Recruitment of Aurora B to a single kinetochore of a bi-oriented pair triggers microtubule release. (A) Schematic of bipolar spindle assay. The bi-oriented sisters are under a tug of war between the two attached K-fibers. Microtubule release or depolymerization at the activated kinetochore (red circles) leads to movement in opposite directions. (B and C) Images from representative experiments showing INBox (B; Video 6) or 2×INBox (C; Video 8) recruitment after activation of a single kinetochore (yellow triangles) at t = 0. Insets show the targeted kinetochore pair at higher magnification. (D and E) Displacement of the activated kinetochore from the metaphase plate over time: Each trace represents a single kinetochore after INBox (D; n = 64) or 2×INBox (E; n = 79) recruitment, with the starting location defined as zero. Dashed lines show the range of chromosome dynamics covering 96% of control (Void recruited) kinetochores (Fig. S2, A and B; and Video 5). Histograms show maximal displacement for each trace. (F and G) Analyses of released kinetochores after INBox or 2×INBox recruitment. Example trace of 2×INBox recruitment (F) shows waiting time after activation, followed by movement at steady-state velocity (Fig. S2 E). (G) Waiting time distribution (2×INBox: median = 1.5 min, n = 41; INBox: median = 3.5 min, n = 20). *, P < 0.005. Scale bars, 5 µm or 1 µm in insets.