Fig. 5. Spindle position is actively maintained through the interplay between microtubules and F-actin.

a Live-cell imaging of the first asymmetric division in the gametophore initial. The positions of the nucleus and gametosome (prophase MTOC appeared in the apical cytoplasm) are indicated with yellow circles and red arrowheads, respectively. Cyan lines show the position and orientation of the phragmoplast. Cell borders are outlined with white lines. Live-cell imagining was repeated three times with similar results. Bar, 10 µm. b The frequency and type of spindle defects in gametophore initial mitosis observed in GH (control), TPX2 1-4Δ, and TPX2-5 HM1 lines. Numbers within the columns indicate number of cells with corresponding phenotypes. c Area occupied by the metaphase spindle (spindle size) in gametophore initials measured by manually tracking spindle borders from Z-projection, n = 9, 12, and 15 for GH, TPX2 1-4Δ and TPX2-5 HM1 lines, respectively. (mean ± SEM; **p = 0.0029, two-tailed Student’s t-test). d Tracking of the spindle center position from NEBD to anaphase onset. We assigned the starting position as Y = 0 and different X positions for each sample group. Note, that after 5 µM latrunculin A treatment, spindles never showed motility towards the basal end of the cell, i.e., negative Y-values. Each line represents spindle movement in a single cell. More than 12 cells were observed for each sample group in three or more independent experiments. e Maximum spindle speed (µm/min), f average spindle speed (µm/min), and g distance traveled by the spindle (µm) in the TPX2-5 HM1 cell line under various treatments. Total number of cells observed, including those without spindle motility: n = 23 (10 µM taxol), 16 (200 nM oryzalin), and 20 (control DMSO, with which the stock solution of taxol, oryzalin, and latrunculin A were prepared). Only spindle motility towards the basal end of the cell was analyzed. Bars represent mean ± SEM.