DDA3 controls mitotic progression. (A) HeLa cells were transfected with control (siControl) or DDA3-specific siRNAs (siDDA3-A and -B), synchronized at prometaphase by a thymidine-nocodazole treatment, and analyzed by Western blotting. Hsp70 served as a loading control. (B) HeLa cells were transfected with siRNAs and collected at the indicated times after transfection. Mitotic index was determined by FACS with anti-MPM2 antibody staining. The MPM2 antibody recognizes mitotic phosphoproteins and was used to determine mitotic index. (C) HeLa cells or HeLa cells stably expressing GFP–DDA3 (HeLa/GFP–DDA3) were analyzed by Western blotting to determine the levels of GFP–DDA3 relative to the endogenous DDA3. Mad2 served as a loading control. Arrowheads point to endogenous DDA3. Bands above endogenous DDA3 in the HeLa lane represent cross reacting proteins. (D) HeLa or HeLa/GFP–DDA3 cells were transfected with siControl or siDDA3-A and collected at 56 h after transfection. Mitotic index was determined by FACS analysis. (E–G) HeLa cells were transfected with siRNAs and stained for DNA (E) at 62 h after transfection. Arrows point to normal mitotic cells. Arrowheads point to metaphase cells with unaligned chromosomes. The percentage of metaphase cells over total mitotic cells (F) and the percentage of metaphase cells with unaligned chromosomes over total metaphase cells (G) were quantified and plotted. Bar, 20 μm.

DDA3 is a spindle-associated cell cycle protein. (A–C) Shown are maximum projections from deconvolved z stacks of representative cells stained for DDA3 (A and C, green)/GFP (B, green), β-tubulin (red), and DNA (blue). A, HeLa cells; B, HeLa/GFP–DDA3 cells; C, HeLa cells transfected with siControl or siDDA3-A. Bars, 5 μm. (D) HeLa cells were synchronized by a double-thymidine treatment and released into fresh media. 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at the indicated times. Cells were analyzed by FACS and Western blotting. AS, unsynchronized cells. (E) HeLa cells were transfected with siRNAs, harvested at 62 h as asynchronous cells, and analyzed to identify DDA3-specific bands on Western blot. (F) Lysates of thymidine-nocodazole–treated mitotic HeLa cells were incubated with or without λ-phosphatase (λ-PPase) before immunoblotting to identify phosphorylated DDA3. Arrows point to hyperphosphorylated mitotic DDA3 and arrowheads point to two forms of hypo- or unphosphorylated DDA3.

Depletion of DDA3 prolongs metaphase and activates the spindle checkpoint. (A and B) HeLa/GFP-Histone H2B cells were transfected with siRNAs and imaged for GFP-Histone H2B by time lapse starting from 50 h after transfection. Images were captured every 3 min to monitor mitotic progression. The duration from nuclear envelop breakdown (NEB) to the formation of a bipolar spindle/metaphase plate with some unaligned chromosomes (NEB to unaligned), from the unaligned state to metaphase without unaligned chromosomes (unaligned to metaphase), and from metaphase without unaligned chromosomes to anaphase (metaphase to anaphase) were determined for control and DDA3-depleted cells (n = 30 cells). Still frames from time-lapse movies of representative cells are shown in B. Arrowheads point to unaligned chromosomes. *, P < 0.0013; **, P < 6 × 10⁻⁵; ***, P < 0.0026 (two-tailed t test). (C) HeLa cells were transfected with siRNAs. At 50 h after transfection, cells were incubated at 4°C for 10 min, fixed, and stained for kinetochore