The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis

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 α-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.     

Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP regulates CPC-spindle interaction to ensure proper microtubule dynamics

Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase-anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC-microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.     

Centromere targeting of the chromosomal passenger complex requires a ternary subcomplex of Borealin, Survivin, and the N-terminal domain of INCENP

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.     

Phosphorylation Regulates Kinase and Microtubule Binding Activities of the Budding Yeast Chromosomal Passenger Complex in Vitro

The chromosomal passenger complex (CPC) is a key regulator of mitosis in eukaryotes. It comprises four essential and conserved proteins known in mammals/yeasts as Aurora B/Ipl1, INCENP/Sli15, Survivin/Bir1, and Borealin/Nbl1. These subunits act together in a highly controlled fashion. Regulation of Aurora B/Ipl1 kinase activity and localization is critical for CPC function. Although regulation of CPC localization and kinase activity in vivo has been investigated elsewhere, studies on the complete, four-subunit CPC and its basic biochemical properties are only beginning. Here we describe the biochemical characterization of purified and complete Saccharomyces cerevisiae four-subunit CPC. We determined the affinity of the CPC for microtubules and demonstrated that the binding of CPC to microtubules is primarily electrostatic in nature and depends on the acidic C-terminal tail (E-hook) of tubulin. Moreover, phosphorylation of INCENP/Sli15 on its microtubule binding region also negatively regulates CPC affinity for microtubules. Furthermore, we show that phosphorylation of INCENP/Sli15 is required for activation of the kinase Aurora B/Ipl1 and can occur in trans. Although phosphorylation of INCENP/Sli15 is essential for activation, we determined that a version of the CPC lacking the INCENP/Sli15 microtubule binding region (residues Glu-91 to Ile-631) is able to form an intact complex that retains microtubule binding activity. Thus, we conclude that this INCENP/Sli15 linker domain plays a largely regulatory function and is not essential for complex formation or microtubule binding.     

Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast

The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B-independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B-mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments.     

The Nup107-160 Nucleoporin Complex Promotes Mitotic Events via Control of the Localization State of the Chromosome Passenger Complex

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere.     

A Bir1p Sli15p kinetochore passenger complex regulates septin organization during anaphase

Kinetochore-passenger complexes in metazoans have been proposed to coordinate the segregation of chromosomes in anaphase with the induction of cytokinesis. Passenger protein homologues in the budding yeast Saccharomyces cerevisiae play a critical role early in mitosis, ensuring proper biorientation of kinetochore-microtubule attachments. Our recent work has implicated the passenger protein Bir1p (Survivin) and the inner kinetochore complex centromere binding factor 3 (CBF3) in the regulation of septin dynamics during anaphase. Here, we present data that is consistent with there being multiple passenger protein complexes. Our data show that Bir1p links together a large passenger complex containing Ndc10p, Sli15p (INCENP), and Ipl1p (Aurora B) and that the interaction between Bir1p and Sli15p is specifically involved in regulating septin dynamics during anaphase. Neither conditional alleles nor mutants of BIR1 that disrupt the interaction between Bir1p and Sli15p resulted in mono-attached kinetochores, suggesting that the Bir1p-Sli15p complex functions in anaphase and independently from Sli15p-Ipl1p complexes. We present a model for how discrete passenger complexes coordinate distinct aspects of mitosis.     

The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation

During cell division, kinetochores form the primary chromosomal attachment sites for spindle microtubules. We previously identified a network of 10 interacting kinetochore proteins conserved between Caenorhabditis elegans and humans. In this study, we investigate three proteins in the human network (hDsn1Q9H410, hNnf1PMF1, and hNsl1DC31). Using coexpression in bacteria and fractionation of mitotic extracts, we demonstrate that these proteins form a stable complex with the conserved kinetochore component hMis12. Human or chicken cells depleted of Mis12 complex subunits are delayed in mitosis with misaligned chromosomes and defects in chromosome biorientation. Aligned chromosomes exhibited reduced centromere stretch and diminished kinetochore microtubule bundles. Consistent with this, localization of the outer plate constituent Ndc80HEC1 was severely reduced. The checkpoint protein BubR1, the fibrous corona component centromere protein (CENP) E, and the inner kinetochore proteins CENP-A and CENP-H also failed to accumulate to wild-type levels in depleted cells. These results indicate that a four-subunit Mis12 complex plays an essential role in chromosome segregation in vertebrates and contributes to mitotic kinetochore assembly.     

