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Microtubule detyrosination guides chromosomes during mitosis

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Science  15 May 2015:
Vol. 348, Issue 6236, pp. 799-803
DOI: 10.1126/science.aaa5175
  • Fig. 1 Chromosome congression requires spatially regulated detyrosination of spindle microtubules.

    (A) Microtubule detyrosination was examined by means of immunoblotting with detyrosinated tubulin antibodies. Protein lysates of U2OS cells were obtained 24 hours after TTL–yellow fluorescent protein (YFP) transfection and 4 hours after adding parthenolide (20 μM). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Deconvolved immunofluorescence images of U2OS cells stained for DNA [4′,6-diamidino-2-phenylindole (DAPI), magenta], α-tubulin, and detyrosinated tubulin (green). TTL-YFP signal was detected by means of direct fluorescence. Arrowheads highlight detyrosinated spindle microtubules in control cells. Detyrosination of spindle microtubules is undetectable after TTL-YFP overexpression or treatment with parthenolide. Scale bar, 10 μm.

  • Fig. 2 CENP-E motility is enhanced on detyrosinated microtubules in vitro.

    (A) Immunoblot of purified tubulin from porcine brain or HeLa cells before (T) and after (D) detyrosination with carboxypeptidase A. Antibodies against tyrosinated, detyrosinated, Δ2, glutamylated (GT335), and acetylated tubulin were used to assess posttranslational modifications. Antibody to α-tubulin demonstrates similar amounts of tubulin in different lanes. (B) Mean velocity and characteristic run length of CENP-E for T-MTs (N = 3 independent experiments, n = 257 molecules) and D-MTs (N = 4 independent experiments, n = 401). Error bars are SEM; **P ≤ 0.01 based on unpaired t test with 95% confidence. (C) Typical traces of bead motions on T- and D-MTs, plotted as force versus time. (D) Example trace of CENP-E bead in a laser trap with step-size fit. (E) Step-size histograms were fit by the sum of two Gaussians (lines). Positive steps were away from the trap’s center. Number of forward steps was 5146 and 3427, and number of backward steps was 1731 and 1114, for T- and D-MTs, respectively. (F) Ratio of forward to backward steps for CENP-E in comparison with Kinesin-1 [data from (25, 26)] with theoretical fittings for ratio >1. (G) Characteristic dwell times as a function of force within 2-pN bins. Total number of dwells for all forces was 6877 and 4541 for T- and D-MTs, respectively. (H) CENP-E detachment rate under load. Error bars for detachment rate were calculated by dividing the square root of the number of detachments by the total time within each bin. Error bars for force are SEM for measurements in each bin. (I) Histograms of detachment force with Gaussian fits. Numbers are the mean ± SEM, n = 491 detachments for T-MTs, and 269 for D-MTs. Vertical error bars are square roots of the number of detachments normalized by the total number of detachments.

  • Fig. 3 Chromosomes cannot complete congression in TTL-depleted cells and are randomly transported away from the spindle pole by CENP-E.

    (A) Spinning-disk confocal imaging of live U2OS cells stably expressing CENP-A–GFP and mCherry-tubulin. Chromosome congression was impaired in 100% of cells treated either with CENP-E inhibitor (13 cells, three independent experiments) or parthenolide (nine cells, two independent experiments). Chromosome congression was impaired in 65% of cells depleted of TTL (23 cells, three independent experiments). Arrowheads highlight kinetochores transported away from the spindle pole toward the cell cortex. Enlarged insets highlight polar regions. Scale bar, 5 μm. Time is hours:min. (B) Quantification of the percentage of pole-proximal chromosomes that were transported away from spindle poles in the different conditions. N (CENP-E inh.) = 311 kinetochores from 10 cells, three independent experiments; N (parthenolide) = 388 kinetochores from 7 cells, two independent experiments; N (TTL RNAi) = 311 kinetochores from 18 cells, three independent experiments. N (TTL RNAi+CENP-E inh) = 720 kinetochores from 17 cells, three independent experiments. Asterisks indicate z test significance values; ***P < 0.001.

