1. MLL/WDR5 Complex Regulates Kif2A Localization to Ensure Chromosome Congression and Proper Spindle Assembly during Mitosis 
Aamir Ali, Sailaja Naga Veeranki, Akash Chinchole, Shweta Tyagi 
Developmental Cell 
Volume 41, Issue 6, Pages e7 (June 2017)
DOI: /j.devcel
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2. Developmental Cell 2017 41, 605-622. e7DOI: (10. 1016/j. devcel. 2017
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3. Figure 1
MLL and WDR5 Proteins Localize to the Spindle Apparatus in Mitosis
(A and B) Immunofluorescence staining (IFS) of endogenous MLLC (A) and MLLN (B) in U2OS cells stably expressing H2B-mCherry. The cells were stained with anti-MLLC (green), or anti-MLLN (green), and anti-α-tubulin (amber) antibody. The DNA is shown in red (H2B-mCherry). See also Figure S1.
(C and D) The U2OS cells stably expressing epitope tagged MLLN—MLLN-GFP (C) and FLAG-MLLN (D)—were imaged for GFP fluorescence and anti-FLAG antibody staining, respectively (see Movie S1).
(E) The U2OS cells were transfected with control or MLL siRNA to check for specificity of MLL stain on the spindle microtubules. The DNA was stained with DAPI (red); MLLC (green) and α-tubulin (amber) antibody staining is shown.
(F) U2OS cells were stained with anti-WDR5 (green) and anti-α-tubulin (amber) antibody. DNA is shown in red.
(G) IFS of Control or WDR5 siRNA-treated U2OS cells. Endogenous WDR5 (green), α-tubulin (amber), and DNA (red) are shown.
(H and I) The U2OS cells stably expressing epitope tagged WDR5—WDR5-GFP (H) and FLAG-WDR5 (I)—were imaged for GFP fluorescence and anti-FLAG antibody staining, respectively.
All scale bars, 5 μm. See also Figure S1 and Movie S1.
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4. Figure 2 MLL- or WDR5-Depleted Cells Display Prolonged Mitosis
(A) Phase-contrast time-lapse images of U2OS cells at 40× magnification. Images were captured at 1-min intervals after treatment with control, MLL, or WDR5 siRNA (60 hr after the first transfection). Selected representative images are shown (the entire series is available as Movie S2). Arrows show cells of interest. Time in hh:mm:ss format is shown in lower left corner.
(B) The MLL- or WDR5-depleted cells show delay in mitotic progression. The time spent (in minutes) by control, MLL, or WDR5 siRNA-treated cells to progress from prophase to the start of anaphase during mitosis was quantified and plotted as a box-and-whisker plot. Median: control siRNA = 21; MLL siRNA = 66.32; WDR5 siRNA = (n = 10 movies, m = 4 experiments). ∗∗∗p ≤ (Mann-Whitney two-tailed test).
(C) Control, MLL, or WDR5 RNAi treatment was performed in live U2OS cells stably expressing mCherry-tagged histone 2B (H2B).The MLL- and WDR5-depleted cells, imaged at 100× magnification, exhibited failure of chromosomes to congress at metaphase plate, prolonged pro-metaphase, and metaphase delay. The arrows indicate misaligned or lagging chromosome (or chromosome bridges in telophase). Time in hh:mm:ss format is shown in white inside the panel. The entire series is available as Movie S3. See also Movies S4 and S5; Figure S2A.
(D and E) The MLL- or WDR5-depleted cells spend longer time in pro-metaphase (D) and metaphase (E) stages of mitosis in comparison with the control cells. The time delay is quantified as box-and-whisker plots. (D) Median: control siRNA = 8.5; MLL siRNA = 26.69; WDR5 siRNA = (E) Median: control siRNA = 12; MLL siRNA = 38.33; WDR5 siRNA = (n = 10 movies, m = 3 experiments). ∗∗∗p ≤ , ∗∗p ≤ (Mann-Whitney two-tailed test).
See also Figure S2 and Movies S2, S3, S4, and S5.
