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1.
Cell ; 187(12): 3006-3023.e26, 2024 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-38744280

RESUMEN

Centromeres are scaffolds for the assembly of kinetochores that ensure chromosome segregation during cell division. How vertebrate centromeres obtain a three-dimensional structure to accomplish their primary function is unclear. Using super-resolution imaging, capture-C, and polymer modeling, we show that vertebrate centromeres are partitioned by condensins into two subdomains during mitosis. The bipartite structure is found in human, mouse, and chicken cells and is therefore a fundamental feature of vertebrate centromeres. Super-resolution imaging and electron tomography reveal that bipartite centromeres assemble bipartite kinetochores, with each subdomain binding a distinct microtubule bundle. Cohesin links the centromere subdomains, limiting their separation in response to spindle forces and avoiding merotelic kinetochore-spindle attachments. Lagging chromosomes during cancer cell divisions frequently have merotelic attachments in which the centromere subdomains are separated and bioriented. Our work reveals a fundamental aspect of vertebrate centromere biology with implications for understanding the mechanisms that guarantee faithful chromosome segregation.


Asunto(s)
Centrómero , Cohesinas , Cinetocoros , Mitosis , Animales , Humanos , Ratones , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Pollos , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Segregación Cromosómica , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Huso Acromático/metabolismo
2.
Mol Cell ; 82(3): 696-708.e4, 2022 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-35090599

RESUMEN

We have used a combination of chemical genetics, chromatin proteomics, and imaging to map the earliest chromatin transactions during vertebrate cell entry into mitosis. Chicken DT40 CDK1as cells undergo synchronous mitotic entry within 15 min following release from a 1NM-PP1-induced arrest in late G2. In addition to changes in chromatin association with nuclear pores and the nuclear envelope, earliest prophase is dominated by changes in the association of ribonucleoproteins with chromatin, particularly in the nucleolus, where pre-rRNA processing factors leave chromatin significantly before RNA polymerase I. Nuclear envelope barrier function is lost early in prophase, and cytoplasmic proteins begin to accumulate on the chromatin. As a result, outer kinetochore assembly appears complete by nuclear envelope breakdown (NEBD). Most interphase chromatin proteins remain associated with chromatin until NEBD, after which their levels drop sharply. An interactive proteomic map of chromatin transactions during mitotic entry is available as a resource at https://mitoChEP.bio.ed.ac.uk.


Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Cromosomas , ADN/metabolismo , Linfoma de Células B/metabolismo , Proteínas Nucleares/metabolismo , Profase , Proteoma , Proteómica , Animales , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Pollos , Cromatina/genética , ADN/genética , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/patología , Proteínas Nucleares/genética , Unión Proteica , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Factores de Tiempo
3.
EMBO J ; 37(6)2018 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-29467217

RESUMEN

The chromosomal passenger complex (CPC) is directed to centromeres during mitosis via binding to H3T3ph and Sgo1. Whether and how heterochromatin protein 1α (HP1α) influences CPC localisation and function during mitotic entry is less clear. Here, we alter HP1α dynamics by fusing it to a CENP-B DNA-binding domain. Tethered HP1 strongly recruits the CPC, destabilising kinetochore-microtubule interactions and activating the spindle assembly checkpoint. During mitotic exit, the tethered HP1 traps active CPC at centromeres. These HP1-CPC clusters remain catalytically active throughout the subsequent cell cycle. We also detect interactions between endogenous HP1 and the CPC during G2 HP1α and HP1γ cooperate to recruit the CPC to active foci in a CDK1-independent process. Live cell tracking with Fab fragments reveals that H3S10ph appears well before H3T3 is phosphorylated by Haspin kinase. Our results suggest that HP1 may concentrate and activate the CPC at centromeric heterochromatin in G2 before Aurora B-mediated phosphorylation of H3S10 releases HP1 from chromatin and allows pathways dependent on H3T3ph and Sgo1 to redirect the CPC to mitotic centromeres.


