Usp16 regulates kinetochore localization of Plk1 to promote proper chromosome alignment in mitosis

During the G2 to M phase transition, a portion of mitotic regulator Plk1 localizes to the kinetochores and regulates the initiation of kinetochore–microtubule attachments for proper chromosome alignment. Once kinetochore–microtubule attachment is achieved, this portion of Plk1 is removed from the kinetochores as a result of ubiquitination. However, the crucial molecular mechanism that promotes the localization and the maintenance of Plk1 on the kinetochores until metaphase is still unclear. We report that ubiquitin-specific peptidase 16 (Usp16) plays a key role during this process. Usp16 deubiquitinates Plk1, resulting in an enhanced interaction with kinetochore-localized proteins such as BubR1, and thereby retains Plk1 on the kinetochores to promote proper chromosome alignment in early mitosis. Down-regulation of Usp16 causes increased ubiquitination and decreased kinetochore localization of Plk1. Thus, our data unveil a unique mechanism by which Usp16 promotes the localization and maintenance of Plk1 on the kinetochores for proper chromosome alignment.     

NudC is required for Plk1 targeting to the kinetochore and chromosome congression

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.     

Identification of two novel components of the human NDC80 kinetochore complex

Proper kinetochore function is essential for the accurate segregation of chromosomes during mitosis. Kinetochores provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved NDC80 complex are required for chromosome congression, and their disruption results in mitotic arrest accompanied by multiple spindle aberrations. To better understand the function of the NDC80 complex, we have identified two novel subunits of the human NDC80 complex, termed human SPC25 (hSPC25) and human SPC24 (hSPC24), using an immunoaffinity approach. hSPC25 interacted with HEC1 (human homolog of yeast Ndc80) throughout the cell cycle and localized to kinetochores during mitosis. RNA interference-mediated depletion of hSPC25 in HeLa cells caused aberrant mitosis, followed by cell death, a phenotype similar to that of cells depleted of HEC1. Loss of hSPC25 also caused multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSPC25, MAD1 and HEC1 failed to localize to kinetochores during mitosis, whereas the kinetochore localization of BUB1 and BUBR1 was largely unaffected. Interestingly, the kinetochore localization of MAD1 in cells with a compromised NDC80 function was restored upon microtubule depolymerization. Thus, hSPC25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.     

Plk1 regulates the kinesin-13 protein Kif2b to promote faithful chromosome segregation

Solid tumors are frequently aneuploid, and many display high rates of ongoing chromosome missegregation in a phenomenon called chromosomal instability (CIN). The most common cause of CIN is the persistence of aberrant kinetochore-microtubule (k-MT) attachments, which manifest as lagging chromosomes in anaphase. k-MT attachment errors form during prometaphase due to stochastic interactions between kinetochores and microtubules. The kinesin-13 protein Kif2b promotes the correction of k-MT attachment errors in prometaphase, but the mechanism restricting this activity to prometaphase remains unknown. Using mass spectrometry, we identified multiple phosphorylation sites on Kif2b, some of which are acutely sensitive to inhibition of Polo-like kinase 1 (Plk1). We show that Plk1 directly phosphorylates Kif2b at threonine 125 (T125) and serine 204 (S204), and that these two sites differentially regulate Kif2b function. Phosphorylation of S204 is required for the kinetochore localization and activity of Kif2b in prometaphase, and phosphorylation of T125 is required for Kif2b activity in the correction of k-MT attachment errors. These data demonstrate that Plk1 regulates both the localization and activity of Kif2b during mitosis to promote the correction of k-MT attachment errors to ensure mitotic fidelity.     

Phosphorylation of human Sgo1 by NEK2A is essential for chromosome congression in mitosis

Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Ourrecent study shows that NEK2A interacts with MAD1 at the kinetochore and possibly functions as a novel integrator ofspindle checkpoint signaling. However, it is unclear how NEK2 A regulates kinetochore-microtubule attachment in mitosis.Here we show that NEK2A phosphorylates human Sgol and such phosphorylation is essential for faithful chromosomecongression in mitosis. NEK2A binds directly to HsSgol in vitro and co-distributes with HsSgol to the kinetochore ofmitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgol is a substrate of NEK2A and the phos-phorylation sites were mapped to Ser~(14) and Ser~(507) as judged by the incorporation of ~(32)P. Although such phosphorylation isnot required for assembly of HsSgol to the kinetochore, expression of non-phosphorylatable mutant HsSgol perturbedchromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic andmonotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation of HsSgol in orches-trating dynamic kinetochore-microtubule interaction. We propose that NEK2 A-mediated phosphorylation of human Sgolprovides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.     

