3D time-lapse imaging shows stereotypical biphasic movements of the mitotic spindle in neuroepithelial cells. (a and b) Time-lapse series of a Centrin2-GFP–expressing neuroepithelial cell in metaphase and anaphase. (a) Top row: successive apical projections of a 20-µm-thick Z-stack; bottom row: a schematic of the XY orientation of the spindle at each time point (colored spindle poles) relative to the previous time point (gray spindle poles). Purple arrows indicate the orientation of the movement between time points and reveal numerous changes in orientation during metaphase. (b) Top row: the same cell as in panel a is seen as successive vertical Z-sections along the axis of the mitotic spindle. Bottom row: a schematic of the same sections. Blue arrows indicate the rotation of the spindle relative to the apical/basal (Z) axis between successive time points. Note that within 6 min the spindle, starting from an oblique position, has aligned with the apical surface and remains aligned until anaphase. Left: 3D models of a metaphase neuroepithelial cell imaged from the apical surface (XY plane) or seen along its apical–basal axis (Z axis) are provided. Bar, 5 µm. (c) The orientation of the spindle relative to the apical surface (αZ) is random at metaphase onset (left), but by anaphase onset all the spindles have adopted a planar orientation parallel to the apical surface (right). (d) Dynamics of Z orientation (αZ) during metaphase: all cells in our study quickly align their spindle parallel to the apical surface during the first minutes of metaphase and remain aligned until anaphase. Eight representative cells are shown. (e) Dynamics of xy orientation (αXY) during metaphase: all cells exhibit active and random XY rotation throughout metaphase. The eight cells are the same as in panel b. (f) The time needed for the spindle to reach a planar orientation depends on its initial Z-orientation. (g) Centrosome velocity from 37 Centrin2-GFP+ cells during metaphase shows an inflection between 5 and 7 min. Error bars = SEM. (h) A 3D schematic of the successive movements of the spindle during metaphase. Immediately after metaphase onset, the spindle quickly undergoes a directed Z-rotation leading to planar orientation; Z rotation is then restricted to maintain planar orientation and most of the movement occurs randomly in a plane parallel to the apical surface. Throughout this figure, blue, magenta, or yellow represent movements and their measurements along the Z axis, in the XY plane, and in the three dimensions, respectively.
