Figure 4. GDP-tubulin island stops... | Rockefeller University Press

Figure 4.

$GDP-tubulin island stops shrinkage at the plus-end of GTP-tubulin-grown microtubules. (A) To monitor GDP-tubulin behavior at the plus end of microtubules assembled from GTP-tubulin, we perfused GTP-tubulin (15 µM, pink) then GDP-tubulin (210 µM, green), and then recorded with TIRF. Representative kymograph shows the behavior of a microtubule recorded after GDP-tubulin perfusion (gray square). The star indicates where the GTP-tubulin-grown microtubule paused. (B) To evaluate whether the paused plus-ends remained competent for microtubule assembly, we polymerized GTP-tubulin (15 µM, pink) from seeds, followed by GDP-tubulin (210 µM, green), then GTP-tubulin (10 µM, magenta). Kymograph shows a microtubule recorded after the last GTP-tubulin perfusion (gray square). Arrows indicate rescue events. The star indicates where the GDP-tubulin induced pausing. (C) Histogram shows the distance of rescue events at the plus end from the junction (position 0, same star as in B) where GDP-tubulin was incorporated. The frequency of rescues at the plus end in the control experiment is shown on the right; sequential perfusion of GTP-tubulin (15 µM, pink) followed by GTP-tubulin (10 µM, magenta). The locations of rescue events at the plus end from the junction where GDP-tubulin was incorporated differed from those in the control experiment. n, total number of rescue events from at least three independent experiments. (D) Percentages of depolymerizing plus-end extremities that cross the junction or stop at the junction. n, total number of events from at least three independent experiments. Triangles represent the percentage of depolymerizing microtubules that stop at the junction for each experiment. ****P < 0.0001 (two-sided Fisher’s test). (E) To reveal GDP-tubulin incorporation at microtubule plus ends, we used the same procedure as in A, except with fluorescent GDP-tubulin containing a high fraction of green-labeled tubulin. Tubulin polymerization was stopped 15 and 40 min after GDP-tubulin perfusion, shown in representative kymographs. (F) Length of GDP-tubulin green segments at the plus (top) and minus (bottom) ends. Values represent means ± SD of at least 84 events from three independent experiments. Triangles represent the mean length for each experiment. ns, non-significant; ****P < 0.0001 (Kruskal–Wallis ANOVA followed by post-hoc Dunn’s multiple comparison).$

GDP-tubulin island stops shrinkage at the plus-end of GTP-tubulin-grown microtubules. (A) To monitor GDP-tubulin behavior at the plus end of microtubules assembled from GTP-tubulin, we perfused GTP-tubulin (15 µM, pink) then GDP-tubulin (210 µM, green), and then recorded with TIRF. Representative kymograph shows the behavior of a microtubule recorded after GDP-tubulin perfusion (gray square). The star indicates where the GTP-tubulin-grown microtubule paused. (B) To evaluate whether the paused plus-ends remained competent for microtubule assembly, we polymerized GTP-tubulin (15 µM, pink) from seeds, followed by GDP-tubulin (210 µM, green), then GTP-tubulin (10 µM, magenta). Kymograph shows a microtubule recorded after the last GTP-tubulin perfusion (gray square). Arrows indicate rescue events. The star indicates where the GDP-tubulin induced pausing. (C) Histogram shows the distance of rescue events at the plus end from the junction (position 0, same star as in B) where GDP-tubulin was incorporated. The frequency of rescues at the plus end in the control experiment is shown on the right; sequential perfusion of GTP-tubulin (15 µM, pink) followed by GTP-tubulin (10 µM, magenta). The locations of rescue events at the plus end from the junction where GDP-tubulin was incorporated differed from those in the control experiment. n, total number of rescue events from at least three independent experiments. (D) Percentages of depolymerizing plus-end extremities that cross the junction or stop at the junction. n, total number of events from at least three independent experiments. Triangles represent the percentage of depolymerizing microtubules that stop at the junction for each experiment. ****P < 0.0001 (two-sided Fisher’s test). (E) To reveal GDP-tubulin incorporation at microtubule plus ends, we used the same procedure as in A, except with fluorescent GDP-tubulin containing a high fraction of green-labeled tubulin. Tubulin polymerization was stopped 15 and 40 min after GDP-tubulin perfusion, shown in representative kymographs. (F) Length of GDP-tubulin green segments at the plus (top) and minus (bottom) ends. Values represent means ± SD of at least 84 events from three independent experiments. Triangles represent the mean length for each experiment. ns, non-significant; ****P < 0.0001 (Kruskal–Wallis ANOVA followed by post-hoc Dunn’s multiple comparison).

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