Accurate Chromosome Segregation In Mitosis Research Articles

PLK1 phosphorylation of ZW10 guides accurate chromosome segregation in mitosis.

Stable transmission of genetic information during cell division requires faithful chromosome segregation. Mounting evidence has demonstrated that PLK1 dynamics at kinetochores control correct kinetochore-microtubule attachments and subsequent silencing of the spindle checkpoint. However, the mechanisms underlying PLK1-mediated silencing of the spindle checkpoint remain elusive. Here, we identified a regulatory mechanism by which PLK1-elicited ZW10 phosphorylation regulates spindle checkpoint silencing in mitosis. ZW10 is a cognate substrate of PLK1, and the phosphorylation of ZW10 at Ser12 enables dynamic ZW10-Zwint1 interactions. Inhibition of ZW10 phosphorylation resulted in misaligned chromosomes, while persistent expression of phospho-mimicking ZW10 mutant caused premature anaphase, in which sister chromatids entangled as cells entered anaphase. These findings reveal the previously uncharacterized PLK1-ZW10 interaction through which dynamic phosphorylation of ZW10 fine-tunes accurate chromosome segregation in mitosis.

Organization of microtubule plus-end dynamics by phase separation in mitosis.

In eukaryotes, microtubule polymers are essential for cellular plasticity and fate decisions. End-binding (EB) proteins serve as scaffolds for orchestrating microtubule polymer dynamics and are essential for cellular dynamics and chromosome segregation in mitosis. Here, we show that EB1 forms molecular condensates with TIP150 and MCAK through liquid-liquid phase separation to compartmentalize the kinetochore-microtubule plus-end machinery, ensuring accurate kinetochore-microtubule interactions during chromosome segregation in mitosis. Perturbation of EB1-TIP150 polymer formation by a competing peptide prevents phase separation of the EB1-mediated complex and chromosome alignment at the metaphase equator in both cultured cells and Drosophila embryos. Lys220 of EB1 is dynamically acetylated by p300/CBP-associated factor in early mitosis, and persistent acetylation at Lys220 attenuates the phase separation of the EB1-mediated complex, dissolves droplets in vitro, and harnesses accurate chromosome segregation. Our data suggest a novel framework for understanding the organization and regulation of eukaryotic spindle for accurate chromosome segregation in mitosis.

Sgo1 interacts with CENP-A to guide accurate chromosome segregation in mitosis.

Shugoshin-1 (Sgo1) is necessary for maintaining sister centromere cohesion and ensuring accurate chromosome segregation during mitosis. It has been reported that the localization of Sgo1 at the centromere is dependent on Bub1-mediated phosphorylation of histone H2A at T120. However, it remains uncertain whether other centromeric proteins play a role in regulating the localization and function of Sgo1 during mitosis. Here, we show that CENP-A interacts with Sgo1 and determines the localization of Sgo1 to the centromere during mitosis. Further biochemical characterization revealed that lysine and arginine residues in the C-terminal domain of Sgo1 are critical for binding CENP-A. Interestingly, the replacement of these basic amino acids with acidic amino acids perturbed the localization of Sgo1 and Aurora B to the centromere, resulting in aberrant chromosome segregation and premature chromatid separation. Taken together, these findings reveal a previously unrecognized but direct link between Sgo1 and CENP-A in centromere plasticity control and illustrate how the Sgo1-CENP-A interaction guides accurate cell division.

CENP-E activation by Aurora A and B controls kinetochore fibrous corona disassembly

Accurate chromosome segregation in mitosis depends on multiprotein structures called kinetochores that are built on the centromeric region of sister chromatids and serve to capture mitotic spindle microtubules. In early mitosis, unattached kinetochores expand a crescent-shaped structure called fibrous corona whose function is to facilitate initial kinetochore-microtubule attachments and chromosome transport by microtubules. Subsequently, the fibrous corona must be timely disassembled to prevent segregation errors. Although recent studies provided new insights on the molecular content and mechanism of fibrous corona assembly, it remains unknown what triggers the disassembly of the outermost and dynamic layer of the kinetochore. Here, we show that Aurora A and B kinases phosphorylate CENP-E to release it from an autoinhibited state. At kinetochores, Aurora B phosphorylates CENP-E to prevent its premature removal together with other corona proteins by dynein. At the spindle poles, Aurora A phosphorylates CENP-E to promote chromosome congression and prevent accumulation of corona proteins at the centrosomes, allowing for their intracellular redistribution. Thus, we propose the Aurora A/B-CENP-E axis as a critical element of the long-sought-for mechanism of fibrous corona disassembly that is essential for accurate chromosome segregation.

