RESUMO
During cytokinesis, signals from the anaphase spindle direct the formation and position of a contractile ring at the cell cortex [1]. The chromosomal passenger complex (CPC) participates in cytokinesis initiation by signaling from the spindle midzone and equatorial cortex [2], but the mechanisms underlying the anaphase-specific CPC localization are currently unresolved. Accumulation of the CPC at these sites requires the presence of microtubules and the mitotic kinesin-like protein 2, MKLP2 (KIF20A), a member of the kinesin-6 family [2-7], and this has led to the hypothesis that the CPC is transported along microtubules by MKLP2 [3-5, 7]. However, the structure of the MKLP2 motor domain with its extended neck-linker region suggests that this kinesin might not be able to drive processive transport [8, 9]. Furthermore, experiments in Xenopus egg extracts indicated that the CPC might be transported by kinesin-4, KIF4A [10]. Finally, CPC-MKLP2 complexes might be directly recruited to the equatorial cortex via association with actin and myosin II, independent of kinesin activity [4, 8]. Using microscopy-based assays with purified proteins, we demonstrate that MKLP2 is a processive plus-end directed motor that can transport the CPC along microtubules in vitro. In cells, strong suppression of MKLP2-dependent CPC motility by expression of an MKLP2 P-loop mutant perturbs CPC accumulation at both the spindle midzone and equatorial cortex, whereas a weaker inhibition of MKLP2 motor using Paprotrain mainly affects CPC localization to the equatorial cortex. Our data indicate that control of cytokinesis initiation by the CPC requires its directional MKLP2-dependent transport.
Assuntos
Anáfase/fisiologia , Citocinese , Cinesinas/genética , Família Multigênica , Células HEK293 , Células HeLa , Humanos , Cinesinas/metabolismo , Transporte ProteicoRESUMO
Aurora B kinase is essential for faithful chromosome segregation during mitosis. During (pro)metaphase, Aurora B is concentrated at the inner centromere by the kinases Haspin and Bub1. However, how Haspin and Bub1 collaborate to control Aurora B activity at centromeres remains unclear. Here, we show that either Haspin or Bub1 activity is sufficient to recruit Aurora B to a distinct chromosomal locus. Moreover, we identified a small, Bub1 kinase-dependent Aurora B pool that supported faithful chromosome segregation in otherwise unchallenged cells. Joined inhibition of Haspin and Bub1 activities fully abolished Aurora B accumulation at centromeres. While this impaired the correction of erroneous KT-MT attachments, it did not compromise the mitotic checkpoint, nor the phosphorylation of the Aurora B kinetochore substrates Hec1, Dsn1, and Knl1. This suggests that Aurora B substrates at the kinetochore are not phosphorylated by centromere-localized pools of Aurora B, and calls for a reevaluation of the current spatial models for how tension affects Aurora B-dependent kinetochore phosphorylation.
Assuntos
Aurora Quinase B/metabolismo , Segregação de Cromossomos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinetocoros/enzimologia , Microtúbulos/enzimologia , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas/genética , Cinesinas/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Fatores de TempoRESUMO
Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore-microtubule (KT-MT) attachments and for the stabilization of bi-oriented KT-MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation.
Assuntos
Aurora Quinase B/metabolismo , Centrômero/ultraestrutura , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Anáfase , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Segregação de Cromossomos , Cromossomos/metabolismo , Inativação Gênica , Células HeLa , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Fosforilação , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismoRESUMO
The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.
Assuntos
Baculoviridae/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Genoma Humano/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação Puntual , Proteínas Serina-Treonina Quinases/genéticaRESUMO
The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB) domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP's centromere-targeting domain (CEN box), which increases the MTB affinity of INCENP. In (pro)metaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onsetwhen the histone mark H2AT120 is dephosphorylatedINCENP and Aurora B switch from centromere to microtubule localization.
Assuntos
Aurora Quinase B/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Microtúbulos/metabolismo , Anáfase , Aurora Quinase B/genética , Segregação de Cromossomos , Células HeLa/fisiologia , Humanos , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Correction of faulty kinetochore-microtubule attachments is essential for faithful chromosome segregation and dictated by the opposing activities of Aurora B kinase and PP1 and PP2A phosphatases. How kinase and phosphatase activities are appropriately balanced is less clear. Here, we show that a centromeric pool of PP2A-B56 counteracts Aurora B T-loop phosphorylation and is recruited to centromeres through Shugoshin-1 (Sgo1). In non-transformed RPE-1 cells, Aurora B, Sgo1, and PP2A-B56 are enriched on centromeres and levels diminish as chromosomes establish bi-oriented attachments. Elevating Sgo1 levels at centromeres recruits excess PP2A-B56, and this counteracts Aurora B kinase activity, undermining efficient correction of kinetochore-microtubule attachment errors. Conversely, Sgo1-depleted cells display reduced centromeric localization of Aurora B, whereas the remaining kinase is hyperactive due to concomitant reduction of centromeric PP2A-B56. Our data suggest that Sgo1 can tune the stability of kinetochore-microtubule attachments through recruitment of PP2A-B56 that balances Aurora B activity at the centromere.