  • Fig. 4 CENP-E–dependent transport of pole-proximal chromosomes along ubiquitously detyrosinated spindle microtubules after TTL RNAi.

    (A) U2OS cells treated with the kinesin-5 inhibitor S-Trytil-l-Cysteine (STLC) and immunostained for DNA (DAPI, blue), kinetochores [anti-centromere antibodies (ACA), green] and centrioles (centrin = red). Maximum-intensity projection images of representative examples for each condition are shown. Numbers indicate mean kinetochore-to-pole distances ± SD of the pooled data from at least two independent experiments per condition. Scale bar, 5 μm. (B) Quantification of kinetochore (KT) to pole distances in STLC-treated cells under different conditions; n (control) = 7185 kinetochores from 56 cells, three experiments; n (CENP-E inh) = 3877 kinetochores from 34 cells, two experiments; n (Parthenolide) = 3611 kinetochores from 30 cells, two experiments; n (TTL RNAi) = 8239 kinetochores from 61 cells, four experiments; n (TTL RNAi + CENP-E inh) = 5458 kinetochores from 45 cells, two experiments. Asterisks indicate Mann-Whitney U test significance values; ***P < 0.001. (C) Deconvolved immunofluorescence images of STLC-treated U2OS cells stained for α-tubulin, detyrosinated tubulin, and ACA. Detyrosination of spindle microtubules was highly increased after TTL RNAi. Scale bar, 5 μm.

Supplementary Materials

  • Microtubule detyrosination guides chromosomes during mitosis

    Marin Barisic, Ricardo Silva e Sousa, Suvranta K. Tripathy, Maria M. Magiera, Anatoly V. Zaytsev, Ana L. Pereira, Carsten Janke, Ekaterina L. Grishchuk, Helder Maiato

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S12
    • Table S1
    • Full Reference List

    Images, Video, and Other Other Media

    Movie S1
    Congression of pole-proximal chromosomes is impaired after CENP-E inhibition, TTL overexpression or TCP inhibition. Spinning-disk confocal time-lapse imaging of U2OS cells stably expressing H2B-GFP and mCherry-α-tubulin under the indicated experimental conditions. Cells were recorded every 2 min. Time=h:min.
    Movie S2
    Inhibition of tubulin carboxipeptidase with parthenolide dissociates CENP-E from microtubules in G2 cells. Spinning-disk confocal time-lapse imaging of HeLa cells stably expressing CENP-E-GFP and transfected with mCherry-α-tubulin under the indicated experimental conditions. Cells were recorded every 2 min. Time=h:min.
    Movie S3
    CENP-E motility on tyrosinated and detyrosinated microtubules in vitro. Single molecules of CENP-E-GFP (green) walk on taxol-stabilized microtubules that were polymerized from tyrosinated ("Tyr MT", red) or detyrosinated ("Det MT", purple) human tubulin. Video is played at 30 fps, which is 3 times faster than recorded.
    Movie S4
    Force generation by CENP-E motor. First image is an overlay of the DIC image of 0.54 *μm bead (grayscale), microtubule (red) and CENP-E-GFP (green); "+" symbol indicates microtubule polarity. All subsequent frames show motions of the bead (DIC) in a stationary laser trap. Images were acquired continuously every 10 ms, but the video shows only every 5th frame. Video is played at 30 fps, so the motions appear 1.4 times slower than recorded.
    Movie S5
    Chromosomes cannot complete congression in TTL depleted cells and are randomly transported away from the spindle poles by CENP-E. Spinning-disk confocal time-lapse imaging of U2OS cells stably expressing GFP-CENP-A and mCherry-α-tubulin under the indicated experimental conditions. Cells were recorded every 2 min. Time=h:min.

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