Balancing the kinetochore ledger

Reduction of polo-like kinase-1 (Plk1) at kinetochores as cells progress from prometaphase to metaphase is surprising given that the kinase is thought to stabilize kinetochore-microtubule (kt-MT) attachments. In this issue, Liu et al. (2012. J. Cell Biol. doi:10.1083/jcb.201205090) demonstrate that kinetochore-associated Plk1 is a potent suppressor of microtubule plus-end dynamics. The authors propose that Plk1 activity facilitates the establishment of kt-MT attachments in prometaphase by stabilizing microtubules and that reduction of the kinase in metaphase promotes force generation by dynamic microtubules.     

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.     

Nuf2 and Hec1 are required for retention of the checkpoint proteins Mad1 and Mad2 to kinetochores

Members of the Ndc80/Nuf2 complex have been shown in several systems to be important in formation of stable kinetochore-microtubule attachments and chromosome alignment in mitosis. In HeLa cells, we have shown that depletion of Nuf2 by RNA interference (RNAi) results in a strong prometaphase block with an active spindle checkpoint, which correlates with low but detectable Mad2 at kinetochores that have no or few stable kinetochore microtubules. Another RNAi study in HeLa cells reported that Hec1 (the human Ndc80 homolog) is required for Mad1 and Mad2 binding to kinetochores and that kinetochore bound Mad2 does not play a role in generating and maintaining the spindle assembly checkpoint. Here, we show that depletion of either Nuf2 or Hec1 by RNAi in HeLa cells results in reduction of both proteins at kinetochores and in the cytoplasm. Mad1 and Mad2 concentrate at kinetochores in late prophase/early prometaphase but become depleted by 5-fold or more over the course of the prometaphase block, which is Mad2 dependent. The reduction of Mad1 and Mad2 is reversible upon spindle depolymerization. Our observations support a model in which Nuf2 and Hec1 function to prevent microtubule-dependent stripping of Mad1 and Mad2 from kinetochores that have not yet formed stable kinetochore-microtubule attachments.     

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.     

Formation of stable attachments between kinetochores and microtubules depends on the B56-PP2A phosphatase

Error-free chromosome segregation depends on the precise regulation of phosphorylation to stabilize kinetochore-microtubule attachments (K-fibres) on sister chromatids that have attached to opposite spindle poles (bi-oriented). In many instances, phosphorylation correlates with K-fibre destabilization. Consistent with this, multiple kinases, including Aurora B and Plk1, are enriched at kinetochores of mal-oriented chromosomes when compared with bi-oriented chromosomes, which have stable attachments. Paradoxically, however, these kinases also target to prometaphase chromosomes that have not yet established spindle attachments and it is therefore unclear how kinetochore-microtubule interactions can be stabilized when kinase levels are high. Here we show that the generation of stable K-fibres depends on the B56-PP2A phosphatase, which is enriched at centromeres/kinetochores of unattached chromosomes. When B56-PP2A is depleted, K-fibres are destabilized and chromosomes fail to align at the spindle equator. Strikingly, B56-PP2A depletion increases the level of phosphorylation of Aurora B and Plk1 kinetochore substrates as well as Plk1 recruitment to kinetochores. Consistent with increased substrate phosphorylation, we find that chemical inhibition of Aurora or Plk1 restores K-fibres in B56-PP2A-depleted cells. Our findings reveal that PP2A, an essential tumour suppressor, tunes the balance of phosphorylation to promote chromosome-spindle interactions during cell division.     

The first mitosis of the mouse embryo is prolonged by transitional metaphase arrest

The first mitosis of the mouse embryo is almost twice as long as the second. The mechanism of the prolongation of the first mitosis remains unknown, and it is not clear whether prometaphase or metaphase or both are prolonged. Prometaphase is characterized by dynamic chromosome movements and spindle assembly checkpoint activity, which prevents anaphase until establishment of stable kinetochore-microtubule connections. The end of prometaphase is correlated with checkpoint inactivation and disappearance of MAD2L1 (MAD2) and RSN (CLIP-170) proteins from kinetochores. Spindle assembly checkpoint operates during the early mouse mitoses, but it is not clear whether it influences their duration. Here, we determine the length of prometaphases and metaphases during the first two embryonic mitoses by time-lapse video recording of chromosomes and by immunolocalization of MAD2L1 and RSN proteins. We show that the duration of the two prometaphases does not differ and that MAD2L1 and RSN disappear from kinetochores very early during each mitosis. The first metaphase is significantly longer than the second one. Therefore, the prolongation of the first embryonic mitosis is due to a prolonged metaphase, and the spindle assembly checkpoint cannot be involved in this process. We show also that MAD2L1 staining disappears gradually from kinetochores of oocytes arrested at metaphase of the second meiotic division. This shows a striking similarity between the first embryonic mitosis and metaphase arrest in oocytes. We postulate that the first embryonic mitosis is prolonged by a transient metaphase arrest that is independent of the spindle assembly checkpoint and is similar to metaphase II arrest. The molecular mechanism of this transient arrest remains to be elucidated.     

