Figure S2.
Increasing GDP-/GTP-tubulin ratios favor growth at the minus end. (A) Estimation of GDP-tubulin incorporation in microtubules assembled from 25 µM unlabeled GTP-tubulin and increasing concentrations of red-fluorescent GDP-tubulin. Values represent mean ± SD of 30 lattice intensities from three independent experiments and are fitted with a linear regression model (R2 = 0.96). Triangles represent the mean intensity for each experiment. (B) Percentages of seeds elongating at both ends or only at the minus ends in the presence of increasing ratios of GDP-/GTP-tubulin. n, total number of seeds from at least three independent experiments. Triangles represent the percentage of seeds elongating at both ends for each experiment. ****P < 0.001; ns, non-significant (two-sided Fisher’s test). P values were calculated relative to the control condition (0:1). (C) Violin plot of the percentage of polymerizing time at the plus end of microtubules assembled with increasing GDP-/GTP-tubulin ratios. The central line represents the median and the dashed lines represent the quartiles. At least 47 microtubules from at least three independent experiments have been analyzed for each condition. Triangles represent the mean polymerizing time of each experiment. ****P < 0.001; ns, non-significant (Kruskal–Wallis ANOVA followed by post-hoc Dunn’s multiple comparison). P values were calculated relative to the control condition (0:1). (D) Two-step perfusion experiment (left) to evaluate the stability of microtubules assembled from 80 µM GDP-tubulin and 25 µM GTP-tubulin, with a representative kymograph (right). Microtubules were first assembled from seeds (S) in the presence of a mixture of GDP-/GTP-tubulin in a 3:1 ratio (light pink). After 15 min of elongation, free tubulin was replaced by GTP-tubulin (magenta). White arrows in kymograph indicate rescue events at the junction between the two lattices (star). (E) Percentages of depolymerizing microtubules that cross the junction (gray bar) or stop at the junction (black bar). 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).

Increasing GDP-/GTP-tubulin ratios favor growth at the minus end. (A) Estimation of GDP-tubulin incorporation in microtubules assembled from 25 µM unlabeled GTP-tubulin and increasing concentrations of red-fluorescent GDP-tubulin. Values represent mean ± SD of 30 lattice intensities from three independent experiments and are fitted with a linear regression model (R2 = 0.96). Triangles represent the mean intensity for each experiment. (B) Percentages of seeds elongating at both ends or only at the minus ends in the presence of increasing ratios of GDP-/GTP-tubulin. n, total number of seeds from at least three independent experiments. Triangles represent the percentage of seeds elongating at both ends for each experiment. ****P < 0.001; ns, non-significant (two-sided Fisher’s test). P values were calculated relative to the control condition (0:1). (C) Violin plot of the percentage of polymerizing time at the plus end of microtubules assembled with increasing GDP-/GTP-tubulin ratios. The central line represents the median and the dashed lines represent the quartiles. At least 47 microtubules from at least three independent experiments have been analyzed for each condition. Triangles represent the mean polymerizing time of each experiment. ****P < 0.001; ns, non-significant (Kruskal–Wallis ANOVA followed by post-hoc Dunn’s multiple comparison). P values were calculated relative to the control condition (0:1). (D) Two-step perfusion experiment (left) to evaluate the stability of microtubules assembled from 80 µM GDP-tubulin and 25 µM GTP-tubulin, with a representative kymograph (right). Microtubules were first assembled from seeds (S) in the presence of a mixture of GDP-/GTP-tubulin in a 3:1 ratio (light pink). After 15 min of elongation, free tubulin was replaced by GTP-tubulin (magenta). White arrows in kymograph indicate rescue events at the junction between the two lattices (star). (E) Percentages of depolymerizing microtubules that cross the junction (gray bar) or stop at the junction (black bar). 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).

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