MT motor proteins regulate THN speed. (A) Triple-citrine-tagged p50 colocalizes with the centrosome at the URL. Scale bar, 5 µm in overview, 1 µm in the magnified region on the right. Box indicates the magnified region. (B) Similarly, p50 is often found at the origin of Map4MTB-decorated MTs in phase 2 THNs. Scale bars represent 20 µm in the overview and 5 µm in magnified images. Box indicates the magnified region. (C) Blocking either dynein by overexpression of p50 or Kif5C in a rigor mutant (T94N) reduces THN speed. Dots trace individual examples. Cyan boxes indicate the magnified region in the stills. Elapsed time in minutes. Scale bars, 20 µm. See also Video 4. (D) Loss of active dynein approximately halves THN speed, while Kif5C rigor mutant overexpression reduces THN speed by 20–30%. n = 7 embryos/85 tracks for control; n = 8 embryos/69 tracks for p50-expressing embryos; n = 25 embryos/155 tracks for Kif5C-expressing embryos; n = 31 embryos/250 tracks for Kif5C T94N-expressing embryos; P = 3.69 × 10−12 p50/control, P = 0.016 Kif5C/control, P = 1.08 × 10−8 Kif5C/Kif5C T94N, and P = 0.002 control/Kif5C T94N. (E) FISH for kif5c expression in 30-hpf embryos demonstrates that Kif5C is found at the MHB at the time of THN migration. Arrows indicate MHBs and arrowheads URLs. Boxes indicate magnified regions on the right. Scale bars represent 200 µm in the overview and 50 µm in magnified regions on the right. (F) WISH for kif5c confirms the expression of the gene at the MHB at 30 hpf. Top: Antisense. Bottom: Sense for control. Magnified regions on the right are indicated by boxes in overview images. Arrows indicates MHBs and arrowheads URLs. Scale bar represents 100 µm or 50 µm in magnified images. Elapsed time in minutes. MHB, solid line; URL, dotted line. Boxes in graphs represent 25–75% of all values and whiskers 1.5 times the quartile. Median is shown as a horizontal bar and mean as a square box. Significance level in Kruskal–Wallis ANOVA: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