hNuf2 inhibition blocks stable kinetochore-microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore-microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.     

Aurora B kinase-dependent recruitment of hZW10 and hROD to tensionless kinetochores

The mitotic checkpoint ensures proper chromosome segregation by monitoring two critical events during mitosis. One is kinetochore attachment to the mitotic spindle, and the second is the alignment of chromosomes at the metaphase plate, resulting in tension across sister kinetochores (reviewed in [1, 2]). Mitotic-checkpoint proteins are known to accumulate at unaligned chromosomes that have not achieved proper kinetochore-microtubule attachments or established an adequate level of tension across sister kinetochores. Here, we report that hZW10 and hROD, two components of the evolutionarily conserved RZZ complex, accumulate at kinetochores in response to the loss of tension. By using live-cell imaging and FRAP, we showed that the accumulation of hZW10 at tensionless kinetochores stems from a 4-fold reduction of kinetochore turnover rate. We also found that cells lacking hZW10 escape loss-of-tension-induced mitotic-checkpoint arrest more rapidly than those arrested in response to the lack of kinetochore-microtubule attachments. Furthermore, we show that pharmacological inhibition of Aurora B kinase activity with ZM447439 in the absence of tension, but not in the absence of kinetochore-microtubule attachments, results in the loss of hZW10, hROD, and hBub1 from kinetochores. We therefore conclude that Aurora B kinase activity is required for the accumulation of tension-sensitive mitotic-checkpoint components, such as hZW10 and hROD, in order to maintain mitotic-checkpoint arrest.     

Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation.

We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 "dynamitin" protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.     

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.     

AKAP95 interacts with nucleoporin TPR in mitosis and is important for the spindle assembly checkpoint

Faithful chromosome segregation during mitosis relies on a proofreading mechanism that monitors proper kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) is based on the concerted action of numerous components that maintain a repressive signal inhibiting transition into anaphase until all chromosomes are attached. Here we show that A-Kinase Anchoring Protein 95 (AKAP95) is necessary for proper SAC function. AKAP95-depleted HeLa cells show micronuclei formed from lagging chromosomes at mitosis. Using a BioID proximity-based proteomic screen, we identify the nuclear pore complex protein TPR as a novel AKAP95 binding partner. We show interaction between AKAP95 and TPR in mitosis, and an AKAP95-dependent enrichment of TPR in the spindle microtubule area in metaphase, then later in the spindle midzone area. AKAP95-depleted cells display faster prometaphase to anaphase transition, escape from nocodazole-induced mitotic arrest and show a partial delocalization from kinetochores of the SAC component MAD1. Our results demonstrate an involvement of AKAP95 in proper SAC function likely through its interaction with TPR.     

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.     

How do kinetochores CLASP dynamic microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizes to the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

How Do Kinetochores CLASP Dynamic Microtubules?

Maintenance of genetic stability during cell division requires binding of chromosomes to the mitotic spindle, a process that involves attachment of spindle microtubules to kinetochores. This enables chromosomes to move to the metaphase plate, to satisfy the spindle checkpoint and finally to segregate during anaphase. Recent studies on the function MAST in Drosophila and its human homologue CLASP1, have revealed that these microtubule-associated proteins play an essential role for the kinetochore-microtubule interaction. CLASP1 localizesto the plus ends of growing microtubules and to the most external kinetochore domain. Depletion of CLASP1 causes abnormal chromosome congression, collapse of the mitotic spindle and attachment of kinetochores to very short microtubules that do not show dynamic behavior. These results suggest that CLASP1 is required at kinetochores to regulate the dynamic behavior of attached microtubules.     

