Dynein function is required for the poleward transport response. Time-lapse live images of a metaphase GFP–α-tubulin PtK2 spindle (phase contrast, blue; GFP–α-tubulin, yellow) response to k-fiber laser ablation when dynein cargo binding is inhibited by transfection of a dominant-negative p150 fragment. After laser ablation (X), the targeted k-fiber rotates (00:00–00:10) and splays (e.g., 02:03). No significant poleward movement of the newly generated k-fiber minus ends (arrowhead) and attached chromosome (kinetochore marked by asterisk) is observed. Minus ends are not reincorporated into the spindle by 02:24 (compare with delays in Fig. S1 B) despite nearby microtubule populations (e.g., 00:46). The spindle is fragmented and multipolar, as described after transfection with this p150 fragment (Quintyne and Schroer, 2002). Dotted line indicates the position of the kinetochore end of the ablated k-fiber immediately before ablation. Time is in min:s, with frame captured immediately after ablation set to 00:00. Bars, 2 µm. See also Video 5.