Aurora B interacts with a distinct class of spindle microtubules. (A) Xenopus S3 cells immunostained for INCENP and tubulin (Tub) and the close proximity of tubulin and Aurora B (AurB) are shown by PLA. Insets are single z sections of Aurora B–tubulin interactions at the centromere (yellow boxes) and at the kinetochores (orange boxes). The bottom shows the effect of ice treatment on S3 cells before fixation. Bars: (main images) 11 µm; (insets) 0.51 µm. (B) Monastrol-treated Xenopus S3 cells immunostained for Ndc80 and tubulin and the close proximity of tubulin and Aurora B shown by PLA—projection of four z sections. Bars: (main images) 1.6 μm; (insets) 1 µm. (C) Nocodazole washout experiments were performed to show Aurora B specifically enriched on pre–K-fibers. Xenopus S3 cells were fixed and stained 5 min (projection of seven z sections) and 15 min (projection of nine z sections) after nocodazole washout. Bars: (main images) 2.3 μm; (insets) 1.4 µm. B and C insets are zoomed in views of the areas highlighted by the boxes showing Aurora B–tubulin PLA spots on pre–K-fibers. Aurora B–tubulin PLA signals are shown with Ndc80 and tubulin costaining. (Individual z slices of whole cell projections are shown in Videos 1–4.)