Sensing Chromosome Bi-Orientation by Spatial Separation of Aurora B Kinase from Kinetochore Substrates

Successful cell division requires that chromosomes attach to opposite poles of the mitotic spindle (bi-orientation). Aurora B kinase regulates chromosome-spindle attachments by phosphorylating kinetochore substrates that bind microtubules. Centromere tension stabilizes bi-oriented attachments, but how physical forces are translated into signaling at individual centromeres is unknown. Using fluorescence resonance energy transfer-based biosensors to measure localized phosphorylation dynamics in living cells, we found that phosphorylation of an Aurora B substrate at the kinetochore depended on its distance from the kinase at the inner centromere. Furthermore, repositioning Aurora B closer to the kinetochore prevented stabilization of bi-oriented attachments and activated the spindle checkpoint. Thus, centromere tension can be sensed by increased spatial separation of Aurora B from kinetochore substrates, which reduces phosphorylation and stabilizes kinetochore microtubules

Shugoshin-1 balances Aurora B kinase activity via PP2A to promote chromosome bi-orientation

Correction of faulty kinetochore-microtubule attachments is essential for faithful chromosome segregation and dictated by the opposing activities of Aurora B kinase and PP1 and PP2A phosphatases. How kinase and phosphatase activities are appropriately balanced is less clear. Here, we show that a centromeric pool of PP2A-B56 counteracts Aurora B T-loop phosphorylation and is recruited to centromeres through Shugoshin-1 (Sgo1). In non-transformed RPE-1 cells, Aurora B, Sgo1, and PP2A-B56 are enriched on centromeres and levels diminish as chromosomes establish bi-oriented attachments. Elevating Sgo1 levels at centromeres recruits excess PP2A-B56, and this counteracts Aurora B kinase activity, undermining efficient correction of kinetochore-microtubule attachment errors. Conversely, Sgo1-depleted cells display reduced centromeric localization of Aurora B, whereas the remaining kinase is hyperactive due to concomitant reduction of centromeric PP2A-B56. Our data suggest that Sgo1 can tune the stability of kinetochore-microtubule attachments through recruitment of PP2A-B56 that balances Aurora B activity at the centromere

Inner centromere localization of the CPC maintains centromere cohesion and allows mitotic checkpoint silencing

Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore-microtubule (KT-MT) attachments and for the stabilization of bi-oriented KT-MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation

Inner centromere localization of the CPC maintains centromere cohesion and allows mitotic checkpoint silencing

Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore-microtubule (KT-MT) attachments and for the stabilization of bi-oriented KT-MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation

Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules

The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB) domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP's centromere-targeting domain (CEN box), which increases the MTB affinity of INCENP. In (pro)metaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onset—when the histone mark H2AT120 is dephosphorylated—INCENP and Aurora B switch from centromere to microtubule localization

MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during Anaphase

During cytokinesis, signals from the anaphase spindle direct the formation and position of a contractile ring at the cell cortex [1]. The chromosomal passenger complex (CPC) participates in cytokinesis initiation by signaling from the spindle midzone and equatorial cortex [2], but the mechanisms underlying the anaphase-specific CPC localization are currently unresolved. Accumulation of the CPC at these sites requires the presence of microtubules and the mitotic kinesin-like protein 2, MKLP2 (KIF20A), a member of the kinesin-6 family [2-7], and this has led to the hypothesis that the CPC is transported along microtubules by MKLP2 [3-5, 7]. However, the structure of the MKLP2 motor domain with its extended neck-linker region suggests that this kinesin might not be able to drive processive transport [8, 9]. Furthermore, experiments in Xenopus egg extracts indicated that the CPC might be transported by kinesin-4, KIF4A [10]. Finally, CPC-MKLP2 complexes might be directly recruited to the equatorial cortex via association with actin and myosin II, independent of kinesin activity [4, 8]. Using microscopy-based assays with purified proteins, we demonstrate that MKLP2 is a processive plus-end directed motor that can transport the CPC along microtubules in vitro. In cells, strong suppression of MKLP2-dependent CPC motility by expression of an MKLP2 P-loop mutant perturbs CPC accumulation at both the spindle midzone and equatorial cortex, whereas a weaker inhibition of MKLP2 motor using Paprotrain mainly affects CPC localization to the equatorial cortex. Our data indicate that control of cytokinesis initiation by the CPC requires its directional MKLP2-dependent transport