Dynamic phosphorylation of CENP-N by CDK1 guides accurate chromosome segregation in mitosis.

In mitosis, accurate chromosome segregation depends on the kinetochore, a supermolecular machinery that couples dynamic spindle microtubules to centromeric chromatin. However, the structure-activity relationship of the constitutive centromere-associated network (CCAN) during mitosis remains uncharacterized. Building on our recent cryo-electron microscopic analyses of human CCAN structure, we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation. Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1, which modulates the CENP-L-CENP-N interaction for accurate chromosome segregation and CCAN organization. Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint. These analyses provide mechanistic insight into a previously undefined link between the centromere-kinetochore network and accurate chromosome segregation.

Acetylation of Nup62 by TIP60 ensures accurate chromosome segregation in mitosis.

Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. In eukaryotic cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Although a list of mitotic kinases has been implicated in NEBD, how they coordinate their activity to dissolve the nuclear envelope and protein machinery such as nuclear pore complexes was unclear. Here, we identified a regulatory mechanism in which Nup62 is acetylated by TIP60 in human cell division. Nup62 is a novel substrate of TIP60, and the acetylation of Lys432 by TIP60 dissolves nucleoporin Nup62-Nup58-Nup54 complex during entry into mitosis. Importantly, this acetylation-elicited remodeling of nucleoporin complex promotes the distribution of Nup62 to the mitotic spindle, which is indispensable for orchestrating correct spindle orientation. Moreover, suppression of Nup62 perturbs accurate chromosome segregation during mitosis. These results establish a previously uncharacterized regulatory mechanism in which TIP60-elicited nucleoporin dynamics promotes chromosome segregation in mitosis.

Phosphorylation of CENP-R by Aurora B regulates kinetochore-microtubule attachment for accurate chromosome segregation.

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules via a fine structure called the centromere that is epigenetically specified by the enrichment of CENP-A nucleosomes. Centromere maintenance during mitosis requires CENP-A-mediated deposition of constitutive centromere-associated network that establishes the inner kinetochore and connects centromeric chromatin to spindle microtubules during mitosis. Although previously proposed to be an adaptor of retinoic acid receptor, here, we show that CENP-R synergizes with CENP-OPQU to regulate kinetochore-microtubule attachment stability and ensure accurate chromosome segregation in mitosis. We found that a phospho-mimicking mutation of CENP-R weakened its localization to the kinetochore, suggesting that phosphorylation may regulate its localization. Perturbation of CENP-R phosphorylation is shown to prevent proper kinetochore-microtubule attachment at metaphase. Mechanistically, CENP-R phosphorylation disrupts its binding with CENP-U. Thus, we speculate that Aurora B-mediated CENP-R phosphorylation promotes the correction of improper kinetochore-microtubule attachment in mitosis. As CENP-R is absent from yeast, we reasoned that metazoan evolved an elaborate chromosome stability control machinery to ensure faithful chromosome segregation in mitosis.

Role of ubiquitin-protein ligase UBR5 in the disassembly of mitotic checkpoint complexes

