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Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) have been described in many cell types. The functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans, additional MTOCs have been discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates the activities of the SPB outer plaque and the sMTOC. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest that the polo-like kinase is a regulator of MTOC activities and acts as a scaffolding unit through interaction with γ-TuRC receptors.

Breast cancer gene 1 (BRCA1) contributes to the regulation of centrosome number. We previously identified receptor for activated C kinase 1 (RACK1) as a BRCA1-interacting partner. RACK1, a scaffold protein that interacts with multiple proteins through its seven WD40 domains, directly binds to BRCA1 and localizes to centrosomes. RACK1 knockdown suppresses centriole duplication, whereas RACK1 overexpression causes centriole overduplication in a subset of mammary gland-derived cells. In this study, we showed that RACK1 binds directly to polo-like kinase 1 (PLK1) and Aurora A, and promotes the Aurora A-PLK1 interaction. RACK1 knockdown decreased phosphorylated PLK1 (p-PLK1) levels and the centrosomal localization of Aurora A and p-PLK1 in S phase, whereas RACK1 overexpression increased p-PLK1 level and the centrosomal localization of Aurora A and p-PLK1 in interphase, resulting in an increase of cells with abnormal centriole disengagement. Overexpression of cancer-derived RACK1 variants failed to enhance the Aurora A-PLK1 interaction, PLK1 phosphorylation and the centrosomal localization of p-PLK1. These results suggest that RACK1 functions as a scaffold protein that promotes the activation of PLK1 by Aurora A in order to promote centriole duplication.This article has an associated First Person interview with the first author of the paper.

Inactivation of cyclin-dependent kinase (Cdk) and reversal of Cdk phosphorylation are universally required for mitotic exit. In budding yeast (Saccharomyces cerevisiae), Cdc14 is essential for both and thought to be the major Cdk-counteracting phosphatase. However, Cdc14 is not required for mitotic exit in many eukaryotes, despite highly conserved biochemical properties. The question of how similar enzymes could have such disparate influences on mitotic exit prompted us to re-examine the contribution of budding yeast Cdc14. By using an auxin-inducible degron, we show that severe Cdc14 depletion has no effect on the kinetics of mitotic exit and bulk Cdk substrate dephosphorylation, but causes a cell separation defect and is ultimately lethal. Phosphoproteomic analysis revealed that Cdc14 is highly selective for distinct Cdk sites in vivo and does not catalyze widespread Cdk substrate dephosphorylation. We conclude that additional phosphatases likely contribute substantially to Cdk substrate dephosphorylation and coordination of mitotic exit in budding yeast, similar to in other eukaryotes, and the critical mitotic exit functions of Cdc14 require trace amounts of enzyme. We propose that Cdc14 plays very specific, and often different, roles in counteracting Cdk phosphorylation in all species.

When cells are exposed to stress they delay entry into mitosis. The most extensively studied mechanism behind this delay is the DNA-damage-induced G2/M checkpoint. Here, we show the existence of an additional stress-response pathway in Schizosaccharomyces pombe that is independent of the classic ATR/Rad3-dependent checkpoint. This novel mechanism delays entry mitosis independently of the spindle assembly checkpoint and the mitotic kinases Fin1, Ark1 and Plo1. The pathway delays activation of the mitotic cyclin-dependent kinase (CDK) Cdc2 after UV irradiation. Furthermore, we demonstrate that translation of the mitotic cyclin Cdc13 is selectively downregulated after UV irradiation, and we propose that this downregulation of Cdc13 contributes to the delayed activation of Cdc2 and the delayed mitosis.

Clathrin, a protein best known for its role in membrane trafficking, has been recognised for many years as localising to the spindle apparatus during mitosis, but its function at the spindle remained unclear. Recent work has better defined the role of clathrin in the function of the mitotic spindle and proposed that clathrin crosslinks the microtubules (MTs) comprising the kinetochore fibres (K-fibres) in the mitotic spindle. This mitotic function is unrelated to the role of clathrin in membrane trafficking and occurs in partnership with two other spindle proteins: transforming acidic coiled-coil protein 3 (TACC3) and colonic hepatic tumour overexpressed gene (ch-TOG; also known as cytoskeleton-associated protein 5, CKAP5). This review summarises the role of clathrin in mitotic spindle organisation with an emphasis on the recent discovery of the TACC3–ch-TOG–clathrin complex.

The mitotic spindle in eukaryotic cells is composed of a bipolar array of microtubules (MTs) and associated proteins that are required during mitosis for the correct partitioning of the two sets of chromosomes to the daughter cells. In addition to the well-established functions of MT-associated proteins (MAPs) and MT-based motors in cell division, there is increasing evidence that the F-actin-based myosin motors are important mediators of F-actin–MT interactions during mitosis. Here, we report the functional characterization of the long-tailed class-1 myosin myosin-1C from Dictyostelium discoideum during mitosis. Our data reveal that myosin-1C binds to MTs and has a role in maintenance of spindle stability for accurate chromosome separation. Both myosin-1C motor function and tail-domain-mediated MT–F-actin interactions are required for the cell-cycle-dependent relocalization of the protein from the cell periphery to the spindle. We show that the association of myosin-1C with MTs is mediated through the tail domain. The myosin-1C tail can inhibit kinesin motor activity, increase the stability of MTs, and form crosslinks between MTs and F-actin. These data illustrate that myosin-1C is involved in the regulation of MT function during mitosis in D. discoideum.

