Two Histone Marks Establish the Inner Centromere and Chromosome Bi-Orientation

Aurora B is a component of the chromosomal passenger complex (CPC) required for correct spindle-kinetochore attachments during chromosome segregation and for cytokinesis. The chromatin factors that recruit the CPC to centromeres are unknown, however. Here we show that phosphorylation of histone H3 threonine 3 (H3T3ph) by Haspin is necessary for CPC accumulation at centromeres and that the CPC subunit Survivin binds directly to H3T3ph. A nonbinding Survivin-D70A/D71A mutant does not support centromeric CPC concentration, and both Haspin depletion and Survivin-D70A/D71A mutation diminish centromere localization of the kinesin MCAK and the mitotic checkpoint response to taxol. Survivin-D70A/D71A mutation and microinjection of H3T3ph-specific antibody both compromise centromeric Aurora B functions but do not prevent cytokinesis. Therefore, H3T3ph generated by Haspin positions the CPC at centromeres to regulate selected targets of Aurora B during mitosis.

A hallmark of mitosis is the appearance of high levels of histone phosphorylation, yet the roles of these modifications remain largely unknown. Here, we demonstrate that histone H3 phosphorylated at threonine 3 is directly recognized by an evolutionarily conserved binding pocket in the BIR domain of Survivin, which is a member of the chromosomal passenger complex (CPC). This binding mediates recruitment of the CPC to chromosomes and the resulting activation of its kinase subunit Aurora B. Consistently, modulation of the kinase activity of Haspin, which phosphorylates H3T3, leads to defects in the Aurora B-dependent processes of spindle assembly and inhibition of nuclear reformation. These findings establish a direct cellular role for mitotic histone H3T3 phosphorylation, which is read and translated by the CPC to ensure accurate cell division.

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

Cytokinesis: Placing and Making the Final Cut

Successful partition of replicated genomes at cell division requires chromosome attachment to opposite poles of mitotic spindle (bi-orientation). Any defects in this regulation bring about chromosomal instability, which may accelerate tumour progression in humans. To achieve chromosome bi-orientation at prometaphase, the chromosomal passenger complex (CPC), composed of catalytic kinase Aurora B and regulatory components (INCENP, Survivin and Borealin), must be localized to centromeres to phosphorylate kinetochore substrates. Although the CPC dynamically changes the subcellular localization, the regulation of centromere targeting is largely unknown. Here we isolated a fission yeast cyclin B mutant defective specifically in chromosome bi-orientation. Accordingly, we identified Cdk1 (also known as Cdc2)-cyclin-B-dependent phosphorylation of Survivin. Preventing Survivin phosphorylation impairs centromere CPC targeting as well as chromosome bi-orientation, whereas phosphomimetic Survivin suppresses the bi-orientation defect in the cyclin B mutant. Survivin phosphorylation promotes direct binding with shugoshin, which we now define as a conserved centromeric adaptor of the CPC. In human cells, the phosphorylation of Borealin has a comparable role. Thus, our study resolves the conserved mechanisms of CPC targeting to centromeres, highlighting a key role of Cdk1-cyclin B in chromosome bi-orientation.

Chromosome biorientation is promoted by the four-member chromosomal passenger complex (CPC) through phosphorylation of incorrect kinetochore–microtubule attachments. During chromosome alignment, the CPC localizes to the inner centromere, the inner kinetochore, and spindle microtubules. Here we show that a small domain of the CPC subunit INCENP/Sli15 is required to target the complex to all three of these locations in budding yeast. This domain, the single alpha helix (SAH), is essential for phosphorylation of outer kinetochore substrates, chromosome segregation, and viability. By restoring the CPC to each of its three locations through targeted mutations and fusion constructs, we determined their individual contributions to chromosome biorientation. We find that only the inner centromere localization is sufficient for cell viability on its own. However, when combined, the inner kinetochore and microtubule binding activities are also sufficient to promote accurate chromosome segregation. Furthermore, we find that the two pathways target the CPC to different kinetochore attachment states, as the inner centromere-targeting pathway is primarily responsible for bringing the complex to unattached kinetochores. We have therefore discovered that two parallel localization pathways are each sufficient to promote CPC activity in chromosome biorientation, both depending on the SAH domain of INCENP/Sli15.

During meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly toward the poles is not known. To address these questions, we focused on the chromosomal passenger complex (CPC). We have found that the CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods that dramatically increase the effectiveness of RNA interference in the germline, we show that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome biorientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data we propose that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the biorientation of homologous chromosomes.

