Transient loss of microtubules establishes ectopic cortical polarity that drives cell division once microtubule dynamics are restored. (A–D) Still images from time-lapse recordings of larval NBs dividing twice. Fluorescent reporter constructs are indicated to the left of each row. White arrows mark apical centrosomes; white arrowheads refer to motile interphase centrosomes and to the basal centrosome in cells in metaphase. GMCs are highlighted by circles and are colored and numbered according to birth order (yellow, first; and green, second). (A–C) Yellow and green lines refer to pre- and posttreatment division orientation, respectively, in the transient microtubule depolymerization experiments followed by colcemid inactivation during (A and B) or before (C) mitosis. (A) The first GMC is delivered basally opposing the apical aster (t₀). Microtubule depolymerization mispositions the centrosomes (t₁). After microtubule polymerization is restored (t₂), the cell enters metaphase (t₃) and divides asymmetrically, delivering the second GMC ectopically (t₄). Then one centrosome organizes an aster at the ectopic apical pole, whereas the other is down-regulated and motile (colcemid added, 19:06:12; effect detectable, 19:20:22; UV pulse delivered, 20:13:17; and total time exposed, 67 min; Video 3). (B) Transient colcemid treatment produces an ectopic basal cortex (t₀–t₃; yellow and green arrowheads outline pre- and posttreatment crescents, respectively). Upon colcemid inactivation, the spindle, reflected by the centrosomes at the spindle poles, aligns with this crescent (t₂) that is segregated to the ectopically delivered GMC (t₃ and t₄; colcemid added, 13:35:43; effect detectable, 14:00:43; UV pulse delivered, 14:51:54; and total time exposed, 76 min). (C) The control division delivers the GMC basally (t₀). 25 min later, the apical centrosome has detached, yet microtubules remain detectable (t₁ and t₂, red arrow). When the UV pulse is delivered shortly thereafter, before entry into mitosis, microtubules regrow only over the ectopically localized apical centrosome (t₃), and once assembled, the spindle rotates, and the following division occurs at the pretreatment orientation (t₄; colcemid added,16:00:46; effect detectable, 16:09:12; UV pulse delivered, 16:32:25; and total exposure time, 32 min; Video 4). (D) Apical crescents form before (t₀, yellow asterisk) and after the addition of Taxol (t₄, green asterisk) at roughly the same sector of the cortex (Video 5). (E) Plot of GMC budding site variations induced by transient microtubule depolymerization (microtubule dynamics restored after entry into mitosis, green; two consecutive control divisions, yellow). (F) Plot of variations of the place of apical crescent assembly after the addition of Taxol (green) compared with two successive control divisions (yellow). Time is shown in hours:minutes:seconds. Bar, 10 µm.