Research ArticleMitosis

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

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Science Signaling  13 Aug 2019:
Vol. 12, Issue 594, eaaw2939
DOI: 10.1126/scisignal.aaw2939
  • Fig. 1 EML4 exhibits reduced affinity for microtubules in mitosis.

    (A) Schematic representation of full-length (FL) human EML4 indicating the trimerization (TD; pink), basic region (brown), and TAPE (blue) domains. Epitopes recognized by the two commercial EML4 antibodies (red) map to residues 150 to 200 (N-terminal antibody) and 951 to 981 (C-terminal antibody). (B) Lysates prepared from U2OS cells that were transfected with either control siRNA (siMock) or one of two EML4-targeted siRNAs for 72 hours were analyzed by Western blot with the antibodies indicated. Blots are representative of three independent experiments. (C and D) Confocal microscopy of cells transfected as described in (B) and fluorescently stained for N-terminal (C) or C-terminal (D) EML4 (green) and α-tubulin (red). Scale bars, 5 μm. Images are representative of three independent experiments. (E and F) Confocal microscopy of U2OS cells captured in interphase or metaphase and fluorescently stained for N-terminal (E) or C-terminal (F) EML4 (green) and α-tubulin (red). “Zoom” panels are magnified views of the merged imaging. Scale bars, 5 μm. Images are representative of three independent experiments. (G and H) Colocalization, assessed as the mean Pearson’s correlation coefficient (R; y axis) between EML4 and microtubules for cells shown in (E) and (F). Data are means ± SD from five lines per cell in 10 cells per each of three independent experiments. I, interphase; M, metaphase. (I and J) Confocal microscopy of U2OS cells captured in interphase and metaphase that were transfected with YFP-EML4 24 hours and then stained for GFP (green), α-tubulin (red), and Hoechst 33258 (for DNA; blue). Scale bars, 5 μm. Colocalization between the GFP and microtubules for cells shown in (I) was calculated as in (G) and (H). Images are representative of and data are means ± SD from 30 cells pooled from three independent experiments. **P < 0.01 and ****P < 0.0001 by a one-tailed unpaired Student’s t test.

  • Fig. 2 The EML4-NTD binds microtubules and is phosphorylated in mitosis.

    (A) Western blotting for EML4, cyclin B1, phospho-histone H3 (pHH3), and α-tubulin in HeLa and U2OS cells that were either untreated (I) or treated with nocodazole (500 ng/ml) for 16 hours to arrest them in mitosis (M) before being lysed. Blots are representative of three independent experiments. (B) Confocal microscopy of U2OS cells transfected with YFP-tagged FL or isolated peptide (NTD, TAPE) EML4 constructs and then fixed and stained for GFP (green), α-tubulin (red), and Hoeschst 33258 (for DNA; blue). Scale bars, 5 μm. Images are representative of three independent experiments. (C) Colocalization, assessed as the mean Pearson’s correlation coefficient (R; y axis), between the EML4 construct and microtubules (GFP and α-tubulin signals) in cells represented in (B). Data are means ± SD from five lines per cell in 10 cells per each of three independent experiments. (D) Western blotting as described in (A) in U2OS cells transfected with YFP or YFP-EML4-NTD for 24 hours. Blots are representative of three independent experiments. (E) Western blotting for GFP in U2OS cells transfected with YFP-EML4-NTD for 24 hours from which lysates were treated with the indicated units of λ-PPase for 30 min. Blots are representative of three independent experiments. ****P < 0.0001 by a one-tailed unpaired Student’s t test.

  • Fig. 3 NEK6 and NEK7 regulate association of EML4 with microtubules.

    (A) Schematic representation of FL EML4 with TD and TAPE domains for reference, indicating the phosphorylation sites detected by mass spectrometry after incubation in vitro with NEK6 (blue) or NEK7 (green). Sites phosphorylated by both kinases are indicated in red. (B) Sequence alignment from species across the five vertebrate classes of the EML4-NTD region spanning the three identified phosphorylation sites (in red). (C) Western blotting in U2OS cells transfected with wild-type (WT) or mutant YFP-EML4-NTD for 24 hours and then treated with nocodazole for 16 hours to arrest cells in mitosis. Blots are representative of three independent experiments. (D and E) Confocal microscopy (D) and analysis of Pearson’s correlation coefficient for colocalization (E) of U2OS cells transfected with wild-type or double-mutant YFP-tagged FL EML4 for 24 hours and then stained for GFP (green), α-tubulin (red), and Hoechst 33258 (for DNA; blue). Scale bar, 5 μm. Images are representative of three independent experiments. Data are means ± SD from five lines per cell in 10 cells per each of three independent experiments. (F) Western blotting for the indicated proteins in U2OS cells transfected with YFP-EML4-NTD and either control siRNA (siMock) or NEK6-, NEK7- or NEK9-targeted siRNA for 48 hours and then treated with nocodazole (500 ng/ml) for 16 hours to arrest cells in mitosis. Blots are representative of three independent experiments. (G and H) Confocal microscopy (G) and analysis of Pearson’s correlation coefficient for colocalization (H) of U2OS cells transfected as in (F) for 72 hours and then stained with EML4 (green), α-tubulin (red), and Hoechst 33258 (for DNA; blue). Scale bar, 5 μm. Images are representative of and data are means ± SD from five lines per cell in 10 cells per each of three independent experiments. ***P < 0.001 and ****P < 0.0001 by a one-tailed unpaired Student’s t test.

