Relocation of Aurora B from centromeres to the central spindle at the metaphase to anaphase transition requires MKlp2

Mitotic kinases of the Polo and Aurora families are key regulators of chromosome segregation and cytokinesis. Here, we have investigated the role of MKlp1 and MKlp2, two vertebrate mitotic kinesins essential for cytokinesis, in the spatial regulation of the Aurora B kinase. Previously, we have demonstrated that MKlp2 recruits Polo-like kinase 1 (Plk1) to the central spindle in anaphase. We now find that in MKlp2 but not MKlp1-depleted cells the Aurora B-INCENP complex remains at the centromeres and fails to relocate to the central spindle. MKlp2 exerts dual control over Aurora B localization, because it is a binding partner for Aurora B, and furthermore for the phosphatase Cdc14A. Cdc14A can dephosphorylate INCENP and may contribute to its relocation to the central spindle in anaphase. We propose that MKlp2 is involved in the localization of Plk1, Aurora B, and Cdc14A to the central spindle during anaphase, and that the integration of signaling by these proteins is necessary for proper cytokinesis.     

Roles and mechanisms of Kinesin-6 KIF20A in spindle organization during cell division

Mitotic kinesin is crucial for spindle assembly and chromosome segregation in cell division. KIF20A/MKlp2, a member of kinesin-6 subfamily, plays important roles in the central spindle organization at anaphase and cytokinesis. In this review, we briefly introduce the discovery and classification of kinesin-6 motors in model organisms, and summarize the biochemical features and mechanics of KIF20A proteins. We emphasize the complicated interactions of KIF20A with partner proteins, including MKlp1, Plk1 and Rab6. Particularly, we highlight the regulation of Cdk1 and chromosomal passenger complex on kinesin-6 KIF20A at late stage of mitosis. We summarized the multiple functions of KIF20A in central spindle assembly and the formation of cleavage furrow in both mitosis and meiosis. In addition, we conclude the expression patterns of KIF20A in tumorigenesis and its applications in tumor therapy.     

KIF4A and PP2A–B56 form a spatially restricted feedback loop opposing Aurora B at the anaphase central spindle

The mitotic kinase Aurora B is concentrated at the anaphase central spindle by the kinesin MKlp2 during mitotic exit and cytokinesis. This pool of Aurora B phosphorylates substrates including the kinesin KIF4A to regulate central spindle length. In this paper, we identify a counteracting system in which PP2A–B56γ and -ε, but not PP2A–B56α, -β, and -δ, are maintained at the central spindle by KIF4A. Biochemical assays show that PP2A–B56γ can dephosphorylate the T799 Aurora B site on KIF4A and thereby counteract the Aurora B– and microtubule-stimulated ATPase activity of KIF4A. In agreement with these observations, combined silencing of PP2A–B56γ and -ε resulted in increased phosphorylation of KIF4A T799 and decreased central spindle growth in anaphase B. Furthermore, reduced turnover of regulatory phosphorylation on another Aurora B substrate MKlp1 was observed, suggesting that PP2A–B56γ and -ε play a general role opposing Aurora B at the central spindle. KIF4A and PP2A–B56γ and -ε therefore create a spatially restricted negative feedback loop counteracting Aurora B in anaphase.     

Late mitotic functions of Aurora kinases

The coordination between late mitotic events such as poleward chromosome motion, spindle elongation, DNA decondensation, and nuclear envelope reformation (NER) is crucial for the completion of chromosome segregation at the anaphase-telophase transition. Mitotic exit is driven by a decrease of Cdk1 kinase activity and an increase of PP1/PP2A phosphatase activities. More recently, Aurora kinases have also emerged as master regulators of late mitotic events and cytokinesis. Aurora A is mainly associated with spindle poles throughout mitosis and midbody during telophase, whereas Aurora B re-localizes from centromeres in early mitosis to the spindle midzone and midbody as cells progress from anaphase to the completion of cytokinesis. Functional studies, together with the identification of a phosphorylation gradient during anaphase, established Aurora B as a major player in the organization of the spindle midzone and in the spatiotemporal coordination between chromosome segregation and NER. Aurora A has been less explored, but a cooperative role in spindle midzone stability has also been proposed, implying that both Aurora A and B contribute to accurate chromosome segregation during mitotic exit. Here, we review the roles of the Aurora kinases in the regulation of late mitotic events and discuss how they work together with other mitotic players to ensure an error-free mitosis.     

