Dynactin and NuMA identify new minus ends within seconds and escort them to spindle poles. (A and E) Representative time-lapse live images of PtK2 cells expressing GFP-Arp1A (A) or GFP-NuMA (E). Arp1A and NuMA (arrowheads) are recruited to the sites of ablation (X) within seconds and move rapidly and processively poleward. GFP-Arp1A and GFP-NuMA puncta move poleward until they are indistinguishable from poles. Time is in min:s, with frame captured immediately after ablation set to 00:00. See also Videos 8 and 10. (B and F) Kymographs along poleward path of GFP-Arp1A (B) or GFP-NuMA (F) puncta, between dashed lines in A and E. Note that the spindle pole itself (bright signal along bottom of kymograph) moves upward during minus end poleward transport, consistent with a reactive force on the spindle pole as the ablated k-fiber is pulled downward via a pole-connected track. (C) Representative time-lapse live images of cells expressing mCherry-tubulin and GFP-Arp1A reveal that recruited Arp1A (arrowheads) localizes at and moves with new microtubule minus ends after ablation (at X). The kinetochore of the ablated k-fiber is marked by an asterisk and a neighboring non-ablated kinetochore is marked by o. See also Video 9. Bars, 2 µm. (D) Kymograph along poleward path between dashed lines in C of ablated mCherry-tubulin k-fiber and GFP-Arp1A puncta. (G) Distance of GFP-NuMA puncta from ablation site as puncta move processively poleward after ablation. In some cases, stationary GFP-NuMA is detectable at the ablation site for up to 40 s before it moves poleward (see also Fig. S4). Red trace indicates the mean (error bars represent SEM) of 43 individual responses (blue traces). On average, the recruited GFP-NuMA first appears ∼0.5 µm farther away from the pole than the site of ablation, which is expected given an ablation area of ∼1 µm (see Materials and methods).