Chromosome-spindle Attachments Research Articles

Dicentric chromosome breakage in Drosophila melanogaster is influenced by pericentric heterochromatin and occurs in nonconserved hotspots.

Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster, we find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of 3 different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all 3 chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the 3 chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment vary significantly between the 3 chromosomes and are correlated with the origin of the centromere and the amount of pericentric heterochromatin. We suggest that different centromere strengths could account for this.

PBAF chromatin remodeler complexes that mediate meiotic transitions in mouse.

Gametogenesis in mammals encompasses highly regulated developmental transitions. These are associated with changes in transcription that cause characteristic patterns of gene expression observed during distinct stages of gamete development, which include specific activities with critical meiotic functions. SWI/SNF chromatin remodelers are recognized regulators of gene transcription and DNA repair, but their composition and functions in meiosis are poorly understood. We have generated gamete-specific conditional knockout mice for ARID2, a specific regulatory subunit of PBAF, and have compared its phenotype with BRG1 knockouts, the catalytic subunit of PBAF/BAF complexes. While Brg1Δ/Δ knockout acts at an early stage of meiosis and causes cell arrest at pachynema, ARID2 activity is apparently required at the end of prophase I. Striking defects in spindle assembly and chromosome-spindle attachment observed in Arid2Δ/Δ knockouts are attributed to an increase in aurora B kinase, a master regulator of chromosome segregation, at centromeres. Further genetic and biochemical analyses suggest the formation of a canonical PBAF and a BRG1-independent complex containing ARID2 and PBRM1 as core components. The data support a model in which different PBAF complexes regulate different stages of meiosis and gametogenesis.

Loss of BubR1 acetylation provokes replication stress and leads to complex chromosomal rearrangements.

Accurate chromosomal segregation during mitosis is regulated by the spindle assembly checkpoint (SAC). SAC failure results in aneuploidy, a hallmark of cancer. However, many studies have suggested that aneuploidy alone is not oncogenic. We have reported that BubR1 acetylation deficiency in mice (K243R/+) caused spontaneous tumorigenesis via weakened SAC signaling and unstable chromosome-spindle attachment, resulting in massive chromosomal mis-segregation. In addition to aneuploidy, cells derived from K243R/+ mice exhibited moderate genetic instability and chromosomal translocation. Here, we investigated how the loss of BubR1 acetylation led to genetic instability and chromosomal rearrangement. To rescue all chromosomal abnormalities generated by the loss of BubR1 acetylation during development, K243R/+ mice were crossed with p53-deficient mice. Genome-wide sequencing and spectral karyotyping of tumors derived from these double-mutant mice revealed that BubR1 acetylation deficiency was associated with complex chromosomal rearrangements, including Robertsonian-like whole-arm translocations. By analyzing the telomeres and centromeres in metaphase chromosome spreads, we found that BubR1 acetylation deficiency increased the collapse of stalled replication forks, commonly referred to as replication stress, and led to DNA damage and chromosomal rearrangements. BubR1 mutations that are critical in interacting with PCAF acetyltransferase and acetylating K250, L249F and A251P, were found from human cancers. Furthermore, a subset of human cancer cells exhibiting whole-arm translocation also displayed defects in BubR1 acetylation, supporting that defects in BubR1 acetylation in mitosis contributes to tumorigenesis. Collectively, loss of BubR1 acetylation provokes replication stress, particularly at the telomeres, leading to genetic instability and chromosomal rearrangement.

Differential requirement for Bub1 and Bub3 in regulation of meiotic versus mitotic chromosome segregation.

Accurate chromosome segregation depends on the proper attachment of kinetochores to spindle microtubules before anaphase onset. The Ipl1/Aurora B kinase corrects improper attachments by phosphorylating kinetochore components and so releasing aberrant kinetochore-microtubule interactions. The localization of Ipl1 to kinetochores in budding yeast depends upon multiple pathways, including the Bub1-Bub3 pathway. We show here that in meiosis, Bub3 is crucial for correction of attachment errors. Depletion of Bub3 results in reduced levels of kinetochore-localized Ipl1 and concomitant massive chromosome missegregation caused by incorrect chromosome-spindle attachments. Depletion of Bub3 also results in shorter metaphase I and metaphase II due to premature localization of protein phosphatase 1 (PP1) to kinetochores, which antagonizes Ipl1-mediated phosphorylation. We propose a new role for the Bub1-Bub3 pathway in maintaining the balance between kinetochore localization of Ipl1 and PP1, a balance that is essential for accurate meiotic chromosome segregation and timely anaphase onset.

