Survivin mediates targeting of the chromosomal passenger complex to the centromere and midbody.
ABSTRACT: The chromosomal passenger complex (CPC) coordinates chromosomal and cytoskeletal events of mitosis. The enzymatic core of this complex (Aurora-B) is guided through the mitotic cell by its companion chromosomal passenger proteins, inner centromere protein (INCENP), Survivin and Borealin/Dasra-B, thereby allowing it to act at the right place at the right time. Here, we addressed the individual contributions of INCENP, Survivin and Borealin to the proper functioning of this complex. We show that INCENP has an important role in stabilizing the complex, and that Borealin acts to promote binding of Survivin to INCENP. Importantly, when Survivin is directly fused to INCENP, this hybrid can restore CPC function at the centromeres and midbody, even in the absence of Borealin and the centromere-targeting domain of INCENP. Thus, Survivin is an important mediator of centromere and midbody docking of Aurora-B during mitosis.
Project description:Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1 (HP1) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1. This borealin-HP1 interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1 interaction, demonstrating the spatial requirement of HP1 in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1 and CPC localization in the centromere and illustrate the critical role of borealin-HP1 interaction in orchestrating an accurate cell division.
Project description:The chromosomal passenger complex (CPC), composed of inner centromere protein (INCENP), Survivin, Borealin, and the kinase Aurora B, contributes to the activation of the mitotic checkpoint. The regulation of CPC function remains unclear. Here, we reveal that in addition to Survivin and Borealin, the single ?-helix (SAH) domain of INCENP supports CPC localization to chromatin and the mitotic checkpoint. The INCENP SAH domain also mediates INCENP's microtubule binding, which is negatively regulated by Cyclin-dependent kinase-mediated phosphorylation of segments flanking the SAH domain. The microtubule-binding capacity of the SAH domain is important for mitotic arrest in conditions of suppressed microtubule dynamics, and the duration of mitotic arrest dictates the probability, but not the timing, of cell death. Although independent targeting of INCENP to microtubules or the kinetochore/centromere promotes the mitotic checkpoint, it is insufficient for a robust mitotic arrest. Altogether, our results demonstrate that dual recognition of chromatin and microtubules by CPC is important for checkpoint maintenance and determination of cell fate in mitosis.
Project description:The chromosomal passenger complex (CPC) of Aurora-B, Borealin, INCENP (inner centromere protein) and Survivin coordinates essential chromosomal and cytoskeletal events during mitosis. Here, we show that the nuclear export receptor Crm1 is crucially involved in tethering the CPC to the centromere by interacting with a leucine-rich nuclear export signal (NES), evolutionarily conserved in all mammalian Survivin proteins. We show that inhibition of the Survivin-Crm1 interaction by treatment with leptomycin B or by RNA-interference-mediated Crm1 depletion prevents centromeric targeting of Survivin. The genetic inactivation of the Survivin-Crm1 interaction by mutation of the NES affects the correct localization and function of Survivin and the CPC during mitosis. By contrast, CPC assembly does not seem to require the Survivin-Crm1 interaction. Our report shows the functional significance of the Survivin-Crm1 interface and provides a novel link between the mitotic effector Crm1 and the CPC.
Project description:The chromosomal passenger complex (CPC), consisting of the serine/threonine kinase Aurora B, the inner centromere protein INCENP, Survivin, and Borealin/DasraB, has essential functions at the centromere in ensuring correct chromosome alignment and segregation. Despite observations that small interfering RNA-mediated knockdown of any one member of the CPC abolishes localization of the other subunits, it remains unclear how the complex is targeted to the centromere. We have now identified a ternary subcomplex of the CPC comprising Survivin, Borealin, and the N-terminal 58 amino acids of INCENP in vitro and in vivo. This subcomplex was found to be essential and sufficient for targeting to the centromere. Notably, Aurora B kinase, the enzymatic core of the CPC, was not required for centromere localization of the subcomplex. We demonstrate that CPC targeting to the centromere does not depend on CENP-A and hMis12, two core components for kinetochore/centromere assembly, and provide evidence that the CPC may be directed to centromeric DNA directly via the Borealin subunit. Our findings thus establish a functional module within the CPC that assembles on the N terminus of INCENP and controls centromere recruitment.
