NuMA and dynactin are recruited specifically to newly generated minus ends. (A) Live images of a GFP–α-tubulin PtK2 spindle immediately before and after k-fiber ablation at targeted sites (X). (B) Representative immunofluorescence image of NuMA, α-tubulin, and DNA (Hoescht) in cell from A, fixed after ablation. NuMA (arrowheads) localizes to new minus ends. (C) Live images of a GFP–α-tubulin PtK2 monopolar spindle (phase contrast [blue] to identify chromosome locations; α-tubulin, red) immediately before (left) and after (right) ablation of non–k-fiber bundles (each ablation site marked by white X). One k-fiber is also ablated (gray X). (D) Representative immunofluorescence image of NuMA, α-tubulin, and kinetochores (CREST) in two planes of same cell from C, fixed after ablation. NuMA (arrowheads) localizes to new minus ends. In Plane 1, new minus ends are not yet reincorporated into the spindle; in Plane 2, the new minus ends appear to have moved poleward along another microtubule bundle (note contact between the microtubule bundles at the new NuMA-marked minus ends). (E) Live images of a GFP–α-tubulin PtK2 spindle immediately before and after k-fiber ablation at targeted sites (X). (F) Representative immunofluorescence images of NuMA, dynactin subunit p150, and α-tubulin in cell from E, fixed after ablation. Arrowheads mark NuMA and p150 recruited to new minus ends. The ablated k-fiber on the left has associated with other microtubules before fixation, whereas the minus ends of the ablated k-fiber on the right remain unattached. (G) Line scan analysis of NuMA, dynactin subunit p150, and α-tubulin intensity along dashed line in F. NuMA and p150 colocalize at new microtubule minus ends, and loss of tubulin intensity confirms ablation. Representative example of five ablations in four cells. Bars: (A–E) 2 µm; (F) 1 µm.