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FIGURE 1:. Aurora kinases phosphorylate Kif2a on T70. (A) Alignment of putative Aurora consensus sites on Kif2a with their homologous MCAK phosphorylation sites. The putative phosphorylated residue is shown in red. (B) Immunoblot of anti-pT70 Kif2a antibody against three Xenopus extracts. CSF, cytostatic factorâarrested egg extract; IE, interphase extract; MOE, mature ooctye extract. Note that that the antibody weakly recognizes a protein at the expected molecular weight of Kif2a at 85 kDa in CSF, but this is much stronger in the MOE, where phosphatases are inhibited by the addition of microcystin. Our interpretation is that only a small portion of Kif2a is phosphorylated in CSF, which lack centromeres. (C) Kinase assay using phosphoantibodies against pT70 shows that Aurora A in this case can phosphorylate Kif2a more efficiently than Aurora B/INCENPin box. This assay also shows that when these two kinases are mixed, the substrate is phosphorylated less than with Aurora A alone. (D) Immunofluorescence in Xenopus S3 cells shows localization of pT70 Kif2a to spindle poles during prometaphase and metaphase and to the spindle midzone during anaphase. Green, pT70; red, Ndc80 (kinetochores). Note that Kif2a localizes to centromeres in these cells (Supplemental Figure 8 in Knowlton et al., 2009), and therefore the absence of staining is not due to absence of Kif2a at these sites. (E) Sperm were added to Xenopus CSFs, and the resulting spindles were spun onto coverslips and stained for pT70. In extracts, pT70 Kif2a is localized to spindle poles and chromatin and enriched at centromeres. pT70 is dependent on Aurora activity, as this staining pattern is lost in the presence of Hesperadin. Red, pT70; blue, DNA; green, microtubules.
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FIGURE 2:. Kif2a is required to complete gastrulation. (A) MO (Kif2a)-injected and MilliQ H2Oâinjected (control) embryos were collected at stages 9, 10, and 10.5, and lysates were examined by immunoblot analysis using Kif2a and tubulin antibodies. Nearly complete depletion of maternal Kif2a occurred by stage 10, and partial depletion was observed at stage 9. (B) Two cell embryos were injected with water (control), Kif2a morpholino (MO), the MO plus RNA encoding human Kif2a (Rescue 2a), or the MO plus RNA encoding human Kif2b (Rescue 2b), and progression of blastopore closure was monitored by time lapse, starting around stage 10 to stage 15. Still frames of the movie are shown at the indicated times postfertilization. (Bâ²) Quantification of completed gastrulation in embryos injected with water (control), Kif2a morpholino (MO), the MO plus RNA encoding human Kif2a (Rescue WT Kif2a), or the MO plus RNA encoding Kif2a phospho-null mutant (T70A) or treated with hydroxyurea and aphidicolin at stage 9 (hydroxyurea). ***p < 0.001 (Bâ²â²) Blastopore diameter was measured at control stage 12 of embryos injected with Kif2a morpholino, morpholino with human Kif2a RNA (Rescue WT Kif2a), or morpholino with human Kif2b RNA (Rescue WT Kifb). ***p < 0.001. (C) Time-lapse movies of epiboly were made from stages 9â14. Representative images; the dot indicates the ending point of the track. (Câ²) Animal cap cells were tracked using the Manual Tracking plug-in of ImageJ (National Institutes of Health, Bethesda, MD). Quantification of the distances spread is shown for control embryos, Kif morphant embryos, and Kif MO rescues (T70A and T70E mutants and human Kif2a and 2b). n = 4.
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FIGURE 3:. Removal of animal cap tissue rescues Kif2a MO gastrulation phenotypes. Embryos were injected with water (Control) or Kif2a morpholino (Kif2aMO) at two-cell stage. At stage 8, animal caps of each treatment were removed microsurgically, and the blastopore closure was monitored through low-light time-lapse microscopy (Control-Capless, Kif2aMO-Capless). One embryo was left intact for staging purposes (Control). Failure of blastopore closure in the morphant phenotypes were rescued by removal of the cap, although closure was slightly delayed compared with the control.
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FIGURE 4:. Kif2a localization and T70 phosphorylation during mitosis. Kif2a MOâtreated caps at stages 10, 10.5, and 11 of development. (A) Confocal micrographs of interphase and mitotic cells in X. laevis animal caps double immunostained for Kif2a (red) and tubulin (green). DNA is stained blue with 4â²,6-diamidino-2-phenylindole. For the phosphorylated form, the T70 protein is immunostained green and α-tubulin localized by a specific (DM1A) antibody red, and the DNA stains blue. During prophase and metaphase, Kif2a is localized to centromeres. During anaphase and telophase, Kif2a became localized to the spindle poles of the cell. The T70 phosphorylated form of Kif2a also localized to the centromeres during prophase and metaphase, and during anaphase, it is localized to the spindle. T-70 was also localized to the poles during metaphase, with slight localization at the poles during anaphase. In addition, pT70 exhibits strong localization to the midbody during telophase. (B) Single-cell embryos were microinjected with MilliQ H2O or the antisense MO for Kif2a (40 nl at 1 mg/ml) and allowed to develop in 0.3à MMR. At stages 10, 10.5, and 11, both groups were dissected, fixed, and stained for Kif2a (red), α-tubulin (green), and DNA (blue). There was an accumulation of multipolar spindles, with development within the Kif2a morphant groups between stages 10 and 11. (C) Quantification of cellular mitotic spindle types (bipolar vs. multipolar) within different, staged animal caps demonstrated an increase or accumulation of multipolar spindles after stage 10 within the MO-injected embryos compared with controls. No multipolar spindles were observed within controls or stage 10 MO-injected groups. (D) The percentage of animal cap cells that are undergoing mitosis and the percentage of mitotic cells within each phase of mitosis are illustrated for each stage and experimental group. ***p < 0.001. Error bars indicate SEM.