CK2 is a serine/threonine kinase with many substrates, largely unknown modes of regulation and essential roles in mitotic progression. CK2α, a catalytic subunit of CK2, is phosphorylated in mitosis, and here we examine the effect of phosphorylation on CK2α localization. Using phosphospecific antibodies, we show that CK2α localizes to the mitotic spindle in a phosphorylation-dependent manner. Mitotic spindle localization requires the unique C-terminus of CK2α, and involves a novel regulatory mechanism in which phosphorylation of CK2α facilitates binding to the peptidyl-prolyl isomerase Pin1, which is required for CK2α mitotic spindle localization. This could explain how the constitutive activity of CK2α might be targeted towards mitotic substrates. Furthermore, because Pin1 has many important spindle substrates, this might represent a general mechanism for localization of mitotic signalling proteins.

Dynamic control of protein phosphorylation is necessary for the regulation of many cellular processes, including mitosis and cytokinesis. Indeed, although the central role of protein kinases is widely appreciated and intensely studied, the importance of protein phosphatases is often overlooked. Recent studies, however, have highlighted the considerable role of protein phosphatases in both the spatial and temporal control of protein kinase activity, and the modulation of substrate phosphorylation. Here, we will focus on recent advances in our understanding of phosphatase structure, and the importance of phosphatase function in the control of mitotic spindle formation, chromosome architecture and cohesion, and cell division.

The p38 mitogen-activated protein kinase (p38 MAPK) family, which is comprised of four protein isoforms, p38α, p38β, p38γ and p38δ, forms one of the key MAPK pathways. The p38 MAPKs are implicated in many cellular processes including inflammation, differentiation, cell growth, cell cycle and cell death. The function of p38 MAPKs in mitotic entry has been well established, but their role in mitotic progression has remained controversial. We identify p38γ MAPK as a modulator of mitotic progression and mitotic cell death. In HeLa cells, loss of p38γ results in multipolar spindle formation and chromosome misalignment, which induce a transient M phase arrest. The majority of p38γ-depleted cells die at mitotic arrest or soon after abnormal exit from M-phase. We show that p38 MAPKs are activated at the kinetochores and spindle poles throughout mitosis by kinase(s) that are stably bound to these structures. Finally, p38γ is required for the normal kinetochore localization of polo-like kinase 1 (Plk1), and this contributes to the activity of the p38 MAPK pathway. Our data suggest a link between mitotic regulation and the p38 MAPK pathway, in which p38γ prevents chromosomal instability and supports mitotic cell viability.

International audience; Recent results indicate that regulating the balance between cyclin-B-Cdc2 kinase, also known as M-phase-promoting factor (MPF), and protein phosphatase 2A (PP2A) is crucial to enable correct mitotic entry and exit. In this work, we studied the regulatory mechanisms controlling the cyclin-B-Cdc2 and PP2A balance by analysing the activity of the Greatwall kinase and PP2A, and the different components of the MPF amplification loop (Myt1, Wee1, Cdc25) during the first embryonic cell cycle. Previous data indicated that the Myt1-Wee1-Cdc25 equilibrium is tightly regulated at the G2-M and M-G1 phase transitions; however, no data exist regarding the regulation of this balance during M phase and interphase. Here, we demonstrate that constant regulation of the cyclin-B-Cdc2 amplification loop is required for correct mitotic division and to promote correct timing of mitotic entry. Our results show that removal of Cdc25 from metaphase-II-arrested oocytes promotes mitotic exit, whereas depletion of either Myt1 or Wee1 in interphase egg extracts induces premature mitotic entry. We also provide evidence that, besides the cyclin-B-Cdc2 amplification loop, the Greatwall-PP2A pathway must also be tightly regulated to promote correct first embryonic cell division. When PP2A is prematurely inhibited in the absence of cyclin-B-Cdc2 activation, endogenous cyclin-A-Cdc2 activity induces irreversible aberrant mitosis in which there is, first, partial transient phosphorylation of mitotic substrates and, second, subsequent rapid and complete degradation of cyclin A and cyclin B, thus promoting premature and rapid exit from mitosis.

Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates ∼3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5[Thr926Ala]. Despite substantial overexpression, Eg5[Ser1033Ala] rescued 50% of cells compared with wild-type Eg5, whereas an Eg5[Ser1033Asp] mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.

The protein kinase Cdc2p is the master regulator of cell cycle progression in the fission yeast Schizosaccharomyces pombe. It is required both for entry into mitosis and for onset of DNA replication. Cdc2p must be inactivated to permit exit from mitosis, licensing of replication origins and cytokinesis. To study the role of Cdc2p in greater detail, we generated a cdc2 allele that is sensitive to an inhibitory ATP analogue. We show that the inhibitor-induced cell cycle arrest is reversible and examine the effect of inhibiting Cdc2p on the regulation of the septation initiation network (SIN), which controls the initiation of cytokinesis in S. pombe. We found that specific inactivation of Cdc2p in a mitotically arrested cell promotes the asymmetrical recruitment of SIN proteins to the spindle poles and the recruitment of the most downstream SIN components and β-(1,3) glucan synthase to the contractile ring. Thus, we conclude that inactivation of Cdc2p is sufficient to activate the SIN and promote cytokinesis.