Chmp4c is required for stable kinetochore-microtubule attachments

Formation of stable kinetochore-microtubule attachments is essential for accurate chromosome segregation in human cells and depends on the NDC80 complex. We recently showed that Chmp4c, an endosomal sorting complex required for transport protein involved in membrane remodelling, localises to prometaphase kinetochores and promotes cold-stable kinetochore microtubules, faithful chromosome alignment and segregation. In the present study, we show that Chmp4c associates with the NDC80 components Hec1 and Nuf2 and is required for optimal NDC80 stability and Hec1-Nuf2 localisation to kinetochores in prometaphase. However, Chmp4c-depletion does not cause a gross disassembly of outer or inner kinetochore complexes. Conversely, Nuf2 is required for Chmp4c kinetochore targeting. Constitutive Chmp4c kinetochore tethering partially rescues cold-stable microtubule polymers in cells depleted of the endogenous Nuf2, showing that Chmp4c also contributes to kinetochore-microtubule stability independently of regulating Hec1 and Nuf2 localisation. Chmp4c interacts with tubulin in cell extracts, and binds and bundles microtubules in vitro through its highly basic N-terminal region (amino acids 1–77). Furthermore, the N-terminal region of Chmp4c is required for cold-stable kinetochore microtubules and efficient chromosome alignment. We propose that Chmp4c promotes stable kinetochore-microtubule attachments by regulating Hec1–Nuf2 localisation to kinetochores in prometaphase and by binding to spindle microtubules. These results identify Chmp4c as a novel protein that regulates kinetochore-microtubule interactions to promote accurate chromosome segregation in human cells.     

Tex14, a Plk1-regulated protein, is required for kinetochore-microtubule attachment and regulation of the spindle assembly checkpoint

Proper assembly of kinetochores (KTs) during mitosis is required for bipolar attachment of spindle microtubules (MTs) and the accumulation of spindle assembly checkpoint (SAC) components. Here we show that testis-expressed protein 14 (Tex14), which has been implicated in midbody function, is recruited to KTs by Plk1 in a Cdk1-dependent manner during early mitosis. Exclusion of Tex14 from kinetochores results in an inability to efficiently localize outer KT components, impaired KT-MT attachment, chromosome congression defects, and whole-chromosome instability. In addition, we demonstrate that phosphorylation of Tex14 by Plk1 during metaphase promotes APC(Cdc20)-mediated Tex14 degradation. Inhibition of this phosphorylation event causes retention of Tex14 at KTs and results in delayed metaphase-to-anaphase transition and chromosome segregation defects. Our findings identify Tex14 as an important mediator of KT structure and function and the fidelity of chromosome separation.     

Phosphorylation of Ran-binding protein-1 by Polo-like kinase-1 is required for interaction with Ran and early mitotic progression

Polo-like kinase-1 (Plk1) is essential for progression of mitosis and localizes to centrosomes, central spindles, midbody, and kinetochore. Ran, a small GTPase of the Ras superfamily, plays a role in microtubule dynamics and chromosome segregation during mitosis. Although Ran-binding protein-1 (RanBP1) has been reported as a regulator of RanGTPase for its mitotic functions, the action mechanism between Ran and RanBP1 during mitosis is still unknown. Here, we demonstrated in vitro and in vivo phosphorylation of RanBP1 by Plk1 as well as the importance of phosphorylation of RanBP1 in the interaction between Plk1 and Ran during early mitosis. Both phosphorylation-defective and N-terminal deletion mutant constructs of RanBP1 disrupted the interaction with Ran, and depletion of Plk1 also disrupted the formation of a complex between Ran and RanBP1. In addition, the results from both ectopic expression of phosphorylation-defective mutant construct and a functional complementation on RanBP1 deficiency with this mutant indicated that phosphorylation of RanBP1 by Plk1 might be crucial to microtubule nucleation and spindle assembly during mitosis.     

Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing

Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.     

The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis

Successful mitosis requires that kinetochores stably attach to the plus ends of spindle microtubules. Central to generating these attachments is the NDC80 complex, made of the four proteins Spc24, Spc25, Nuf2, and Hec1/Ndc80. Structural studies have revealed that portions of both Hec1 and Nuf2 N termini fold into calponin homology (CH) domains, which are known to mediate microtubule binding in certain proteins. Hec1 also contains a basic, positively charged stretch of amino acids that precedes its CH domain, referred to as the "tail." Here, using a gene silence and rescue approach in HeLa cells, we show that the CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contributes to kinetochore-microtubule attachment in distinct ways. The most severe defects in kinetochore-microtubule attachment were observed in cells rescued with a Hec1 CH domain mutant, followed by those rescued with a Hec1 tail domain mutant. Cells rescued with Nuf2 CH domain mutants, however, generated stable kinetochore-microtubule attachments but failed to generate wild-type interkinetochore tension and failed to enter anaphase in a timely manner. These data suggest that the CH and tail domains of Hec1 generate essential contacts between kinetochores and microtubules in cells, whereas the Nuf2 CH domain does not.     

Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis

In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.     

Hec1 and nuf2 are core components of the kinetochore outer plate essential for organizing microtubule attachment sites

A major goal in the study of vertebrate mitosis is to identify proteins that create the kinetochore-microtubule attachment site. Attachment sites within the kinetochore outer plate generate microtubule dependent forces for chromosome movement and regulate spindle checkpoint protein assembly at the kinetochore. The Ndc80 complex, comprised of Ndc80 (Hec1), Nuf2, Spc24, and Spc25, is essential for metaphase chromosome alignment and anaphase chromosome segregation. It has also been suggested to have roles in kinetochore microtubule formation, production of kinetochore tension, and the spindle checkpoint. Here we show that Nuf2 and Hec1 localize throughout the outer plate, and not the corona, of the vertebrate kinetochore. They are part of a stable "core" region whose assembly dynamics are distinct from other outer domain spindle checkpoint and motor proteins. Furthermore, Nuf2 and Hec1 are required for formation and/or maintenance of the outer plate structure itself. Fluorescence light microscopy, live cell imaging, and electron microscopy provide quantitative data demonstrating that Nuf2 and Hec1 are essential for normal kinetochore microtubule attachment. Our results indicate that Nuf2 and Hec1 are required for organization of stable microtubule plus-end binding sites in the outer plate that are needed for the sustained poleward forces required for biorientation at kinetochores.     

Correcting improper chromosome-spindle attachments during cell division

For accurate segregation of chromosomes during cell division, microtubule fibres must attach sister kinetochores to opposite poles of the mitotic spindle (bi-orientation). Aurora kinases are linked to oncogenesis and have been implicated in the regulation of chromosome-microtubule attachments. Although loss of Aurora kinase activity causes an accumulation of mal-orientated chromosomes in dividing cells, it is not known how the active kinase corrects improper chromosome attachments. The use of reversible small-molecule inhibitors allows activation of protein function in living vertebrate cells with temporal control. Here we show that by removal of small-molecule inhibitors, controlled activation of Aurora kinase during mitosis can correct chromosome attachment errors by selective disassembly of kinetochore-microtubule fibres, rather than by alternative mechanisms involving initial release of microtubules from either kinetochores or spindle poles. Observation of chromosomes and microtubule dynamics with real-time high-resolution microscopy showed that mal-orientated, but not bi-orientated, chromosomes move to the spindle pole as both kinetochore-microtubule fibres shorten, followed by alignment at the metaphase plate. Our results provide direct evidence for a mechanism required for the maintenance of genome integrity during cell division.     

Molecular architecture of a kinetochore-microtubule attachment site

Kinetochore attachment to spindle microtubule plus-ends is necessary for accurate chromosome segregation during cell division in all eukaryotes. The centromeric DNA of each chromosome is linked to microtubule plus-ends by eight structural-protein complexes. Knowing the copy number of each of these complexes at one kinetochore-microtubule attachment site is necessary to understand the molecular architecture of the complex, and to elucidate the mechanisms underlying kinetochore function. We have counted, with molecular accuracy, the number of structural protein complexes in a single kinetochore-microtubule attachment using quantitative fluorescence microscopy of GFP-tagged kinetochore proteins in the budding yeast Saccharomyces cerevisiae. We find that relative to the two Cse4p molecules in the centromeric histone, the copy number ranges from one or two for inner kinetochore proteins such as Mif2p, to 16 for the DAM-DASH complex at the kinetochore-microtubule interface. These counts allow us to visualize the overall arrangement of a kinetochore-microtubule attachment. As most of the budding yeast kinetochore proteins have homologues in higher eukaryotes, including humans, this molecular arrangement is likely to be replicated in more complex kinetochores that have multiple microtubule attachments.     

Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis

Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Cyclin B1 binds CDK1, a cy-clin-dependent kinase catalytic subunit, forming a complex that orchestrates mitosis through phosphorylation of key proteins. Cyclin B1 regulates both the activation of CDK1 and its subcellular localization, which may be critical for substrate selection. Here, we demonstrate that cyclin B1 is concentrated on the outer plate of the kinetochore during prometaphase. This localization requires the cyclin box region of the protein. Cyclin B1 is displaced from individual kinetochores to the spindle poles by microtubule attachment to the kinetochores, and this displacement is dependent on the dynein/dynactin complex. Depletion of cyclin B1 by vector-based siRNA causes inefficient attachment between kinetochores and microtubules, and chromosome alignment defects, and delays the onset of anaphase. We conclude that cyclin B1 accumulates at kinetochores during prometaphase, where it contributes to the correct attachment of mi- crotubules to kinetochores and efficient alignment of the chromosomes, most likely through localized phosphorylation of specific substrates by cyclin B1-CDK1. Cyclin B1 is then transported from each kinetochore as microtubule attachment is completed, and this relocalization may redirect the activity of cyclin B1-CDK1 and contribute to inactivation of the spindle assembly checkpoint.     