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation in mitosis by preventing the onset of anaphase until correct bipolar attachment of sister chromosomes to the mitotic spindle is attained. It acts by promoting the assembly of a mitotic checkpoint complex (MCC), composed of mitotic checkpoint proteins BubR1, Bub3, Mad2, and Cdc20. MCC binds to and inhibits the action of ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), which targets for degradation regulators of anaphase initiation. When the checkpoint system is satisfied, MCCs are disassembled, allowing the recovery of APC/C activity and initiation of anaphase. Many of the pathways of the disassembly of the different MCCs have been elucidated, but the mode of their regulation remained unknown. We find that UBR5 (ubiquitin-protein ligase N-recognin 5) is associated with the APC/C*MCC complex immunopurified from extracts of nocodazole-arrested HeLa cells. UBR5 binds to mitotic checkpoint proteins BubR1, Bub3, and Cdc20 and promotes their polyubiquitylation invitro. The dissociation of a Bub3*BubR1 subcomplex of MCC is stimulated by UBR5-dependent ubiquitylation, as suggested by observations that this process in mitotic extracts requires UBR5 and α-β bond hydrolysis of adenosine triphosphate. Furthermore, a system reconstituted from purified recombinant components carries out UBR5- and ubiquitylation-dependent dissociation of Bub3*BubR1. Immunodepletion of UBR5 from mitotic extracts slows down the release of MCC components from APC/C and prolongs the lag period in the recovery of APC/C activity in the exit from mitotic checkpoint arrest. We suggest that UBR5 may be involved in the regulation of the inactivation of the mitotic checkpoint.

Polo regulates Spindly to prevent premature stabilization of kinetochore–microtubule attachments

Accurate chromosome segregation in mitosis requires sister kinetochores to bind to microtubules from opposite spindle poles. The stability of kinetochore-microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing kinetochore-microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Here, resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, we identified a strong genetic interaction between Polo and the Rod-ZW10-Zwilch (RZZ) complex, whose kinetochore accumulation has been shown to antagonize microtubule stability. We find that Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from kinetochores and consequently delays the formation of stable end-on attachments. We propose that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. Our findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation.

Structure of the inner kinetochore CCAN complex assembled onto a centromeric nucleosome.

In eukaryotes, accurate chromosome segregation in mitosis and meiosis maintains genome stability and prevents aneuploidy. Kinetochores are large protein complexes, that by assembling onto specialized Cenp-A nucleosomes 1,2, function to connect centromeric chromatin to microtubules of the mitotic spindle 3,4. Whereas the centromeres of vertebrate chromosomes comprise Mb of DNA and attach to multiple microtubules, the simple point centromeres of budding yeast are connected to individual microtubules 5,6. All 16 budding yeast chromosomes assemble complete kinetochores using a single Cenp-A nucleosome (Cenp-ANuc), each of which is perfectly centred on its cognate centromere 7–9. The inner and outer kinetochore modules are responsible for interacting with centromeric chromatin and microtubules, respectively. Here, we describe the cryo-EM structure of the S. cerevisiae inner kinetochore module - the constitutive centromere associated network (CCAN) complex, assembled onto a Cenp-A nucleosome (CCAN–Cenp-ANuc). The structure explains the inter-dependency of CCAN’s constituent sub-complexes and shows how the ‘Y’-shaped opening of CCAN accommodates Cenp-ANuc to allow specific CCAN subunits to contact the nucleosomal DNA and histone subunits. Interactions with the unwrapped DNA duplex at the two termini of Cenp-ANuc are mediated predominantly by a DNA-binding groove present in the Cenp-LN sub-complex. Disruption of these interactions impairs assembly of CCAN onto Cenp-ANuc. Our data indicate a mechanism of Cenp-A nucleosome recognition by CCAN and how CCAN acts as a platform for assembly of the outer kinetochore for linking centromeres to the mitotic spindle for chromosome segregation.

NDP52 tunes cortical actin interaction with astral microtubules for accurate spindle orientation.

Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. Here, we show that NDP52 regulates spindle orientation via remodeling the polar cortical actin cytoskeleton. siRNA-mediated NDP52 suppression surprisingly revealed a ring-like compact subcortical F-actin architecture surrounding the spindle in prophase/prometaphase cells, which resulted in severe defects of astral microtubule growth and an aberrant spindle orientation. Remarkably, NDP52 recruited the actin assembly factor N-WASP and regulated the dynamics of the subcortical F-actin ring in mitotic cells. Mechanistically, NDP52 was found to bind to phosphatidic acid-containing vesicles, which absorbed cytoplasmic N-WASP to regulate local filamentous actin growth at the polar cortex. Our TIRFM analyses revealed that NDP52-containing vesicles anchored N-WASP and shortened the length of actin filaments in vitro. Based on these results we propose that NDP52-containing vesicles regulate cortical actin dynamics through N-WASP to accomplish a spatiotemporal regulation between astral microtubules and the actin network for proper spindle orientation and precise chromosome segregation. In this way, intracellular vesicles cooperate with microtubules and actin filaments to regulate proper mitotic progression. Since NDP52 is absent from yeast, we reason that metazoans have evolved an elaborate spindle positioning machinery to ensure accurate chromosome segregation in mitosis.