Orderly progression through the eukaryotic cell cycle is a complex process involving both regulation of cyclin dependent kinase activity and control of specific substrate-Cdk interactions. In Saccharomyces cerevisiae, the mitotic cyclin Clb2 has a central role in regulating the onset of anaphase and in maintaining the cellular shape of the bud by inhibiting growth polarization induced in G1. However, how Clb2 and the partially redundant cyclin Clb1 confer specificity to Cdk1 in these processes still remains unclear. Here, we show that Clb2 mutants impaired in nuclear import or export are differentially affected for subsets of Clb2 functions while remaining fully functional for others. Our data support a direct role of the cytoplasmic pool of Clb1,2-Cdk1 in terminating cytoskeleton and growth polarization, independently of G1 cyclin transcriptional regulation. By contrast, the nuclear form of the cyclin is required for timely initiation of anaphase. Clb2 localization influences its stage-specific degradation as well. We report that Clb2 trapped in the cytoplasm is stabilized during anaphase but not at the time of mitotic exit. Altogether, our results demonstrate that the subcellular localization of the mitotic cyclin Clb2 is one of the key determinants of its biological function.

Two relatives of the GIN4 protein kinase family, Cdr1p and Cdr2p, exist in the yeast Schizosaccharomyces pombe. Although in Saccharomyces cerevisiae GIN4-related kinases influence septin ring organization and septin rings influence the localization and function of GIN4-related protein kinases, it is unknown whether this relationship is conserved in S. pombe. Here, we have probed the relationship between Cdr2p activity and septins and find that Cdr2p and septins are functionally independent. Cdr2p localizes in a cortical band overlying the nucleus during interphase, whose dimension is proportional to cell length, and to a medial ring structure in late mitosis. Both localizations are septin-independent and disrupted by treatment with filipin. Structure/function analysis indicates that the intracellular targeting domain of Cdr2p is largely contained within its non-catalytic C-terminus. Cdr2 protein kinase activity, while unimportant for its localization, is critical for its cell cycle function. Our data indicate that Cdr2p functions at two positions within the cell at discrete cell cycle stages to influence the timing of mitotic entry and cytokinesis, respectively.

Protein kinase CK2, a vital, pleiotropic and highly conserved serine/threonine phosphotransferase is involved in transcription-directed signaling, gene control and cell cycle regulation and is suspected to play a role in global processes. Searching for these global roles, we analyzed the involvement of CK2 in gene expression at cell cycle entry by using genome-wide screens. Comparing expression profiles of Saccharomyces cerevisiaewild-type strains with strains with regulatory or catalytic subunits of CK2 deleted, we found significant alterations in the expression of genes at all cell cycle phases and often in a subunit- and isoform-specific manner. Roughly a quarter of the genes known to be regulated by the cell cycle are affected. Functionally, the genes are involved with cell cycle entry, progression and exit, including spindle pole body formation and dynamics. Strikingly, most CK2-affected genes exhibit no common transcriptional control features, and a considerable proportion of temporarily altered genes encodes proteins involved in chromatin remodeling and modification, including chromatin assembly,(anti-)silencing and histone (de-)acetylation. In addition, various metabolic pathway and nutritional supply genes are affected. Our data are compatible with the idea that CK2 acts at different levels of cellular organization and that CK2 has a global role in transcription-related chromatin remodeling.

The centrosome acts as the major microtubule-organizing center (MTOC) for cytoskeleton maintenance in interphase and mitotic spindle assembly in vertebrate cells. It duplicates only once per cell cycle in a highly spatiotemporally regulated manner. When the cell undergoes mitosis, the duplicated centrosomes separate to define spindle poles and monitor the assembly of the bipolar mitotic spindle for accurate chromosome separation and the maintenance of genomic stability. However, centrosome abnormalities occur frequently and often lead to monopolar or multipolar spindle formation, which results in chromosome instability and possibly tumorigenesis. A number of studies have begun to dissect the role of mitotic kinases, including NIMA-related kinases (Neks), cyclin-dependent kinases (CDKs), Polo-like kinases (Plks) and Aurora kinases, in regulating centrosome duplication, separation and maturation and subsequent mitotic spindle assembly during cell cycle progression. In this Commentary, we review the recent research progress on how these mitotic kinases are coordinated to couple the centrosome cycle with the cell cycle, thus ensuring bipolar mitotic spindle fidelity. Understanding this process will help to delineate the relationship between centrosomal abnormalities and spindle defects.

mDia1 is a mammalian homolog of Drosophila diaphanous and works as an effector of the small GTPase Rho. It is a member of the formin homology (FH) proteins and contains the Rho-binding domain and an FH3 region in its N terminus, an FH1 region containing polyproline stretches in the middle and an FH2 region in the C terminus. Several lines of evidence indicate that mDia1 and diaphanous are essential in cytokinesis. mDia1 is present in a large amount in the cytoplasm of both interphase and mitotic cells. Using the instantaneous fixation method that preferentially extracts soluble components, we have analyzed localization of mDia1 in mitotic HeLa cells. Immunocytochemistry using polyclonal anti-mDia1 antibody revealed specific immunofluorescence localized to the mitotic spindle. This localization was seen from prophase to telophase. Western blot analysis also detected anti-mDia1 immunoreactivity in the mitotic spindle fraction isolated from mitotic HeLa cells. Consistently, expression of full-length mDia1 as a fusion protein with green fluorescence protein (GFP) revealed the GFP fluorescence again in the mitotic spindle in HeLa cells. Expression of GFP fusions of various truncated mutants of mDia1 identified that this localization is determined by a 173 amino acid-long sequence between the Rho-binding domain and the FH1 region, which contains the C-terminal part of the FH3 region. Point mutation analysis revealed that Leu(434) and Leu(455) in the FH3 region are essential in localization to the mitotic spindle. Neither electroporation of botulinum C3 exoenzyme nor microinjection of Val14RhoA into mitotic cells affected the localization of endogenous mDia1 to the mitotic spindle, suggesting that mDia1 localizes to the mitotic spindle independent of Rho activity. The present study has thus established the mDia1 localization in the mitotic spindle. This localization suggests a role of mDia1 in the spindle-cleavage furrow interaction during cell division.

