The CPC (chromosomal passenger complex) performs essential roles in the regulation and co-ordination of chromosomal and cytoskeletal events during mitosis and meiosis. The first functional analyses showed evidence of a role of the CPC in the regulation of cytokinesis. In this review, I summarize what we have learned since then about the role of the CPC in the late stages of mitosis and cytokinesis.
Introduction: the start of the journey
Over 20 years ago, a screen for components of the chicken chromosome scaffold led to the discovery of a new kind of protein with a highly dynamic distribution in mitosis . These new chromosomal antigens associated generally with chromatin in early mitosis, and by prometaphase they concentrated in the inner centromere of the mitotic chromosomes. This localization gave the name to the INCENPs (inner centromere proteins). Most surprisingly, despite having been isolated as tightly bound chromosome scaffold components, the INCENPs left the chromosomes altogether during the metaphase to anaphase transition, transferred to the midzone microtubules and then concentrated in the midbody . This particular distribution led Earnshaw and Cooke  to hypothesize that the INCENPs travelled on chromosomes in order to be transported to the spindle midzone and eventually the cleavage furrow, “where they performed essential functions in the late stages of mitosis and cytokinesis”. The chromosomal passengers had started their journey.
There was indeed evidence for a role of the chromosomal passengers in cytokinesis from the start. In the earliest studies it was already noticed that during anaphase the INCENPs accumulated subjacent to the plasma membrane in the position where the contractile ring would eventually form but before there was any evidence of furrowing  or accumulation of myosin heavy chain . However, the first functional evidence for a role in cytokinesis came from expressing a chimaeric protein in which INCENP was fused to the centromeric protein CENP-B (centromere protein B): when INCENP was tethered to the centromere it caused a dominant cytokinesis failure . In cells overexpressing an N-terminal fragment of INCENP, the cleavage furrow started constricting but then regressed and cytokinesis failed . There was also an early cytokinesis connection for Aurora B kinase: AIM-1, a rat homologue of the Drosophila Aurora kinase, was localized similarly to INCENP in the midzone and midbody. Overexpression of a kinase-dead version of AIM-1 led to cleavage furrow disruption, failed cytokinesis and polyploidy . Also Caenorhabditis elegans embryos in which AIR-2 (the Aurora B orthologue) levels were depleted by RNAi (RNA interference) showed defects in the completion of cytokinesis: the cleavage furrow formed and initiated in the correct place, but then regressed prematurely .
We now know that INCENP is a targeting and regulatory subunit of Aurora B kinase, and that both of them are part of a protein complex termed the CPC (chromosomal passenger complex), which also comprises the proteins Survivin and Borealin/Dasra B . The CPC is a key regulator of mitosis and meiosis whose activity is essential for the accurate distribution of genetic material. The three targeting and activating subunits of the complex (INCENP, Survivin and Borealin/Dasra B) guide the Aurora B kinase to its substrates and modulate its activity in the different stages of cell division . CPC function is required in various processes from early mitosis to metaphase, including regulation of chromosome structure and cohesion, kinetochore maturation, kinetochore-microtubule attachment, chromosome alignment and control of the spindle checkpoint . In this review, I summarize what we have learned about the role of the CPC in the late stages of mitosis and cytokinesis in the last 20 years.
Transfer to the spindle
During the metaphase to anaphase transition of mitosis (but not meiosis, see [8,9]), the CPC leaves the inner centromere and transfers to the spindle midzone microtubules. However, association of the complex with centromeres is not a prerequisite for the transfer of the complex to the central spindle and execution of cytokinesis. The Drosophila mutants fusolo and solofuso show severe defects in chromosome segregation in meiosis, producing secondary spermatocytes that are devoid of chromosomes. These secondary spermatocytes recruit Aurora B normally to the central spindle in the second meiotic division despite the absence of chromosomes . In mammalian cells, a Survivin mutant lacking an intact BIR (baculovirus inhibitor of apoptosis protein repeat) domain does not target properly to inner centromeres, and does not recruit Aurora B, Borealin/Dasra B or BubRI. However, the CPC localizes correctly to central spindle and midbody and cytokinesis proceeds normally . The protein deubiquitinating enzyme hFAM regulates the dynamic association of Survivin with the centromeres . Depletion of hFAM by RNAi disrupts normal centromeric localization of Survivin and Aurora B; however, targeting to the central spindle microtubules and midbody is unaffected . The dynamic properties of some CPC components change when the complex moves to the spindle midzone in anaphase . The association of Survivin with centromeres until metaphase is highly dynamic; however, it becomes almost immobile upon association with microtubules in anaphase .
