Related to Figs. 2 and 3. (A and B) Representative images (A) and displacements (B) for Void recruitment to a single kinetochore (yellow triangles) of a bi-oriented pair, as a negative control. Displacements, plotted as in Fig. 2, D and E, were used to define the range of naturally occurring chromosome oscillations (dashed lines cover 45 of 47 traces, 96%; Video 5). (C) Representative images showing kinetochore movement indicating microtubule depolymerization after recruiting INBox to a single kinetochore (white triangles; Video 7). (A and C) Inset: The targeted kinetochore pair at higher magnification. Scale bars in A and C: 5 µm or 1 µm in insets. (D) The fraction of release vs. depolymerization events from Fig. 2, D and E (2×INBox: 87 ± 5% mean ± SEM, n = 47; INBox: 67 ± 9% mean ± SEM, n = 30; *, P < 0.05, one tailed). (E and F) Steady-state velocities (E) and interkinetochore distances (F) for released kinetochores. Distances are plotted before activation and at steady-state after 2×INBox recruitment. Each data point represents a single kinetochore or pair of sisters. Black lines: Mean ± SEM (n = 41 for 2×INBox, n = 20 for INBox; *, P < 0.05). (G) INBox recruitment on monopolar and bipolar spindles, measured as mCherry intensity (mean ± SEM). (H) Higher magnification of the image shown in Fig. 3 B. Full series of sections through the centromeres of chromosomes within yellow boxes are shown in H′ and H′′. (H') The activated kinetochore Ka and and its non-activated sister kinetochore Kb. Microtubules have been released from Ka but are present at Kb. Arrowheads denote kinetochore plates. (H′′) Non-irradiated sister kinetochores of a properly aligned chromosome within the metaphase plate. Similar number of microtubules are attached to both kinetochores.