Figure 3.
Reduction of MTs leads to THN migration decrease.(A) After 10 rounds of PST-1P activation every 3 min, most EB3-GFP signals are lost and do not reappear in subsequent cycles. In contrast, Map4MTB-GFP signal appears weakened, but not completely lost (arrows) after 1 h of PST-1P activation. Elapsed time in minutes. Scale bar, 20 µm. (B) An example of a PST-1P-treated 34-hpf embryo with the activation region outlined in blue and control regions in red. Orange box indicates region magnified in B. (C) Map4MTB-GFP shows that MTs in the activated region are reduced, but some resist depolymerization. Scale bar, 5 µm. (D) Stills from B showing that THNs within the activated region (top row) are impaired in their migration, while control cells (bottom row) are able to move. Dots follow individual examples, and progress is indicated on the right; arrows indicate start position and arrowheads the end point after 3 h. Scale bar, 20 µm. See also Video 3. (E) THNs with reduced MTs generate half the speed of THNs recorded with the same illumination setting without the drug, and one third of the speed of THNs migrating in the control regions of PST-1P-treated embryos. n = 8 embryos/63 tracks for PST-1P activated embryos, n = 7 embryos/39 tracks for PST-1P nonactivated embryos, n = 6 embryos/62 tracks for UV-illuminated embryos; P = 2.59 × 10−6 PST-1P activated/UV only, 5.37 × 10−10 PST-1P activated/nonactivated. (F) Overexpression of Katanin P60 impairs THN migration when combined with aTat1-mediated MT acetylation. n = 17 embryos/103 tracks for Map4MTB + Katanin P60-expressing embryos, n = 20 embryos/101 tracks for aTat1 + Katanin P60-expressing embryos; P = 2.37 × 10−8. (G) THNs tend to escape coexpression of Katanin P60, Map4MTB, and aTat1. Arrows indicate examples of coexpression in phase 2 THNs. Note that these THNs appear to have formed very short leading processes. Scale bar, 20 µm. (H) In examples of embryos coexpressing Katanin P60 with either Map4MTB or aTat1, coexpression is low in aTat1 and Katanin P60-expressing embryos, and significant debris is visible in the tissue. Dots indicate several examples, with orange following a Katanin P60-only expressing THN. Cyan boxes indicate magnified regions. Scale bars, 20 µm. Elapsed time in min. 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 < 0.001.

Reduction of MTs leads to THN migration decrease. (A) After 10 rounds of PST-1P activation every 3 min, most EB3-GFP signals are lost and do not reappear in subsequent cycles. In contrast, Map4MTB-GFP signal appears weakened, but not completely lost (arrows) after 1 h of PST-1P activation. Elapsed time in minutes. Scale bar, 20 µm. (B) An example of a PST-1P-treated 34-hpf embryo with the activation region outlined in blue and control regions in red. Orange box indicates region magnified in B. (C) Map4MTB-GFP shows that MTs in the activated region are reduced, but some resist depolymerization. Scale bar, 5 µm. (D) Stills from B showing that THNs within the activated region (top row) are impaired in their migration, while control cells (bottom row) are able to move. Dots follow individual examples, and progress is indicated on the right; arrows indicate start position and arrowheads the end point after 3 h. Scale bar, 20 µm. See also Video 3. (E) THNs with reduced MTs generate half the speed of THNs recorded with the same illumination setting without the drug, and one third of the speed of THNs migrating in the control regions of PST-1P-treated embryos. n = 8 embryos/63 tracks for PST-1P activated embryos, n = 7 embryos/39 tracks for PST-1P nonactivated embryos, n = 6 embryos/62 tracks for UV-illuminated embryos; P = 2.59 × 10−6 PST-1P activated/UV only, 5.37 × 10−10 PST-1P activated/nonactivated. (F) Overexpression of Katanin P60 impairs THN migration when combined with aTat1-mediated MT acetylation. n = 17 embryos/103 tracks for Map4MTB + Katanin P60-expressing embryos, n = 20 embryos/101 tracks for aTat1 + Katanin P60-expressing embryos; P = 2.37 × 10−8. (G) THNs tend to escape coexpression of Katanin P60, Map4MTB, and aTat1. Arrows indicate examples of coexpression in phase 2 THNs. Note that these THNs appear to have formed very short leading processes. Scale bar, 20 µm. (H) In examples of embryos coexpressing Katanin P60 with either Map4MTB or aTat1, coexpression is low in aTat1 and Katanin P60-expressing embryos, and significant debris is visible in the tissue. Dots indicate several examples, with orange following a Katanin P60-only expressing THN. Cyan boxes indicate magnified regions. Scale bars, 20 µm. Elapsed time in min. 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 < 0.001.

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