MKLP2 Is a Motile Kinesin that Transports the Chromosomal Passenger Complex during Anaphase

During cytokinesis, signals from the anaphase spindle direct the formation and position of a contractile ring at the cell cortex [1]. The chromosomal passenger complex (CPC) participates in cytokinesis initiation by signaling from the spindle midzone and equatorial cortex [2], but the mechanisms underlying the anaphase-specific CPC localization are currently unresolved. Accumulation of the CPC at these sites requires the presence of microtubules and the mitotic kinesin-like protein 2, MKLP2 (KIF20A), a member of the kinesin-6 family [2-7], and this has led to the hypothesis that the CPC is transported along microtubules by MKLP2 [3-5, 7]. However, the structure of the MKLP2 motor domain with its extended neck-linker region suggests that this kinesin might not be able to drive processive transport [8, 9]. Furthermore, experiments in Xenopus egg extracts indicated that the CPC might be transported by kinesin-4, KIF4A [10]. Finally, CPC-MKLP2 complexes might be directly recruited to the equatorial cortex via association with actin and myosin II, independent of kinesin activity [4, 8]. Using microscopy-based assays with purified proteins, we demonstrate that MKLP2 is a processive plus-end directed motor that can transport the CPC along microtubules in vitro. In cells, strong suppression of MKLP2-dependent CPC motility by expression of an MKLP2 P-loop mutant perturbs CPC accumulation at both the spindle midzone and equatorial cortex, whereas a weaker inhibition of MKLP2 motor using Paprotrain mainly affects CPC localization to the equatorial cortex. Our data indicate that control of cytokinesis initiation by the CPC requires its directional MKLP2-dependent transport

Mps1 promotes rapid centromere accumulation of Aurora B

Aurora B localization to mitotic centromeres, which is required for proper chromosome alignment during mitosis, relies on Haspin-dependent histone H3 phosphorylation and on Bub1-dependent histone H2A phosphorylation—which interacts with Borealin through a Shugoshin (Sgo) intermediate. We demonstrate that Mps1 stimulates the latter recruitment axis. Mps1 activity enhances H2A-T120ph and is critical for Sgo1 recruitment to centromeres, thereby promoting Aurora B centromere recruitment in early mitosis. Importantly, chromosome biorientation defects caused by Mps1 inhibition are improved by restoring Aurora B centromere recruitment. As Mps1 kinetochore localization reciprocally depends on Aurora B, we propose that this Aurora B-Mps1 recruitment circuitry cooperates with the Aurora B-Haspin feedback loop to ensure rapid centromere accumulation of Aurora B at the onset of mitosis

A role for Aurora C in the chromosomal passenger complex during human preimplantation embryo development

BACKGROUND: Human embryos generated by IVF demonstrate a high incidence of chromosomal segregation errors during the cleavage divisions. To analyse underlying molecular mechanisms, we investigated the behaviour of the chromosomal passenger complex (CPC) in human oocytes and embryos. This important mitotic regulatory complex comprises the inner centromere protein (INCENP), survivin, borealin and Aurora B, or the meiotic kinase Aurora C. METHODS: We analysed mRNA expression by quantitative RT-PCR of all CPC members in human oocytes, tripronuclear (3PN) zygotes, 2-cell and 4-cell embryos developed from 3PN zygotes, plus good-quality cryopreserved 8-cell, morula and blastocyst stage embryos. Protein expression and localization of CPC members were investigated by immunofluorescence in oocytes and embryos arrested at prometaphase. Histone H3S10 phosphorylation was investigated as an indicator of a functional CPC. RESULTS: INCENP, survivin and borealin were detected at the inner centromere of prometaphase chromosomes in all stages investigated. Whereas Aurora B and C are both present in oocytes, Aurora C becomes the most prominent kinase in the CPC during the first three embryonic cell cycles. Moreover, Aurora C mRNA was up-regulated with Aurora B after activation of the embryonic genome and both proteins were detected in early Day 4 embryos. Subsequently, only Aurora B was detected in blastocysts. CONCLUSIONS: In contrast to somatic cells, our results point to a specific role for Aurora C in the CPC during human preimplantation embryo development. Although, the presence of Aurora C in itself may not explain the high chromosome segregation error rate, the data presented here provide novel information regarding possible mechanisms