The Phosphatase PP1 Promotes Mitotic Slippage Through Mad3 Dephosphorylation

Accurate chromosome segregation in mitosis requires the bipolar attachment of kinetochores to spindle microtubules. A conserved surveillance mechanism, the Spindle Assembly Checkpoint (SAC), responds to lack of kinetochore-microtubule connections and delays the onset of anaphase until all chromosomes are bipolarly attached. SAC signalling fires at kinetochores and involves the formation of a soluble Mitotic Checkpoint Complex (MCC) made by Mad2, Mad3/BubR1, Bub3 and Cdc20, which inhibits the ubiquitin ligase activity of the Anaphase Promoting Complex (APC). The mitotic delay imposed by SAC, however, is not everlasting. After a variable time during which kinetochores have failed to establish bipolar connections, cells escape from the SAC-induced mitotic arrest through a process called mitotic slippage. Mitotic slippage occurs in the presence of SAC signalling at kinetochores, but whether and how MCC stability and APC inhibition are actively controlled during slippage is not known. The PP1 phosphatase has emerged as a key factor in SAC silencing once all kinetochores are bipolarly attached. PP1 turns off SAC signalling through dephosphorylation of the SAC scaffold Knl1/Blinkin at kinetochores. Here we show that in budding yeast PP1 is also required for mitotic slippage. However, its involvement in this process is not linked to kinetochores but rather to MCC stability. We identify S268 of Mad3 as a critical target of PP1 in this process. Mad3 S268 dephosphorylation destabilises the MCC, but in spite of that, does not affect the initial SAC-induced mitotic arrest. Conversely, it accelerates mitotic slippage and overcomes the slippage defect of PP1 mutants. Thus, slippage is not the mere consequence of incomplete APC inactivation that brings about mitotic exit, as originally proposed, but involves the exertive antagonism between kinases and phosphatases.

Dynamic acetylation of the kinetochore-associated protein HEC1 ensures accurate microtubule–kinetochore attachment

Faithful chromosome segregation during mitosis is critical for maintaining genome integrity in cell progeny and relies on accurate and robust kinetochore-microtubule attachments. The NDC80 complex, a tetramer comprising kinetochore protein HEC1 (HEC1), NDC80 kinetochore complex component NUF2 (NUF2), NDC80 kinetochore complex component SPC24 (SPC24), and SPC25, plays a critical role in kinetochore-microtubule attachment. Mounting evidence indicates that phosphorylation of HEC1 is important for regulating the binding of the NDC80 complex to microtubules. However, it remains unclear whether other post-translational modifications, such as acetylation, regulate NDC80-microtubule attachment during mitosis. Here, using pulldown assays with HeLa cell lysates and site-directed mutagenesis, we show that HEC1 is a bona fide substrate of the lysine acetyltransferase Tat-interacting protein, 60 kDa (TIP60) and that TIP60-mediated acetylation of HEC1 is essential for accurate chromosome segregation in mitosis. We demonstrate that TIP60 regulates the dynamic interactions between NDC80 and spindle microtubules during mitosis and observed that TIP60 acetylates HEC1 at two evolutionarily conserved residues, Lys-53 and Lys-59. Importantly, this acetylation weakened the phosphorylation of the N-terminal HEC1(1-80) region at Ser-55 and Ser-62, which is governed by Aurora B and regulates NDC80-microtubule dynamics, indicating functional cross-talk between these two post-translation modifications of HEC1. Moreover, the TIP60-mediated acetylation was specifically reversed by sirtuin 1 (SIRT1). Taken together, our results define a conserved signaling hierarchy, involving HEC1, TIP60, Aurora B, and SIRT1, that integrates dynamic HEC1 acetylation and phosphorylation for accurate kinetochore-microtubule attachment in the maintenance of genomic stability during mitosis.

