Asp-GFP at the intraspindle MT end is translocated to the pole by spindle flux. (A) Kymographs generated along spindle MTs indicate that Asp-GFP signals move poleward in control cells (a plus sign indicates the chromosomal side). Bars: (left) 5 µm; (right) 2 µm and 1 min. (B) Asp-GFP signals after Ncd/Dhc64C, Klp10A/CLASP, or Klp61F RNAi. An intraspindle MT bundle decorated with Asp-GFP in the Ncd/Dhc64C RNAi sample is magnified. Bars: 5 µm; (inset) 2 µm. (C) Velocity distribution of moving Asp-GFP signals. Mean velocities were significantly reduced after Klp10A/CLASP or Klp61F RNAi (P < 0.0001; Mann–Whitney U test), but not subsequent to Ncd/Dhc64C RNAi (P > 0.29). (D) Quantification of the Asp-GFP spot numbers in the absence of Dgt6, an augmin subunit (±SEM). We selected the first frame of the video in which a metaphase spindle was observed and counted the number of Asp-GFP spots in the spindle (a single focal plane). The numbers were then divided by the MT intensity in the central 60% area of the spindle (polar regions that had Asp-GFP clusters were excluded). Only the cells with similar total GFP intensity were selected for analysis (GFP intensity: 5.3 ± 1.4 [SD] for four control cells; 5.5 ± 1.5 [SD] for four Dgt6-depleted cells). Significant reduction (P < 0.021) was observed after Dgt6 RNAi. Bar, 5 µm. a.u., arbitrary units.