  • Fig. 4 Subtilisin treatment of microtubules leads to loss of association of EML4 and EML1 in vitro.

    (A) Microtubules (MTs) were either untreated or incubated with subtilisin before repurification to remove the enzyme. These were then incubated with purified EML4-NTD before sedimentation to generate a supernatant (S) and pellet (P) fraction. Samples were then analyzed by SDS-PAGE and Coomassie Blue stain. A representative example of three independent experiments is shown. (B and C) The relative fraction of the EML4-NTD protein present in the pellet fraction represented in (A) is shown for two different concentrations of the EML4-NTD protein. Data are means ± SD from three independent experiments. *P < 0.05; n.s., nonsignificant; by a one-tailed unpaired Student’s t test. (D) Representative TIRF image of fluorescently labeled microtubules (left) treated with (magenta) or without (green) subtilisin before incubation with YFP-EML1-NTD (residues 1 to 174) (right). Images are representative of three independent experiments. (E) Box plots reveal YFP intensity (a.u., arbitrary units) associated with X-rhodamine (561) and Hilyte640-labeled microtubules that were either untreated or treated with subtilisin. Boxes represent quartiles; whiskers show 10/90% data ± SD (n > 200 microtubules, pooled from two independent experiments).

  • Fig. 5 Superresolution and cryo–electron microscopy reveal EML4-NTD binding microtubules through a flexible interaction with α- and β-tubulin C-terminal tails.

    (A and B) U2OS cells were stained for EML4 (C-terminal antibody; magenta) and α-tubulin (green) and imaged by SIM. Boxed region in (A) is magnified in (B), as an example of clusters of EML4 foci that colocalize with microtubules. Scale bar, 5 μm. Images are representative of three independent experiments. (C) Section through a cryo-tomogram of an EML4-decorated microtubule. The microtubule lumen is indicated in light blue false color. Red arrows indicate clear microtubule-bound densities, with sizes consistent with EML4-NTD trimers. Red arrowhead notes where, in some regions, a periodicity of 4 nm for extra densities was observed, consistent with binding to both α- and β-tubulin. Scale bar, 30 nm. (D) C1 single-particle cryo–electron microscopy reconstruction of EML4-NTD–decorated 13 protofilament microtubules (resolution, 4.4 Å), low-pass–filtered to 20 Å. Density within 9 Å of the fitted α- and β-tubulin atomic models is indicated with light and dark gray, respectively, and defined, connected density > 9 Å away is indicated with red. (E) Symmetrized reconstruction (resolution, 3.6 Å), showing two tubulin dimers within a single protofilament. Local resolution–filtered α- and β-tubulin density is shown as transparent light and dark gray density, respectively. The fitted H12 atomic model is shown as ribbons, with the flexible C-terminal tails indicated by dashed lines. The reconstruction low-pass–filtered to 20 Å is also shown as mesh, with extra densities associated with α- and β-tubulin colored in red and orange, respectively.

  • Fig. 6 Displacement of EML4 from microtubules reduces their stability in mitosis.

    (A) U2OS cells were transfected with either control siRNA (siMock) or one of two siRNAs targeting EML4 (siEML4.1 or siEML4.2) for 72 hours and then either left untreated or treated with 75 nM nocodazole for 2 hours. Cells were fixed and stained with α-tubulin antibodies. Scale bars, 5 μm. Images are representative of three independent experiments. DMSO, dimethyl sulfoxide. (B) Microtubule sedimentation assay performed with lysates prepared from U2OS cells that were transfected as in (A) and Western blotting the pellet (P) and supernatant (S) fractions for α-tubulin. (C) Quantification of the % tubulin in the pellet fraction represented in (B). Data are means ± SD from three independent experiments. (D to F) Western blotting and analysis for acetylated (Ac.), detyrosinated (Detyro.), and total tubulin in lysates from U2OS cells transfected as in (A). Blots are representative of three independent experiments. Data are means ± SD from three independent experiments. (G) U2OS cells were transfected with YFP-EML4-WT or YFP-EML4-S144/146A and incubated with SiR-Tubulin to visualize microtubules, the intensity of which was measured every 60 s after addition of nocodazole (200 μg/ml). Stills from representative movies are shown at 0, 7, and 14 min. Scale bar, 5 μm. (H) The relative SiR-Tubulin intensity of cells described in (G) is shown. Data are means ± SD from three independent experiments with a minimum of 10 positions per condition. ***P < 0.001 and ****P < 0.0001 (C, E, and F) and the P value in (H) assessed by a one-tailed unpaired Student’s t test.

  • Fig. 7 A phospho-null EML4 mutant interferes with chromosome congression and activates the SAC.