The Ndc80 complex targets Bod1 to human mitotic kinetochores.

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of protein phosphatase 2A containing the B56 regulatory subunit (PP2A-B56). This phosphatase controls the amount of phosphorylation of several kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. Like PP2A-B56, Bod1 directly localizes to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localization of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1's binding to and inhibition of PP2A-B56, peaks in prometaphase when PP2A-B56 localization to kinetochores is highest. We demonstrate here that kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Crucially, Bod1 depletion functionally affects Ndc80 phosphorylation at the N-terminal serine 55 (S55), as well as a number of other phosphorylation sites within the outer kinetochore, including Knl1 at serine 24 and 60 (S24, S60), and threonine T943 and T1155 (T943, T1155). Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80, and Knl1 phosphorylation, including sites that mediate the attachment of microtubules to kinetochores.

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation

The spindle assembly checkpoint kinase Mps1 notonly inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemicallytuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (Kfibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers invivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding invitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

Spindle Size Scaling Contributes to Robust Silencing of Mitotic Spindle Assembly Checkpoint

Chromosome segregation during mitosis hinges on proper assembly of the microtubule spindle that establishes bipolar attachment to each chromosome. Experiments demonstrate allometry of mitotic spindles and a universal scaling relationship between spindle size and cell size across metazoans, which indicates a conserved principle of spindle assembly at play during evolution. However, the nature of this principle is currently unknown. Researchers have focused on deriving the mechanistic underpinning of the size scaling from the mechanical aspects of the spindle assembly process. In this work we take a different standpoint and ask: What is the size scaling for? We address this question from the functional perspectives of spindle assembly checkpoint (SAC). SAC is the critical surveillance mechanism that prevents premature chromosome segregation in the presence of unattached or misattached chromosomes. The SAC signal gets silenced after and only after the last chromosome-spindle attachment in mitosis. We previously established a model that explains the robustness of SAC silencing based on spindle-mediated spatiotemporal regulation of SAC proteins. Here, we refine the previous model, and find that robust and timely SAC silencing entails proper size scaling of mitotic spindle. This finding provides, to our knowledge, a novel, function-oriented angle toward understanding the observed spindle allometry, and the universal scaling relationship between spindle size and cell size in metazoans. In a broad sense, the functional requirement of robust SAC silencing could have helped shape the spindle assembly mechanism in evolution.

Sororin actively maintains sister chromatidcohesion.

Cohesion between sister chromatids is established during DNA replication but needs to be maintained to enable proper chromosome–spindle attachments in mitosis or meiosis. Cohesion is mediated by cohesin, but also depends on cohesin acetylation and sororin. Sororin contributes to cohesion by stabilizing cohesin on DNA. Sororin achieves this by inhibiting WAPL, which otherwise releases cohesin from DNA and destroys cohesion. Here we describe mouse models which enable the controlled depletion of sororin by gene deletion or auxin‐induced degradation. We show that sororin is essential for embryonic development, cohesion maintenance, and proper chromosome segregation. We further show that the acetyltransferases ESCO1 and ESCO2 are essential for stabilizing cohesin on chromatin, that their only function in this process is to acetylate cohesin's SMC3 subunit, and that DNA replication is also required for stable cohesin–chromatin interactions. Unexpectedly, we find that sororin interacts dynamically with the cohesin complexes it stabilizes. This implies that sororin recruitment to cohesin does not depend on the DNA replication machinery or process itself, but on a property that cohesin acquires during cohesion establishment.

Sequential Multisite Phospho-Regulation of KNL1-BUB3 Interfaces at Mitotic Kinetochores

Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface.

Loss of BRCA1 impairs centromeric cohesion and triggers chromosomal instability.

