Activated kinetochores lack end-on microtubule attachments. (A) Live imaging showing 2×INBox recruitment and chromosome movement after activation of a single kinetochore (yellow triangles) at t = 0, as in Fig. 2 C. (B) The same cell was fixed at 3.5 min and examined by fluorescence and serial-section EM. Top: A single focal plane shows 2×INBox overlaying SPC25 at the activated kinetochore (white arrow) and chromosomes by fluorescence, and the corresponding section from the 3D EM dataset. Kinetochores of the activated chromosome pair (B’) and a nonactivated aligned chromosome (B′′) are shown at higher magnification below (blue: DNA; green: microtubules; magenta: kinetochores). Microtubules are bound to the leading kinetochore (Kb), but not the activated lagging kinetochore (Ka). Full EM series through these two kinetochores are shown in Fig. S2 H.