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1.
Sci China Life Sci ; 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38763998

RESUMO

Proper function of the centromeres ensures correct attachment of kinetochores to spindle microtubules and faithful chromosome segregation in mitosis. Defects in the integrity and function of centromeres can result in chromosome missegregation and genomic instability. Bub1 is essential for the mitotic centromere dynamics, yet the underlying molecular mechanisms remain largely unclear. Here, we demonstrate that TIP60 acetylates Bub1 at K424 and K431 on kinetochores in early mitosis. This acetylation increases the kinase activity of Bub1 to phosphorylate centromeric histone H2A at T120 (H2ApT120), which recruits Aurora B and Shugoshin 1 (Sgo1) to regulate centromere integrity, protect centromeric cohesion, and ensure the subsequent faithful chromosome segregation. Expression of the non-acetylated Bub1 mutant reduces its kinase activity, decreases the level of H2ApT120, and disrupts the recruitment of centromere proteins and chromosome congression, leading to genomic instability of daughter cells. When cells exit mitosis, HDAC1-regulated deacetylation of Bub1 decreases H2ApT120 levels and thereby promotes the departure of centromeric CPC and Sgo1, ensuring timely centromeres disassembly. Collectively, our results reveal a molecular mechanism by which the acetylation and deacetylation cycle of Bub1 modulates the phosphorylation of H2A at T120 for recruitment of Aurora B and Sgo1 to the centromeres, ensuring faithful chromosome segregation during mitosis.

2.
J Cell Biol ; 223(2)2024 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-38117947

RESUMO

Precise chromosome congression and segregation requires the proper assembly of a steady-state metaphase spindle, which is dynamic and maintained by continuous microtubule flux. NuSAP is a microtubule-stabilizing and -bundling protein that promotes chromosome-dependent spindle assembly. However, its function in spindle dynamics remains unclear. Here, we demonstrate that NuSAP regulates the metaphase spindle length control. Mechanistically, NuSAP facilitates kinetochore capture and spindle assembly by promoting Eg5 binding to microtubules. It also prevents excessive microtubule depolymerization through interaction with Kif2A, which reduces Kif2A spindle-pole localization. NuSAP is phosphorylated by Aurora A at Ser-240 during mitosis, and this phosphorylation promotes its interaction with Kif2A on the spindle body and reduces its localization with the spindle poles, thus maintaining proper spindle microtubule flux. NuSAP knockout resulted in the formation of shorter spindles with faster microtubule flux and chromosome misalignment. Taken together, we uncover that NuSAP participates in spindle assembly, dynamics, and metaphase spindle length control through the regulation of microtubule flux and Kif2A localization.


Assuntos
Segregação de Cromossomos , Cinesinas , Proteínas Associadas aos Microtúbulos , Fuso Acromático , Humanos , Células HeLa , Cinesinas/genética , Cinesinas/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Mitose , Fuso Acromático/genética , Fuso Acromático/metabolismo
3.
J Biol Chem ; 298(12): 102658, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36356903

RESUMO

Cartwheel assembly is considered the first step in the initiation of procentriole biogenesis; however, the reason for persistence of the assembled human cartwheel structure from S phase to late mitosis remains unclear. Here, we demonstrate mainly using cell synchronization, RNA interference, immunofluorescence and time-lapse-microscopy, biochemical analysis, and methods that the cartwheel persistently assembles and maintains centriole engagement and centrosome integrity during S phase to late G2 phase. Blockade of the continuous accumulation of centriolar Sas-6, a major cartwheel protein, after procentriole formation induces premature centriole disengagement and disrupts pericentriolar matrix integrity. Additionally, we determined that during mitosis, CDK1-cyclin B phosphorylates Sas-6 at T495 and S510, disrupting its binding to cartwheel component STIL and pericentriolar component Nedd1 and promoting cartwheel disassembly and centriole disengagement. Perturbation of this phosphorylation maintains the accumulation of centriolar Sas-6 and retains centriole engagement during mitotic exit, which results in the inhibition of centriole reduplication. Collectively, these data demonstrate that persistent cartwheel assembly after procentriole formation maintains centriole engagement and that this configuration is relieved through phosphorylation of Sas-6 by CDK1-cyclin B during mitosis in human cells.


