Figure S1.
Characterization of GDP-tubulin-assembled microtubules. (A) Mass spectrometry–based proteomic analysis of tubulin batches (four independent experiments). Enrichment of tubulin over all detected proteins is indicated. (B) Mass spectrometry-based proteomic analysis of microtubules assembled from GDP-tubulin and GTP-tubulin (three independent experiments). Enrichment of tubulin over all detected proteins is indicated. No MAP was identified in either tubulin (A) or microtubule (B) samples (details in dataset PXD049371, PRIDE partner repository). (C) Relative abundance of main tubulin isotypes in GDP-and GTP-tubulin-assembled microtubules (three independent experiments) determined by mass spectrometry. ns, non-significant (non-parametric Mann–Whitney test). (D) Quantitative analysis of immunoblots of GDP- and GTP-tubulin-assembled microtubules for various posttranslational modifications of tubulin (four independent experiments). Tyr, tyrosinated; DeTyr, detyrosinated; Ac, acetylated; PolyGlu, Polyglutaminated. ns, non-significant (non-parametric Mann–Whitney test). (E) GDP and GTP nucleotide content of purified tubulin (see also Materials and methods). (F) To determine whether the 7% GTP-tubulin contamination of the GDP-tubulin stock (as determined in E) might account for our results, we tested microtubule assembly under several conditions. We combined 3.75 µM red-fluorescent GDP-tubulin with 21.25 µM GTP-tubulin with (condition 1) and without (condition 2) added GTP. The fluorescence intensity of the resulting lattices was very similar (100.00 ± 21.08 a.u. and 90.44 ± 27.73 a.u. for condition 1 and 2, respectively). Condition 3 (24.75 µM unlabeled GTP-tubulin and 0.25 µM red-fluorescent GTP-tubulin) corresponds to the intensity of the lattice if only 7% of the initial fluorescent GDP-tubulin (i.e., 7% of 3.75 µM GDP-tubulin) had been incorporated (7.90 ± 1.90 a.u). Values represent the mean ± SD of 102, 108, and 60 microtubule lattices from at least three independent experiments. Triangles represent the mean lattice intensity for each experiment normalized to condition 1. ****P < 0.001; ns, non-significant (Kruskal–Wallis ANOVA followed by post-hoc Dunn’s multiple comparison). (G) Kymograph showing the growth of GDP-tubulin (210 µM) at the two extremities of a microtubule. GDP-tubulin is in green and the GMPCPP seed is in magenta. (H) Percentages of seeds nucleating GDP-tubulin at one or both ends. n, total number of seeds from at least three independent experiments. (I) Growth rates and seeded nucleation lag times at the minus and plus ends of GDP-tubulin-assembled microtubules. Values indicate the mean ± SD. n, number of growth events from at least three independent experiments. (J) Percentages of microtubules growing from the minus end or both ends of GMPCPP seeds incubated with 210, 100, or 50 µM of GDP-tubulin with or without 0.5 µM DCX or tau. n, total number of seeds from at least three independent experiments. For control conditions with 100 and 50 µM GDP-tubulin, no nucleating seeds were observed. Triangles represent the percentage of one-end nucleating seeds for each experiment. (K) Time series showing dilution-induced depolymerization of GTP-tubulin- and GDP-tubulin-assembled microtubules. Arrowheads indicate minus ends. S, seed. Histogram shows dilution-induced shrinkage rates of microtubule minus ends. n, total number of microtubules from three independent experiments. ****P < 0.0001 (non-parametric Mann–Whitney test). Source data are available for this figure: SourceData FS1.

Characterization of GDP-tubulin-assembled microtubules. (A) Mass spectrometry–based proteomic analysis of tubulin batches (four independent experiments). Enrichment of tubulin over all detected proteins is indicated. (B) Mass spectrometry-based proteomic analysis of microtubules assembled from GDP-tubulin and GTP-tubulin (three independent experiments). Enrichment of tubulin over all detected proteins is indicated. No MAP was identified in either tubulin (A) or microtubule (B) samples (details in dataset PXD049371, PRIDE partner repository). (C) Relative abundance of main tubulin isotypes in GDP-and GTP-tubulin-assembled microtubules (three independent experiments) determined by mass spectrometry. ns, non-significant (non-parametric Mann–Whitney test). (D) Quantitative analysis of immunoblots of GDP- and GTP-tubulin-assembled microtubules for various posttranslational modifications of tubulin (four independent experiments). Tyr, tyrosinated; DeTyr, detyrosinated; Ac, acetylated; PolyGlu, Polyglutaminated. ns, non-significant (non-parametric Mann–Whitney test). (E) GDP and GTP nucleotide content of purified tubulin (see also Materials and methods). (F) To determine whether the 7% GTP-tubulin contamination of the GDP-tubulin stock (as determined in E) might account for our results, we tested microtubule assembly under several conditions. We combined 3.75 µM red-fluorescent GDP-tubulin with 21.25 µM GTP-tubulin with (condition 1) and without (condition 2) added GTP. The fluorescence intensity of the resulting lattices was very similar (100.00 ± 21.08 a.u. and 90.44 ± 27.73 a.u. for condition 1 and 2, respectively). Condition 3 (24.75 µM unlabeled GTP-tubulin and 0.25 µM red-fluorescent GTP-tubulin) corresponds to the intensity of the lattice if only 7% of the initial fluorescent GDP-tubulin (i.e., 7% of 3.75 µM GDP-tubulin) had been incorporated (7.90 ± 1.90 a.u). Values represent the mean ± SD of 102, 108, and 60 microtubule lattices from at least three independent experiments. Triangles represent the mean lattice intensity for each experiment normalized to condition 1. ****P < 0.001; ns, non-significant (Kruskal–Wallis ANOVA followed by post-hoc Dunn’s multiple comparison). (G) Kymograph showing the growth of GDP-tubulin (210 µM) at the two extremities of a microtubule. GDP-tubulin is in green and the GMPCPP seed is in magenta. (H) Percentages of seeds nucleating GDP-tubulin at one or both ends. n, total number of seeds from at least three independent experiments. (I) Growth rates and seeded nucleation lag times at the minus and plus ends of GDP-tubulin-assembled microtubules. Values indicate the mean ± SD. n, number of growth events from at least three independent experiments. (J) Percentages of microtubules growing from the minus end or both ends of GMPCPP seeds incubated with 210, 100, or 50 µM of GDP-tubulin with or without 0.5 µM DCX or tau. n, total number of seeds from at least three independent experiments. For control conditions with 100 and 50 µM GDP-tubulin, no nucleating seeds were observed. Triangles represent the percentage of one-end nucleating seeds for each experiment. (K) Time series showing dilution-induced depolymerization of GTP-tubulin- and GDP-tubulin-assembled microtubules. Arrowheads indicate minus ends. S, seed. Histogram shows dilution-induced shrinkage rates of microtubule minus ends. n, total number of microtubules from three independent experiments. ****P < 0.0001 (non-parametric Mann–Whitney test). Source data are available for this figure: SourceData FS1.

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