Regulation of microtubule dynamic instability by the carboxy-terminal tail of β-tubulin

(A) Coomassie-stained SDS–PAGE gel of tubulin after a time course of subtilisin digestion. (B) Plot of line scans of lanes 2 (black), 4 (red), and 6 (blue). (C) Plot of sum of pixel intensity of respective bands in the Coomassie-stained gel in (A). (D) Western blot of the same samples as in (A), probed with an antibody to α-tubulin (4A1; see the Materials and Methods section). This blot is the same as in Fig 1A (middle). (E) Plot of line scans of lanes 2 (black), 4 (red), and 6 (blue), from the blot in (D). (F) Western blot of the same samples as in (A, B), probed with an antibody to β-tubulin (9F3; see the Materials and Methods section). This blot is the same as in Fig 1A (top). (G) Plot of line scans of lanes 2 (black), 4 (red), and 6 (blue), from the blot in (F). (H) Western blots of the two independent S-tubulin samples made after subtilisin digestion for 5 min at 30ºC, probed for α-tubulin (top; DM1A) and β-tubulin (bottom; 9F3). These samples were snap-frozen and stored at −80ºC in single-use aliquots. Source data are available for this figure.

(A) Coomassie-stained SDS–PAGE gel of tubulin after subtilisin digestion for 0 or 5 min. Boxes indicate the bands SF1 (black), SF4 (blue), and SF5 (red) that were excised and analyzed by mass spectrometry. (B) Amino acid sequence of residues 393–445 of Sus scrofa TUBB2B shown for reference alongside peptides identified in the SF5 band. We were not able to identify peptides corresponding to the β-tubulin carboxy termini in any of the other bands analyzed, likely because of high levels of heterogeneity introduced by posttranslational modifications. (C) Amino acid sequence of residues 403–451 of Sus scrofa TUBA1B shown for reference alongside peptides identified in the SF1 (black), SF4 (blue), and SF5 (red) bands.

(A) Representative image of a microtubule assembled from the untreated tubulin (6.9 μM). Dashed white line is the coverage of the line scan across the microtubule with intensities plotted in (C). Scale bar = 1 μm. (B) Same as (A) assembled with S-tubulin (4.0 μM). (C) Intensity measurements plotted as a function of pixel position of the microtubules in (A) (black) and (B) (red). (D) Mean tip SD plotted as a function of polymerization rate for tubulin (black) and S-tubulin (red). N = 355 time points per polymerization rate, from at least five different microtubules. Data points are mean ± 95% CI plotted for each concentration.

(A) Schematic of the washout experiment. (B) Representative kymographs of untreated tubulin (6.3 μM) and S-tubulin (4.3 μM) in the washout experiment. Labels denote aspects analyzed for the washout experiment, including P—polymerization rate, D—delay time to catastrophe, S—slow depolymerization rate, and F—fast depolymerization rate. S-tubulin kymograph does not exhibit the D and S states. (C) Percentage of microtubules without a slow depolymerization rate (B, labeled “S”) for all washout experiments (number of microtubules without S state/total number of microtubules × 100). N = 180 tubulin and n = 94 S-tubulin microtubules, pooled from at least four different experiments for each. Error bars are standard error of the proportion. **P « 0.001, significance determined by Fisher’s exact test. (D) Time to catastrophe (B, labeled “D”) in seconds for tubulin and S-tubulin microtubules after washout. N = 179 tubulin and n = 51 S-tubulin microtubules, pooled from at least four different experiments. Lines represent the median. Microtubules without a detectable slow depolymerization phase were excluded from this analysis. *P = 0.04, significance determined by the Mann–Whitney U test. (E) Slow depolymerization rate (B, labeled “S”) for tubulin and S-tubulin microtubules after washout. N = 162 tubulin and n = 41 S-tubulin microtubules, pooled from at least four different experiments. Lines represent the median. Microtubules without a detectable slow depolymerization phase and/or a rate calculated to be zero were excluded from this analysis. *P = 0.05, significance determined by the Mann–Whitney U test. (F) Fast depolymerization rate (B, labeled “F”) for tubulin and S-tubulin microtubules after washout. N = 179 tubulin and n = 92 S-tubulin microtubules, pooled from at least four different experiments for each. **P « 0.001, significance determined by the Mann–Whitney U test.

