Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle

Abstract

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Here, we describe Borealin, a novel member of the complex. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin; and Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex.

Figure 1.

Borealin is a novel chromosomal passenger protein. (A) Localization of Borealin-GFP transiently expressed in HeLa cells at prophase (A), metaphase (A'), and telophase (A”). (B) Aurora B colocalizes with Borealin as detected by affinity-purified anti-Borealin antibody R1647 in HeLa cells. (C) Immunoblot of HeLa cell lysate probed with R1647. (D) Immunoblot showing Borealin levels in S-phase and M-phase cells. (E) R1647 staining confirming that Borealin is a chromosomal passenger protein. Insets in A and E show higher magnification views of Borealin in the inner centromere with ACA staining in red and Borealin in green. Bars, 5 μm.

Figure 2.

Borealin is conserved among vertebrates with distantly related proteins in insects and nematodes. Multiple sequence alignment and secondary structure (A; Ginalski et al., 2003) plus tentative tree (B; Brodskii et al., 1995) for the vertebrate Borealin family. Colors indicate similarity of >60% (yellow) and >80% (green). Secondary structure predicted consistently for Borealin 1 (blue) is indicated (H, helix; E, strand), with plausible core elements underlined based on their length (H >3; E >2) and presence in all sequences. NCBI/SPTrEMBL accession codes: Human, Hs_Bor, cdc-associated protein 8, NP_060571/Q9NVW5 [cDNA, chromosome 1]; Hs_hypBor, likely pseudogene, Hs7_34123 (5467829–5468473) [genomic DNA chromosome 7]. Mouse, Mm_Bor, BAC37258/Q8BHQ5 [cDNA chromosome 4]. Chicken, Gg_Bor1, (BU135762+BU317198) [EST]; Gg_Bor2, (BU315188+BU328206+ BU324297+BU225870) [EST]. X. laevis, Xl_Bor1 (BJ626030+BJ642424) [EST]; Xl_Bor2, CA973941+CA982691 [EST]. Zebrafish, Dr_Bor1, CK683767 [cDNA]; Dr_Bor2, (BM777478+BM080944). Fugu, Fr_Bor, SINFRUP00000052671 corrected in one exon [pred. protein]. Caenorhabditis, Ce_CSC-1, O45952/CAB07696. Mosquito, Ag_Borealin-related1, XP_309425 [pred. protein]; Ag_Borealin-related2, XP_309424 [pred. protein]. Drosophila, Dm_Borealin- related, CG4454-PB [pred. protein].

Figure 3.

Borealin localization is dependent on the correct localization of other chromosomal passengers. (A and B) Depletion of Survivin by RNA interference results in mislocalization of Aurora B and Borealin from centromeres. (C–F) In cells expressing INCENP_1–405, Borealin-GFP and TD-60 are abnormally localized at metaphase and anaphase. Bars, 5 μm.

Figure 4.

Borealin interacts with subunits of the chromosomal passenger complex in vivo and in vitro. (A) Chromosomal passengers coimmunoprecipitate with endogenous Borealin. Mitotic HeLa cell lysate was incubated with affinity-purified R1647 (IP: Borealin) or preimmune serum R1647 (mock IP) cross-linked to protein A beads. Chromosomal passenger proteins in the unbound (U) and bound (B) fractions were visualized by immunoblotting. The unbound fraction after immunoprecipitation of Borealin was further incubated with anti-Aurora B antibody (IP: Aurora B) cross-linked to protein A beads. Protein loading per lane is equivalent to mitotic lysate from 1.5 × 10⁵ cells. The asterisk denotes residual rabbit IgG heavy chain coeluted from the beads due to incomplete cross-linking. (B) Borealin binds to Survivin, INCENP, and itself in vitro. Proteins were translated in the presence of [³⁵S]methionine and incubated with bacterially expressed GST-Borealin or GST alone bound to glutathione sepharose beads. Bound (B) and unbound (U) fractions were separated by SDS-PAGE, and the proteins were visualized by phosphorimaging. Luciferase was included to check for nonspecific binding of GST-Borealin. (B') GST-Borealin and GST used in the binding reactions (input) stained with Coomassie brilliant blue. (C) Quantification of the binding experiments shown in B. The bars represent the percentage of total translated protein bound to GST-Borealin (black) or GST (gray; n ≥ 3). (D) The NH₂- and COOH-terminal halves of Borealin show differential binding properties in vitro. Experiments were performed as described in B, but included GST fusions of Borealin_1–141 and Borealin_142–280 in addition to full-length Borealin_1–280. Equal loading (input) was checked by CBB staining (D').