In contrast to its well-known role in the DNA damage response during interphase, the function of BRCA1 in the maintenance of chromosomal stability during mitosis remains to be defined. In this study, we uncover a novel role of BRCA1 in preserving centromere integrity in mitotic human cells. Using immunofluorescence and chromatin immunoprecipitation approaches, we report BRCA1 association with centromeric chromatin during mitosis. BRCA1 depletion impairs centromeric cohesion, leading to an increase in interkinetochore distance and in unpaired sister-chromatids frequency during prometaphase. Moreover, BRCA1 loss partially decreased accumulation of the Aurora B kinase at the centromere. We found that proper recruitment of the DNMT3b DNA methyltransferase to satellite sequences is BRCA1-dependent during mitosis, suggesting that DNA hypomethylation contributes to Aurora B mislocalization. BRCA1-deficient cells exhibited decreased ability to correct improper Aurora B-dependent chromosome-spindle attachments and to align chromosomes at metaphase. Finally, we show that BRCA1 disruption promotes merotelic kinetochore attachments that represent a major mechanism of aneuploidy in human cells. In summary, we report here a novel function of BRCA1 in maintaining chromosomal stability through its contribution to the mitotic centromere integrity necessary for faithful segregation of sister-chromatids during cell division.

Conditional targeting of MAD1 to kinetochores is sufficient to reactivate the spindle assembly checkpoint in metaphase

Fidelity of chromosome segregation is monitored by the spindle assembly checkpoint (SAC). Key components of the SAC include MAD1, MAD2, BUB1, BUB3, BUBR1, and MPS1. These proteins accumulate on kinetochores in early prometaphase but are displaced when chromosomes attach to microtubules and/or biorient on the mitotic spindle. As a result, stable attachment of the final chromosome satisfies the SAC, permitting activation of the anaphase promoting complex/cyclosome (APC/C) and subsequent anaphase onset. SAC satisfaction is reversible, however, as addition of taxol during metaphase stops cyclin B1 degradation by the APC/C. We now show that targeting MAD1 to kinetochores during metaphase is sufficient to reestablish SAC activity after initial silencing. Using rapamycin-induced heterodimerization of FKBP-MAD1 to FRB-MIS12 and live monitoring of cyclin B1 degradation, we show that timed relocalization of MAD1 during metaphase can stop cyclin B1 degradation without affecting chromosome-spindle attachments. APC/C inhibition represented true SAC reactivation, as FKBP-MAD1 required an intact MAD2-interaction motif and MPS1 activity to accomplish this. Our data show that MAD1 kinetochore localization dictates SAC activity and imply that SAC regulatory mechanisms downstream of MAD1 remain functional in metaphase.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-014-0458-9) contains supplementary material, which is available to authorized users.

Partial Hepatectomy in Acetylation-Deficient BubR1 Mice Corroborates that Chromosome Missegregation Initiates Tumorigenesis.

BackgroundAneuploidy has been suggested as one of the major causes of cancer from the time of Boveri. In support of this notion, many studies have shown that cancer cells exhibit aneuploidy. However, there are evidences that do not support the aneuploidy hypothesis. We have previously reported that the spindle assembly checkpoint protein BubR1 is acetylated in mitosis and that the acetylation of BubR1 is crucial for checkpoint maintenance and chromosome-spindle attachment. Mice heterozygous for acetylation-deficient BubR1 (K243R/+) spontaneously develop cancer with chromosome instability. As K243R/+ mice develop hepatocellular carcinoma, we set out to test if chromosome mis-segregation was the cause of their liver cancer.MethodsPrimary hepatocytes in the regenerating liver after partial hepatectomy (PH) were analyzed and compared for various mitotic parameters.ResultsPrimary hepatocytes isolated from K243R/+ mice after PH displayed a marked increase of chromosome misalignment, accompanied by an increase of micronuclei. In comparison, the number of nuclei per cell and the centrosome numbers were not different between wild-type and K243R/+ mice. Taken together, chromosome mis-segregation provokes tumorigenesis in mouse liver.ConclusionOur results corroborate that PH provides a reliable tool for assessing mitotic infidelity and cancer in mice.