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5. Figure 3
MLL- or WDR5-Depleted Cells Show Chromosome Misalignment and Spindle Defects
(A and B) MLL-depleted (A) or WDR5-depleted (B) cells display chromosome misalignment (panels b, c, e and f), abnormal spindle apparatus with either long microtubule rich spindles (panels b and e), or spindles with microtubule of poor intensity (panel c) or multipolar spindles (panel f). The cells were stained with anti-MLLC (A, green), anti-WDR5 (B, green), and anti-α-tubulin (A and B, amber) antibody. The DNA was stained with DAPI (red) (see also Figure S2).
(C) MLL- or WDR5-depleted cells were stained with anti-CENPA (red) and anti-α-tubulin (green) antibodies. The DNA was stained with DAPI (blue). Arrows indicate misaligned chromatid pairs with intact sister kinetochores.
(A–C) Scale bars, 5 μm.
(D) The percentage of the cells with misaligned chromosomes (a), abnormal spindle apparatus (b), and multipolar spindles (c) were quantified in cells treated with control, MLL, or WDR5 siRNA (n = 100 cells, m = 4 experiments). Data are represented as mean ± SD. ∗p ≤ 0.05, ∗∗p ≤ 0.01 (Student’s unpaired t test).
(E) Unusually long spindles with severe chromosome misalignment were observed in MLL siRNA-treated or WDR5 siRNA-treated cells. Cells stained for α-tubulin (red) and DAPI (blue) are shown. The interpolar distances were measured using LSM and ZEN software. White arrow shows the length of the spindle and values are shown for representative images. The average interpolar distance in control cells was 7–8 μm. Proportion of cells displaying value ≥10 μm is shown as percentage on the right. Control cells did not show cells with ≥10 μm interpolar distance. Scale bars, 5 μm.
(F) The pole-to pole distances were quantified and plotted as a box-and-whisker plot. Median: control siRNA = 7.51; MLL siRNA = 9.93; WDR5 siRNA = 9.82 (n = 50 cells, m = 4 experiments). ∗∗∗p ≤ (Mann-Whitney two-tailed test).
See also Figure S2.
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6. Figure 4 Mapping Domain of MLL for Chromosome Misalignment Defect
(A) To calculate the extent of chromosome misalignment, we measured the DNA spread parallel to spindle pole axis using LSM and ZEN software. White arrow shows the extent of spread and values (μm) are shown inset.
(B) Extent of chromosome (Chr.) misalignment was quantified and plotted as a box-and-whisker plot in wild-type U2OS cells, U2OS cells stably expressing MLL full length (MLLFL), MLLN representing the N subunit, and MLLC representing the C subunit, upon control (C) or MLL (T) siRNA treatment. Median: sample 1 = 6.05; sample 2 = 10.59; sample 3 = 6.53; sample 4 = 5.92; sample 5 = 6.55; sample 6 = 10.55; sample 7 = 6.56; sample 8 = 7.43 (n = 100 cells, m = 2 experiments). ∗∗∗p ≤ (Mann-Whitney two-tailed test).
(C) Extent of chromosome (Chr.) misalignment was quantified and plotted as a box-and-whisker plot in U2OS cells stably expressing MLL ΔSET lacking the SET domain, MLLCΔTAD lacking the transcriptional activation domain (TAD), and MLL ΔSETΔWin lacking the SET domain and point mutation in Win motif (R3765A) upon control (C) or MLL (T) siRNA treatment. Median: sample 1 = 6.46; sample 2 = 6.8; sample 3 = 6.22; sample 4 = 7.16; sample 5 = 6.2; sample 6 = (n = 100 cells, m = 2 experiments). ∗∗∗p ≤ (Mann-Whitney two-tailed test).
(D) Extent of chromosome (Chr.) misalignment was quantified upon WDR5 or control siRNA treatment in U2OS cells and plotted as a box-and-whisker plot. Median: control siRNA = 6.67; WDR5 siRNA = (n = 100 cells, m = 2 experiments). ∗∗∗p ≤ (Mann-Whitney two-tailed test).
See also Figures S2 and S3.