The transfer of the CPC to the central spindle microtubules in anaphase is a highly regulated process that probably involves changes in the ubiquitylation and phosphorylation state of different CPC subunits and requires the activity of several motor proteins.
Ubiquitylation may influence the transfer of Aurora B to the central spindle microtubules. In human cells, the Cul3 E3 ligase complex (Cul3/KLHL9/KLHL13) is required to remove Aurora B kinase from mitotic chromosomes . In the absence of Cul3/KLHL9/KLHL13 activity, Aurora B spreads along chromosomes and fails to dissociate from them during anaphase. Aurora B binds the substrate-recognition modules of the E3 ligase complex, KLHL9 and KLHL13, and is ubiquitylated in vivo in a Cul3- and KLHL9/KLHL13-dependent manner. However the possibility that the Cul3 E3 ligase complex regulates another member of the CPC has not been totally ruled out .
Transfer of Aurora B/INCENP (Ipl1/Sli15) to the central spindle in budding yeast depends on the dephosphorylation of INCENP by Cdc14 phosphatase upon activation by separase . In higher eukaryotes, the kinesin 6 motor protein MKLP2 [mitotic KLP (kinesin-like protein) 2] is required to transfer the CPC, Plk1 (Polo-like kinase 1) [17,18] and CDC14  to the spindle midzone at the onset of anaphase. Plk1 phosphorylates MKLP2, and this phosphorylation is in turn required for targeting Plk1 to the midzone (Figure 1).
Diagram showing the interactions of the CPC in the late stages of mitosis and cytokinesis
In human cells, MKLP2-mediated transfer of Aurora B to the central spindle is required for Aurora B to phosphorylate another kinesin, MKLP1 (see below; ). This specific phosphorylation site (Ser911) overlaps a nuclear localization sequence . A non-phosphorylatable MKLP1 mutant is imported to the nucleus prematurely and does not rescue the cytokinesis defects produced by MKLP1 depletion .
Regulation of central spindle formation and actomyosin ring formation
The central spindle is instrumental in the positioning and formation of the actomyosin ring. Essential for central spindle formation is the centralspindlin complex, a hetero-tetramer composed of the kinesin 6 superfamily member MKLP1 and MgcRacGAP [a Rho family GAP (GTPase-activating protein)] . Centralspindlin's ability to bundle microtubules is negatively regulated by CDK1 (cyclin-dependent kinase 1) phosphorylation until metaphase . MKLP1 transports MgcRacGAP to the equator where it binds Ect2 [a Rho GEF (guanine-nucleotide-exchange factor)] [22,23]. Ect2 is required to activate the small GTPase RhoA in a localized way in the equatorial region of the cell membrane [22,23a]. This activation of RhoA in turn promotes actin polymerization and myosin II activation, and therefore causes contractile ring formation (Figure 1)
In order to understand how Aurora B regulates different aspects of cytokinesis, it is essential to analyse the way in which it regulates the centralspindlin complex. The localization of the CPC partially overlaps with centralspindlin in the central spindle. CPC function is required for stable association of centralspindlin with the spindle midzone in C. elegans [24,25]. In human cells treated with Aurora B inhibitors, centralspindlin is delocalized and central spindle assembly is defective . Aurora B phosphorylates MKLP1 on Ser708. A non-phosphorylatable mutant localizes properly to the central spindle, but impairs completion of cytokinesis . In Drosophila cells, depletion of CPC components by RNAi led to conflicting results [28,29]; however, analysis of Incenp mutants in meiosis revealed defects in central spindle formation and Pavarotti-KLP (MKLP1 homologue) localization (M. Carmena, unpublished work; Figure 2)
Drosophila Incenp mutants show defects in central spindle organization and Pavarotti-MKLP localization in male meiosis
Aurora B may also regulate MgcRacGAP directly. Aurora B phosphorylates MgcRacGAP, and overexpression of a non-phosphorylatable mutant disrupts the final stages of cytokinesis. MgcRacGAP phosphorylation by Aurora B was reported to induce GAP activity towards RhoA in vitro, and may therefore be important for the regulation of MgcRacGAP activity in vivo .