Present in organisms ranging from yeast to man, homologues of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme CDC34 have been shown to play important roles in the regulation of cell cycle progression and checkpoint function. Here we analyze the expression and intracellular localization of endogenous CDC34 during mammalian cell cycle progression. We find that CDC34 protein is constitutively expressed during all stages of the cell cycle. Immunofluorescence experiments reveal that during interphase, endogenous CDC34 is localized to distinct speckles in both the nucleus and the cytoplasm. The presence of CDC34 in these compartments has also been established by biochemical fractionation experiments. Interestingly, nuclear localization depends on the presence of specific carboxy-terminal CDC34 sequences that have previously been shown to be required for CDC34's cell cycle function in Saccharomyces cerevisiae. Finally, we find that in anaphase and not during early stages of mitosis, CDC34 colocalizes with (beta)-tubulin at the mitotic spindle, implying that it may contribute to spindle function at later stages of mitosis. Taken together, these results support a model in which CDC34 ubiquitin-conjugating enzyme functions in the regulation of nuclear and cytoplasmic activities as well as in the process of chromosome segregation at the onset of anaphase in mammalian cells.

Schizosaccharomyces pombe cdc16p is required to limit the cell to forming a single division septum per cell cycle; the heat-sensitive loss-of-function mutant cdc16-116 completes mitosis, and then undergoes multiple rounds of septum formation without cell cleavage. cdc16p is a homologue of Saccharomyces cerevisiae BUB2p, and has also been implicated in the spindle assembly checkpoint function in S. pombe. To identify other proteins involved in regulating septum formation, we have screened for multicopy suppressors of the cdc16-116 mutation. In this paper, we describe one of these suppressors, zfs1. The null allele (zfs1-D1) is viable. However, at low temperatures it divides at a reduced size, while at higher temperatures, it partially suppresses heat sensitive mutants in genes signalling the onset of septum formation. Zfs1-D1 cells show an increased rate of chromosome loss during exponential growth. Moreover, if assembly of the spindle is prevented, zfs1-D1 cells do not arrest normally, but the activity of cdc2p kinase decays, and cells form a division septum without completing a normal mitosis. We conclude that zfs1 function is required to prevent septum formation and exit from mitosis if the mitotic spindle is not assembled. The suppression of cdc16-116 by zfs1 is independent of dma1 function and the spindle assembly checkpoint genes mad2 and mph1. The genetic interactions of zfs1 with genes regulating septum formation suggest that it may be a modulator of the signal transduction network controlling the onset of septum formation and exit from mitosis.

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

The molecular identification of an evolutionarily conserved set of cell cycle regulators in yeast, Xenopus egg extracts, and vertebrate cell culture has opened up a new perspective for understanding the mechanisms that regulate cell proliferation during metazoan development. Now we can study how the crucial regulators of eukaryotic cell cycle progression, the various cyclin/cdk complexes (for a recent review see Nigg (1995) BioEssays 17, 471–480), are turned on or off during development. In Drosophila, this analysis is most advanced, in particular in the case of the rather rigidly programmed embryonic cell cycles that generate the cells of the larvae. In addition, this analysis has revealed how the mitotic cycle is transformed into an endocycle which allows the extensive growth of larvae and oocytes. In contrast, we know little about cyclin/cdk regulation during the imaginal proliferation that generates the cells of the adult. Nevertheless, we will also consider this second developmental phase with its conspicuous regulative character, because it will be of great interest for the analysis of the molecular mechanisms that integrate growth and proliferation during development.

Mitotic spindle assembly in animal cells is orchestrated by a chromosome-dependent pathway that directs microtubule stabilization. RanGTP generated at chromosomes releases spindle assembly factors from inhibitory complexes with importins, the nuclear transport factors that facilitate protein import into the nucleus during interphase. In addition, the nuclear export factor Crm1 has been proposed to act as a mitotic effector of RanGTP through the localized assembly of protein complexes on the mitotic spindle, notably at centrosomes and kinetochores. It has been unclear, however, how the functions of nuclear transport factors are controlled during mitosis. Here, we report that human Crm1 is phosphorylated at serine 391 in mitosis by CDK1-cyclin-B (i.e. the CDK1 and cyclin B complex). Expression of Crm1 with serine 391 mutated to either non-phosphorylated or phosphorylation-mimicking residues indicates that phosphorylation directs the localization of Crm1 to the mitotic spindle and facilitates spindle assembly, microtubule stabilization and chromosome alignment. We find that phosphorylation of Crm1 at serine 391 enhances its RanGTP-dependent interaction with RanGAP1-RanBP2 and promotes their recruitment to the mitotic spindle. These results show that phosphorylation of Crm1 controls its molecular interactions, localization and function during mitosis, uncovering a new mechanism for the control of mitotic spindle assembly by CDK1-cyclin-B. We propose that nuclear transport factors are controlled during mitosis through the selection of specific molecular interactions by protein phosphorylation.