Aurora at the pole and equator: overlapping functions of Aurora kinases in the mitotic spindle

The correct assembly and timely disassembly of the mitotic spindle is crucial for the propagation of the genome during cell division. Aurora kinases play a central role in orchestrating bipolar spindle establishment, chromosome alignment and segregation. In most eukaryotes, ranging from amoebas to humans, Aurora activity appears to be required both at the spindle pole and the kinetochore, and these activities are often split between two different Aurora paralogues, termed Aurora A and B. Polar and equatorial functions of Aurora kinases have generally been considered separately, with Aurora A being mostly involved in centrosome dynamics, whereas Aurora B coordinates kinetochore attachment and cytokinesis. However, double inactivation of both Aurora A and B results in a dramatic synergy that abolishes chromosome segregation. This suggests that these two activities jointly coordinate mitotic progression. Accordingly, recent evidence suggests that Aurora A and B work together in both spindle assembly in metaphase and disassembly in anaphase. Here, we provide an outlook on these shared functions of the Auroras, discuss the evolution of this family of mitotic kinases and speculate why Aurora kinase activity may be required at both ends of the spindle microtubules.     

Dictyostelium Aurora kinase has properties of both Aurora A and Aurora B kinases

Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain two kinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial region respectively. It is not currently known whether the specialized functions of the two kinases arose after their duplication in animal cells or were already present in their ancestral kinase. We show that Dictyostelium discoideum contains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B. Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at the spindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes on centromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end of cytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres in anaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes at the cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and the motor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to the different Aurora kinases in animals and also those in other organisms.     

Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis

We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.     

Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase central spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to central spindle size control in anaphase.     

Mad2 inhibits the mitotic kinesin MKlp2

We identified the mitotic kinesin-like protein 2 (MKlp2), a kinesin required for chromosome passenger complex (CPC)-mediated cytokinesis, as a target of the mitotic checkpoint protein Mad2. MKlp2 possesses a consensus Mad2-binding motif required for Mad2 binding. Mad2 prevents MKlp2 from loading onto the mitotic spindle, a prerequisite step for its function as a mitotic kinesin. Furthermore, Mad2 inhibits the ability of MKlp2 to relocate the CPC from centromeres, an essential step to promote cytokinesis. An MKlp2 mutant that is refractory to Mad2-mediated inhibition prematurely translocates to the mitotic spindle and mislocalizes the CPC component Aurora B from the midbody of dividing cells. This correlates with an increased incidence of cytokinesis failure. Together, these findings reveal that MKlp2 is a novel mitotic target of Mad2 necessary for proper mitotic progression and cytokinesis.     

Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP regulates CPC-spindle interaction to ensure proper microtubule dynamics

Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase-anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC-microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.     

Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference

Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail.     

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.     

Targeting Aurora B to the Equatorial Cortex by MKlp2 Is Required for Cytokinesis

Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.     

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin-independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.     

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G₂/M transition. We find that activation of Aurora A first occurs at centrosomes at late G₂ and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.     

Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis

Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila (Glover, D.M., M.H. Leibowitz, D.A. McLean, and H. Parry. 1995. Cell. 81:95-105), the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, M.A., A.V. Philp, D.M. Glover, and H.J. Bellen. 1996. Cell. 87:1103-1114.). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.     

Aurora B kinases restrict chromosome decondensation to telophase of mitosis

The Aurora kinases comprise a family of evolutionary conserved serine/threonine kinases that have important functions in centrosome duplication, mitotic spindle assembly, chromosome condensation, chromosome biorientation on the spindle and chromosome segregation. Vertebrates have three Aurora kinases, Aurora-A, -B and -C, while invertebrates have only Aurora-A and -B and yeasts have a single Aurora kinase, Ipl1 in S. cerevisiae and Ark1 in S. pombe. Recently, the role of Aurora kinases in chromosome condensation has been defined; Aurora B plays a crucial role in the axial shortening of chromosomes during anaphase, presumably in order to prevent chromosome arms from becoming trapped within the cytokinetic plate.     

The roles of fission yeast ase1 in mitotic cell division, meiotic nuclear oscillation, and cytokinesis checkpoint signaling

The Ase1/Prc1 proteins constitute a conserved microtubule-associated protein family that is implicated in central spindle formation and cytokinesis. Here we characterize a role for fission yeast Ase1. Ase1 localizes to microtubule overlapping zones and displays dynamic alterations of localization during the cell cycle. In particular, its spindle localization during metaphase is reduced substantially, followed by robust appearance at the spindle midzone in anaphase. ase1 deletions are viable but defective in nuclear and septum positioning and completion of cytokinesis, which leads to diploidization and chromosome loss. Time-lapse imaging shows that elongating spindles collapse abruptly in the middle of anaphase B. Either absence or overproduction of Ase1 results in profound defects on microtubule bundling in an opposed manner, indicating that Ase1 is a dose-dependent microtubule-bundling factor. In contrast microtubule nucleating activities are not noticeably compromised in ase1 mutants. During meiosis astral microtubules are not bundled and oscillatory nuclear movement is impaired significantly. The Aurora kinase does not correctly localize to central spindles in the absence of Ase1. Finally Ase1 acts as a regulatory component in the cytokinesis checkpoint that operates to inhibit nuclear division when the cytokinesis apparatus is perturbed. Ase1, therefore, couples anaphase completion with cytokinesis upon cell division.     

Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

Background

Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro.

Methodology/Principal Findings

We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation.

Conclusions/Significance

These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.