Aurora B colocalizes with MKlp2 at the central spindle. Antibodies to Aurora B, MKlp2, and MKlp1 were preincubated for 1 h in the presence or absence of a 50-fold excess of the appropriate recombinant protein antigen. They were then used to (A) probe Western blots of 20 μg extracts from interphase, I, and nocodazole arrested, M, HeLa cells, or (B) stain HeLa cells. The asterisks indicate cross-reactions of the rabbit anti-MKlp1 antibody that are not competed by the antigen, and the arrowhead marks MKlp1. (C and D) HeLa cells were fixed and stained with antibodies to Aurora B (red), and MKlp1 and MKlp2 (green). The two panels show representative staining in (C) anaphase and (D) telophase cells. DNA was stained with DAPI. Bar, 10 μm.

Aurora B targeting to the central spindle requires MKlp2. HeLa cells were treated with siRNA duplexes for (A and B) lamin A or (C and D) MKlp2 for 30 h and then stained with antibodies to (A and C) Aurora B (red) and tubulin (green) or (B and D) INCENP (red) and MKlp2 (green). The images show representative staining in early and late anaphase, and telophase cells. (E) Western blots of HeLa cells transfected with siRNA duplexes for MKlp1, MKlp2, and lamin A for 30 h. The open arrowhead marks MKlp1 and an asterisk indicates a further protein cross reacting with this antibody. (F) HeLa cells transfected with MKlp2 siRNA duplexes for 30 h were stained with antibodies to MKlp1 (red) and MKlp2 (green). DNA was stained with DAPI. Bar, 10 μm.

Rescue of the MKlp2 siRNA phenotype. HeLa cells treated with siRNA duplexes targeting MKlp2 for 6 h, were then transfected with plasmids encoding full-length MKlp2 (myc-MKlp2f), the MKlp2 motor domain (myc-MKlp2N^t) or the MKlp2 stalk and tail region (myc-MKlp2C^t) for a further 24 h. Cells were stained with antibodies to Aurora B (green) and the myc-epitope to detect the transfected MKlp2 (red). Aurora B and myc-MKlp2 localization, filled and open arrowheads mark, respectively, is shown in (A) anaphase and (B) telophase cells. DNA was stained with DAPI. Bar, 10 μm. (C) Western blots of HeLa cells transfected with siRNA duplexes for lamin A and MKlp2 for 30 h, and MKlp2 siRNA cells rescued with myc-MKlp2f and myc-MKlp2C^t for 24 h.

Targeting of the Aurora B–MKlp2 complex is independent of MKlp1. HeLa cells were treated with siRNA duplexes for (A) lamin A or (B) MKlp1 for 48 h and then stained with antibodies to Aurora B (red), and MKlp1 and MKlp2 (green). The images in the top panels show representative staining for Aurora B and MKlp1 in anaphase cells. The bottom panels show the effects of depleting MKlp1 on MKlp2 localization. DNA was stained with DAPI. Bar, 10 μm. (C) MKlp1 and MKlp2 were immune precipitated from anaphase HeLa cell extracts, and then Western blotted. The asterisk on the rabbit anti-INCENP blot marks a cross-reaction of the donkey anti–-rabbit secondary antibody with the rabbit antibody used to immune precipitate MKlp1. (D) Purified His-tagged MKlp1 and MKlp2, 5 μg, were incubated with GST, GST-tagged Aurora B, or Cdc14A for 1 h at 4°C, and then recovered on glutathione agarose. Recovered complexes were analyzed by Western blotting, half of the input fraction is loaded, and all of the bound fractions. The double asterisk marks a degradation product of MKlp2 lacking the COOH terminus that does not bind to Aurora B or Cdc14A. The single asterisks mark degradation products of MKlp1.

Cdc14A targeting to the central spindle requires MKlp2. (A) Extracts, 5 μg, from HeLa cells left untransfected or transfected with plasmids encoding untagged Cdc14A, myc-Cdc14A, and Cdc14B were Western blotted with affinity purified rabbit anti-Cdc14A antibodies or a mAb to the myc-epitope. (B) U2OS cells were fixed and stained with antibodies to Aurora B (red) and Cdc14A (green). A representative staining in anaphase cells is shown. (C) U2OS cells were treated with siRNA duplexes for lamin A or MKlp2 for 36 h and then costained with antibodies to MKlp2 and Cdc14A. (D) U2OS cells treated with siRNA duplexes targeting MKlp2 for 6 h, were then transfected with myc-MKlp2f for a further 30 h. Cells were stained with antibodies to Cdc14A and the myc-epitope to detect the transfected MKlp2. Arrowheads indicated central spindle staining. DNA was stained with DAPI. Bars: (C and D) 10 μm. (E) Bait constructs comprising the full-length MKlp2, the MKlp2 motor domain, or the COOH-terminal stalk and tail region were tested against full-length human Cdc14A for the ability to grow on selective medium (QDO), compared with nonselective medium (−LW) in the yeast two-hybrid system. (F) Aurora B–INCENP complexes were immune precipitated from mitotic HeLa cell extracts with antibodies to Aurora B. Samples before and after dephosphorylation with buffer, 50 ng human Cdc14A, or phosphatase dead Cdc14A (Cdc14A^PD) were then blotted for INCENP and Cdc14A. Note that dephosphorylated form of INCENP is detected more efficiently than the phosphorylated form with the antibody used for this experiment.