Assuntos
Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Pareamento Cromossômico , HumanosRESUMO
Aurora B localization to mitotic centromeres, which is required for proper chromosome alignment during mitosis, relies on Haspin-dependent histone H3 phosphorylation and on Bub1-dependent histone H2A phosphorylation--which interacts with Borealin through a Shugoshin (Sgo) intermediate. We demonstrate that Mps1 stimulates the latter recruitment axis. Mps1 activity enhances H2A-T120ph and is critical for Sgo1 recruitment to centromeres, thereby promoting Aurora B centromere recruitment in early mitosis. Importantly, chromosome biorientation defects caused by Mps1 inhibition are improved by restoring Aurora B centromere recruitment. As Mps1 kinetochore localization reciprocally depends on Aurora B, we propose that this Aurora B-Mps1 recruitment circuitry cooperates with the Aurora B-Haspin feedback loop to ensure rapid centromere accumulation of Aurora B at the onset of mitosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Células HeLa , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , FosforilaçãoRESUMO
To understand how the chromosomal passenger complex ensures chromosomal stability, it is crucial to identify its substrates and to find ways to specifically inhibit the enzymatic core of the complex, Aurora B. We therefore developed a chemical genetic approach to selectively inhibit human Aurora B. By mutating the gatekeeper residue Leu-154 in the kinase active site, the ATP-binding pocket was enlarged, but kinase function was severely disrupted. A unique second site suppressor mutation was identified that rescued kinase activity in the Leu-154 mutant and allowed the accommodation of bulky N(6)-substituted adenine analogs. Using this analog-sensitive Aurora B kinase, we found that retention of the chromosomal passenger complex at the centromere depends on Aurora B kinase activity. Furthermore, analog-sensitive Aurora B was able to use bulky ATPγS analogs and could thiophosphorylate multiple proteins in cell extracts. Utilizing an unbiased approach for kinase substrate mapping, we identified several novel substrates of Aurora B, including the nucleosomal-binding protein HMGN2. We confirmed that HMGN2 is a bona fide Aurora B substrate in vivo and show that its dynamic association to chromatin is controlled by Aurora B.
Assuntos
Proteína HMGN2/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Aurora Quinase B , Aurora Quinases , Domínio Catalítico , Extratos Celulares/química , Linhagem Celular Tumoral , Centrômero , Sequência Conservada , Difosfatos/farmacologia , Histonas/metabolismo , Humanos , Leucina/genética , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Mutagênese Sítio-Dirigida , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
BACKGROUND: Human embryos generated by IVF demonstrate a high incidence of chromosomal segregation errors during the cleavage divisions. To analyse underlying molecular mechanisms, we investigated the behaviour of the chromosomal passenger complex (CPC) in human oocytes and embryos. This important mitotic regulatory complex comprises the inner centromere protein (INCENP), survivin, borealin and Aurora B, or the meiotic kinase Aurora C. METHODS: We analysed mRNA expression by quantitative RT-PCR of all CPC members in human oocytes, tripronuclear (3PN) zygotes, 2-cell and 4-cell embryos developed from 3PN zygotes, plus good-quality cryopreserved 8-cell, morula and blastocyst stage embryos. Protein expression and localization of CPC members were investigated by immunofluorescence in oocytes and embryos arrested at prometaphase. Histone H3S10 phosphorylation was investigated as an indicator of a functional CPC. RESULTS: INCENP, survivin and borealin were detected at the inner centromere of prometaphase chromosomes in all stages investigated. Whereas Aurora B and C are both present in oocytes, Aurora C becomes the most prominent kinase in the CPC during the first three embryonic cell cycles. Moreover, Aurora C mRNA was up-regulated with Aurora B after activation of the embryonic genome and both proteins were detected in early Day 4 embryos. Subsequently, only Aurora B was detected in blastocysts. CONCLUSIONS: In contrast to somatic cells, our results point to a specific role for Aurora C in the CPC during human preimplantation embryo development. Although, the presence of Aurora C in itself may not explain the high chromosome segregation error rate, the data presented here provide novel information regarding possible mechanisms.