    (A) HeLa:EGFP-Lamin A/mCherry-H2B cells were either untransfected (left column) or transfected with YFP-EML4 wild-type protein (WT; middle column) or YFP-EML4-S144/146A (right column) for 24 hours before time-lapse confocal imaging. Stills from representative movies are shown with time (in minutes) indicated from mitotic entry. Scale bar, 7.5 μm. Images are representative of three independent experiments. (B and C) Quantification of cells described in (A) indicating time from nuclear envelope breakdown (NEBD) to last chromosome congressed (B) and time from last chromosome congressed to anaphase onset (C). Data are means of cumulative frequencies ± SD; n = 20. (D) U2OS cells were either untransfected or transfected with YFP-EML4-FL, EML4-WT, or EML4-S144/146A for 24 hours before being treated with RO-3306 for 16 hours, followed by 4 hours with MG132. Cells were then fixed and stained for GFP (green), α-tubulin (red), and Hoechst 33258 (DNA; blue). Scale bar, 5 μm. Images are representative of three independent experiments. (E) Quantification of the percentage of cells described and represented in (D) with normal or uncongressed chromosomes in mitosis. Data are means ± SD from at least 30 cells pooled from three independent experiments. (F) U2OS cells were either untransfected or transfected with EML4-S144/146A for 24 hours before being fixed and stained for GFP (green), BUBR1 (red), and Hoechst 33258 (DNA; blue). Scale bar, 5 μm. Images are representative of three independent experiments. (G) The relative intensity of BUBR1 was quantified and plotted relative to that in control samples. Data are means ± SD from 30 cells pooled from three independent experiments. ****P < 0.0001. (H) Histogram shows the mitotic index upon MPS1 inhibition (MPS1i) in U2OS cells transfected with either EML4-WT or EML4-S144/146A constructs. Data are means ± SD from three independent experiments. *P < 0.05 and **P < 0.01 by a one-tailed unpaired Student’s t test.

  • Fig. 8 Phospho-dependent regulation of EML4 microtubule binding affinity.

    (A) Schematic model illustrating how the NEK9-NEK6-NEK7 kinases regulate affinity of EML4 for microtubules at the G2-M transition. Phosphorylation of two sites within the EML4-NTD as a result of activation of these kinases reduces electrostatic interaction of this basic region of EML4 for the acidic microtubule surface. (B) Cartoon showing electrostatic interaction of the basic NTD of the trimeric EML4 protein with the acidic tubulin C-terminal tails (green).

  • Table 1 Cryo-EM data collection, refinement, and validation statistics.

    Data collection and processing information for the EML4-NTD–bound 13-protofilament HeLa cell tubulin microtubule dataset and refinement statistics for the HeLa cell tubulin dimer model asymmetric unit built into the symmetrized electron density map.

    (EMD-0331, PDB ID: 6I2I)
    Data collection and processing
    Pixel size (Å)1.37
    Symmetry imposed*Pseudo-helical
    Number of micrographs1967
    Initial particle images (no.)44,946
    Final particle images (no.)19,542
    Symmetrized map resolution (Å)3.58
    FSC thresholdIndependent half-map FSC 0.143
    Map resolution range (Å)3.5–4.3
    Refinement
    Initial models used6DPU, 5SYF
    Refinement resolution (Å)3.7
    FSCaverage0.84
    Map local sharpening B-factor (Å2)−90
    Model composition
    Nonhydrogen atoms41,058
    Protein residues5256
    Ligands18
    rms deviations§
    Bond lengths (Å)0.01
    Bond angles (°)1.20
    Validation
    MolProbity score1.91
    Clashscore8.24
    Poor rotamers (%)0.5
    Ramachandran plot
    Favored (%)92.73
    Allowed (%)7.27
    Outliers (%)0

    *Microtubules exhibit pseudo-helical symmetry due to a symmetry break at the seam. Thirteen-fold pseudo-helical symmetry was applied to the 13 protofilament microtubule reconstructions.

    †The resolution value at the gold-standard Fourier shell correlation (FSC) 0.143 criterion between independently refined half-maps is shown for the central 15% of the reconstruction along the helical axis (isolated with a soft mask).

    ‡Average FSC between the model and the symmetrized electron density map of the asymmetric unit (calculated around model atoms only).

    §Root mean square (rms) deviations of bond lengths or angles in the model.

    ‖As defined by the MolProbity validation server (59).

    Supplementary Materials

    • stke.sciencemag.org/cgi/content/full/12/594/eaaw2939/DC1

      Fig. S1. Mass spectrometry profiles of EML4 phosphorylation sites.

      Fig. S2. Activated NEK6, NEK7, and NEK9 reduce the association of EML4 with interphase microtubules.

      Fig. S3. Cryo–electron microscopy diagnostics and resolution estimation.

      Fig. S4. EML4 depletion leads to reduced microtubule acetylation.

    • This PDF file includes:

      • Fig. S1. Mass spectrometry profiles of EML4 phosphorylation sites.
      • Fig. S2. Activated NEK6, NEK7, and NEK9 reduce the association of EML4 with interphase microtubules.
      • Fig. S3. Cryo–electron microscopy diagnostics and resolution estimation.
      • Fig. S4. EML4 depletion leads to reduced microtubule acetylation.

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