ARTICLE Received 2 Dec 2014 | Accepted 26 Feb 2015 | Published 9 Apr 2015 DOI: 10.1038/ncomms7775 Borealin dimerization mediates optimal CPC checkpoint function by enhancing localization to centromeres and kinetochores Michael E. Bekier1,w, Travis Mazur1, Maisha S. Rashid1 & William R. Taylor1 The chromosomal passenger complex (CPC) localizes to centromeres where it activates the mitotic checkpoint in response to inappropriate inter-kinetochore tension. This error correction function is essential for proper chromosome segregation. Here we define several critical features of CPC localization and function. First, the Borealin dimerization domain suppresses dynamic exchange at the centromere to allow optimal CPC function. Second, Borealin dimerization is essential to target a subpopulation of CPC proximal to the kinetochore when the mitotic spindle is disrupted. This subpopulation is also needed for full CPC checkpoint function. The existence of a pool of CPC at the kinetochore suggests that error correction is more complicated than predicted from the Aurora B phosphorylation gradient model. Finally, Haspin kinase plays a key role in maintaining the slowly exchanging centromere Borealin pool, while Aurora B and Mps1 play minimal roles in maintaining CPC localization once cells are in mitosis. 1 Department of Biological Sciences, University of Toledo, 2801 West Bancroft Street, MS 601, Toledo, Ohio 43606, USA. w Present address: Department of Molecular, Cellular and Developmental Biology, University of Michigan, 2127A Natural Science, 830 North University, Ann Arbor, Michigan 48109, USA. Correspondence and requests for materials should be addressed to W.R.T. (email: [email protected]). NATURE COMMUNICATIONS | 6:6775 | DOI: 10.1038/ncomms7775 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7775 ancer cells are commonly aneuploid, a condition defined Sgo1/2 binds pH2AT120, both the N-terminal and central regions by the gain or loss of whole chromosomes or chromo- of Borealin are implicated in centromere targeting of the Csomal segments1,2. Aneuploidy can result from errors in CPC17,18,25,27. We analysed truncated forms of Borealin lacking cell division, such as mis-segregation of individual chromosomes either the dimerization domain (Borealin1–221) or both the 3 during mitosis . Therefore, cell division is monitored by multiple central region and the dimerization domain (Borealin1–110). cell cycle checkpoints to prevent aneuploidy4,5. For example, the Immunofluorescence analysis of FLAG-tagged Borealin spindle assembly checkpoint (SAC) prevents anaphase onset until truncations was performed in cells blocked in mitosis with all chromosomes achieve biorientation on the mitotic spindle6. taxol. Full-length Borealin localized to centromeres with little to 7 The SAC is activated by unattached kinetochores . Anaphase no detectable cytoplasmic staining (Fig. 1a,b). Both Borealin1–221 onset is also delayed in response to insufficient tension generated and Borealin1–110 displayed punctate foci between Hec1-stained between sister kinetochores, for example, when both kinetochores kinetochores; however, truncated forms of Borealin showed attach to the same spindle pole. Insufficient tension triggers elevated cytoplasmic staining compared with full-length the release of bound microtubules, generating unattached Borealin (Fig. 1a,b). Quantification of the centromere/cytoplasm kinetochores that activate the SAC8,9. ratio of Borealin staining indicated a value of 4.5±1.2 The tension-sensing system that regulates kinetochore– (average±s.d.) for Borealin1–280. In contrast, this ratio was microtubule interactions depends on the chromosomal passenger significantly lower for both Borealin1–221 (2.0±0.4, Po0.001 6 complex (CPC) , composed of Borealin, Survivin, INCENP and using a Student’s t-test) and Borealin1–110 (1.7±0.3, Po0.001). Aurora B8. Prior to anaphase, the CPC localizes to inner Punctate staining of truncated forms of Borealin was also centromeres, adjacent to sister kinetochores, to monitor inter- observed when expressed in cells in which the endogenous kinetochore tension. The prevailing model that explains the role protein was knocked down using a untranslated region-targeted of Aurora B in regulating the SAC posits that under low tension, short hairpin RNA (shRNA) (Supplementary Fig. 1). When either Aurora B phosphorylates microtubule-binding kinetochore Borealin1–221 or Borealin1–110 were expressed in cells with proteins resulting in reduced affinity for microtubules leading endogenous Borealin intact, the level of INCENP on to dissociation of kinetochore–microtubule interactions10–14.As chromosomes was significantly reduced compared with cells chromosomes become bioriented