Assuntos
Blastocisto/enzimologia , Proteínas Cromossômicas não Histona/fisiologia , Desenvolvimento Embrionário/genética , Proteínas Serina-Treonina Quinases/fisiologia , Aurora Quinase B , Aurora Quinase C , Aurora Quinases , Blastocisto/metabolismo , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/análise , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas Inibidoras de Apoptose/análise , Proteínas Inibidoras de Apoptose/metabolismo , Masculino , Oócitos/enzimologia , Oócitos/metabolismo , Gravidez , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/metabolismo , Espermatócitos/enzimologia , SurvivinaRESUMO
Error-free chromosome segregation requires that all chromosomes biorient on the mitotic spindle. The motor protein Centromere-associated protein E (CENP-E) facilitates chromosome congression by mediating the lateral sliding of sister chromatids along existing K-fibers, while the mitotic kinase Aurora B detaches kinetochore-microtubule interactions that are not bioriented. Whether these activities cooperate to promote efficient chromosome biorientation and timely anaphase onset is not known. We here show that the chromosomes that fail to congress after CENP-E depletion displayed high centromeric Aurora B kinase activity. This activity destabilized spindle pole proximal kinetochore-microtubule interactions resulting in a checkpoint-dependent mitotic delay that allowed CENP-E-independent chromosome congression, thus reducing chromosome segregation errors. This shows that Aurora B keeps the mitotic checkpoint active by destabilizing kinetochore fibers of polar chromosomes to permit chromosome congression in CENP-E-compromised cells and implies that this kinase normally prevents pole proximal syntelic attachments to allow CENP-E-mediated congression of mono-oriented chromosomes.
Assuntos
Anáfase/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos Humanos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Células HeLa , Humanos , Cinetocoros/metabolismo , Microscopia de Fluorescência/métodos , Microtúbulos/metabolismo , Mitose , RNA Interferente Pequeno , Fuso Acromático/metabolismoRESUMO
Successful cell division requires that chromosomes attach to opposite poles of the mitotic spindle (bi-orientation). Aurora B kinase regulates chromosome-spindle attachments by phosphorylating kinetochore substrates that bind microtubules. Centromere tension stabilizes bi-oriented attachments, but how physical forces are translated into signaling at individual centromeres is unknown. Using fluorescence resonance energy transfer-based biosensors to measure localized phosphorylation dynamics in living cells, we found that phosphorylation of an Aurora B substrate at the kinetochore depended on its distance from the kinase at the inner centromere. Furthermore, repositioning Aurora B closer to the kinetochore prevented stabilization of bi-oriented attachments and activated the spindle checkpoint. Thus, centromere tension can be sensed by increased spatial separation of Aurora B from kinetochore substrates, which reduces phosphorylation and stabilizes kinetochore microtubules.
Assuntos
Centrômero/metabolismo , Cromossomos Humanos/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Aurora Quinase B , Aurora Quinases , Autoantígenos/metabolismo , Técnicas Biossensoriais , Linhagem Celular Tumoral , Centrômero/enzimologia , Proteína Centromérica A , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Mitose , Modelos Biológicos , Fosforilação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The chromosomal passenger complex (CPC) is a critical regulator of chromosome segregation during mitosis by correcting nonbipolar microtubule-kinetochore interactions. By severing these interactions, the CPC is thought to create unattached kinetochores that are subsequently sensed by the spindle assembly checkpoint (SAC) to prevent premature mitotic exit. We now show that spindle checkpoint function of the CPC and its role in eliminating nonbipolar attachments can be uncoupled. Replacing the chromosomal passenger protein INCENP with a mutant allele that lacks its coiled-coil domain results in an overt defect in a SAC-mediated mitotic arrest in response to taxol treatment, indicating that this domain is critical for CPC function in spindle checkpoint control. Surprisingly, this mutant could restore alignment and cytokinesis during unperturbed cell divisions and was capable of resolving syntelic attachments. Also, Aurora-B kinase was localized and activated normally on centromeres in these cells, ruling out a role for the coiled-coil domain in general Aurora-B activation. Thus, mere microtubule destabilization of nonbipolar attachments by the CPC is insufficient to install a checkpoint-dependent mitotic arrest, and additional, microtubule destabilization-independent CPC signaling toward the spindle assembly checkpoint is required for this arrest, potentially through amplification of the unattached kinetochore-derived checkpoint signal.