NuMA is a 236 kDa intranuclear protein that is distributed into each daughter cell during mitosis through association with the pericentrosomal region of the mitotic spindle. NuMA's interaction with the microtubules of the mitotic spindle is mediated through its 45 kDa carboxyl-terminal globular tail, and there is indirect evidence suggesting that NuMA's interaction with the mitotic spindle is controlled in a mitosis-specific manner. Consistent with this evidence is the fact that all four of the predicted p34cdc2 consensus phosphorylation sites in the NuMA protein are located in the carboxyl-terminal globular domain, and we demonstrate here that NuMA is phosphorylated in a mitosis-specific fashion in vivo. To test if the predicted p34cdc2 phosphorylation sites are necessary for NuMA's mitosis-specific interaction with the mitotic spindle, we have introduced mutations into the human NuMA cDNA that convert these predicted p34cdc2 phosphorylation sites from threonine or serine residues into alanine residues, and subsequently determined the cell cycle-dependent localization of these altered NuMA proteins following their expression in tissue culture cells. While none of these specific mutations in the NuMA sequence alters the faithful targeting of the protein into the interphase nucleus, mutation of threonine residue 2040 alone or in combination with mutations in other potential p34cdc2 phosphorylation sites abolishes NuMA's ability to associate normally with the microtubules of the mitotic spindle. Instead of binding to the mitotic spindle these mutant forms of NuMA concentrate at the plasma membrane of the mitotic cell. Cells expressing these mutant forms of NuMA have disorganized mitotic spindles, fail to complete cytokinesis normally, and assemble micronuclei in the subsequent interphase. These data suggest that NuMA's interaction with the microtubules of the mitotic spindle is controlled by cell cycle-dependent phosphorylation in addition to differential subcellular compartmentalization, and the characteristics of the dominant negative phenotype induced by these mutant forms of NuMA support a role for NuMA in the organization of the mitotic spindle apparatus.

SUMMARY The eukaryotic cell nucleus is a highly dynamic organelle. This is illustrated most dramatically during mitosis, when the nuclear envelope breaks down, the nuclear lamina disassembles, chromosomes condense, and a microtubule-based spindle apparatus distributes sister chromatids to the dividing daughter cells. Many of these dramatic changes in nuclear architecture and microtubule organization are controlled by phosphorylation and dephosphorylation events. Whereas the cardinal role of cyclin-dependent kinases (CDKs) in the regulation of mitosis is well established, there is now clear evidence for the requirement of additional mitotic protein kinases. Studies into the regulation of CDKs and other mitotic kinases have revealed that these enzymes undergo cell cycle dependent changes in subcellular distribution, suggesting that localization may contribute to regulating their activities. This article describes some recent findings relating to the nucleocytoplasmic translocation of CDK/cyclin complexes at the onset of mitosis. In addition, it summarizes recent information on two novel human protein kinases which have been implicated in the control of mitotic progression.

The impact of aberrant centrosomes and/or spindles on asymmetric cell division in embryonic development indicates the tight regulation of bipolar spindle formation and positioning that is required for mitotic progression and cell fate determination. WD40-repeat protein 62 (WDR62) was recently identified as a spindle pole protein linked to the neurodevelopmental defect of microcephaly but its roles in mitosis have not been defined. We report here that the in utero electroporation of neuroprogenitor cells with WDR62 siRNAs induced their cell cycle exit and reduced their proliferative capacity. In cultured cells, we demonstrated cell-cycle-dependent accumulation of WDR62 at the spindle pole during mitotic entry that persisted until metaphase–anaphase transition. Utilizing siRNA depletion, we revealed WDR62 function in stabilizing the mitotic spindle specifically during metaphase. WDR62 loss resulted in spindle orientation defects, decreased the integrity of centrosomes displaced from the spindle pole and delayed mitotic progression. Additionally, we revealed JNK phosphorylation of WDR62 is required for maintaining metaphase spindle organization during mitosis. Our study provides the first functional characterization of WDR62 and has revealed requirements for JNK/WDR62 signaling in mitotic spindle regulation that may be involved in coordinating neurogenesis.

Polo-like kinases play an important role in a variety of mitotic events in mammalian cells, ranging from centriole separation and chromosome congression to abscission. To fulfill these roles, PLK homologs move to different cellular locations as the cell cycle progresses, starting at the centrosome, progressing to the spindle poles and then the midbody. In the protist parasite Trypanosoma brucei, the single polo-like kinase homolog TbPLK is essential for cytokinesis and is necessary for the correct duplication of a centrin-containing cytoskeletal structure known as the bilobe. We show that TbPLK has a dynamic localization pattern during the cell cycle. The kinase localizes to the basal body, which nucleates the flagellum, and then successively localizes to a series of cytoskeletal structures that regulate the position and attachment of the flagellum to the cell body. The kinase localizes to each of these structures as they are duplicating. TbPLK associates with a specialized set of microtubules, known as the microtubule quartet, which may transport the kinase during its migration. Depletion of TbPLK causes defects in basal body segregation and blocks the duplication of the regulators that position the flagellum, suggesting that its presence on these structures might be necessary for their proper biogenesis. The ability of PLKs to migrate throughout the cell is preserved in T. brucei, but the specific locations to which it targets and functions are geared towards the inheritance of a properly positioned and attached flagellum.

polo and CDC5 are two genes required for passage through mitosis in Drosophila melanogaster and Saccharomyces cerevisiae, respectively. Both genes encode structurally related protein kinases that have been implicated in regulating the function of the mitotic spindle. Here, we report the characterization of a human protein kinase that displays extensive sequence similarity to Drosophila polo and S. cerevisiae Cdc5; we refer to this kinase as Plk1 (for polo-like kinase 1). The largest open reading frame of the Plk1 cDNA encodes a protein of 68,254 daltons, and a protein of this size is detected by immunoblotting of HeLa cell extracts with monoclonal antibodies raised against the C-terminal part of Plk1 expressed in Escherichia coli. Northern blot analysis of RNA isolated from human cells and mouse tissues shows that a single Plk1 mRNA of 2.3 kb is highly expressed in tissues with a high mitotic index, consistent with a possible function of Plk1 in cell proliferation. The Plk1 gene maps to position p12 on chromosome 16, a locus for which no associations with neoplastic malignancies are known. The Plk1 protein levels and its distribution change during the cell cycle, in a manner consistent with a role of Plk1 in mitosis. Thus, like Drosophila polo and S. cerevisiae Cdc5, human Plk1 is likely to function in cell cycle progression.