Project description:Chromosome association of the chromosomal passenger complex (CPC; consisting of Borealin, Survivin, INCENP, and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres, failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.
Project description: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.
Project description:The kinase Aurora B forms the chromosomal passenger complex (CPC) together with Borealin, INCENP, and Survivin to mediate chromosome condensation, the correction of erroneous spindle-kinetochore attachments, and cytokinesis. Phosphorylation of histone H3 Thr3 by Haspin kinase and of histone H2A Thr120 by Bub1 concentrates the CPC at the centromere. However, how the CPC is recruited to chromosome arms upon mitotic entry is unknown. Here, we show that asymmetric dimethylation at Arg2 on histone H3 (H3R2me2a) by protein arginine methyltransferase 6 (PRMT6) recruits the CPC to chromosome arms and facilitates histone H3S10 phosphorylation by Aurora B for chromosome condensation. Furthermore, in vitro assays show that Aurora B preferentially binds to the H3 peptide containing H3R2me2a and phosphorylates H3S10. Our findings indicate that the long-awaited key histone mark for CPC recruitment onto mitotic chromosomes is H3R2me2a, which is indispensable for maintaining appropriate CPC levels in dynamic translocation throughout mitosis.
Project description:The chromosomal passenger complex (CPC) acts as a key regulator of mitosis, preventing asymmetric segregation of chromosomal material into daughter cells. The CPC is composed of three non-enzymatic components termed Survivin, the inner centromere protein (INCENP) and Borealin, and an enzymatic component, Aurora B kinase. Survivin is necessary for the appropriate separation of sister chromatids during mitosis and is involved in liver regeneration, but its role in regenerative processes is incompletely elucidated. Whether Survivin, which is classified as an inhibitor of apoptosis protein (IAP) based on domain composition, also has a role in apoptosis is controversial. The present study examined the in vivo effects of Survivin ablation in the liver and during liver regeneration after 70% hepatectomy in a hepatocyte-specific knockout mouse model. The absence of Survivin caused a reduction in the number of hepatocytes in the liver, together with an increase in cell volume, macronucleation and polyploidy, but no changes in apoptosis. During liver regeneration, mitosis of hepatocytes was associated with mislocalization of the members of the CPC, which were no longer detectable at the centromere despite an unchanged protein amount. Furthermore, the loss of survivin in regenerating hepatocytes was associated with reduced levels of phosphorylated Histone H3 at serine 28 and abolished phosphorylation of CENP-A and Hec1 at serine 55, which is a consequence of decreased Aurora B kinase activity. These data indicate that Survivin expression determines hepatocyte number during liver development and liver regeneration. Lack of Survivin causes mislocalization of the CPC members in combination with reduced Aurora B activity, leading to impaired phosphorylation of its centromeric target proteins and inappropriate cytokinesis.
Project description:During mitosis, the chromosomal passenger complex (CPC) ensures the faithful transmission of the genome. The CPC is composed of the enzymatic component Aurora B (AURKB) and the three regulatory and targeting components borealin, INCENP, and survivin (also known as BIRC5). Although the CPC is known to be involved in diverse mitotic events, it is still unclear how CPC function terminates after mitosis. Here we show that borealin is ubiquitylated by the anaphase promoting complex/cyclosome (APC/C) and its cofactor Cdh1 (also known as FZR1) and is subsequently degraded in G1 phase. Cdh1 binds to regions within the N terminus of borealin that act as a non-canonical degron. Aurora B has also been shown previously to be degraded by the APC/CCdh1 from late mitosis to G1. Indeed, Cdh1 depletion sustains an Aurora B activity with stable levels of borealin and Aurora B throughout the cell cycle, and causes reduced efficiency of DNA replication after release from serum starvation. Notably, inhibition of Aurora B kinase activity improves the efficiency of DNA replication in Cdh1-depleted cells. We thus propose that APC/CCdh1 terminates CPC activity upon mitotic exit and thereby contributes to proper control of DNA replication.
Project description:The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ? 32-nm-long single ?-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ? 80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible "dog leash," allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.