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.     

BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.     

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.     

Polo-like kinase1 is required for recruitment of dynein to kinetochores during mitosis

Kinetochore dynein has been implicated in microtubule capture, correcting inappropriate microtubule attachments, chromosome movement, and checkpoint silencing. It remains unclear how dynein coordinates this diverse set of functions. Phosphorylation is responsible for some dynein heterogeneity (Whyte, J., Bader, J. R., Tauhata, S. B., Raycroft, M., Hornick, J., Pfister, K. K., Lane, W. S., Chan, G. K., Hinchcliffe, E. H., Vaughan, P. S., and Vaughan, K. T. (2008) J. Cell Biol. 183, 819-834), and phosphorylated and dephosphorylated forms of dynein coexist at prometaphase kinetochores. In this study, we measured the impact of inhibiting polo-like kinase 1 (Plk1) on both dynein populations. Phosphorylated dynein was ablated at kinetochores after inhibiting Plk1 with a small molecule inhibitor (5-Cyano-7-nitro-2-(benzothiazolo-N-oxide)-carboxamide) or chemical genetic approaches. The total complement of kinetochore dynein was also reduced but not eliminated, reflecting the presence of some dephosphorylated dynein after Plk1 inhibition. Although Plk1 inhibition had a profound effect on dynein, kinetochore populations of dynactin, spindly, and zw10 were not reduced. Plk1-independent dynein was reduced after p150(Glued) depletion, consistent with the binding of dephosphorylated dynein to dynactin. Plk1 phosphorylated dynein intermediate chains at Thr-89 in vitro and generated the phospho-Thr-89 phospho-epitope on recombinant dynein intermediate chains. Finally, inhibition of Plk1 induced defects in microtubule capture and persistent microtubule attachment, suggesting a role for phosphorylated dynein in these functions during prometaphase. These findings suggest that Plk1 is a dynein kinase required for recruitment of phosphorylated dynein to kinetochores.     

The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1

BACKGROUND: The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified. RESULTS: We describe the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabled us to study controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. CONCLUSIONS: Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.     

Chk2 prevents mitotic exit when the majority of kinetochores are unattached

The spindle checkpoint delays exit from mitosis in cells with spindle defects. In this paper, we show that Chk2 is required to delay anaphase onset when microtubules are completely depolymerized but not in the presence of relatively few unattached kinetochores. Mitotic exit in Chk2-deficient cells correlates with reduced levels of Mps1 protein and increased Cdk1–tyrosine 15 inhibitory phosphorylation. Chk2 localizes to kinetochores and is also required for Aurora B–serine 331 phosphorylation in nocodazole or unperturbed early prometaphase. Serine 331 phosphorylation contributed to prometaphase accumulation in nocodazole after partial Mps1 inhibition and was required for spindle checkpoint establishment at the beginning of mitosis. In addition, expression of a phosphomimetic S331E mutant Aurora B rescued chromosome alignment or segregation in Chk2-deficient cells. We propose that Chk2 stabilizes Mps1 and phosphorylates Aurora B–serine 331 to prevent mitotic exit when most kinetochores are unattached. These results highlight mechanisms of an essential function of Chk2 in mitosis.     

Roles of polo-like kinase 1 in the assembly of functional mitotic spindles

BACKGROUND: The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation. RESULTS: We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not associated with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Experiments with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces. CONCLUSIONS: Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.     

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.     

Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.     

Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint

The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.     

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.     

BubR1 is an effector of multiple mitotic kinases that specifies kinetochore: Microtubule attachments and checkpoint

BubR1 is a critical component of the mitotic checkpoint but has also been shown to play an essential role in establishing kinetochore:microtubule attachments. BubR1 is hyperphosphorylated in mitosis and recent studies in human and Xenopus have identified 9 phosphorylation sites. Plk1-dependent phosphorylations (T792, T1008 and S676) were reported to stimulate BubR1 kinase activity, promote kinetochore microtubule attachments, monitor kinetochore tension, as well as the recruitment of Mad2 checkpoint protein to kinetochores. Plk1-independent sites (S435, S543, S670 and S1043) were also identified and some of these were found to be sensitive to the loss of microtubule attachment but not tension. Functional studies showed that phosphorylation of S670 is critical for correcting aberrant attachments. Once end-on attachments are established, dephosphorylation of S670 appeared to be important for generating tension to signal anaphase onset. The collective data when combined with early EM studies that showed BubR1 is present at both the inner and outer kinetochore plates suggest that BubR1 maybe an effector of multiple kinases that specifies its roles in microtubule attachments and checkpoint functions.     

Liaisons between Survivin and Plk1 during Cell Division and Cell Death

Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser²⁰ by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.     

Loading of the 3F3/2 antigen onto kinetochores is dependent on the ordered assembly of the spindle checkpoint proteins

Accurate chromosome segregation is controlled by the spindle checkpoint, which responds to the lack of microtubule-kinetochore attachment or of tension across sister kinetochores through phosphorylation of kinetochore proteins by the Mps1, Bub1, BubR1, Aurora B, and Plk1/Plx1 kinases. The presence of the 3F3/2 phosphoepitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immunodepletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phosphoepitope is dependent on the checkpoint activation but also the loading of the 3F3/2 substrate to kinetochores requires the prior assembly of Mps1, Bub1 and BubR1 onto kinetochores. Interestingly, generation of the 3F3/2 epitope in checkpoint extracts requires the kinase activities of Mps1 and Bub1 but not that of BubR1. Furthermore, we demonstrate that checkpoint proteins in Xenopus extracts are assembled onto kinetochores in a highly ordered pathway consisting of three steps. Mps1 and Bub1 are loaded first, and BubR1 and Plx1 second, followed by Mad1 and Mad2. The characterization of this ordered assembly pathway provides a framework for the biochemical mechanism of the checkpoint signaling and will aid in the eventual identification of the 3F3/2 substrate.     

