Knockdown of Myo10 leads to mitotic spindle defects. (a) Western blot showing Myo10 protein levels in uninjected (U), Myo10 MO (MO), and 5-mispair control MO (Ctrl MO) embryos. Lysates were prepared from embryos 24 h after microinjection. (b) Confocal micrographs of α-tubulin staining in embryos microinjected with nuclease-free water or Myo10 MO (MO) fixed at 12, 16, or 24 h after microinjection. (c) Quantification of mitotic spindles in water (H2O)-, Myo10 MO (MO)-, and 5-mispair control MO (Ctrl MO)-injected embryos 16 and 24 h after microinjection. At 16 and 24 h, Myo10 morphants have significantly more multipolar spindles than the mispair control (red). An increased number of bipolar spindles is seen in the morphant at 24 h, which is suggestive of a delay in mitosis (n = 15, 11, 14, 17, 18, and 18 embryos for water 16 h, MO 16 h, Ctrl MO 16 h, water 24 h, MO 24 h, and Ctrl MO 24 h, respectively). Error bars represent the standard error of the mean. (d) Box and whisker plots displaying metaphase spindle length in water (n = 92 spindles)- and Myo10 MO (n = 50 spindles)-injected embryos 24 h after injection. Spindle length measurements are shown as a percentage of total cell length to control for differences in cell size. Metaphase spindles in the Myo10 morphant are significantly longer than in water-injected controls. (e) Propidium iodide (red) and α-tubulin (green) staining of control and morphant spindles showing that chromosomes localize to the metaphase plate relatively normally in morphant spindles. (f) Confocal micrographs of α-tubulin–stained spindles assembled in vitro in the presence of either a control antibody or an anti-Myo10 antibody. In control conditions, normal bipolar spindles assemble, whereas the inhibition of Myo10 by antibody addition leads to multipolar spindles. For significance testing, unpaired Student's t tests were performed: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
