Microtubule pair sliding driven by purified Eg5 in buffer. (a) Schematic of Eg5 within a pair of antiparallel microtubules. The red microtubule is biotinylated and immobilized to a chemically functionalized glass surface via biotin–neutravidin links. The green microtubule is not biotinylated and mobile. (b) Fluorescence microscopy images of Alexa 568 microtubule pairs formed with Eg5-paGFP. Dimly labeled microtubules immobilized on a biotin-PEG glass surface supporting Eg5-mediated binding of brightly labeled microtubules (see Materials and methods). (c) Time series of confocal fluorescence microscopy images of a Cy5-labeled microtubule (green) moved by Eg5 along an immobilized Alexa 568–labeled microtubule (red). Arrows indicate the initial position of the Cy5-labeled microtubule. The bottom panel shows a kymograph of a line along the microtubule pair during the first 5 min of the time series. (d) Histograms showing distributions for speeds of microtubule sliding driven by Eg5 and Eg5-paGFP.