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.     

p31(comet) acts to ensure timely spindle checkpoint silencing subsequent to kinetochore attachment

The spindle assembly checkpoint links the onset of anaphase to completion of chromosome-microtubule attachment and is mediated by the binding of Mad and Bub proteins to kinetochores of unattached or maloriented chromosomes. Mad2 and BubR1 traffic between kinetochores and the cytosol, thereby transmitting a "wait anaphase" signal to the anaphase-promoting complex. It is generally assumed that this signal dissipates automatically upon kinetochore-microtubule binding, but it has been shown that under conditions of nocodazole-induced arrest p31(comet), a Mad2-binding protein, is required for mitotic progression. In this article we investigate the localization and function of p31(comet) during normal, unperturbed mitosis in human and marsupial cells. We find that, like Mad2, p31(comet) traffics on and off kinetochores and is also present in the cytosol. Cells depleted of p31(comet) arrest in metaphase with mature bipolar kinetochore-microtubule attachments, a satisfied checkpoint, and high cyclin B levels. Thus p31(comet) is required for timely mitotic exit. We propose that p31(comet) is an essential component of the machinery that silences the checkpoint during each cell cycle.     

Polo-like kinase-1 is required for bipolar spindle formation but is dispensable for anaphase promoting complex/Cdc20 activation and initiation of cytokinesis

Polo-like kinase-1 (Plk1) performs multiple essential functions during the cell cycle. Here we show that human Plk1-deficient cells are unable to separate their centrosomes, fail to form a bipolar spindle, and undergo a Mad2/BubR1-dependent prometaphase arrest. However, electron microscopy demonstrates that kinetochore-microtubule interactions can be established in cells lacking Plk1. In addition, co-depletion of Plk1 and survivin allows mitotic exit. This indicates that Plk1 depletion does not prevent microtubule attachment, but specifically interferes with the generation of tension, as a consequence of a failure to form a bipolar spindle. Moreover, we find that after silencing of the spindle assembly checkpoint, degradation of cyclin B1 is unaffected in cells lacking Plk1. These data indicate that activation of the anaphase promoting complex or cyclosome (APC/C)-Cdc20 complex that is under control of the spindle assembly checkpoint does not require Plk1 activity. Finally, we find that translocation of chromosome passengers and initiation of cleavage furrow ingression is unaffected in cells depleted of Plk1. Thus, our data confirm an important role of Plk1 in bipolar spindle formation, and also demonstrate that Plk1 is dispensable for APC/C-Cdc20 activation and the initiation of cytokinesis.     

Inhibition of aurora B kinase blocks chromosome segregation,overrides the spindle checkpoint,and perturbs microtubule dynamics in mitosis

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.     

Choice of Plk1 docking partners during mitosis and cytokinesis is controlled by the activation state of Cdk1

Spatial and temporal coordination of polo-like kinase 1 (Plk1) activity is necessary for mitosis and cytokinesis, and this is achieved through binding to phosphorylated docking proteins with distinct subcellular localizations. Although cyclin-dependent kinase 1 (Cdk1) creates these phosphorylated docking sites in metaphase, a general principle that explains how Plk1 activity is controlled in anaphase after Cdk1 inactivation is lacking. Here, we show that the microtubule-associated protein regulating cytokinesis (PRC1) is an anaphase-specific binding partner for Plk1, and that this interaction is required for cytokinesis. In anaphase, Plk1 creates its own docking site on PRC1, whereas in metaphase Cdk1 phosphorylates PRC1 adjacent to this docking site and thereby prevents binding of Plk1. Mutation of these Cdk1-sites results in a form of PRC1 that prematurely recruits Plk1 to the spindle during prometaphase and blocks mitotic progression. The activation state of Cdk1, therefore, controls the switch of Plk1 localization from centrosomes and kinetochores during metaphase, to the central spindle during anaphase.     

Dynein at the kinetochore: Timing,Interactions and Functions

Kinetochores have been proposed to play multiple roles in mitotic chromosome alignment, including initial microtubule (MT) capture, monitoring MT attachments, prometaphase and anaphase chromosome movement and tension generation at metaphase. In addition, kinetochores are essential components of the spindle assembly checkpoint (SAC), and couple chromosome alignment with SAC silencing at metaphase. Although the molecular details of these activities remain under investigation, cytoplasmic dynein has been implicated in several aspects of MT and SAC regulation. Recent work clarifies the contribution of dynein to MT interactions and to events that drive anaphase onset. This review summarizes these studies and provides new models for dynein function.