MTs. Shown are maximum projections from deconvolved z stacks of representative cells stained for CenpA (green), β-tubulin (red), and DNA (blue). Insets show single focal planes of boxed regions. (D and E) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Mad2 (D, red)/ BubR1 (E, red), and DNA (blue). Insets show single focal planes of boxed regions. Mad2 and BubR1 signals on kinetochores were quantified in five control or DDA3-depleted cells at prometaphase (PM) or metaphase (M; n > 100 kinetochores for each quantification). For unaligned chromosomes (UC) in DDA3-depleted metaphase cells, 40 kinetochores on unaligned chromosomes from 10 metaphase cells were quantified. *, P < 6 × 10⁻³²; **, P < 6 × 10⁻¹⁰ (two-tailed t test relative to siControl metaphase cells). AU, arbitrary units. (F and G) Shown are maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells stained for CREST (green), Hec1 (red), and DNA (blue). Images were collected with a 100× objective lens without binning. Interkinetochore (Inter-KT) distance in prometaphase and metaphase cells was quantified from >100 kinetochore pairs in five cells for each quantification. For unaligned chromosomes in DDA3-depleted metaphase cells, 10 kinetochore pairs were quantified. Boxes 1 and 2 in D–F represent aligned and unaligned chromosomes in DDA3-depleted metaphase cells, respectively. *, P < 5 × 10⁻⁵⁷; **, P < 1.1⁻⁴ (two-tailed t test relative to siControl metaphase cells). (H) HeLa cells stably expressing GFP-CenpA were transfected with siRNAs and imaged for GFP-CenpA by time lapse. Images were acquired every 6 s on a spinning disc confocal microscope using a 63× objective lens with 2 × 2 binning. Interkinetochore distance of individual kinetochore pairs from chromosomes aligned at the metaphase plate was tracked and plotted over 60 s. Error bars show SEM. Bars: (B) 10 μm; (C–F) 5 μm.

DDA3 controls spindle dynamics. (A–C) HeLa cells were transfected with siRNAs and stained for pericentrin (green), β-tubulin (red), and DNA (blue). Maximum projections from deconvolved z stacks of representative control or DDA3-depleted HeLa cells are shown in A. Images were acquired under a constant exposure time for the β-tubulin channel. Pole-to-pole distances (B) were quantified by pericentrin immunofluorescence in control (n = 10) or DDA3-depleted (n = 10) metaphase cells. Total immunofluorescence intensity for β-tubulin on the metaphase spindle (n = 10 cells for each quantification) was quantified and plotted in C. In addition, siRNA-transfected cells were treated with 1 μg/ml nocodazole for 4 min at 37°C or incubated at 4°C for 35 min, and MT fluorescence intensity of metaphase cells (n = 10 cells for each quantification) was quantified and normalized to their respective siControl samples (C). *, P < 3 × 10⁻⁵; **, P < 10⁻³ (B; two-tailed t test). *, P < 3 × 10⁻⁴; **, P < 10⁻⁵; ***, P < 4 × 10⁻⁴ (C; two-tailed t test relative to their respective siControl cells). (D) HeLa and HeLa/GFP–DDA3 cells were not treated, treated with 1 μg/ml nocodazole for 2 min at 37°C, or incubated at 4°C for 20 min. Cells were then stained for GFP, β-tubulin, and DNA, and MT fluorescence intensity of metaphase cells (n = 10 cells for each quantification) was quantified and normalized to their respective HeLa samples. *, P < 5 × 10⁻³; **, P < 4 × 10⁻²; ***, P < 6 × 10⁻³ (two-tailed t test relative to their respective HeLa cells). (E–G) Control or DDA3-depleted HeLa cells were