Phosphorylation of CLIP-170 by Plk1 and CK2 promotes timely formation of kinetochore�Cmicrotubule attachments

CLIP‐170 is implicated in the formation of kinetochore–microtubule attachments through direct interaction with the dynein/dynactin complex. However, whether this important function of CLIP‐170 is regulated by phosphorylation is unknown. Herein, we have identified polo‐like kinase 1 (Plk1) and casein kinase 2 (CK2) as two kinases of CLIP‐170 and mapped S195 and S1318 as their respective phosphorylation sites. We showed that a CK2 unphosphorylatable mutant lost its ability to bind to dynactin and to localize to kinetochores during prometaphase, indicating that the CK2 phosphorylation of CLIP‐170 is involved in its dynactin‐mediated kinetochore localization. Furthermore, we provide evidence that Plk1 phosphorylation of CLIP‐170 at S195 enhances its association with CK2. Finally, we detected defects in the formation of kinetochore fibres in cells expressing the CLIP‐S195A and ‐S1318A, but not the CLIP‐S195E and ‐S1318D, confirming that Plk1‐ and CK2‐associated phosphorylations of CLIP‐170 are involved in the timely formation of kinetochore–microtubule attachments in mitosis.     

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule- and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20- to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three- to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.     

A role for Plk1 phosphorylation of NudC in cytokinesis

Polo-like kinase 1 (Plk1) plays essential roles at multiple events during cell division, yet little is known about its physiological substrates. In a cDNA phage display screen using Plk1 C-terminal affinity columns, we identified NudC (nuclear distribution gene C) as a Plk1 binding protein. Here, we characterize the interaction between Plk1 and NudC, show that Plk1 phosphorylates NudC at conserved S274 and S326 residues in vitro, and present evidence that NudC is also a substrate for Plk1 in vivo. Downregulation of NudC by RNA interference results in multiple mitotic defects, including multinucleation and cells arrested at the midbody stage, which are rescued by ectopic expression of wild-type NudC, but not by NudC with mutations in the Plk1 phosphorylation sites. These results suggest that Plk1 phosphorylation of NudC may influence cytokinesis.     

Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension

Genetic evidence is mounting that survivin plays a crucial role in mitosis, but its exact role in human cell division remains elusive. We show that mammalian cells lacking survivin are unable to align their chromosomes, fail to recruit Aurora B to kinetochores and become polyploid at a very high frequency. Survivin-depleted cells enter mitosis with normal kinetics, but are delayed in prometaphase in a BubR1/Mad2-dependent fashion. Nonetheless, these cells exit mitosis prior to completion of chromosome congression and without sister chromatid segregation, indicating that the spindle assembly checkpoint is not fully functional. Indeed, in survivin-depleted cells, BubR1 and Mad2 are prematurely displaced from kinetochores, yet no tension is generated at kinetochores. Importantly, these cells fail to respond to drugs that prevent tension, but do arrest in mitosis after depolymerization of the mitotic spindle. This demonstrates that survivin is not required for initial checkpoint activation, or for sustained checkpoint activation by loss of microtubules. However, stable association of BubR1 to kinetochores and sustained checkpoint signalling in response to lack of tension crucially depend on survivin.     

BubR1 is a spindle assembly checkpoint protein regulating meiotic cell cycle progression of mouse oocyte

BubR1 (Bub1-related kinase or MAD3/Bub1b) is an essential component of the spindle assembly checkpoint (SAC) and plays an important role in kinetochore localization of other spindle checkpoint proteins in mitosis. But its roles in mammalian oocyte meiosis are unclear. In the present study, we examined the expression, localization and function of BubR1 during mouse oocyte meiotic maturation. The expression level of BubR1 increased progressively from germinal vesicle to metaphase II stages. Immunofluorescent analysis showed that BubR1 localized to kinetochores from the germinal vesicle breakdown to the prometaphase I stages, co-localizing with polo-like kinase 1, while it disappeared from the kinetochores at the metaphase I stage. Spindle disruption by nocodazole treatment caused relocation of BubR1 to kinetochores at metaphase I, anaphase I and metaphase II stages; spindle microtubules were disrupted by low temperature treatment in the BubR1-depleted oocytes in meiosis I, suggesting that BubR1 monitors kinetochore-microtubule (K-MT) attachments. Over-expression of exogenous BubR1 arrested oocyte meiosis maturation at the M I stage or earlier; in contrast, dominant-negative BubR1 and BubR1 depletion accelerated meiotic progression. In the BubR1-depleted oocytes, higher percentage of chromosome misalignment was observed and more oocytes overrode the M I stage arrest induced by low concentration of nocodazole. Our data suggest that BubR1 is a spindle assembly checkpoint protein regulating meiotic progression of oocytes.     

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.