An essential step of kinetochore formation controlled by the SNARE protein Snap29.

The kinetochore is an essential structure that mediates accurate chromosome segregation in mitosis and meiosis. While many of the kinetochore components have been identified, the mechanisms of kinetochore assembly remain elusive. Here, we identify a novel role for Snap29, an unconventional SNARE, in promoting kinetochore assembly during mitosis in Drosophila and human cells. Snap29 localizes to the outer kinetochore and prevents chromosome mis‐segregation and the formation of cells with fragmented nuclei. Snap29 promotes accurate chromosome segregation by mediating the recruitment of Knl1 at the kinetochore and ensuring stable microtubule attachments. Correct Knl1 localization to kinetochore requires human or Drosophila Snap29, and is prevented by a Snap29 point mutant that blocks Snap29 release from SNARE fusion complexes. Such mutant causes ectopic Knl1 recruitment to trafficking compartments. We propose that part of the outer kinetochore is functionally similar to membrane fusion interfaces.

Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation.

Faithful chromosome segregation in mammalian cells requires the bi-orientation of sister chromatids which relies on sensing correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation that triggers mitotic entry is extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via acetyltransferase TIP60 (Tat-interactive protein 60 kDa) in human cell division. CDK1-cyclin B phosphorylated Ser90 of TIP60, which elicited TIP60-dependent acetylation of Aurora B and promoted accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protected the phosphorylation of its activation loop from PP2A reactivation-elicited dephosphorylation to ensure a robust, error-free metaphase-anaphase transition. These findings delineated a conserved signaling cascade that integrates protein phosphorylation and acetylation to cell cycle progression for maintenance of genomic stability.

Control of genetic stability by a new heterochromatin compaction pathway involving the Tip60 histone acetyltransferase.

Pericentric heterochromatin is a highly compacted structure required for accurate chromosome segregation in mitosis. In mammals, it relies on methylation of histone H3K9 by Suv39H enzymes, which provides a docking site for HP1 proteins, therefore mediating heterochromatin compaction. Here we show that, when this normal compaction pathway is defective, the histone acetyltransferase Tip60 is recruited to pericentric heterochromatin, where it mediates acetylation of histone H4K12. Furthermore, in such a context, depletion of Tip60 leads to derepression of satellite transcription, decompaction of pericentric heterochromatin, and defects in chromosome segregation in mitosis. Finally, we show that depletion of BRD2, a double bromodomain-containing protein that binds H4K12ac, phenocopies the Tip60 depletion with respect to heterochromatin decompaction and defects in chromosome segregation. Taking the results together, we identify a new compaction pathway of mammalian pericentric heterochromatin relying on Tip60 that might be dependent on BRD2 recruitment by H4K12 acetylation. We propose that the underexpression of Tip60 observed in many human tumors can promote genetic instability via defective pericentric heterochromatin.

Constitutive heterochromatin formation and transcription in mammals.

Constitutive heterochromatin, mainly formed at the gene-poor regions of pericentromeres, is believed to ensure a condensed and transcriptionally inert chromatin conformation. Pericentromeres consist of repetitive tandem satellite repeats and are crucial chromosomal elements that are responsible for accurate chromosome segregation in mitosis. The repeat sequences are not conserved and can greatly vary between different organisms, suggesting that pericentromeric functions might be controlled epigenetically. In this review, we will discuss how constitutive heterochromatin is formed and maintained at pericentromeres in order to ensure their integrity. We will describe the biogenesis and the function of main epigenetic pathways that are involved and how they are interconnected. Interestingly, recent findings suggest that alternative pathways could substitute for well-established pathways when disrupted, suggesting that constitutive heterochromatin harbors much more plasticity than previously assumed. In addition, despite of the heterochromatic nature of pericentromeres, there is increasing evidence for active and regulated transcription at these loci, in a multitude of organisms and under various biological contexts. Thus, in the second part of this review, we will address this relatively new aspect and discuss putative functions of pericentromeric expression.