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess a unique set of kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles or how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. By employing biochemical, structural, and cell biological approaches, we demonstrate that KIN-A and KIN-B serve as the scaffold for the assembly of the remaining CPC subunits. Kinetochore localization of the CPC depends on the KKT7 – KKT8 complex pathway, with the C-terminal unstructured tail of KIN-A serving as a key interaction partner for the KKT8 complex. Our data therefore show that, unlike other eukaryotes that take advantage of histone modifications for centromere recruitment, trypanosomes rely on kinetochore proteins to recruit the CPC onto kinetochores. Furthermore, the ATPase activity of KIN-A promotes chromosome alignment in prometaphase and CPC translocation to the central spindle upon anaphase onset. Thus, KIN-A constitutes a unique ‘two-in-one’ CPC localization module in complex with KIN-B, which directs the CPC to kinetochores (from S phase until metaphase) via its C-terminal tail, and to the central spindle (in anaphase) via its N-terminal kinesin motor domain. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora B kinases in early eukaryotes.

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess a unique set of kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles or how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. By employing biochemical, structural, and cell biological approaches, we demonstrate that KIN-A and KIN-B serve as the scaffold for the assembly of the remaining CPC subunits. Kinetochore localization of the CPC depends on the KKT7 – KKT8 complex pathway, with the C-terminal unstructured tail of KIN-A serving as a key interaction partner for the KKT8 complex. Our data therefore show that, unlike other eukaryotes that take advantage of histone modifications for centromere recruitment, trypanosomes rely on kinetochore proteins to recruit the CPC onto kinetochores. Furthermore, the ATPase activity of KIN-A promotes chromosome alignment in prometaphase and CPC translocation to the central spindle upon anaphase onset. Thus, KIN-A constitutes a unique ‘two-in-one’ CPC localization module in complex with KIN-B, which directs the CPC to kinetochores (from S phase until metaphase) via its C-terminal tail, and to the central spindle (in anaphase) via its N-terminal kinesin motor domain. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora B kinases in early eukaryotes.

The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here we have identified multiple sites of CPC autophosphorylation on yeast Sli15 that are located within its central microtubule-binding domain and examined the functional significance of their phosphorylation by Ipl1 through mutation of these sites, either to non-phosphorylatable alanine (sli15-20A) or to acidic residues to mimic constitutive phosphorylation (sli15-20D). Both mutant sli15 alleles confer chromosome instability, but this is mediated neither by changes in the capacity of Sli15 to activate Ipl1 kinase nor by decreased efficiency of chromosome biorientation, a key process in cell division that requires CPC function. Instead, we find that mimicking constitutive phosphorylation of Sli15 on the Ipl1 phosphorylation sites causes delocalization of the CPC in metaphase, whereas blocking phosphorylation of Sli15 on the Ipl1 sites drives excessive localization of Sli15 to the mitotic spindle in pre-anaphase cells. Consistent with these results, direct interaction of Sli15 with microtubules in vitro is greatly reduced either following phosphorylation by Ipl1 or when constitutive phosphorylation at the Ipl1-dependent phosphorylation sites is mimicked by aspartate or glutamate substitutions. Furthermore, we find that mimicking Ipl1 phosphorylation of Sli15 interferes with the ‘tension checkpoint’ – the CPC-dependent mechanism through which cells activate the spindle assembly checkpoint to delay anaphase in the absence of tension on kinetochore-microtubule attachments. Ipl1-dependent phosphorylation of Sli15 therefore inhibits its association with microtubules both in vivo and in vitro and may negatively regulate the tension checkpoint mechanism.

The chromosomal passenger complex (CPC) regulates various events in cell division. This complex is composed of a catalytic subunit, Aurora B kinase, and three nonenzymatic subunits, INCENP, Survivin, and Borealin. Together, these four subunits interdependently regulate CPC function, and they are highly conserved among eukaryotes. However, a Borealin homologue has never been characterized in the fission yeast, Schizosaccharomyces pombe. Here, we isolate a previously uncharacterized S. pombe protein through association with the Cdc14 phosphatase homologue, Clp1/Flp1, and identify it as a Borealin-like member of the CPC. Nbl1 (novel Borealin-like 1) physically associates with known CPC components, affects the kinase activity and stability of the S. pombe Aurora B homologue, Ark1, colocalizes with known CPC subunits during mitosis, and shows sequence similarity to human Borealin. Further analysis of the Clp1-Nbl1 interaction indicates that Clp1 requires CPC activity for proper accumulation at the contractile ring (CR). Consistent with this, we describe negative genetic interactions between mutant alleles of CPC and CR components. Thus, this study characterizes a fission yeast Borealin homologue and reveals a previously unrecognized connection between the CPC and the process of cytokinesis in S. pombe.