Assuntos
Centríolos , Centrossomo , Humanos , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Centrossomo/metabolismo , Mitose , Fosforilação , Proteínas/metabolismo , Ciclina B
4.
Nat Commun ; 12(1): 7157, 2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34887424

RESUMO

A functional mitotic spindle is essential for accurate chromosome congression and segregation during cell proliferation; however, the underlying mechanisms of its assembly remain unclear. Here we show that NuMA regulates this assembly process via phase separation regulated by Aurora A. NuMA undergoes liquid-liquid phase separation during mitotic entry and KifC1 facilitates NuMA condensates concentrating on spindle poles. Phase separation of NuMA is mediated by its C-terminus, whereas its dynein-dynactin binding motif also facilitates this process. Phase-separated NuMA droplets concentrate tubulins, bind microtubules, and enrich crucial regulators, including Kif2A, at the spindle poles, which then depolymerizes spindle microtubules and promotes poleward spindle microtubule flux for spindle assembly and structural dynamics. In this work, we show that NuMA orchestrates mitotic spindle assembly, structural dynamics and function via liquid-liquid phase separation regulated by Aurora A phosphorylation.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Fuso Acromático/química , Fuso Acromático/metabolismo , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Dineínas/genética , Dineínas/metabolismo , Células HeLa , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Fuso Acromático/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
5.
Proc Natl Acad Sci U S A ; 118(28)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260378

RESUMO

Centrosome duplication and DNA replication are two pivotal events that higher eukaryotic cells use to initiate proliferation. While DNA replication is initiated through origin licensing, centrosome duplication starts with cartwheel assembly and is partly controlled by CP110. However, the upstream coordinator for both events has been, until now, a mystery. Here, we report that suppressor of fused protein (Sufu), a negative regulator of the Hedgehog (Hh) pathway playing a significant role in restricting the trafficking and function of glioma-related (Gli) proteins, acts as an upstream switch by facilitating CP110 phosphorylation by CDK2, promoting intranuclear Cdt1 degradation and excluding prereplication complex (pre-RC) components from chromosomes, independent of its canonical function in the Hh pathway. We found that Sufu localizes to both the centrosome and the nucleus and that knockout of Sufu induces abnormalities including centrosome amplification, increased nuclear size, multipolar spindle formation, and polyploidy. Serum stimulation promotes the elimination of Sufu from the centrosome by vesicle release at the ciliary tip and from the nucleus via protein degradation, which allows centrosome duplication and DNA replication to proceed. Collectively, this work reveals a mechanism through which Sufu negatively regulates the G1-S transition.


Assuntos
Centrossomo/metabolismo , Replicação do DNA , Proteínas Repressoras/metabolismo , Animais , Proteínas de Ligação a Calmodulina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Núcleo Celular/metabolismo , Cílios/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Vesículas Citoplasmáticas/metabolismo , Fibroblastos/metabolismo , Fase G1 , Células HEK293 , Células HeLa , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Mitose , Mutação/genética , Fosforilação , Proteólise , Proteínas Repressoras/genética , Fase S
6.
J Cell Biol ; 220(1)2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33351100

RESUMO

Centrosome duplication occurs under strict spatiotemporal regulation once per cell cycle, and it begins with cartwheel assembly and daughter centriole biogenesis at the lateral sites of the mother centrioles. However, although much of this process is understood, how centrosome duplication is initiated remains unclear. Here, we show that cartwheel assembly followed by daughter centriole biogenesis is initiated on the NEDD1-containing layer of the pericentriolar material (PCM) by the recruitment of SAS-6 to the mother centriole under the regulation of PLK4. We found that PLK4-mediated phosphorylation of NEDD1 at its S325 amino acid residue directly promotes both NEDD1 binding to SAS-6 and recruiting SAS-6 to the centrosome. Overexpression of phosphomimicking NEDD1 mutant S325E promoted cartwheel assembly and daughter centriole biogenesis initiations, whereas overexpression of nonphosphorylatable NEDD1 mutant S325A abolished the initiations. Collectively, our results demonstrate that PLK4-regulated NEDD1 facilitates initiation of the cartwheel assembly and of daughter centriole biogenesis in mammals.