Figure 5.

Transient expression of Borealin deletion mutants identifies functional domains in vivo. (A–E) Localization of deletion mutants expressed as GFP fusions in interphase (left), prometaphase/metaphase (middle), and anaphase (right). HeLa cells were transfected with GFP constructs containing Borealin amino acids 1–280 (A), 1–141 (B), 142–280 (C), 1–88 (D), and 89–280 (E) and processed for fluorescence microscopy after 36 h. (F) Summary of Borealin deletion and endogenous chromosomal passenger localization in mitosis. Cen, centromere; mdz, anaphase spindle midzone. (G–J) Effect of Borealin deletion expression on the localization of endogenous chromosomal passengers at prometaphase/metaphase. Transfected cells were stained for Survivin (G and I) and TD-60 (H and J) 36 h after transfection. Bars, 5 μm.

Figure 6.

Depletion of Borealin by RNA interference disrupts mitotic progression. (A) Immunoblot showing substantial depletion of Borealin in HeLa cells exposed to Borealin siRNA and effect on the levels of other selected proteins. (B) Distribution of mitotic stages showing an accumulation in prometaphase of cells exposed to Borealin siRNA (n = 3; total number of cells scored: control siRNA, 361; Borealin siRNA, 404). (C) Quantitation of time-lapse experiments examining the effects of Borealin depletion on mitotic progression. Low magnification images of synchronized cells were taken at 1-h intervals 6 h after the release from an S-phase block, and individual cells were categorized according to their fate during the course of the experiment (n = 3; total number of cells scored: control, 158; siRNA, 204). Aurora B (D), Survivin (E), INCENP (F), and TD-60 (G) are mislocalized in Borealin-depleted cells. Bar, 5 μm.

Figure 7.

Borealin depletion promotes kinetochore–spindle misattachments and the formation of ectopic spindle poles. (A–D) Mitotic cells exposed to control siRNA with normal bipolar attachment of kinetochores to the spindle (A), and cells treated with Borealin siRNA (B–D) showing syntelic (B and D) and merotelic (C–C”) attachments. (E) Quantitation of anaphases with kinetochores lagging in the spindle midzone (number of cells scored: control siRNA, 41; Borealin siRNA, 21). (F–H) Aurora A staining of cells exposed to Borealin (G and H) or control (F) siRNAs. Bars: (all except D) 5 μm; (D) 1 μm.

Figure 8.

Live imaging of Borealin-depleted cells reveals severe defects in the partitioning of chromosomes in anaphase. (A–C) β- and γ-tubulin costaining of Borealin-transfected cells (B and C) and a control cell (A). (D) Quantification of multipolar mitotic cells (n = 3; total number of cells scored: control siRNA, 138; Borealin siRNA, 144). (E) Selected frames of a representative live cell imaging experiment performed on mitotic HeLa cells expressing histone H2B-GFP treated with Borealin siRNA. Bar, 5 μm.

Figure 9.

Borealin is a substrate for Aurora B kinase in vitro. (A) Kinase activity toward MBP of GST-Aurora B/GST-INCENP/His-Survivin copurified from insect cells using glutathione sepharose beads compared with GST-Aurora B. Black line indicates that intervening lanes have been spliced out. (B) Kinase activity of GST-Aurora B/GST-INCENP/His-Survivin with or without His-Borealin, which was either copurified with the other subunits (left) or added separately in increasing amounts to GST-Aurora B/GST-INCENP/His-Survivin (right). (C) Phosphorylation of GST-Borealin_1–280 and GST-Borealin_142–280, but not GST-Borealin_1–141, by Aurora B. (C') The input of GST-Aurora B/GST-INCENP/His-Survivin was checked by immunoblotting.