It has recently been reported that Aurora B may also participate in the activation of RhoA through GEF-H1, a microtubule-binding Rho GEF. Aurora A/Aurora B and Cdk1/cyclin B are involved in the inhibitory phosphorylation of GEF-H1; dephosphorylation of GEF-H1 promotes the activation of Rho A .
Septins are GTP-binding cytoskeletal proteins that assemble filamentous structures and are required for diverse functions in development and cytokinesis . In budding yeast, septins are essential for polarized growth, and during cytokinesis they provide a scaffold for the assembly of the contractile ring. Studies in budding yeast have led to the proposal that a CPC subcomplex consisting of Bir1p-Sli15 (Survivin-INCENP homologues) is involved in the regulation of septin dynamics in anaphase . There is no evidence of a similar regulatory mechanism in higher eukaryotes, though it has been shown that septin 1 is an Aurora B substrate in vitro and the two proteins interact in vivo in human cells .
Abcission: IF (intermediate filament) regulation
Protein phosphorylation and dephosphorylation of IFs in the cleavage furrow is important for the efficient segregation of IFs into the daughter cells (for a review see ). Both Aurora B and Rho kinases are involved in these phosphorylation events, which are believed to play a role in the local breakdown of the filaments [36,37]. Some of the phosphorylation sites in IF proteins are shared by Aurora B and Rho kinase [Thr7, Ser13 and Ser38 in GFAP (glial fibrillar acidic protein) and Thr16 in Desmin]; others are specific for Aurora B kinase (Ser59 in Desmin). Mutation of the Aurora B or Rho kinase phosphorylation sites in Desmin lead to defects in filament separation between daughter cells . Aurora B also phosphorylates vimentin at Ser72. Mutation of the Aurora B phosphorylation site leads to formation of IF bridges and multinucleation .
Abcission: the NoCut pathway
Aurora B may also be involved in the inhibition of abcission. In budding yeast, Ipl1 is required for a signalling pathway that is proposed to delay cytokinesis while there is still chromatin in the midzone. The NoCut pathway involves the anillin-like proteins Boi1 and Boi2 which depend on Ipl1 for their correct localization .
Conclusions and perpectives
Almost two decades of research into CPC function have confirmed the early indications that the complex plays an important role in cytokinesis. Aurora B is required for the regulation of the components of the centralspindlin complex, which is essential for central spindle formation and assembly of the actomyosin ring through RhoA activation. Aurora B may also contribute to the activation of RhoA through the regulation of GEF-H1. During abscission, Aurora B phosphorylates IF proteins in the midbody, contributing to the local breakdown of filaments. In yeast, Ipl1 is also involved in the regulation of septins.
In the near future, it will be of special interest to find out if Aurora regulates anillin-like proteins in animal cells by a mechanism similar to the NoCut pathway in yeast. It will also be important to determine whether Aurora B has a role in regulating components involved in the membrane trafficking events of abcission. Future research will undoubtedly reveal further targets of Aurora B in cytokinesis.
Mechanics and Control of Cytokinesis: Biochemical Society Focused Meeting held at Royal College of Surgeons, Edinburgh, U.K., 9–12 January 2008. Organized and Edited by Gwyn Gould (Glasgow, U.K.) and Iain Hagan (Manchester, U.K.).
I thank William C. Earnshaw, Sandrine Ruchaud, Kumiko Samejima and Richard R. Adams for critical reading of the manuscript and comments. M.C.'s work is supported by a Wellcome Trust Programme Grant to W.C. Earnshaw.