TPX2 regulates Eg5 activity and accumulation on microtubules in vitro. (A, top) A diagram of gliding assay. (bottom) Microtubule gliding velocity of Eg5-513 alone (control) and with the addition of 50, 250, and 500 nM TPX2 or TPX2-710. *, P < 0.05; **, P < 0.01. n = 174–618 microtubules from at least three independent experiments. Error bars represent SEM. (B, top) A diagram of TIRF assay. (bottom) Mean fluorescence intensity of Eg5-EGFP in arbitrary units (a.u.) along the length of the microtubule. **, P < 0.01. n = 290–374 microtubules from three independent experiments. Error bars represent SEM. (C, top) A diagram of microtubule–microtubule sliding assay. (bottom) Overlay from time-lapse imaging of immobilized microtubules (green) and mobile microtubules (red). The arrow indicates when TPX2 was added. Dashed lines show the location of the trailing microtubule end over time (time = minutes/seconds). n = 3 microtubules from two independent experiments. Bar, 1 µm. (D) A schematic model of the contribution of the TPX2–Eg5 interaction to spindle formation. Boxed regions depict how the TPX2–Eg5 interaction could regulate Eg5 behavior on antiparallel and parallel microtubules.