Acanthamoeba castellanii cells become growth arrested at different positions of the G2 phase of the cell cycle. Cells arresting at the competence position of G2 either develop into cysts in response to starvation or enter the mitotic division cycle in response to fresh nutrient medium. In cells arresting elsewhere in G2 growth can be initiated. The cell cycle position at which cells become growth arrested is dependent on the mode of cell cycle progression which, in turn, is strongly dependent on physical parameters of the cultivation conditions. Cells arresting at the competence position progress through cell cycles in which developmental competence and the so-called ‘growth-associated’ or ‘M phase-specific’ histone H1 kinase activity oscillate in levels. H1 kinase activity increases rapidly at the onset of mitosis and decreases to its basal level immediately after the completion of mitosis. In this case, peak levels of developmental competence slightly precede peak levels of H1 kinase activity. In contrast, cells arresting elsewhere in G2 are advanced through their cell cycle, i.e. undergo mitosis at reduced G2 phase length and smaller cell size compared with normal. The premature mitosis correlates in time with a rapid increase of H1 kinase activity. However, the extent of its activation is increased and its Lnactivation is slower so that H1 kinase activity does not decrease to its basal level during the shortened G2 phase. In this case a cell cycle period in which establishment of developmental competence takes place is lacking. In response to cycloheximide or vanadate, which delay the onset of premature mitosis, the competence period can be restored. H1 kinase activity is likely to represent p34cdc2 protein kinase activity, which has been shown in a wide variety of eukaryotic cell types to play a key role in regulating mitosis. Therefore, the results indicate that the mechanisms regulating development are coupled to controls acting over mitosis. In vitro translation patterns suggest that the length of G2 and/or a low basal level of H1 kinase activity is (are) important for regulating the cellular content of competence- and development-specific RNA species.

Spindle morphogenesis is regulated by cyclin-dependent kinases and monitored by checkpoint pathways to accurately coordinate chromosomal segregation with other events in the cell cycle. We have previously dissected the contribution of individual B-type cyclins to spindle morphogenesis in Saccharomyces cerevisiae. We showed that the S-phase cyclin Clb5p is required for coupling spindle assembly and orientation. Loss of Clb5p-dependent kinase abolishes intrinsic asymmetry between the spindle poles resulting in lethal translocation of the spindle into the bud with high penetrance in diploid cells. This phenotype was exploited in a screen for high dosage suppressors that yielded spc110(Delta)(13), encoding a truncation of the spindle pole body component Spc110p (the intranuclear receptor for the gamma-tubulin complex). We found that Clb5p-GFP was localised to the spindle poles and intranuclear microtubules and that Clb5p-dependent kinase promoted cell cycle dependent phosphorylation of Spc110p contributing to spindle integrity. Two cyclin-dependent kinase consensus sites were required for this phosphorylation and were critical for the activity of spc110(Delta)(13) as a suppressor. Together, our results point to the function of cyclin-dependent kinase phosphorylation of Spc110p and provide, in addition, support to a model for Clb5p control of spindle polarity at the level of astral microtubule organisation.

Cdc37 is a molecular chaperone whose clients are predominantly protein kinases, many of which are important in cell-cycle progression. Temperature-sensitive mutants of cdc37 in Schizosaccharomyces pombe are lethal at the restrictive temperature, arresting cell division within a single cell cycle. These mutant cells elongate during incubation at the restrictive temperature, consistent with a cell-cycle defect. The cell-cycle arrest arises from defective function of the mutant Cdc37 proteins rather than a reduction in Cdc37 protein levels. Around 80% of the arrested, elongated cells contain a single nucleus and replicated (2C) DNA content, indicating that these mutants arrest the cell cycle in G2 or mitosis (M). Cytological observations show that the majority of cells arrest in G2. In fission yeast, a G2 cell-cycle arrest can arise by inactivation of the cyclin-dependent kinase (Cdk) Cdc2 that regulates entry into mitosis. Studies of the cdc37 temperature-sensitive mutants show a genetic interaction with some cdc2 alleles and overexpression of cdc2 rescues the lethality of some cdc37 alleles at the restrictive temperature, suggesting that Cdc2 is a likely client for the Cdc37 molecular chaperone. In cdc37 temperature-sensitive mutants at the restrictive temperature, the level of Cdc2 protein remains constant but Cdc2 protein kinase activity is greatly reduced. Inactivation of Cdc2 appears to result from the inability to form complexes with its mitotic cyclin partner Cdc13. Further evidence for Cdc2 being a client of Cdc37 in S. pombe comes from the identification of genetic and biochemical interactions between these proteins.

Polo-like kinases and kinesin-like motor proteins are among the many proteins implicated in the execution of cytokinesis. Polo-like-kinase 1 (Plk1) interacts with the mitotic kinesin-like motor protein CHO1/MKLP-1 during anaphase and telophase, and CHO1/MKLP-1 is a Plk1 substrate in vitro. Here, we explore the molecular interactions of these two key contributors to mitosis and cytokinesis. Using the transient transfection approach, we show that the C-terminus of Plk1 binds CHO1/MKLP-1 in a Polo-box-dependent manner and that the stalk domain of CHO1/MKLP-1 is responsible for its binding to Plk1. The stalk domain was found to localize with Plk1 to the mid-body, and Plk1 appears to be mislocalized in CHO1/MKLP-1-depleted cells during late mitosis. We showed that Ser904 and Ser905 are two major Plk1 phosphorylation sites. Using the vector-based RNA interference approach, we showed that depletion of CHO1/MKLP-1 causes the formation of multinucleate cells with more centrosomes, probably because of a defect in the early phase of cytokinesis. Overexpression of a non-Plk1-phosphorylatable CHO1 mutant caused cytokinesis defects, presumably because of dominant negative effect of the construct. Finally, CHO1-depletion-induced multinucleation could be partially rescued by co-transfection of a non-degradable hamster wild-type CHO1 construct, but not an unphosphorylatable mutant. These data provide more detailed information about the interaction between Plk1 and CHO1/MKLP-1, and the significance of this is discussed.