treated with 1 μg/ml nocodazole for 10 min at 37°C to completely depolymerize the mitotic spindle. Nocodazole-treated cells were washed twice with prewarmed PBS, released into fresh media, and fixed at 4 (E–G), 6 (F), and 10 min (F) after release. Shown in E are maximum projections from deconvolved z stacks of representative cells stained for β-tubulin (red) and DNA (blue). Images for β-tubulin were acquired under a constant exposure time. β-tubulin immunofluorescence intensity on metaphase spindles was quantified and normalized to their respective siControl sample at the 4-min time point (n = 10 cells for each quantification in F and G). *, P < 9 × 10⁻⁴; **, P < 5 × 10⁻⁴; ***, P < 3 × 10⁻² (F; two-tailed t test relative to their respective siControl cells at the corresponding time points). *, P < 3 × 10⁻³ (G; two-tailed t test). (H) HeLa/GFP and HeLa/GFP–DDA3 cells were treated with 1 μg/ml nocodazole for 10 min at 37°C, washed with prewarmed PBS, and then released into fresh media for the indicated time. The β-tubulin immunofluorescence intensity on metaphase spindles was measured as described in F. *, P < 0.047; **, P < 0.022 (two-tailed t test relative to their respective GFP cells at the corresponding time points). (I) HeLa cells stably expressing GFP–α-tubulin were transfected with siRNAs. GFP fluorescence intensity was acquired every 0.58 s while a photobleaching laser was focused to a diffraction-limited spot in the cytoplasm away from the spindle. 10 half-spindles from 10 metaphase cells were quantified and fluorescence signals for each half-spindle were normalized to their intensity at 0 s. The 10 half-spindles were then averaged at each time point to generate mean traces. Turnover half-lives for GFP–α-tubulin on the spindle were calculated from the averaged fluorescence signal traces. The standard errors were calculated every fifth time point and are shown in the plots. P-value is from two-tailed t test. (J) Velocity of sister chromatid segregation at anaphase A was measured in control and DDA3 knockdown cells from time-lapse images shown in Fig 3 (A and B; n = 100 anaphase cells for each quantification). *, P < 3 × 10⁻²; **, P < 3 × 10⁻³ (two-tailed t test). AU, arbitrary units. Error bars show SEM. Bars, 5 μm.

DDA3 recruits Kif2a to the mitotic spindle and spindle poles. (A–D) HeLa cells were transfected with siRNAs and stained at 62 h after transfection for various markers of the mitotic spindle and spindle poles. Shown in A and B are maximum projections from deconvolved z stacks of representative cells stained for Kif2a (A, green)/Aurora A (B, green), β-tubulin (red), and DNA (blue). Images were acquired under a constant exposure time for each channel. Immunofluorescence intensities for Kif2a, Aurora A, NuMA, HURP, and Kif2c in metaphase cells were quantified and normalized to their respective siControl samples (n = 10 cells for each quantification in C). The effect of DDA3 knockdown on the levels of the Kif2a protein was analyzed by Western blotting (D). *, P < 10⁻⁵ (two-tailed t test). (E and F) Shown in E are maximum projections from deconvolved z stacks of representative HeLa/GFP or HeLa/GFP–DDA3 stained for Kif2a and DNA. Images were acquired under a constant exposure time in the Kif2a channel and Kif2a immunofluorescence intensity in metaphase cells was quantified and plotted (n = 10 cells in F). *, P < 0.05 (two-tailed t test). AU, arbitrary units. Error bars show SEM. Bars, 5 μm.