Phosphorylation tunes elongation propensity and cohesiveness of INCENP’s intrinsically disordered region

The inner centromere protein, INCENP, is crucial for correct chromosome segregation during mitosis. It connects the kinase Aurora B to the inner centromere allowing this kinase to dynamically access its kinetochore targets. However, the function of its central, 440-residue long intrinsically disordered region (IDR) and its multiple phosphorylation sites is unclear. Here, we determined the conformational ensemble of INCENP’s IDR, systematically varying the level of phosphorylation, using all-atom and coarse-grain molecular dynamics simulations. Our simulations show that phosphorylation expands INCENP’s IDR, both locally and globally, mainly by increasing its overall net charge. The disordered region undergoes critical globule-to-coil conformational transitions and the transition temperature non-monotonically depends on the degree of phosphorylation, with a mildly phosphorylated case of neutral net charge featuring the highest collapse propensity. The IDR transitions from a multitude of globular states, accompanied by several specific internal contacts that reduce INCENP length by loop formation, to weakly interacting and highly extended coiled conformations. Phosphorylation critically shifts the population between these two regimes. It thereby influences cohesiveness and phase behavior of INCENP IDR assemblies, a feature presumably relevant for INCENP’s function in the chromosomal passenger complex. Overall, we propose the disordered region of INCENP to act as a phosphorylation-regulated and length-variable component, within the previously defined “dog-leash” model, that thereby regulates how Aurora B reaches its targets for proper chromosome segregation.     

Regulation of Sli15/INCENP, kinetochore, and Cdc14 phosphatase functions by the ribosome biogenesis protein Utp7

The Sli15–Ipl1–Bir1 chromosomal passenger complex is essential for proper kinetochore–microtubule attachment and spindle stability in the budding yeast Saccharomyces cerevisiae. During early anaphase, release of the Cdc14 protein phosphatase from the nucleolus leads to the dephosphorylation of Sli15 and redistribution of this complex from kinetochores to the spindle. We show here that the predominantly nucleolar ribosome biogenesis protein Utp7 is also present at kinetochores and is required for normal organization of kinetochore proteins and proper chromosome segregation. Utp7 associates with and regulates the localization of Sli15 and Cdc14. Before anaphase onset, it prevents the premature nucleolar release of Cdc14 and the premature concentration of Sli15 on the spindle. Furthermore, Utp7 can regulate the localization and phosphorylation status of Sli15 independent of its effect on Cdc14 function. Thus, Utp7 is a multifunctional protein that plays essential roles in the vital cellular processes of ribosome biogenesis, chromosome segregation, and cell cycle control.     

INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the inner centromere protein (INCENP [2]), the Aurora-related serine-threonine protein kinase AIRK2 [3,4] and the unidentified human autoantigen TD-60 [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with AIRK2 function primarily affects cytokinesis [3,8]. Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.     

Exploring the functional interactions between Aurora B,INCENP, and survivin in mitosis

The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. Herein, we have investigated the regulation of human Aurora B by its complex partners inner centromere protein (INCENP) and survivin. We found that overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impaired the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbed mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, we found that Aurora B kinase activity was stimulated by INCENP and that the C-terminal region of INCENP was sufficient for activation. Under identical assay conditions, survivin did not detectably influence kinase activity. Human INCENP was a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893-895AAA) was a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation.     