Phosphorylation of Microtubule-binding Protein Hec1 by Mitotic Kinase Aurora B Specifies Spindle Checkpoint Kinase Mps1 Signaling at the Kinetochore

The spindle assembly checkpoint (SAC) is a quality control device to ensure accurate chromosome attachment to spindle microtubule for equal segregation of sister chromatid. Aurora B is essential for SAC function by sensing chromosome bi-orientation via spatial regulation of kinetochore substrates. However, it has remained elusive as to how Aurora B couples kinetochore-microtubule attachment to SAC signaling. Here, we show that Hec1 interacts with Mps1 and specifies its kinetochore localization via its calponin homology (CH) domain and N-terminal 80 amino acids. Interestingly, phosphorylation of the Hec1 by Aurora B weakens its interaction with microtubules but promotes Hec1 binding to Mps1. Significantly, the temporal regulation of Hec1 phosphorylation orchestrates kinetochore-microtubule attachment and Mps1 loading to the kinetochore. Persistent expression of phosphomimetic Hec1 mutant induces a hyperactivation of SAC, suggesting that phosphorylation-elicited Hec1 conformational change is used as a switch to orchestrate SAC activation to concurrent destabilization of aberrant kinetochore attachment. Taken together, these results define a novel role for Aurora B-Hec1-Mps1 signaling axis in governing accurate chromosome segregation in mitosis.

SUV39H1 Orchestrates Centromeric Methylation Dynamics in Mitosis

Histone methylation performs multiple functions such as DNA replication, transcription regulation, heterochromatin formation, and chromatin condensation. How this methylation gradient is orchestrated in the centromere during chromosome segregation is not known. Here we examine the temporal dynamics of protein methylation in the centromere by SUV39H1 methyltransferase, a key mitotic regulator, using fluorescence resonance energy transfer‐based sensors in living HeLa cells and immunofluorescence of native SUV39H1 substrates. A quantitative analysis of methylation dynamics, using centromere‐targeted sensors, reveals a temporal change during chromosome segregation. These dynamics result in an accurate chromosome congression to and alignment at the equator as an inhibition of methylation dynamics using SUV39H1 inhibitor perturbs chromosome congression in living HeLa cells. Surprisingly, this inhibition of methylation results in a brief increase in Aurora B kinase activity and an enrichment of microtubule depolymerase MCAK in the centromere with a concomitant kinetochore‐microtubule destabilization and a reduced tension across the sister kinetochores with ultimate chromosome misalignments. We reason that SUV39H1 generates a gradient of methylation marks at the kinetochore that provides spatiotemporal information essential for accurate chromosome segregation in mitosis. Currently, we are uncovering the kinetochore substrates of SUV39H1 and understand how methylation of non‐histone proteins regulates kinetochore microtubule dynamics and chromosome plasticity in mitosis.

SUV39H1 orchestrates temporal dynamics of centromeric methylation essential for faithful chromosome segregation in mitosis

Histone methylation performs multiple functions such as DNA replication, transcription regulation, heterochromatin formation, and chromatin condensation. How this methylation gradient is orchestrated in the centromere during chromosome segregation is not known. Here we examine the temporal dynamics of protein methylation in the centromere by SUV39H1 methyltransferase, a key mitotic regulator, using fluorescence resonance energy transfer-based sensors in living HeLa cells and immunofluorescence of native SUV39H1 substrates. A quantitative analysis of methylation dynamics, using centromere-targeted sensors, reveals a temporal change during chromosome segregation. These dynamics result in an accurate chromosome congression to and alignment at the equator as an inhibition of methylation dynamics using SUV39H1 inhibitor perturbs chromosome congression in living HeLa cells. Surprisingly, this inhibition of methylation results in a brief increase in Aurora B kinase activity and an enrichment of microtubule depolymerase MCAK in the centromere with a concomitant kinetochore-microtubule destabilization and a reduced tension across the sister kinetochores with ultimate chromosome misalignments. We reason that SUV39H1 generates a gradient of methylation marks at the kinetochore that provides spatiotemporal information essential for accurate chromosome segregation in mitosis.