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FIGURE 5:. Kif2a morphant cellular phenotypes are rescued by Kif2a but not Kif2a(T70A). (A) Confocal micrographs of control, Kif2a-MO, and WT rescue morphants (Rescue Kif2a WT) stained for α-tubulin (green) and DNA (blue). In the control and MO-rescue animal caps, spindles were bipolar, whereas within the Kif2a-MO group, multipolar spindles were abundant. (B) Confocal images of control and Kif2a morphants rescued with a phospho-null Kif RNA (Kif2a T70A) show that Kif2a is mislocalized in the absence of T70 phosphorylation. Animal caps were stained for α-tubulin (green), Kif2a (red), and DNA (blue). (C) Confocal measurements of spindle lengths were calibrated as a pole-to-pole length (note spindle lines). The stage 10.5 Kif2a morphant cells had significantly greater metaphase and anaphase spindle lengths compared with the control and rescue animal cap cells.
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FIGURE 6:. Kif2a morphant phenotypes caused by lagging chromosomes and failure of cytokinesis. (A) Embryos were injected at two-cell stage with GAP43 RFP:RNA and H2B:GFP RNA (Control) and coinjected with Kif2a morpholino (Kif2aMO), MO plus human 2a RNA (Rescue 2a), or MO plus human 2b RNA (Rescue 2b). The animal caps were microdissected at stage 9 and placed in an imaging chamber for confocal microscopy. A time-lapse movie was made on a Zeiss 780 Confocal Microscope with the 25à objective and a framing rate of 30 s. Still frames of these movies are shown from the indicated time postfertilization. The morphant embryos display lagging chromosomes and failure of cytokinesis (indicated by white or yellow arrows). Scale bar, 20 μm. (B) From time-lapse movies taken from stages 9â12 (similar to those described earlier), quantification of divisions with lagging chromosomes was made for control embryos, Kif2a morphants at stage 9.5, Kif2a morphants at stage 10.5, and morphants rescued with either human Kif2a or Kif2b. Bars represent the percentage of divisions imaged with lagging chromosomes. n = 20 for each category of embryo. Control embryos displayed no divisions with lagging chromosomes. Error bars, SEM. (C) Percentage of divisions imaged with failed cytokinesis. n = 20 for the categories of embryos in B. Error bars, SEM.
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FIGURE 7:. Multipolar spindles have multiple centrosomes. Multipolarity within Kif 2a morphants results in an abnormal distribution of chromosomes to daughter cells. (A) Confocal images of animal caps stained with Cep57 (red), α-tubulin (green), and DNA (blue). Cep 57 is localized to the centrosome area within bipolar (control) and multipolar (MO) spindles. Note that the multipolar spindles generated by Kif2a depletion have additional centrosomes in each pole, and occasionally a pole has two centrosomes (white arrow tip). (B) The percentages of cells with two or fewer, three or four, or more than four centrosomes were quantified for both control and Kif2a morphants. A minimum number of 100 mitotic figures were counted from >10 different caps within each (control and MO-injected) group. **p < 0.01. (C) Confocal images of control, stage 10.5, and stage 11 morphant animal cap cells. Ndc80 immunostaining is red, α-tubulin staining is green, and DNA staining is blue. (D) Scatter plot of Ndc80 paired foci. Ndc80 paired foci increase in number in Kif2a multipolar morphants, indicating an increase in cellular chromosomes. Fifty percent of the Kif 2a morphant cells (n = 50) had Ndc80 foci number in excess of the average number of control cells (36 pairs) and exhibited a diploid (2N) increase in number.
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FIGURE 8:. Kif 2a-depleted cells poorly coalesce centrosomes. (A) Embryos were treated with 80 μM cytochalasin B at stages 8/9 and washed out of the drug after 1 h. At stage 10 embryos were fixed and imaged. Caps were immunostained for α-tubulin (green) and Cep57 (red) and stained for DNA (blue). Arrows (cytochalasin group) indicate centrosomes and multicentrosomes within cells. (B) Inhibition of cytokinesis generates multipolar spindles within stage 10 embryonic caps at a stage before their normal appearance in untreated MO-injected caps. Both cytochalasin-treated groups had multipolar spindles, but they were more abundant within the MO-injected group treated with DMSO instead of cytochalasin (control), depleted of Kif2a and DMSO (MO), control MO treated with cytochalasin B (CytoC control), or Kif2a-depleted embyros treated with cytochalasin (CytoC-MO). (C) Measuring pole coalescence after cytochalasin treatment by quantifying centrosomes at poles. Top, whole-cell micrographs; bottom, confocal micrographs, enlarged half-spindles of the same micrographs. Arrows indicate centrosomes within the spindles. (D) Quantification of C. For each group, 25 spindles from 10 caps were assessed.
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