Kinetochore-bound Mps1 regulates kinetochore–microtubule attachments via Ndc80 phosphorylation

Dividing cells detect and correct erroneous kinetochore–microtubule attachments during mitosis, thereby avoiding chromosome missegregation. The Aurora B kinase phosphorylates microtubule-binding elements specifically at incorrectly attached kinetochores, promoting their release and providing another chance for proper attachments to form. However, growing evidence suggests that the Mps1 kinase is also required for error correction. Here we directly examine how Mps1 activity affects kinetochore–microtubule attachments using a reconstitution-based approach that allows us to separate its effects from Aurora B activity. When endogenous Mps1 that copurifies with kinetochores is activated in vitro, it weakens their attachments to microtubules via phosphorylation of Ndc80, a major microtubule-binding protein. This phosphorylation contributes to error correction because phospho-deficient Ndc80 mutants exhibit genetic interactions and segregation defects when combined with mutants in other error correction pathways. In addition, Mps1 phosphorylation of Ndc80 is stimulated on kinetochores lacking tension. These data suggest that Mps1 provides an additional mechanism for correcting erroneous kinetochore–microtubule attachments, complementing the well-known activity of Aurora B.     

Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1

Background

Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.

Methodology/Principal Findings

Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.

Conclusions/Significance

Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.     

CLIP-170 facilitates the formation of kinetochore-microtubule attachments

CLIP-170 is a microtubule 'plus end tracking' protein involved in several microtubule-dependent processes in interphase. At the onset of mitosis, CLIP-170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP-170 during mitosis, the molecular function of CLIP-170 in mitosis has not yet been revealed. Here, we used a combination of high-resolution microscopy and RNAi-mediated depletion to study the function of CLIP-170 in mitosis. We found that CLIP-170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP-170 is transported along kinetochore-microtubules by the dynein/dynactin complex. Interference with CLIP-170 expression results in defective chromosome congression and diminished kinetochore-microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore-microtubule stability. Taken together, our results indicate that CLIP-170 facilitates the formation of kinetochore-microtubule attachments, possibly through direct capture of microtubules at the kinetochore.     

Dynactin helps target Polo‐like kinase 1 to kinetochores via its left‐handed beta‐helical p27 subunit

Dynactin is a protein complex required for the in vivo function of cytoplasmic dynein, a microtubule (MT)‐based motor. Dynactin binds both dynein and MTs via its p150^Glued subunit, but little is known about the ‘pointed‐end complex’ that includes the protein subunits Arp11, p62 and the p27/p25 heterodimer. Here, we show that the p27/p25 heterodimer undergoes mitotic phosphorylation by cyclin‐dependent kinase 1 (Cdk1) at a single site, p27 Thr186, to generate an anchoring site for polo‐like kinase 1 (Plk1) at kinetochores. Removal of p27/p25 from dynactin results in reduced levels of Plk1 and its phosphorylated substrates at kinetochores in prometaphase, which correlates with aberrant kinetochore–MT interactions, improper chromosome alignment and abbreviated mitosis. To investigate the structural implications of p27 phosphorylation, we determined the structure of human p27. This revealed an unusual left‐handed β‐helix domain, with the phosphorylation site located within a disordered, C‐terminal segment. We conclude that dynactin plays a previously undescribed regulatory role in the spindle assembly checkpoint by recruiting Plk1 to kinetochores and facilitating phosphorylation of important downstream targets.     

Biochemical heterogeneity and developmental varieties in T-cell leukemia

During mitosis, chromosomes undergo dynamic structural changes that include condensation of chromosomes – the formation of individual compact chromosomes necessary for faithful segregation of sister chromatids in anaphase. Polo-like kinase 1 (Plk1) regulates multiple mitotic events by binding to targeting factors at different mitotic structures in a phosphorylation dependent manner. In this study, we report the identification of a putative ATPase that targets Plk1 to chromosome arms during mitosis. PICH (Plk1-interacting checkpoint “helicase”) displays a temporal localization on chromosome arms and kinetochores during early mitosis. Interaction with PICH recruits Plk1 to chromosome arms and disruption of this interaction abolishes Plk1 localization on chromosome arms. Moreover, depletion of PICH or overexpression of PICH mutant that is defective in Plk1 binding or ATP binding causes defects in mitotic chromosome compaction, formation of anaphase bridge and cytokinesis failure. We provide data to show that both PICH phosphorylation and its ATPase activity are required for mitotic chromosome compaction. Our study provides a mechanism for targeting Plk1 to chromosome arms and suggests that the PICH ATPase activity is important for the regulation of mitotic chromosome architecture.