In vitro motility assays

For all in vitro motility experiments, the motility chamber was perfused with 0.5 mg ml⁻¹ streptavidin for immobilizing polarity-marked HyLite 647 microtubules. GFP-tagged kinesin molecules (GFP-KlpA and GFP-KlpA-Δtail) were then diluted in motility buffer (BRB50 supplemented with 25 mM KCl, 1 mM ATP, 25 μM taxol, 1.3 mg ml⁻¹ casein and an oxygen scavenger system⁶⁶) and added to the chamber. Time-lapse images were acquired at 1 frame per second. The setting was 100-ms exposure and 5-min duration for the high-concentration flux experiments and 200-ms exposure and 10 min for single-molecule experiments to determine the velocity and run-length of GFP-KlpA. Kymographs were generated and analysed in ImageJ (NIH) for determining directionality, velocity and run-length information of GFP-tagged kinesin motors. Velocity and run-length were determined by fitting the histograms to a Gaussian distribution and an exponential distribution, respectively, in Matlab (MathWorks)