and sister kinetochores are transfected with full-length Borealin (Fig. 1b,c). under high tension, Aurora B is spatially separated from the To better understand the elevated cytoplasmic staining of kinetochore and can no longer destabilize kinetochore– Borealin truncations and the reduced INCENP levels, we microtubule interactions. According to this spatial gradient measured centromere-binding dynamics of green fluorescent model, inner centromere localization of the CPC is paramount protein (GFP)-tagged Borealin in nocodazole-arrested mitotic for error correction15. In yeast, however, INCENP can cells, which also exhibit punctate localization of Borealin regulate chromosome segregation independent of centromere truncations (our unpublished data). Full-length Borealin-GFP localization16. recovered to B40% of pre-bleach levels within 60 s with a rate B À 1 CPC localization during metaphase is thought to rely on the constant of 0.020 s (Fig. 1d–f). Borealin1–110 recovered to intersection of two histone modifications at the centromere. B90% of pre-bleach levels with a rate constant of B0.068 s À 1 T3 B Haspin kinase phosphorylates histone H3 on threonine 3 (pH3 ) (Fig. 1d–f). Similarly, Borealin1–221 recovered to 90% of pre- along chromosome arms and at centromeres, which is bound bleach levels with a rate constant of 0.056 s À 1 (Fig. 1d–f). by Survivin17,18. The kinetochore-localized kinase Bub1 A Borealin mutant in which its dimerization domain was fused to T120 phosphorylates histone H2A on threonine 120 (pH2A )at amino acids 1–110 (Borealin110 þ DD) recovered to B90% of pericentromeric chromatin, which recruits Sgo1/2 (refs 18,19). pre-bleach levels with a rate constant of 0.054 s À 1 (Fig. 1d–f). Sgo1/2 can bind CDK1-phosphorylated Borealin, which also These observations suggest that although the N-terminus of contributes to CPC localization to centromeres18,20. Furthermore, Borealin is sufficient for localization to centromeres, the Aurora B regulates pH3T3 by directly activating Haspin via C-terminus suppresses dynamic exchange to maintain stable phosphorylation. Also, both Aurora B and Mps1 regulate Bub1 CPC binding at this location. Furthermore, because Borealin1–221 17,21–23 recruitment to kinetochores . We have analysed CPC and Borealin1–110 behave similarly, the N-terminus likely plays a localization and function using a combination of Borealin dominant role in localization under these conditions. structure–function studies, an FK506-binding protein (FKBP)- Because all Borealin truncations could be detected at mediated dimerization system and kinase inhibitors to deplete centromeres, we tested whether these mutants retained CPC known CPC receptors at the centromere. Our results suggest that function during mitosis. Cells were transfected with Borealin monomeric CPC interacts transiently with the centromere via shRNA along with shRNA-resistant Borealin C-terminal trunca- binding to pH3T3, while dimerization enhances centromere tions and analysed by western blotting to confirm knockdown binding to provide optimal CPC function. Selective reduction of (Fig. 1g,h) and by flow cytometry to determine DNA content pH3T3 reveals a kinetochore-proximal pool of CPC that partly co- (Fig. 1i). Borealin shRNA alone increased the percentage of localizes with pH2AT120, and depends on dimerization for polyploid cells to B13% compared with 1% in control cells, localization. Additional experiments suggest that both the inner indicating impaired CPC function (Fig. 1i). Expression of full- centromere and kinetochore-proximal pool of CPC contribute to length Borealin reduced the incidence of polyploidy to B3.5%, activation of the SAC. while expression of any Borealin truncation partially rescued the incidence of polyploidy to B8% (Fig. 1i). Next, we measured the frequency of mitotic phases in cells expressing Borealin shRNA, Results Borealin truncations and H2BGFP as a screenable marker. Domains of Borealin required for stable centromere binding. Borealin shRNA alone increased the percentage of prometaphase Borealin can be divided into three functional regions, the cells to B95% compared with B20% in control cells, indicating N-terminal alpha helices that interact with Survivin and INCENP impaired mitotic progression and defective CPC function (amino acids 15–76), an unstructured central region that contains (Fig. 1j). Co-expression of full-length Borealin with Borealin CDK1 phosphorylation sites required for interaction with Sgo1/2 shRNA reduced the percentage of prometaphase cells to 60% (amino acids 106–219) and a C-terminal dimerization domain (Fig. 1j). Co-expression of any Borealin C-terminal truncation (amino acids 224–280)24–26. Since Survivin binds pH3T3 and with Borealin