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. 2015 May 15;348(6236):799-803.
doi: 10.1126/science.aaa5175. Epub 2015 Apr 23.

Mitosis. Microtubule detyrosination guides chromosomes during mitosis

Marin Barisic  1 , Ricardo Silva e Sousa  2 , Suvranta K Tripathy  2 , Maria M Magiera  3 , Anatoly V Zaytsev  4 , Ana L Pereira  1 , Carsten Janke  3 , Ekaterina L Grishchuk  5 , Helder Maiato  6
Affiliations

Mitosis. Microtubule detyrosination guides chromosomes during mitosis

Marin Barisic et al. Science. .

Abstract

Before chromosomes segregate into daughter cells, they align at the mitotic spindle equator, a process known as chromosome congression. Centromere-associated protein E (CENP-E)/Kinesin-7 is a microtubule plus-end-directed kinetochore motor required for congression of pole-proximal chromosomes. Because the plus-ends of many astral microtubules in the spindle point to the cell cortex, it remains unknown how CENP-E guides pole-proximal chromosomes specifically toward the equator. We found that congression of pole-proximal chromosomes depended on specific posttranslational detyrosination of spindle microtubules that point to the equator. In vitro reconstitution experiments demonstrated that CENP-E-dependent transport was strongly enhanced on detyrosinated microtubules. Blocking tubulin tyrosination in cells caused ubiquitous detyrosination of spindle microtubules, and CENP-E transported chromosomes away from spindle poles in random directions. Thus, CENP-E-driven chromosome congression is guided by microtubule detyrosination.

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Conflict of interest statement

The authors declare no conflict of interests.

Figures

Fig. 1
Fig. 1. Chromosome congression requires spatially regulated detyrosination of spindle microtubules
(A) Microtubule detyrosination was examined by immunoblotting with detyrosinated tubulin antibodies. Protein lysates of U2OS cells were obtained 24h after TTL-YFP transfection and 4h after adding parthenolide (20 µM). GAPDH was used as loading control. (D) Deconvolved immunofluorescence images of U2OS cells stained for DNA (DAPI=magenta), α-tubulin and detyrosinated tubulin (green). TTL-YFP signal was detected by direct fluorescence. Arrowheads highlight detyrosinated spindle microtubules in control cells. Detyrosination of spindle microtubules is undetectable after TTL-YFP over-expression or treatment with parthenolide. Scale bar = 10 µm.
Fig. 2
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 post-translational modifications. Anti-α-tubulin antibody demonstrates similar amounts of tubulin in different lanes. (B) Mean velocity and characteristic run length of CENP-E for D-MTs (N=4 independent experiments, n=401) and T-MTs (N=3, n=257 molecules). Error bars are SEMs; **p≤0.01 based on unpaired t-test with 95% confidence. (C) Typical traces of bead motions on D- and T-MTs, plotted as force vs. 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 centre. Number of forward steps was 3427 and 5146; backward steps 1114 and 1731 for D- and T-MTs, respectively. (F) Ratio of forward to backward steps for CENP-E in comparison to 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 4541 and 6877 for D- and T-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=269 for D-MTs, and 491 for T-MTs. Vertical error bars are square roots of the number of detachments normalized by the total number of detachments.
Fig. 3
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, 3 independent experiments) or parthenolide (9 cells, 2 independent experiments). Chromosome congression was impaired in 65% of cells depleted of TTL (23 cells, 3 independent experiments). Arrowheads highlight kinetochores transported away from the spindle pole towards the cell cortex. Enlarged insets highlight polar regions. Scale bar = 5 µm. Time = h: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, 3 independent experiments; N(parthenolide)=388 kinetochores from 7 cells, 2 independent experiments; N(TTL RNAi)=311 kinetochores from 18 cells, 3 independent experiments. N(TTL RNAi+CENP-E inh)=720 kinetochores from 17 cells, 3 independent experiments. Asterisks indicate z test significance values;***p<0.001.
Fig. 4
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 (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, 3 experiments; n(CENP-E inh)=3877 kinetochores from 34 cells, 2 experiments; n(Parthenolide)=3611 kinetochores from 30 cells, 2 experiments; n(TTL RNAi)=8239 kinetochores from 61 cells, 4 experiments; n(TTL RNAi + CENP-E inh)=5458 kinetochores from 45 cells, 2 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.
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