Assuntos
Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Centrossomo/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismo
7.
J Biol Chem ; 295(43): 14780-14788, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-32938714

RESUMO

The mitotic kinase Aurora B regulates the condensation of chromatin into chromosomes by phosphorylating chromatin proteins during early mitosis, whereas the phosphatase PP1γ performs the opposite function. The roles of Aurora B and PP1γ must be tightly coordinated to maintain chromosomes at a high phosphorylation state, but the precise mechanisms regulating their function remain largely unclear. Here, mainly through immunofluorescence microscopy and co-immunoprecipitation assays, we find that dissociation of PP1γ from chromosomes is essential for maintaining chromosome phosphorylation. We uncover that PP1γ is recruited to mitotic chromosomes by its regulatory subunit Repo-Man in the absence of Aurora B activity and that Aurora B regulates dissociation of PP1γ by phosphorylating and disrupting PP1γ-Repo-Man interactions on chromatin. Overexpression of Repo-Man mutants that cannot be phosphorylated or inhibition of Aurora B kinase activity resulted in the retention of PP1γ on chromatin and prolonged the chromatin condensation process; a similar outcome was caused by the ectopic targeting of PP1γ to chromatin. Together, our findings reveal a novel regulation mechanism of chromatin condensation in which Aurora B counteracts PP1γ activity by releasing PP1γ from Repo-Man and may have important implications for understanding the regulations of dynamic structural changes of the chromosomes in mitosis.


Assuntos
Aurora Quinase B/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteína Fosfatase 1/metabolismo , Cromatina/metabolismo , Cromossomos Humanos/metabolismo , Células HeLa , Humanos , Mitose , Fosforilação , Mapas de Interação de Proteínas
8.
J Cell Sci ; 132(18)2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31434716

RESUMO

Importin-α serves as an adaptor linking importin-ß to proteins carrying a nuclear localization sequence (NLS). During interphase, this interaction enables nuclear protein import, while in mitosis it regulates spindle assembly factors (SAFs) and controls microtubule nucleation, stabilization and spindle function. Here, we show that human importin-α1 is regulated during the cell cycle and is phosphorylated at two sites (threonine 9 and serine 62) during mitosis by the major mitotic protein kinase CDK1-cyclin B. Mutational analysis indicates that the mitotic phosphorylation of importin-α1 inhibits its binding to importin-ß and promotes the release of TPX2 and KIFC1, which are then targeted like importin-ß to the spindle. Loss of importin-α1 or expression of a non-phosphorylated mutant of importin-α1 results in the formation of shortened spindles with reduced microtubule density and induces a prolonged metaphase, whereas phosphorylation-mimicking mutants are functional in mitosis. We propose that phosphorylation of importin-α1 is a general mechanism for the spatial and temporal control of mitotic spindle assembly by CDK1-cyclin B1 that acts through the release of SAFs such as TPX2 and KIFC1 from inhibitory complexes that restrict spindle assembly.


Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B1/metabolismo , alfa Carioferinas/metabolismo , Proteínas de Ciclo Celular , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Imunoprecipitação , Microtúbulos/metabolismo , Mitose/genética , Mitose/fisiologia , Fosforilação , Fuso Acromático/genética , Fuso Acromático/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismo
9.
J Biol Chem ; 294(27): 10383-10391, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31152066

RESUMO

In higher eukaryotic cells, the nuclear envelope (NE) is composed of double nuclear membranes studded with nuclear pore complexes (NPCs) and undergoes dynamic disassembly and reassembly during the cell cycle. However, how the NE and NPC reassemble remains largely unclear. Here, using HeLa, HEK293, and Drosophila cells, along with immunofluorescence microscopy and transmission EM methods, we found that postmitotic annulate lamellae (AL) assembly contributes to NE and NPC assembly. We observed that the AL are parallel membrane-pair stacks and possess regularly spaced AL pore complexes (ALPCs) that are morphologically similar to the NPCs. We found that the AL assemble in the cytoplasm during mitotic exit simultaneously with NE re-formation in daughter cells. Then, the assembled AL either bound the decondensing chromatin to directly transform into the NE or bound and fused with the outer nuclear membrane to join the assembling NE. The AL did not colocalize with sheet and tubular endoplasmic reticulum (ER) marker proteins on the ER or the lamin B receptor-localized membrane in the cytoplasm, suggesting that postmitotic AL assembly occurs independently of the chromatin and ER. Collectively, our results indicate that postmitotic AL assembly is a common cellular event and an intermediate step in NE and NPC assembly and in NE expansion in higher eukaryotic cells.