The 'anaphase problem': how to disable the mitotic checkpoint when sisters split

Two closely connected mechanisms safeguard the fidelity of chromosome segregation in eukaryotic cells. The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays anaphase onset until all sister kinetochores have become attached to opposite poles. In addition, an error correction mechanism destabilizes erroneous attachments that do not lead to tension at sister kinetochores. Aurora B kinase, the catalytic subunit of the CPC (chromosomal passenger complex), acts as a sensor and effector in both pathways. In this review we focus on a poorly understood but important aspect of mitotic control: what prevents the mitotic checkpoint from springing into action when sister centromeres are split and tension is suddenly lost at anaphase onset? Recent work has shown that disjunction of sister chromatids, in principle, engages the mitotic checkpoint, and probably also the error correction mechanism, with potentially catastrophic consequences for cell division. Eukaryotic cells have solved this 'anaphase problem' by disabling the mitotic checkpoint at the metaphase-to-anaphase transition. Checkpoint inactivation is in part due to the reversal of Cdk1 (cyclin-dependent kinase 1) phosphorylation of the CPC component INCENP (inner centromere protein; Sli15 in budding yeast), which causes the relocation of the CPC from centromeres to the spindle midzone. These findings highlight principles of mitotic checkpoint control: when bipolar chromosome attachment is reached in mitosis, the checkpoint is satisfied, but still active and responsive to loss of tension. Mitotic checkpoint inactivation at anaphase onset is required to prevent checkpoint re-engagement when sister chromatids split.     

Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity

Cells have evolved a signaling pathway called the spindle assembly checkpoint (SAC) to increase the fidelity of chromosome segregation by generating a “wait anaphase” signal until all chromosomes are properly aligned within the mitotic spindle. It has been proposed that tension generated by the stretch of the centromeric chromatin of bioriented chromosomes stabilizes kinetochore microtubule attachments and turns off SAC activity. Although biorientation clearly causes stretching of the centromeric chromatin, it is unclear whether the kinetochore is also stretched. To test whether intrakinetochore stretch occurs and is involved in SAC regulation, we developed a Drosophila melanogaster S2 cell line expressing centromere identifier–mCherry and Ndc80–green fluorescent protein to mark the inner and outer kinetochore domains, respectively. We observed stretching within kinetochores of bioriented chromosomes by monitoring both inter- and intrakinetochore distances in live cell assays. This intrakinetochore stretch is largely independent of a 30-fold variation in centromere stretch. Furthermore, loss of intrakinetochore stretch is associated with enhancement of 3F3/2 phosphorylation and SAC activation.     

The kinetochore is an enhancer of pericentric cohesin binding

The recruitment of cohesins to pericentric chromatin in some organisms appears to require heterochromatin associated with repetitive DNA. However, neocentromeres and budding yeast centromeres lack flanking repetitive DNA, indicating that cohesin recruitment occurs through an alternative pathway. Here, we demonstrate that all budding yeast chromosomes assemble cohesin domains that extend over 20–50 kb of unique pericentric sequences flanking the conserved 120-bp centromeric DNA. The assembly of these cohesin domains requires the presence of a functional kinetochore in every cell cycle. A similar enhancement of cohesin binding was also observed in regions flanking an ectopic centromere. At both endogenous and ectopic locations, the centromeric enhancer amplified the inherent levels of cohesin binding that are unique to each region. Thus, kinetochores are enhancers of cohesin association that act over tens of kilobases to assemble pericentric cohesin domains. These domains are larger than the pericentric regions stretched by microtubule attachments, and thus are likely to counter microtubule-dependent forces. Kinetochores mediate two essential segregation functions: chromosome movement through microtubule attachment and biorientation of sister chromatids through the recruitment of high levels of cohesin to pericentric regions. We suggest that the coordination of chromosome movement and biorientation makes the kinetochore an autonomous segregation unit.     

Functional cooperation of Dam1, Ipl1, and the inner centromere protein (INCENP)-related protein Sli15 during chromosome segregation.

We have shown previously that Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. We show here that Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1-Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules.     

The chromosomal passenger complex activates Polo kinase at centromeres

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.     

Sensing centromere tension: Aurora B and the regulation of kinetochore function

Maintaining genome integrity during cell division requires regulated interactions between chromosomes and spindle microtubules. To ensure that daughter cells inherit the correct chromosomes, the sister kinetochores must attach to opposite spindle poles. Tension across the centromere stabilizes correct attachments, whereas phosphorylation of kinetochore substrates by the conserved Ipl1/Aurora B kinase selectively eliminates incorrect attachments. Here, we review our current understanding of how mechanical forces acting on the kinetochore are linked to biochemical changes to control chromosome segregation. We discuss models for tension sensing and regulation of kinetochore function downstream of Aurora B, and mechanisms that specify Aurora B localization to the inner centromere and determine its interactions with substrates at distinct locations.     

