Comt/NSF, Rtnl2/RTN2, and Shrub/CHMP4B are required for efficient acentric reintegration into daughter nuclei. (A–D) Stills from videos of mitotic neuroblasts expressing I-CreI and H2Av-RFP (gray) only (A; Video 6), or expressing I-CreI and H2Av-RFP in combination with RNAi against Comt (B; Video 7), Rtnl2 (C; Video 8), or Shrub (D; Video 9). Each panel is composed of a row of maximum projections (top row) above their corresponding sum projections that are pseudo-colored to illustrate differences in Z-position (bottom row; red, upper Z-planes; blue, lower Z-planes). Acentrics (arrows) segregated poleward and reentered daughter nuclei in control videos. However, acentrics in dividing neuroblasts expressing RNAi against Comt, Rtnl2, or Shrub initially segregated but failed to reintegrate into daughter nuclei and instead formed micronuclei (arrowheads). Time is written in seconds after initial acentric poleward movement. Scale bars are 2 µm. Yellow dashed boxes indicate magnified regions. Asterisks indicate statistical significance to control as determined by γ2 tests (B; P = 0.016; C, P = 0.005; D, P = 0.006). (A′–D′) Distance of acentrics from main nuclei in control (A′–D′; black line; n = 29), Comt-depleted (B′; purple line; n = 27), Rtnl2-depleted (C′; purple line; n = 20), and Shrub-depleted (D′; purple line; n = 14) neuroblasts. Lines represent averages. Dark-shaded regions represent ± the standard error. Light-shaded regions represent ± twice the standard error. Dashed green lines represent the previously measured average time when acentrics begin passing through channels (155 s after acentric segregation). Although initially following a similar trajectory as acentrics in control neuroblasts, acentrics in both Comt- and Rtnl2-depleted neuroblasts ultimately remained farther apart from daughter nuclei at 400 s after initial acentric segregation than those in control divisions. The point of divergence was approximately the measured time when acentrics begin passing through channels. In contrast, the trajectory of acentrics in Shrub-depleted neuroblasts, which likewise remained farther apart from daughter nuclei at 400 s after initial acentric segregation than those in control divisions, diverged from acentrics in control neuroblasts soon after initial acentric segregation. See also Fig. S3.