Loss of BubR1 acetylation causes defects in spindle assembly checkpoint signaling and promotes tumor formation

BubR1 acetylation is essential in mitosis. Mice heterozygous for the acetylation-deficient BubR1 allele (K243R/+) spontaneously developed tumors with massive chromosome missegregations. K243R/+ mouse embryonic fibroblasts (MEFs) exhibited a weakened spindle assembly checkpoint (SAC) with shortened mitotic timing. The generation of the SAC signal was intact, as Mad2 localization to the unattached kinetochore (KT) was unaltered; however, because of the premature degradation of K243R-BubR1, the mitotic checkpoint complex disassociated prematurely in the nocodazole-treated condition, suggesting that maintenance of the SAC is compromised. BubR1 acetylation was also required to counteract excessive Aurora B activity at the KT for stable chromosome-spindle attachments. The association of acetylation-deficient BubR1 with PP2A-B56α phosphatase was reduced, and the phosphorylated Ndc80 at the KT was elevated in K243R/+ MEFs. In relation, there was a marked increase of micronuclei and p53 mutation was frequently detected in primary tumors of K243R/+ mice. Collectively, the combined effects of failure in chromosome-spindle attachment and weakened SAC cause genetic instability and cancer in K243R/+ mice.

Spatial-Temporal Regulation of Spindle Assembly Checkpoint

During mitosis, microtubule spindle organized by the two spindle poles (SP) need to properly capture and align chromosomes at the cell equator to ensure faithful chromosome segregation. Improper chromosome-spindle attachment will activate spindle assembly checkpoint proteins (SAC) emanated from this chromosome. Active SAC prevents cyclin B degradation in the cytoplasm and, consequently halts the progression of mitosis. Only after the last chromosome gets properly attached can chromosome segregation begin. Given the tens of chromosomes and everlasting fluctuations in the cell, it remains murky how the silencing of SAC faithfully couples with the last chromosome attachment. We established a theoretical model that describes the mitotic spindle structure (chromosomes, mitotic spindles, and spindle poles) as a coherent transport system. Depending on whether the chromosome is properly attached, SAC and cyclin B circulate within the transport system via dyneins, and exchange with the cytoplasm in accordance to their well-known biochemical regulations. The basis of such coherent transport is the compartmentalization underlied by direct and/or indirect protein binding affinities with these mitotic structures. Our model results show that such transport system recapitulates the observed spatial-temporal pattern of SAC and cyclin B. More importantly, the transport system establishes a robust and sensitive mechanism for silencing SAC activity in accordance with the status of chromosome-spindle attachments.

MiR-125b promotes cell death by targeting spindle assembly checkpoint gene MAD1 and modulating mitotic progression

The spindle assembly checkpoint (SAC) is a 'wait-anaphase' mechanism that has evolved in eukaryotic cells in response to the stochastic nature of chromosome-spindle attachments. In the recent past, different aspects of the SAC regulation have been described. However, the role of microRNAs in the SAC is vaguely understood. We report here that Mad1, a core SAC protein, is repressed by human miR-125b. Mad1 serves as an adaptor protein for Mad2 - which functions to inhibit anaphase entry till the chromosomal defects in metaphase are corrected. We show that exogenous expression of miR-125b, through downregulation of Mad1, delays cells at metaphase. As a result of this delay, cells proceed towards apoptotic death, which follows from elevated chromosomal abnormalities upon ectopic expression of miR-125b. Moreover, expressions of Mad1 and miR-125b are inversely correlated in a variety of cancer cell lines, as well as in primary head and neck tumour tissues. We conclude that increased expression of miR-125b inhibits cell proliferation by suppressing Mad1 and activating the SAC transiently. We hypothesize an optimum Mad1 level and thus, a properly scheduled SAC is maintained partly by miR-125b.

POLO ensures chromosome bi-orientation by preventing and correcting erroneous chromosome–spindle attachments

Correct chromosome segregation during cell division requires bi-orientation at the mitotic spindle. Cells possess mechanisms to prevent and correct inappropriate chromosome attachment. Sister kinetochores assume a 'back-to-back' geometry on chromosomes that favors amphitelic orientation but the regulation of this process and molecular components are unknown. Abnormal chromosome-spindle interactions do occur but are corrected through the activity of Aurora B, which destabilizes erroneous attachments. Here, we address the role of Drosophila POLO in chromosome-spindle interactions and show that, unlike inhibition of its activity, depletion of the protein results in bipolar spindles with most chromosomes forming stable attachments with both sister kinetochores bound to microtubules from the same pole in a syntelic orientation. This is partly the result of impaired localization and activity of Aurora B but also of an altered centromere organization with abnormal distribution of centromeric proteins and shorter interkinetochore distances. Our results suggests that POLO is required to promote amphitelic attachment and chromosome bi-orientation by regulating both the activity of the correction mechanism and the architecture of the centromere.