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7. Figure 5 MLL Complex Interacts with Kinesin and Dynein Motor Proteins
(A) The HeLa cells expressing transgenes with LAP-tag fusion at C terminus were used. LAP-tagged Kif2A, Kif2B, Kif2C, Kif5A, Kif5B, dynein heavy chain (DYNC1H1), dynein intermediate chain (DYNC1I2), and dynactin subunit (DCTN1) expressing cells were synchronized in mitosis, lysed, and subjected to affinity pull-down using S-protein-agarose beads. The HeLa cells expressing SFB-GFP or Kif11-LAP were used as controls. The recovered bead-bound proteins were analyzed by immunoblot using antibodies indicated on the left. Position of molecular weight markers (in kDa) is shown on the right. IN, input; PD, pull-down. See also Figure S4.
(B) HeLa spinner cells were lysed and subjected to endogenous immunoprecipitation (IP) using antibodies against endogenous WDR5, RbBP5, MLLC, and MLL2. The anti-immunoglobulin G antibody was used as control. The immunoblots (IB) were probed with antibodies indicated on the right. Black line indicates that intervening lanes have been spliced out.
(C) HeLa cells stably expressing SFB-GFP, SFB-WDR5, and SFB-MLLC were subjected to pull-downs using S-protein-agarose beads. The immunoblots were probed with indicated antibodies to detect endogenous Kif2A and exogenous GFP, WDR5 (using anti-FLAG), and MLLC, respectively. G, SFB-GFP; W, SFB-WDR5.
(D) HeLa cells stably expressing Kif11-LAP were synchronized in mitosis, lysed, and subjected to affinity pull-down using GST and GST-WDR5-bound beads. The immunoblots were probed with anti-Kif2A (panel a) and anti-GFP (panel b) antibodies to detect endogenous Kif2A and transgenic Kif11-LAP proteins, respectively. Panel c shows the bead-bound GST or GST-WDR5 proteins stained with Coomassie brilliant blue (CBB).
(E) Asynchronous and mitotically synchronized U2OS cells stably expressing GFP-Kif2A were subjected to affinity pull-down. The immunoblots were probed with anti-Kif2A (panel a) and anti-GAPDH (panel b) antibodies. I, interphase; M, mitosis.
(F) U2OS cells stably expressing GFP-Kif2A were treated with 100 ng/mL nocodazole for 14 hr, harvested as such (+), or released into fresh medium (R) and harvested after 60 min, to obtain cells with depolymerized (+) or intact microtubules (R). GST- and GST-WDR5-bound beads were used for affinity pull-down. The blot was probed with anti-Kif2A. In (E) and (F), bead-bound GST or GST-WDR5 proteins stained with CBB are shown in the bottom panel. G, GFP-Kif2A; E, endogenous Kif2A.
(G) The upper figure shows schematic representation of N-terminal GST fused fragments of MLLC subunit (D1 to D3; indicated in bold lines) used for interaction in the study shown below. Numbers indicate amino acid (aa) residues. TAD, transcriptional activation domain; FYRN and FYRC, “FY-rich” domain N terminus (FYRN) and “FY-rich” domain C terminus (FYRC), respectively; SET, SET domain. Line with star denotes the WDR5-interacting (Win) motif. The lower figure shows mitotically synchronized U2OS cells stably expressing GFP-Kif2A and subjected to affinity pull-down using GST, and deletions of MLLC. Immunoblot was probed using anti-GFP. Proteins stained with CBB are shown at the bottom.
(B–G) Position of molecular weight markers (in kDa) is shown on the left.
(D–G) Arrowheads indicate GST proteins. See also Figure S4.
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8. Figure 6
MLL Complex Regulates Spindle Microtubule Recruitment of Kif2A
(A) U2OS cells were treated with control and Kif2A siRNA followed by IFS with anti-Kif2A (green) and anti-α-tubulin (amber) antibody. See also Figure S4.
(B and C) The cells displaying chromosome misalignment and spindles with elongated phenotype were imaged (B) and quantified by calculating the chromosome (Chr.) misalignment (C a) and pole-to-pole distance (C b) in control and Kif2A-depleted cells, respectively. White arrow shows the extent of DNA spread (DAPI) or pole-to-pole distance (α-tubulin), and values are shown in μm inset % cells showed long MT-rich spindles (n = 100 cells, m = 2 experiments). ∗∗∗p ≤ (Mann-Whitney two-tailed test).