Kif2a controls spindle dynamics. (A–C, F, and G) HeLa cells were transfected with a control or a Kif2a-specific siRNA and analyzed at 62 h after transfection by Western blotting (A), FACS, or immunofluorescence staining of β-tubulin and pericentrin. Mitotic index was determined by FACS with anti-MPM2 antibody staining (B), and the percentage of metaphase cells with unaligned chromosomes over total metaphase cells (n = 100 cells; C) was quantified. Pole-to-pole distances (F) and the MT fluorescence intensity (G) in metaphase cells were quantified and plotted (n = 10 cells each). *, P < 4 × 10⁻³ (F). *, P < 0.06 (G; two-tailed t test). (D and E) HeLa/GFP-Histone H2B cells were transfected with siRNAs and imaged for GFP-Histone H2B by time lapse. Quantification of mitotic duration (D) in knockdown cells (n = 30 cells) was determined as described in Fig 3 A, and still frames from time-lapse images of representative cells are shown in E. Arrowheads in E point to unaligned chromosomes. *, P < 0.016; **, P < 0.00012 (two-tailed t test). (H) Control or Kif2a-depleted cells were treated with 1 μg/ml nocodazole for 15 min at 37°C, washed, released into fresh media, and stained for β-tubulin at the indicated time points after release. MT fluorescence intensity in released cells was quantified and normalized to the siControl sample at the 4-min time point (n = 10 cells). *, P < 0.55; **, P < 0.03; ***, P < 0.11 (two-tailed t test relative to their respective siControl cells at the corresponding time points). (I) HeLa cells stably expressing GFP–α-tubulin were transfected with siRNAs, and the half-lives of GFP–α-tubulin on the spindle were measured as described in Fig 4 I. (J) Control or Kif2a-depleted cells were stained for Hec1 and CREST and interkinetochore (Inter-KT) distance in prometaphase and metaphase cells measured from >100 kinetochore pairs in five cells for each quantification. *, P < 9.3 × 10⁻²⁰ (two-tailed t test relative to siControl metaphase cells). (K) Velocity of sister chromatid segregation at anaphase A was measured in control and Kif2a knockdown cells from time-lapse images shown in Fig 6 (D–E; n = 40 anaphase cells for each quantification). *, P < 2.5 × 10⁻¹¹ (two-tailed t test). AU, arbitrary units. Error bars show SEM. Bar, 10 μm.

DDA3 directly interacts with Kif2a. (A) Myc-DDA3 was cotransfected with GFP-Kif2a or GFP into 293T cells. Lysates of transfected 293T cells were immunoprecipitated (IP) with anti-GFP antibodies followed by Western blotting. (B) HeLa/GFP–DDA3 cells were synchronized by a double-thymidine treatment, released, and harvested at 9 h after release (TT9). Alternatively, 1 μM Taxol was added to cells at 8 h after release from the double-thymidine arrest and then harvested at 12 h after release (Taxol sample). Cell lysates were immunoprecipitated with nonspecific IgG (lanes 4, 6, and 8) or with anti-GFP antibodies (lanes 5, 7, and 9) followed by Western blotting. AS, asynchronous cells. (C and D) HeLa/GFP–DDA3 (C, lanes 1–5) or HeLa (C, lane 6) cells were synchronized by a double-thymidine arrest, released, and harvested at 9 h after release. The levels of Kif2a and DDA3 in total cell lysates were determined by Western blotting (D). GFP–DDA3 was immunoprecipitated with anti-GFP antibody beads, incubated with or without the Tev protease, separated into supernatant (sup) and beads, and then assayed by Western blotting (C). (E) HeLa/GFP–DDA3 cells were synchronized by a thymidine arrest and release into fresh media. 1 μM taxol or 150 ng/ml nocodazole was added at 8 h after release and cells were harvested at 12 h after release. GFP–DDA3 was immunoprecipitated with anti-GFP antibodies and associated Kif2a was analyzed by Western blotting. (F and G) Cells stably expressing DDA3-S-GFP were synchronized by a single thymidine arrest and harvested at 8 h after release to enrich G2 and M cells (G2/M). Alternatively, cells were harvested after incubation with 250 ng/ml nocodazole for 18 h (Noc). The DDA3 complex was tandem affinity purified from the DDA3-S-GFP cells (F, lanes 1 and 3) or from the control parental cells (lanes 2 and 4), separated by SDS–PAGE, and visualized by silver staining. Arrowhead points to the precision protease used during purification to cleave its recognition site engineered between S and GFP tags. Peptides of Kif2a identified in the DDA3 complex are listed in G with XCorr and DeltaCN scores indicated. (H) Recombinant GST-DDA3 and GST were incubated with ³⁵S-Kif2a that had been synthesized in reticulocyte lysates in vitro. GST and GST-DDA3 were then purified by glutathione beads and associated Kif2a analyzed by SDS-PAGE. CB, Coomassie blue staining.