Mitotic spindle movements are randomized in Dlg1 knockdown cells. (A) 3D models of a mitotic neuroepithelial cell imaged from the apical surface (xy plane) or seen along its apical–basal axis (z axis). αz represents the angle between the spindle axis and the apical (xy) plane. (B) Mitotic spindle αz measurements reveal an identical phenotype in Dlg1 knockdown between fixed and live conditions. For metaphase measurements, n = 206 time frames from 13 control (Ctrl) cells and 574 time frames from 15 Dlg RNAi cells. Error bars show SEMs. (C and D) Time-lapse series of dividing neuroepithelial cells expressing PACT-mKO1 (PACT-KO) and H2B-GFP without (C) or with (D) Dlg1 RNAi. (top) En face view projection of z stacks encompassing both centrosomes. (bottom) Vertical z section along the mitotic spindle axis. White and orange arrows point to the same centrosome in en face and z views. Dotted lines highlight the spindle axis. Bars, 5 µm. (E) Z rotation dynamics during metaphase for control cells (left) or Dlg1 RNAi cells (right). Each color curve corresponds to one individual cell (nine representative cells). Thick black lines show mean angles of all analyzed cells normalized to metaphase onset. The red lines mark the time of the transition from the phase of directed z rotation to the phase of planar maintenance observed in control cells. (F and G) Absolute and relative (directional) z rotations (means + SEM) for control (F) and Dlg1 RNAi cells (G). See Materials and methods for a definition of absolute and relative rotation. wt, wild type. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