(D) The chromosome (Chr.) misalignment and length of the spindles were quantified by calculating the DNA spread (a) and pole-to-pole distance (b) in partial-knockdown samples as indicated (n = 50 cells, m = 3 experiments). ∗∗∗p ≤ (Mann-Whitney two-tailed test). Contr, control.
(E and F) Control, MLL, or WDR5 RNAi treatment was performed in the U2OS (E) and U2OS stably expressing GFP-Kif2A (F) cells. Following knockdown, the cells were either stained with anti-Kif2A antibody (E) or imaged for GFP fluorescence (F) to check for localization of Kif2A (Figure S4).
(G) Control, MLL, or WDR5 RNAi was performed in HeLa cells stably expressing Kif11-LAP. The cells were stained with anti-GFP antibody (green) to check spindle localization of Kif11 LAP.
(A and E–G) White arrows show the position of spindle poles.
(A, B, and E–G) Tubulin (amber); DNA (blue). Scale bars, 5 μm.
(H) Control, MLL, WDR5, or Kif2A RNAi was performed in U2OS cells. Following fixation the cells were stained with anti-α-Tubulin antibody. The microtubule intensities per unit area in pole proximal region were quantified and plotted (n = 50 cells, m = 3 experiments). Data are represented as mean ± SD. ∗∗∗p ≤ (Mann Whitney two-tailed test). AU, arbitrary units.
See also Figures S4 and S5.
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9. Figure 7 Kif2A Interacts with MLL and WDR5 Proteins
(A) Schematic of Kif2A full-length protein and its functional domains. Evolutionarily conserved Win motif localized in the N terminus of Kif2A is shown in pink. Protein sequence alignment of Win motif (pink, with conserved arginine in blue) from human (NP_ ), mouse (NP_ ), rat (NP_ ), African clawed frog (NP_ ), bovine (NP_ ), chicken (NP_ ), fruit fly (NP_ ), and zebrafish (XP_ ) Kif2A protein is shown. Analogous protein sequence in MLL (NP_ ) and histone 3.1 (NP_ ) is shown at the bottom. Point denotes conserved amino acid. Numbers indicate amino acid position (see also Figure S5).
(B) The HeLa cells nuclear extract was subjected to affinity pull-down using GST, GST-Kif2A (wild-type), and GST-Kif2AΔWin (Win mutant R117A) bound beads. The immunoblot was analyzed with indicated antibodies to detect endogenous WDR5 and the GST-Kif2A proteins.
(C) The HeLa cell lysate was subjected to affinity pull-down using GST, GST-WDR5 (wild-type), and GST-WDR5 F133A bound beads. The immunoblot was probed with anti-Kif2A antibody to detect endogenous Kif2A.
(D) The HeLa cell lysate was used for affinity pull-down using GST, GST-MLLC D3 (wild-type), and GST-MLLC D3ΔWin (R3765A) bound beads. The immunoblot was analyzed by using anti-Kif2A (panel a) and anti-WDR5 (panel b) antibody. In (C) and (D), GST proteins stained with CBB are shown at the bottom, and position of molecular weight markers (in kDa) is shown on the left. (B–D) Arrowheads indicate GST proteins.
(E) Endogenous Kif2A was stained using anti-Kif2A antibody in U2OS cells stably expressing MLLCΔWin and WDR5 F133L following control and MLL, or WDR5 knockdown, respectively.
(F) GFP-Kif2A (Wt) and GFP-Kif2AΔWin were expressed in U2OS cells. The cells were stained with anti-α-tubulin antibody to mark the spindle microtubules. In (E) and (F), the white arrows indicate the position of spindle poles. Scale bars, 5 μm (see also Figures S6 and S7).
(G) The spindle GFP-Kif2A (a) and microtubule (b) fluorescence intensities were quantified in U2OS cells stably expressing GFP-Kif2A or GFP-Kif2AΔWin. Following fixation, the cells were stained with anti-α-tubulin antibody. (a) The spindle-associated GFP fluorescence per unit area was quantified and plotted. (b) The microtubule intensities per unit area in pole proximal region were quantified and plotted (n = 50 cells, m = 3 experiments). Data are represented as mean ± SD. ∗∗∗p ≤ (Mann-Whitney two-tailed test). AU, arbitrary units.
See also Figures S5–S7.
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