Augmin, but not centrosomes or HURP, is critical for interzonal MT generation in unperturbed anaphase cells. (A) Single time frame images (top), 60-s stacked images (middle), and 10-s tracings (bottom) of EB1-GFP in RNAi-treated cells. Absence of the centrosome at the left pole of the monastral bipolar spindle of the Plk4-depleted cell was confirmed by z-axis confocal sectioning of the whole cell (Fig. S2 E). The following comets were distinguished: interzonal comets moving from left to right (acentrosomal to centrosomal side in Plk4-depleted cells; red), those moving in the opposite direction (green), comets moving in the external region on the left side (acentrosomal side in Plk-depleted cells; orange), or those on the opposite side (magenta). Light blue, interzonal comets with unknown directionalities; gray lines, borders between the external region and interzone. (B) Number of comets observed in each region of the half-spindle in control (blue) or Plk4-depleted cells (centrosomal or acentrosomal halves shown in green or red, respectively). Asterisks indicate statistically significant difference from the control (P < 0.008 by t test). The numbers of interzonal comets with unknown directionalities were 9.7 ± 2.3 (SEM) and 9.7 ± 3.5 in control and Plk4-depleted cells, respectively (not depicted). (C) The number of comets observed in each region of the whole spindle in control (blue), hDgt6-depleted (gray), and HURP-depleted cells (yellow) is shown. Means ± SEM of three independent experiments were shown (nine cells were analyzed for each region). Asterisks indicate statistically significant difference from control (P < 0.005 by t test). An identical dataset was used for luciferase RNAi cells in B and C. (D) Immunostaining of MTs (green), γ-tubulin (red), and chromosomes (blue) in control and hDgt6-depleted cells in anaphase. In the control cell, γ-tubulin was concentrated at both ends of the central spindle (arrowheads); however, such concentration was not observed in the augmin-depleted cell. Bars, 5 µm.