Drosophila Mis12 complex acts as a single functional unit essential for anaphase chromosome movement and a robust spindle assembly checkpoint

The kinetochore is a dynamic multiprotein complex assembled at the centromere in mitosis. Exactly how the structure of the kinetochore changes during mitosis and how its individual components contribute to chromosome segregation is largely unknown. Here we have focused on the contribution of the Mis12 complex to kinetochore assembly and function throughout mitosis in Drosophila. We show that despite the sequential kinetochore recruitment of Mis12 complex subunits Mis12 and Nsl1, the complex acts as a single functional unit. mis12 and nsl1 mutants show strikingly similar developmental and mitotic defects in which chromosomes are able to congress at metaphase, but their anaphase movement is strongly affected. While kinetochore association of Ndc80 absolutely depends on both Mis12 and Nsl1, BubR1 localization shows only partial dependency. In the presence of residual centromeric BubR1 the checkpoint still responds to microtubule depolymerization but is significantly weaker. These observations point to a complexity of the checkpoint response that may reflect subpopulations of BubR1 associated with residual kinetochore components, the core centromere, or elsewhere in the cell. Importantly our results indicate that core structural elements of the inner plate of the kinetochore have a greater contribution to faithful chromosome segregation in anaphase than in earlier stages of mitosis.     

Efficient Chromosome Biorientation and the Tension Checkpoint in Saccharomyces cerevisiae both Require Bir1