The dynamic cellular reorganization needed for successful mitosis requires regulatory cues that vary across microns. The chromosomal passenger complex (CPC) is a conserved regulator involved in key mitotic events such as chromosome-microtubule attachment and spindle midzone formation. Recently, spatial phosphorylation gradients have been reported for CPC substrates, raising the possibility that CPC-dependent signaling establishes order on the micron-length scale in dividing cells. However, this hypothesis has not been tested, largely because of incomplete characterization of the CPC-dependent phosphorylation dynamics. Without these data it is difficult to evaluate perturbations of CPC signaling and select one that alters the spatial organization of substrate phosphorylation at a particular stage of mitosis, without changing overall phosphorylation levels. Here we examine the spatiotemporal dynamics of CPC-dependent phosphorylation along microtubules throughout mitosis using a Förster resonance energy transfer-based sensor. We find that a CPC substrate phosphorylation gradient, with highest phosphorylation levels between the two spindle poles, emerges when a cell enters mitosis. Interestingly, this gradient becomes undetectable at metaphase, but can be revealed by partially suppressing CPC activity, suggesting that high substrate phosphorylation levels can mask persistent CPC-dependent spatial patterning. After anaphase onset, the gradient emerges and persists until cell cleavage. Selective mislocalization of the CPC during anaphase suppresses gradient formation, but overall substrate phosphorylation levels remain unchanged. Under these conditions, the spindle midzone fails to organize and function properly. Our findings suggest a model in which the CPC establishes phosphorylation gradients to coordinate the spatiotemporal dynamics needed for error-free cell division.

Traveling through Mitosis with the Chromosomal Passenger Complex

Spatial restriction of Aurora B to T3-phosphorylated histone H3 (H3pT3) nucleosomes adjacent to centromeres during prometaphase and metaphase enables it to phosphorylate proteins necessary for spindle assembly checkpoint signalling and biorientation of chromosomes on the mitotic spindle. Aurora B binding to H3pT3-nucleosomes requires a multivalent targeting module, the chromosomal passenger complex (CPC), consisting of survivin, borealin, and INCENP. To shed light on how these components mediate CPC localisation during prometaphase and metaphase, we determined the structure of the CPC targeting module in complex with haspin-phosphorylated H3pT3-nucleosomes by cryo-electron microscopy. This structure shows how the N-terminus of borealin and the survivin BIR domain act as pivot and flexible tethering points, respectively, to increase CPC affinity for H3pT3 nucleosomes without limiting it to a specific orientation. We demonstrate that this flexible, yet constrained pivot-tether arrangement is important for the control of spindle assembly checkpoint signalling by Aurora B.

Accurate segregation of the replicated genome requires chromosome biorientation on the spindle. Biorientation is ensured by Aurora B kinase, a member of the 4-subunit chromosomal passenger complex (CPC)1,2. Localization of the CPC to the inner centromere is central to the current model for how tension ensures chromosome biorientation—kinetochore-spindle attachments not under tension remain close to the inner centromere and are destabilized by Aurora B phosphorylation, whereas kinetochores under tension are pulled away from the influence of Aurora B, stabilizing their microtubule attachments3–5. Here we show that an engineered truncation of the INCENP/Sli15 subunit of budding yeast CPC that eliminates association with the inner centromere nevertheless supports proper chromosome segregation during both mitosis and meiosis. Truncated INCENP/Sli15 suppresses the deletion phenotypes of the inner centromere-targeting proteins Survivin/Bir1, Borealin/Nbl1, Bub1 and Sgo16. Unlike wildtype INCENP/Sli15, truncated INCENP/Sli15 localizes to pre-anaphase spindle microtubules. Premature targeting of full-length INCENP/Sli15 to microtubules by preventing Cdk1 phosphorylation also suppresses inviability of Survivin/Bir1 deletion. These results suggest that activation of Aurora B/Ipl1 by clustering either on chromatin or on microtubules is sufficient for chromosome biorientation.