Orderly progression through mitosis is essential to reduce segregation errors in the cell's genetic material. We have used a cytological screen to identify a mutant that progresses through mitosis aberrantly and have cloned the complementing gene, nimU, which encodes a protein related to Pot1 and other telomere end-binding proteins. We show that loss of nimU function leads to premature mitotic spindle elongation, premature mitotic exit, errors in chromosome segregation, and failure to delay mitotic exit under conditions that normally evoke the mitotic spindle checkpoint response. Whereas premature mitotic exit is dependent upon anaphase promoting complex function, premature spindle elongation is not. We conclude that nimU is constitutively required for orderly mitotic progression under normal growth conditions and also required for the conditional mitotic spindle checkpoint response.

Metazoans contain three aurora-related kinases. Aurora A is required for spindle formation while aurora B is required for chromosome condensation and cytokinesis. Less is known about the function of aurora C. S. pombe contains a single aurora-related kinase, Ark1. Although Ark1 protein levels remained constant as cells progressed through the mitotic cell cycle, its distribution altered during mitosis and meiosis. Throughout G2 Ark1 was concentrated in one to three nuclear foci that were not associated with the spindle pole body/centromere complex. Following commitment to mitosis Ark1 associated with chromatin and was particularly concentrated at several sites including kinetochores/centromeres. Kinetochore/centromere association diminished during anaphase A, after which it was distributed along the spindle. The protein became restricted to a small central zone that transiently enlarged as the spindle extended. As in many other systems mitotic fission yeast cells exhibit a much greater degree of phosphorylation of serine 10 of histone H3 than interphase cells. A number of studies have linked this modification with chromosome condensation. Ark1 immuno-precipitates phosphorylated serine 10 of histone H3 in vitro. This activity was highest in mitotic extracts. The absence of the histone H3 phospho-serine 10 epitope from mitotic cells in which the ark1+ gene had been deleted (ark1.Δ1); the inability of these cells to resolve their chromosomes during anaphase and the co-localisation of this phospho-epitope with Ark1 early in mitosis, all suggest that Ark1 phosphorylates serine 10 of histone H3 in vivo. ark1.Δ1 cells also exhibited a reduction in kinetochore activity and a minor defect in spindle formation. Thus the enzyme activity, localisation and phenotype arising from our manipulations of this single fission yeast aurora kinase family member suggest that this single kinase is executing functions that are separately implemented by distinct aurora A and aurora B kinases in higher systems.

In Saccharomyces cerevisiae, the phosphoprotein phosphatase Cdc14p plays a central role in exit from mitosis, by promoting B-type cyclin degradation and allowing accumulation of the cyclin-dependent kinase inhibitor Sic1p. Cdc14p is sequestered in the nucleolus during interphase, from where it is released at the end of mitosis, dependent upon mitotic exit network function. The CDC14 gene is essential and loss-of-function mutants arrest at the end of mitosis. We have identified a fission yeast orthologue of CDC14 through database searches. A Schizosaccharomyces pombe flp1 (cdc fourteen-like-phosphatase) null mutant is viable, divides at a reduced size and shows defects in septation. flp1p is not the essential effector of the S. pombe septation initiation network, but may potentiate signalling of the onset of septation. In contrast to S. cerevisiae Cdc14p, flp1p is not required for the accumulation or destruction of the B-type cyclin cdc13p, the cyclin-dependent kinase inhibitor rum1p, or for dephosphorylation of the APC/C specificity factor ste9p in G1. Like its budding yeast counterpart, flp1p is restricted to the nucleolus until mitosis, when it is dispersed through the nucleus. In contrast to S. cerevisiae Cdc14p, flp1p is also present on the mitotic spindle and contractile ring. The potential roles of flp1p in cell cycle control are discussed.

Drosophila 14-3-3 epsilon and 14-3-3 zeta proteins have been shown to function in RAS/MAP kinase pathways that influence the differentiation of the adult eye and the embryo. Because 14-3-3 proteins have a conserved involvement in cell cycle checkpoints in other systems, we asked (1) whether Drosophila 14-3-3 proteins also function in cell cycle regulation, and (2) whether cell proliferation during Drosophila development has different requirements for the two 14-3-3 proteins. We find that antibody staining for 14-3-3 family members is cytoplasmic in interphase and perichromosomal in mitosis. Using mutants of cyclins, Cdk1 and Cdc25(string) to manipulate Cdk1 activity, we found that the localization of 14-3-3 proteins is coupled to Cdk1 activity and cell cycle stage. Relocalization of 14-3-3 proteins with cell cycle progression suggested cell-cycle-specific roles. This notion is confirmed by the phenotypes of 14-3-3 epsilon and 14-3-3 zeta mutants: 14-3-3 epsilon is required to time mitosis in undisturbed post-blastoderm cell cycles and to delay mitosis following irradiation; 14-3-3 zeta is required for normal chromosome separation during syncytial mitoses. We suggest a model in which 14-3-3 proteins act in the undisturbed cell cycle to set a threshold for entry into mitosis by suppressing Cdk1 activity, to block mitosis following radiation damage and to facilitate proper exit from mitosis.