(A) Polymerization rate plotted as a function of tubulin concentration. Crosses represent data from experiments conducted at 1 mM MgCl₂ and dots are data points from Fig 1D from experiments conducted at 5 mM MgCl₂. Black: tubulin and red: S-tubulin. Data points are mean ± 95% CI plotted for each concentration. Each data point represents at least 61 polymerization events from at least 24 microtubules, collected from at least three separate experiments. Linear regressions were fit to the data with at least three different concentrations and plotted as lines. (B) Depolymerization rate plotted as a function of tubulin concentration. Each data point represents at least 45 depolymerization events from at least 24 microtubules, collected from at least three separate experiments. Linear regressions were fit to the data with at least three different concentrations and plotted as lines. (C) Slow depolymerization rate (Fig 3B, labeled “S”) for tubulin and S-tubulin microtubules after washing out free tubulin. The MgCl₂ concentration indicated is for the washout buffer. The 5 mM plots are data from Fig 3E, provided for comparison. Data for the 1 mM MgCl₂ represent 45 microtubules for untreated tubulin (4.8–6.8 μM) and 158 microtubules for S-tubulin (7.8–12.0 μM), pooled from at least four different experiments for each. Microtubules without a detectable slow depolymerization rate and/or a rate calculated to be zero were excluded from this analysis. *P = 0.05 and **P « 0.001, significance determined by the Mann–Whitney U test. (D) Fast depolymerization rate (Fig 3B, labeled “F”) for tubulin and S-tubulin microtubules after washing out free tubulin. The 5-mM plots are data from Fig 3F, provided for comparison. Data for the 1 mM MgCl₂ represent 52 microtubules for untreated tubulin (4.8–6.8 μM) and 260 microtubules for S-tubulin (7.8–12.0 μM), pooled from at least four different experiments for each. N = 179 tubulin and n = 92 S-tubulin microtubules, pooled from at least four different experiments for each. *P = 0.01 and **P « 0.001, significance determined by the Mann–Whitney U test.

(A) Representative growth curves of wild-type cells in different magnesium conditions. Control group was cultured in synthetic media with 4 mM MgSO₄. Other groups were grown in synthetic media without MgSO₄ but supplemented with MgCl₂ at concentrations indicated. All cultures were grown at 30°C with agitation for 21 h, and OD600 was measured every 5 min. (B) Median doubling times of wild-type, mnr2Δ, and tub2-430Δ cells at indicated magnesium conditions, normalized to the doubling time of wild-type cells in 4 mM MgSO₄. Each data point represents at least 11 replicates from four separate experiments. Error bars are ± 95% CI. (C) Representative images of cells in preanaphase expressing GFP-labeled microtubules, grown in synthetic media or without MgSO₄. Scale bar = 1 μm. (D) Distribution of astral microtubule (aMT) lengths measured in preanaphase cells from an asynchronous culture. Lengths were measured every 4 to 5 s for 5 min. Cells were cultured in synthetic media with (+) or without (−) 4 mM MgSO₄. Data pooled from two separate experiments, with at least seven cells and 396 total measurements for each group. **P « 0.001. Significance determined by the Mann–Whitney U test. Lines denote median. (E) Representative images of cells in G1 phase expressing GFP-labeled microtubules, grown in synthetic media or without MgSO₄. Scale bar = 1 μm. (F) Distribution of aMT lengths measured in G1 from an asynchronous culture. Cells were cultured in synthetic media with (+) or without (−) 4 mM MgSO₄. Data pooled from two separate experiments, with at least 60 cells for each group. *P = 0.01, **P « 0.001. Significance determined by the Mann–Whitney U test. Lines denote median.