Assuntos
Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Animais , Citoplasma/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitose , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
10.
J Biol Chem ; 294(14): 5643-5656, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723163

RESUMO

In all eukaryotes, a functional mitotic spindle is essential for distributing duplicated chromosomes into daughter cells. Mitotic spindle assembly involves highly ordered arrangement of microtubules (MTs). The Augmin protein complex recruits γ-tubulin ring complex (γ-TuRC) to MTs and thereby promotes MT-based MT nucleation and mitotic spindle assembly. However, several factors that may promote Augmin recruitment to MTs remain unknown. Here, we show that echinoderm microtubule-associated protein-like 3 (EML3), an MT-associated protein, facilitates binding between MTs and Augmin/γ-TuRC and recruiting the latter to MTs for proper mitotic spindle assembly and kinetochore-MT connections. Using immunofluorescence microscopy, live-cell imaging, and immunoprecipitation assays, we found that EML3 recruits Augmin/γ-TuRC to the MTs to enhance MT-based MT nucleation in both spindle and small acentrosomal asters. We also noted that the EML3-mediated recruitment is controlled by cyclin-dependent kinase 1 (CDK1), which phosphorylated EML3 at Thr-881 and promoted its binding to Augmin/γ-TuRC. RNAi-mediated EML3 knockdown in HeLa cells reduced spindle localization of Augmin/γ-TuRC, which resulted in abnormal spindle assembly and caused kinetochore-MT misconnection. The introduction of exogenous WT or a Thr-881 phosphorylation mimic EML3 variant into the EML3 knockdown cells restored normal Augmin/γ-TuRC localization and spindle assembly. The EML3 knockdown also affected the spindle assembly checkpoint, delaying chromosome congression and cell division. Taken together, our results indicate that EML3 regulates mitotic spindle assembly and the kinetochore-MT connection by regulating MT-based MT nucleation and recruiting Augmin/γ-TuRC to MTs.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Substituição de Aminoácidos , Proteínas de Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Mutação de Sentido Incorreto , Fuso Acromático/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
11.
Proc Natl Acad Sci U S A ; 116(3): 874-879, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30598432

RESUMO

The Sonic Hedgehog (Shh) pathway conducts primarily in the primary cilium and plays important roles in cell proliferation, individual development, and tumorigenesis. Shh ligand binding with its ciliary membrane-localized transmembrane receptor Patched1 results in the removal of Patched1 from and the translocation of the transmembrane oncoprotein Smoothened into the cilium, leading to Shh signaling activation. However, how these processes are coupled remains unknown. Here, we show that the Patched1-ArhGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened. We find that Patched1 interacts with and stabilizes the PKA negative regulator ArhGAP36 to the centrosome. Activating the Shh pathway results in the removal of ArhGAP36 from the mother centriole and the centrosomal PKA accumulation. This PKA then phosphorylates Inversin and promotes its interaction with and the ciliary translocation of Smoothened. Knockdown of Inversin disrupts the ciliary translocation of Smoothened and Shh pathway activation. These findings reveal a regulatory molecular mechanism for the initial step of Shh pathway activation.


Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Receptor Patched-1/metabolismo , Receptor Smoothened/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Camundongos , Fosforilação , Transdução de Sinais
12.
J Cell Sci ; 132(2)2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30578313

RESUMO

Hedgehog (Hh) signaling is a highly conserved cell signaling pathway important for cell life, development and tumorigenesis. Increasing evidence suggests that the Hh signaling pathway functions in certain phases of the cell cycle. However, the coordination between Hh signaling and cell cycle control remains poorly understood. Here, we show that polo-like kinase-1 (Plk1), a critical protein kinase regulating many processes during the cell cycle, also regulates Hh signaling by phosphorylating and inhibiting Gli1, a downstream transcription factor of the Hh signaling pathway. Gli1 expression increases along with Hh signaling activation, leading to upregulation of Hh target genes, including cyclin E, during the G1 and S phases. Gli1 is phosphorylated at S481 by Plk1, and this phosphorylation facilitates the nuclear export and binding of Gli1 with its negative regulator Sufu, leading to a reduction in Hh signaling activity. Inhibition of Plk1 kinase activity led to Gli1 maintaining is role in promoting downstream gene expression. Collectively, our data reveal a novel mechanism regarding the crosstalk between Hh signaling and cell cycle control.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteínas de Ciclo Celular/genética , Células HEK293 , Células HeLa , Proteínas Hedgehog/genética , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Quinase 1 Polo-Like
13.
J Biol Chem ; 292(24): 10131-10141, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28446612