In eukaryotes an abnormal spindle activates a conserved checkpoint consisting of the MAD and BUB genes that results in mitotic arrest at metaphase. Recently, we and others identified a novel Bub2-dependent branch to this checkpoint that blocks mitotic exit. This cell-cycle arrest depends upon inhibition of the G-protein Tem1 that appears to be regulated by Bfa1/Bub2, a two-component GTPase-activating protein, and the exchange factor Lte1. Here, we find that Bub2 and Bfa1 physically associate across the entire cell cycle and bind to Tem1 during mitosis and early G1. Bfa1 is multiply phosphorylated in a cell-cycle-dependent manner with the major phosphorylation occurring in mitosis. This Bfa1 phosphorylation is Bub2-dependent. Cdc5, but not Cdc15 or Dbf2, partly controls the phosphorylation of Bfa1 and also Lte1. Following spindle checkpoint activation, the cell cycle phosphorylation of Bfa1 and Lte1 is protracted and some species are accentuated. Thus, the Bub2-dependent pathway is active every cell cycle and the effect of spindle damage is simply to protract its normal function. Indeed, function of the Bub2 pathway is also prolonged during metaphase arrests imposed by means other than checkpoint activation. In metaphase cells Bub2 is crucial to restrain downstream events such as actin ring formation, emphasising the importance of the Bub2 pathway in the regulation of cytokinesis. Our data is consistent with Bub2/Bfa1 being a rate-limiting negative regulator of downstream events during metaphase.

Dbf2 is a multifunctional protein kinase in Saccharomyces cerevisiae that functions in transcription, the stress response and as part of a network of genes in exit from mitosis. By analogy with fission yeast it seemed likely that these mitotic exit genes would be involved in cytokinesis. As a preliminary investigation of this we have used Dbf2 tagged with GFP to examine intracellular localisation of the protein in living cells. Dbf2 is found on the centrosomes/spindle pole bodies (SPBs) and also at the bud neck where it forms a double ring. The localisation of Dbf2 is cell cycle regulated. It is on the SPBs for much of the cell cycle and migrates from there to the bud neck in late mitosis, consistent with a role in cytokinesis. Dbf2 partly co-localises with septins at the bud neck. A temperature-sensitive mutant of dbf2 also blocks progression of cytokinesis at 37 degrees C. Following cytokinesis some Dbf2 moves into the nascent bud. Localisation to the bud neck depends upon the septins and also the mitotic exit network proteins Mob1, Cdc5, Cdc14 and Cdc15. The above data are consistent with Dbf2 acting downstream in a pathway controlling cytokinesis.

H1 histone kinase activity has been followed in selection-synchronised cultures of fission yeast wild-type and wee1 mutant cells, and in induction-synchronised cells of the mutant cdc2-33. The main conclusions are: (1) in all three cases, the peak of activity is near mitosis. (2) The rise in activity is relatively slow starting in wild type at 0.4 of the cycle before mitosis. It is proposed that the beginning of the rise is the first identified event in the mitotic control. (3) The rise is twice as fast in wee and starts nearer to mitosis. (4) In all cases the beginning of the rise is in G2. (5) The fall in activity is also slow, lasting for 0.25 of the cycle, in wild type. Exit from mitosis happens well before activity has fallen to baseline. (6) In a range of size mutants, activity is roughly proportional to cell size. It is suggested that the kinase may have a cytoplasmic function. (7) Estimates have been made of the timing of mitosis in the mutants. In wee, mitosis is 0.14 of the cycle earlier than in wild type because the cells have a longer septated period at the end of the cycle. (8) A novel method has been developed for eliminating the effects of the partial asynchrony in synchronous cultures, without which the kinetic analysis would have been inaccurate.

Cell divisions are sometimes oriented by extrinsic signals, by mechanisms that are poorly understood. Proteins containing TPR and GoLoco-domains (C. elegans GPR-1/2, Drosophila Pins, vertebrate LGN and AGS3) are candidates for mediating mitotic spindle orientation by extrinsic signals, but the mechanisms by which TPR-GoLoco proteins may localize in response to extrinsic cues are not well defined. The C. elegans TPR-GoLoco protein pair GPR-1/2 is enriched at a site of contact between two cells – the endomesodermal precursor EMS and the germline precursor P2 – and both cells align their divisions toward this shared cell-cell contact. To determine whether GPR-1/2 is enriched at this site within both cells, we generated mosaic embryos with GPR-1/2 bearing a different fluorescent tag in different cells. We were surprised to find that GPR-1/2 distribution is symmetric in EMS, where GPR-1/2 had been proposed to function as an asymmetric cue for spindle orientation. Instead, GPR-1/2 is asymmetrically distributed only in P2. We demonstrate a role for normal GPR-1/2 localization in P2 division orientation. We show that MES-1/Src signaling plays an instructive role in P2 for asymmetric GPR-1/2 localization and normal spindle orientation. We ruled out a model in which signaling localizes GPR-1/2 by locally inhibiting LET-99, a GPR-1/2 antagonist. Instead, asymmetric GPR-1/2 distribution is established by destabilization at one cell contact, diffusion, and trapping at another cell contact. Once the mitotic spindle of P2 is oriented normally, microtubule-dependent removal of GPR-1/2 prevented excess accumulation, in an apparent negative-feedback loop. These results highlight the role of dynamic TPR-GoLoco protein localization as a key mediator of mitotic spindle alignment in response to instructive, external cues.