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

View ORCID ProfileColby P Fees, View ORCID ProfileJeffrey K Moore  Correspondence email
Colby P Fees
Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA
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Jeffrey K Moore
Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA
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  • For correspondence: jeffrey.moore@ucdenver.edu
Published 19 April 2018. DOI: 10.26508/lsa.201800054
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  • Figure 1.
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    Figure 1. β-CTT promotes microtubule dynamics.

    (A) Western blot of tubulin after a time course of subtilisin digestion. Porcine brain tubulin was treated with 1% subtilisin for indicated times at 30°C. Blots were probed with anti-β-tubulin (top), anti-α-tubulin (middle), and anti-α-tyrosinated tubulin (bottom). Arrowhead marks the faster migrating species of β-tubulin produced after 5-min digestion. (B) Representative kymograph of tubulin (green) polymerized from GMPCPP-stabilized microtubule seeds (red), collected at 3-s intervals. Tubulin concentration: 5.6 μM, vertical scale bar = 1 min, and horizontal scale bar = 1 μm. (C) Representative kymograph of S-tubulin as in (B). S-tubulin concentration: 4.9 μM, vertical scale bar = 1 min, and horizontal scale bar = 1 μm. (D) Polymerization rate plotted as a function of tubulin concentration. Data points are mean ± 95% CI plotted for each concentration. Each data point represents at least 197 polymerization events from at least 36 microtubules, collected from four separate experiments. Linear regressions were fit to the data and plotted as solid lines. Black: tubulin; red: S-tubulin. (E) Depolymerization rate plotted as a function of tubulin concentration. Each data point represents at least 62 depolymerization events from at least 28 microtubules, collected from three separate experiments. Data points are mean ± 95% CI plotted for each concentration. (F) Microtubule length at catastrophe plotted as the cumulative fraction of the total population. Tubulin concentrations: 3.4 (black dotted line) and 8.6 μM (solid black line); N = 502 and 125 catastrophe events, respectively. S-tubulin concentrations: 1.6 (red dotted line) and 3.9 μM (solid red line); N = 74 and 112, respectively.

    Source data are available for this figure.

    Source Data for Figure 1[LSA-2018-00054_SdataF1.tif]

  • Figure S1.
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    Figure S1. Limited subtilisin digestion of purified porcine brain 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.

    Source Data for Figure S1[LSA-2018-00054_SdataF1.tif]

  • Figure S2.
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    Figure S2. Proteomic analysis of subtilisin-digested purified porcine brain tubulin.

    (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.

  • Figure 2.
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    Figure 2. β-CTT alters the structure of the microtubule plus end.

    (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.

  • Figure 3.
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    Figure 3. β-CTT promotes microtubule plus-end stability.

    (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.

  • Figure 4.
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    Figure 4. Magnesium regulates tubulin equilibrium through β-CTT.

    (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.

  • Figure 5.
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    Figure 5. β-CTT regulates magnesium sensitivity in vivo.

    (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.

  • Figure 6.
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    Figure 6. β-CTT function requires negatively charged amino acids.

    (A) Amino acid sequences of β-CTTs of mutant yeast strains characterized in Fig 6. The native (TUB2) allele provided for comparison. (B) Representative images of preanaphase yeast strains expressing microtubules labeled with GFP-Tub1 treated with either 1.5 μM nocodazole or DMSO as a control. Arrowheads indicate the plus ends of astral microtubules (aMTs). (C) Percent of cells exhibiting aMTs after exposure to 1.5 μM nocodazole or DMSO for 1 h. Bars represent the pooled mean ± standard error of the proportion of four separate experiments pooled. Each group is composed of n ≥ 735 cells. **P « 0.001. Significance determined by Fisher's exact test. (D) Representative aMT life plots of wild-type and tub2-polyQ cells expressing GFP-labeled tubulin. Microtubule lengths were measured at 3- to 4-s time intervals. (E) Distributions of aMT lengths in living preanaphase cells. Images were collected at 3- to 4-s intervals for 10 min. Results represent pooled data from at least four separate experiments and at least 40 aMTs sampling n ≥ 3,593 time points for each strain. Red bars indicate median values. **P « 0.001. Significance determined by the Mann–Whitney U test. (F) Distributions of aMT polymerization rates, from cells analyzed in (E). (G) Distributions of aMT depolymerization rates, from cells analyzed in (E). (H) Distributions of aMT catastrophe frequency, from cells analyzed in (E).

Tables

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    Table 1.

    Dynamics of astral microtubules measured in yeast cells during preanaphase.

    Polymerization rate (μm/min)Depolymerization rate (μm/min)Polymerization duration (s)Depolymerization duration (s)Catastrophe frequency (events/min)Rescue frequency (events/min)
    Wild type1.59 (1.40–1.78)2.80 (2.52–3.07)40 (35–45)24 (21–27)1.00 (0.78–1.22)1.67 (1.31–2.02)
    tub2-430∆1.57 (1.34–1.80)2.49 (2.19–2.78)44* (38–50)36* (32–41)0.69* (0.52–0.85)0.79* (0.53–1.06)
    tub2-438∆1.61 (1.24–1.98)2.65 (2.30–3.00)40 (33–47)28 (24–32)0.94 (0.55–1.33)1.08* (0.69–1.48)
    tub2-polyQ1.31*(1.15–1.47)2.21* (2.01–2.41)43 (37–49)35* (31–39)0.82 (0.67–0.97)0.91* (0.58–1.23)

    Source Data for Table 1[LSA-2018-00054_SdataT1.xlsx]

    • Source data are available for this table.

    • Image series were collected at 3- to 4-s intervals for 10 min and microtubule lengths were measured at each time point. Values shown are the medians, with 95% CIs in parentheses, of pooled data from at least four separate experiments.

    • ↵* P < 0.05, compared with wild type, based on the Mann–Whitney U test. Number of aMTs measured for each genotype: wild type, n = 45; tub2-430∆, n = 51; tub2-438∆, n = 34; and tub2-polyQ, n = 65.

Supplementary Materials

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  • Table S1 Peptides identified in proteomic analysis of untreated and subtilisin-digested tubulin.

  • Table S2 Yeast strains used in this study.

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β-tubulin tail regulates dynamic instability
Colby P Fees, Jeffrey K Moore
Life Science Alliance Apr 2018, 1 (2) e201800054; DOI: 10.26508/lsa.201800054

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β-tubulin tail regulates dynamic instability
Colby P Fees, Jeffrey K Moore
Life Science Alliance Apr 2018, 1 (2) e201800054; DOI: 10.26508/lsa.201800054
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