithm (solid lines; Kerssemakers et al., 2006). The inset shows a histogram of the step sizes, which averaged 71.8 ± 18 nm. Experiments were performed at 1 µM MgATP. The occasional back steps in this trace are not representative (see also Fig. S3). The green/red displacement versus time data is a single representative trace. A total of 15 traces from three experiments was analyzed to generate the histogram (inset).