Targeting a kinetochore-associated motor protein to kill cancer cells.

Tumors are characterized by excessive proliferation and, therefore, chemotherapeutics that target proteins involved in tumor cell division can be effective anticancer agents. However, as many of these proteins are involved in core mechanisms in dividing and nondividing cells, these therapeutic agents do not selectively kill tumor cells and their efficacy is limited by general toxicity. As new therapeutics that target proteins involved in these core processes are developed, the likelihood increases of identifying a protein that cancer cells depend on more than normal cells. Wood et al. report the discovery and characterization of an inhibitor of a mitotic motor protein, centromere-associated protein-E (CENP-E, kinesin-7) (1). This inhibitor for a protein whose functions are limited to cell division has the potential to lead to improved cancer chemotherapies. Currently used antimitotic therapeutics target the cytoskeletal protein tubulin, which polymerizes to form microtubules (2). During cell division, segregation of chromosomes requires a microtubule-based bipolar spindle. Only after all chromosome pairs have been attached to the opposite ends of the bipolar spindle through microtubules, the spindle assembly checkpoint is satisfied, chromosomes are separated, and the cell cycle progresses. When normal tubulin polymerization dynamics are disrupted, proper chromosome-spindle attachments are not established, and the cell cycle is blocked by the checkpoint. Through poorly understood mechanisms, this cell-cycle arrest can lead to cell death (3). However, microtubules have essential roles in other cellular processes, such as neuronal transport. Therefore, the use of tubulin-targeting antimitotic agents is associated with side effects, including neurotoxicity. A decade ago, a small-molecule inhibitor, monastrol, was reported for … 1To whom correspondence should be addressed. E-mail: kapoor{at}rockefeller.edu.

Cyclin A and Nek2A: APC/C–Cdc20 substrates invisible to the mitotic spindle checkpoint

Active cyclin B1-Cdk1 (cyclin-dependent kinase 1) keeps cells in mitosis, allowing time for spindle microtubules to capture the chromosomes and for incorrect chromosome-spindle attachments to be repaired. Meanwhile, securin, an inhibitor of separase, secures cohesion between sister chromatids, preventing anaphase onset. The spindle checkpoint is a signalling pathway emerging from improperly attached chromosomes that inhibits Cdc20, the mitotic activator of the APC/C (anaphase-promoting complex/cyclosome) ubiquitin ligase. Blocking Cdc20 stabilizes cyclin B1 and securin to delay mitotic exit and anaphase until all chromosomes reach bipolar spindle attachments. Cells entering mitosis in the absence of a functional spindle checkpoint degrade cyclin B1 and securin right after nuclear-envelope breakdown, in prometaphase. Interestingly, two APC/C substrates, cyclin A and Nek2A, are normally degraded at nuclear-envelope breakdown, even when the spindle checkpoint is active. This indicates that the APC/C is activated early in mitosis, whereas cyclin B1 and securin are protected as long as the spindle checkpoint inhibits Cdc20. Remarkably, destruction of cyclin A and Nek2A also depends on Cdc20. The paradox of Cdc20 being both active and inhibited in prometaphase could be explained if cyclin A and Nek2A are either exceptionally efficient Cdc20 substrates, or if they are equipped with 'stealth' mechanisms to effectively escape detection by the spindle checkpoint. In the present paper, we discuss recently emerging models for spindle-checkpoint-independent APC/C-Cdc20 activity, which might even have implications for cancer therapy.

Chromosome congression: on the bi-orient express

Errors in chromosome-spindle attachments during cell division can lead to an irreversible change in chromosome number. Proper connections between chromosomes and spindle microtubules can be promoted by both chromosome-intrinsic and extrinsic mechanisms during mitosis and meiosis.

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.