RESUMO

CDK4 regulates G1/S phase transition in the mammalian cell cycle by phosphorylating retinoblastoma family proteins. However, the mechanism underlying the regulation of CDK4 activity is not fully understood. Here, we show that CDK4 protein is degraded by anaphase-promoting complex/cyclosome (APC/C) during metaphase-anaphase transition in HeLa cells, whereas its main regulator, cyclin D1, remains intact but is sequestered in cytoplasm. CDK4 protein reaccumulates in the following G1 phase and shuttles between the nucleus and the cytoplasm to facilitate the nuclear import of cyclin D1. Without CDK4, cyclin D1 cannot enter the nucleus. Point mutations that disrupt CDK4 and cyclin D1 interaction impair the nuclear import of cyclin D1 and the activity of CDK4. RNAi knockdown of CDK4 also induces cytoplasmic retention of cyclin D1 and G0/G1 phase arrest of the cells. Collectively, our data demonstrate that CDK4 protein is degraded in late mitosis and reaccumulates in the following G1 phase to facilitate the nuclear import of cyclin D1 for activation of CKD4 to initiate a new cell cycle in HeLa cells.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Fase G1 , Mitose , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Ciclina D1/química , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/química , Quinase 4 Dependente de Ciclina/genética , Indução Enzimática , Estabilidade Enzimática , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Camundongos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Estabilidade Proteica , Transporte Proteico , Proteólise , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo
14.
J Cell Biol ; 210(3): 373-83, 2015 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-26240182

RESUMO

A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A-dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fuso Acromático/fisiologia , Animais , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Metáfase/fisiologia , Mitose/fisiologia , Fosforilação , Multimerização Proteica , Tubulina (Proteína)/metabolismo , Xenopus
15.
J Biol Chem ; 290(28): 17546-58, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25987563

RESUMO

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.


Assuntos
Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Sequência de Aminoácidos , Animais , Aurora Quinase A/química , Aurora Quinase A/genética , Aurora Quinase B/química , Aurora Quinase B/genética , Compartimento Celular , Pontos de Checagem do Ciclo Celular , Centrossomo/metabolismo , Cromatina/metabolismo , Evolução Molecular , Células HeLa , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Mitose , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fuso Acromático/metabolismo
16.
Proc Natl Acad Sci U S A ; 110(38): 15295-300, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24003142

RESUMO

Kinetochore capture by dynamic kinetochore microtubule fibers (K fibers) is essential for proper chromosome alignment and accurate distribution of the replicated genome during cell division. Although this capture process has been extensively studied, the mechanisms underlying the initiation of this process and the proper formation of the K fibers remain largely unknown. Here we show that transforming acidic coiled-coil-containing protein 3 (TACC3) is essential for kinetochore capture and proper K-fiber formation in HeLa cells. To observe the assembly of acentrosomal microtubules more clearly, the cells were released from higher concentrations of nocodazole into zero or lower concentrations. We find that small acentrosomal TACC3-microtubule aster formation near the kinetochores and binding of the asters with the kinetochores are the initial steps of the kinetochore capture by the acentrosomal microtubules, and that the sorting of kinetochore-captured acentrosomal microtubules with centrosomal microtubules leads to the capture of kinetochore by centrosomal microtubules from both spindle poles. We demonstrate that the sorting of the TACC3-associated microtubules with the centrosomal microtubules is a crucial process for spindle assembly and chromosome movement. These findings, which are also supported in the unperturbed mitosis without nocodazole, reveal a critical TACC3-dependent acentrosomal microtubule nucleation and sorting process to regulate kinetochore-microtubule connections and provide deep insight into the mechanisms of mitotic spindle assembly and chromosome alignment.


Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/fisiologia , Western Blotting , Imunofluorescência , Células HeLa , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , Fuso Acromático/metabolismo
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