Accurate chromosome segregation requires the capture of sister kinetochores by microtubules from opposite spindle poles prior to the initiation of anaphase, a state termed chromosome biorientation. In the budding yeast Saccharomyces cerevisiae, the conserved protein kinase Ipl1 (Aurora B in metazoans) is critical for ensuring correct chromosomal alignment. Ipl1 associates with its activators Sli15 (INCENP), Nbl1 (Borealin), and Bir1 (Survivin), but while Sli15 clearly functions with Ipl1 to promote chromosome biorientation, the role of Bir1 has been uncertain. Using a temperature-sensitive bir1 mutant (bir1-17), we show that Bir1 is needed to permit efficient chromosome biorientation. However, once established, chromosome biorientation is maintained in bir1-17 cells at the restrictive temperature. Ipl1 is partially delocalized in bir1-17 cells, and its protein kinase activity is markedly reduced under nonpermissive conditions. bir1-17 cells arrest normally in response to microtubule depolymerization but fail to delay anaphase when sister kinetochore tension is reduced. Thus, Bir1 is required for the tension checkpoint. Despite their robust mitotic arrest in response to nocodazole, bir1-17 cells are hypersensitive to microtubule-depolymerizing drugs and show a more severe biorientation defect on recovery from nocodazole treatment. The role of Bir1 therefore may become more critical when spindle formation is delayed.Accurate chromosome segregation during anaphase is vital for ensuring the maintenance of genome integrity during cell division and, in turn, depends critically on the correct attachment of sister chromatids to kinetochore microtubules. For high-fidelity chromosome segregation, kinetochores must capture spindle microtubules such that sister chromatids are connected to opposite spindle poles (termed amphitelic attachment or chromosome biorientation), ensuring that they are pulled in opposite directions during the subsequent anaphase.In the budding yeast Saccharomyces cerevisiae, the majority of sister chromatids remain attached to microtubules from a single pole (mono-oriented) without the intervention of a correction mechanism to promote amphitelic attachment (36), a key element of which is the Ipl1 protein kinase. Ipl1 has been proposed to promote the detachment of incorrect microtubule-kinetochore connections so that correct attachments subsequently can form (35). In the absence of Ipl1 function, at the point of anaphase onset around two-thirds of sister chromatids remain mono-oriented, attached to microtubules originating from a single pole to which they then cosegregate (35). Kinetochore proteins such as Dam1 and Ndc80 have been proposed as key Ipl1 substrates for their role in promoting chromosome biorientation (6, 41). Ipl1 kinase also is required for cells to activate the spindle checkpoint in the absence of tension on kinetochore-microtubule attachments, and hence ipl1 mutant cells fail to delay anaphase despite their many mono-oriented chromosomes (2). Depending on the circumstances, the checkpoint role of Ipl1 involves either the generation of unattached kinetochores (26) or the phosphorylation of the checkpoint protein Mad3 (19). Ipl1 also is required in the absence of the BimC family kinesin Cin8p, probably reflecting a role in spindle assembly (9, 21), and is involved in regulating spindle disassembly following anaphase (5).Ipl1 kinase is highly conserved, and its metazoan ortholog (Aurora B) is involved in both chromosome biorientation and the spindle assembly checkpoint, forming part of the chromosomal passenger complex that also contains INCENP, Survivin, and Borealin (27, 40). The chromosomal passenger complex is so called because although these proteins colocalize throughout the cell cycle, their location changes dynamically from the chromosome arms in G₁ to centromeres in prometaphase and finally to the central spindle in anaphase. Such coordinated behavior is consistent with the recent crystal structure of the complex between INCENP, Survivin, and Borealin, in which they interact via tightly entwined helical domains (16).In budding yeast, Ipl1 interacts with Sli15, Bir1, and Nbl1, which have been proposed to be orthologs of INCENP, Survivin, and Borealin, respectively (6, 18). All three proteins are the products of essential genes. Like INCENP, Sli15 has a conserved C-terminal domain (the IN-box) that is required for Ipl1 kinase activation, and sli15 mutants have a phenotype that is very similar to that of ipl1 mutants (17, 18). Although yeast cells with reduced Bir1 function show chromosome instability, the first-described bir1 mutants failed to reveal a chromosome biorientation defect but instead conferred defects in septin dynamics during anaphase (38). Bir1 interacts with Ndc10 and is responsible for taking Ndc10 to the anaphase spindle (38, 42, 43), a role that may be linked to this septin defect (4). Yeast Bir1 is much larger than its metazoan counterpart (Survivin) and shows little sequence conservation outside the conserved BIR domain, yet this region is nonessential in yeast (42) and therefore unlikely to be involved in chromosome biorientation. Conversely, metazoan Borealin proteins are much larger than yeast Nbl1, which consists of little more than the helical region proposed to form the tight interaction with INCENP/Sli15 and Survivin/Bir1 complexes. Furthermore, a significant fraction of both Sli15 and Bir1 are present in a complex that lacks Ipl1 (29, 38) and that recent work has shown to contain Nbl1 (25), bringing into question the importance of Bir1 for chromosome biorientation. The extent to which Bir1 and Survivin function in conserved or analogous ways within the chromosomal passenger complexes of yeast and metazoans therefore was unclear at the start of our work.The Sli15-Bir1 complex has been proposed to interact both with microtubules (via the central domain of Sli15) and with kinetochores (through the Bir1-Ndc10 interaction) and through these interactions to function as a tension sensor, relaying information concerning the state of microtubule-kinetochore connections to Ipl1 kinase. Thus, when chromosomes are mono-oriented, the Bir1-Sli15-Nbl1 complex might activate Ipl1 in the absence of tension so as to promote chromosome biorientation by detaching incorrect microtubule attachments (29). This model predicts an essential role for Bir1 in promoting chromosome biorientation, but such evidence has been lacking. By generating a temperature-sensitive bir1 allele (bir1-17) and showing that it confers a profound defect in chromosome biorientation, we demonstrate that Bir1 does play a key role in the correction process needed to ensure that all yeast chromosomes become correctly aligned on the mitotic spindle. Furthermore, since the bir1-17 mutant fails to activate the spindle assembly checkpoint properly in response to reduced sister kinetochore tension, like Ipl1 it forms part of the tension checkpoint mechanism. Our data therefore are consistent with a role for Bir1 in conferring tension responsiveness on Ipl1 function.