Ectopic division orientations induced by transient microtubule depolymerization are permanently kept once microtubule dynamics are restored. (A and B) Still images from time-lapse recordings of larval NBs under normal microtubule dynamics (t0), after microtubule depolymerization by colcemid (t1), and after colcemid inactivation (t2–t4). Fluorescent reporter constructs are indicated to the left of each row. Arrows mark apical centrosomes; arrowheads refer to motile interphase centrosomes and to the basal centrosome in cells in metaphase. Colored circles highlight GMCs, and colored lines highlight the division axis. Dashed circles indicate the position where GMCs will be delivered. Each are colored according to birth order (yellow, first; green, second; and red, third). (A) An NB dividing three times (colcemid added, 13:55:46; effect detectable, 14:11:46; UV pulse delivered, 15:05:17; and total time exposed, 70 min). The orientation of the apical crescent and the spindle reflect the polarity axis (t0, yellow line). Under microtubule depolymerization conditions, the apical crescent forms ectopically (t1, green asterisk). After the drug is inactivated, the newly assembled spindle reorients to align with the ectopic crescent (t2, green asterisk/line). The same ectopic orientation is kept in the next cell cycle so that the third GMC is delivered on top of the second, away from the first (t4, red asterisk/line). In this NB, the original apical centrosome ended up in the GMC, and the other remained in the NB (t1 and t2). (B) An NB dividing three times (colcemid added, 15:34:41; UV pulse delivered, 16:45:23; and total time exposed to colcemid, 71 min; Video 6). (t0) The dashed line indicates the control polarity axis, as judged by the place of the most recent GMC. The cell rounds up upon colcemid treatment and upon release from colcemid arrest, divides asymmetrically with an ectopic orientation (t1 and t2, green line). This orientation is kept in the next mitosis, which delivers the third GMC on top of the second, diametrically opposed to the first GMC (t3 and t4). (C) Quantification of GMC budding sites plotting the variation (yellow sector) between the first and second cell division orientation (green and red, respectively) with respect to the control division (dashed line, 0°). Time is shown in hours:minutes:seconds. Bar, 10 µm.