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Heterocromatina/metabolismo , Mitosis , Línea Celular Tumoral , Centrómero/metabolismo , Homólogo de la Proteína Chromobox 5 , Humanos , Fosforilación
4.
J Cell Sci ; 132(21)2019 11 06.
Artículo en Inglés | MEDLINE | ID: mdl-31601613

RESUMEN

Timely and precise control of Aurora B kinase, the chromosomal passenger complex (CPC) catalytic subunit, is essential for accurate chromosome segregation and cytokinesis. Post-translational modifications of CPC subunits are directly involved in controlling Aurora B activity. Here, we identified a highly conserved acidic STD-rich motif of INCENP that is phosphorylated during mitosis in vivo and by Plk1 in vitro and is involved in controlling Aurora B activity. By using an INCENP conditional-knockout cell line, we show that impairing the phosphorylation status of this region disrupts chromosome congression and induces cytokinesis failure. In contrast, mimicking constitutive phosphorylation not only rescues cytokinesis but also induces ectopic furrows and contractile ring formation in a Plk1- and ROCK1-dependent manner independent of cell cycle and microtubule status. Our experiments identify the phospho-regulation of the INCENP STD motif as a novel mechanism that is key for chromosome alignment and cytokinesis.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Microtúbulos/metabolismo , Mutación/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Citocinesis/fisiología , Humanos , Mitosis/fisiología , Quinasas Asociadas a rho/metabolismo , Quinasa Tipo Polo 1
5.
J Cell Sci ; 131(9)2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29618633

RESUMEN

In metazoa, the Nup107 complex (also known as the nucleoporin Y-complex) plays a major role in formation of the nuclear pore complex in interphase and is localised to kinetochores in mitosis. The Nup107 complex shares a single highly conserved subunit, Seh1 (also known as SEH1L in mammals) with the GATOR2 complex, an essential activator of mTORC1 kinase. mTORC1/GATOR2 has a central role in the coordination of cell growth and proliferation. Here, we use chemical genetics and quantitative chromosome proteomics to study the role of the Seh1 protein in mitosis. Surprisingly, Seh1 is not required for the association of the Nup107 complex with mitotic chromosomes, but it is essential for the association of both the GATOR2 complex and nucleoporin Nup153 with mitotic chromosomes. Our analysis also reveals a role for Seh1 at human centromeres, where it is required for efficient localisation of the chromosomal passenger complex (CPC). Furthermore, this analysis detects a functional interaction between the Nup107 complex and the small kinetochore protein SKAP (also known as KNSTRN).


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromosomas Humanos , Mitosis/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mitosis/genética , Proteínas de Complejo Poro Nuclear/genética , Transfección
6.
J Cell Sci ; 131(4)2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29361541

RESUMEN

The requirement for condensin in chromosome formation in somatic cells remains unclear, as imperfectly condensed chromosomes do form in cells depleted of condensin by conventional methodologies. In order to dissect the roles of condensin at different stages of vertebrate mitosis, we have established a versatile cellular system that combines auxin-mediated rapid degradation with chemical genetics to obtain near-synchronous mitotic entry of chicken DT40 cells in the presence and absence of condensin. We analyzed the outcome by live- and fixed-cell microscopy methods, including serial block face scanning electron microscopy with digital reconstruction. Following rapid depletion of condensin, chromosomal defects were much more obvious than those seen after a slow depletion of condensin. The total mitotic chromatin volume was similar to that in control cells, but a single mass of mitotic chromosomes was clustered at one side of a bent mitotic spindle. Cultures arrest at prometaphase, eventually exiting mitosis without segregating chromosomes. Experiments where the auxin concentration was titrated showed that different condensin levels are required for anaphase chromosome segregation and formation of a normal chromosome architecture.This article has an associated First Person interview with the first author of the paper.


Asunto(s)
Adenosina Trifosfatasas/genética , Cromatina/ultraestructura , Cromosomas/ultraestructura , Proteínas de Unión al ADN/genética , Mitosis/genética , Complejos Multiproteicos/genética , Adenosina Trifosfatasas/metabolismo , Animales , Pollos , Cromatina/genética , Cromatina/metabolismo , Aberraciones Cromosómicas , Segregación Cromosómica/genética , Cromosomas/genética , Cromosomas/metabolismo , Proteínas de Unión al ADN/metabolismo , Ácidos Indolacéticos/farmacología , Microscopía Electrónica de Rastreo , Complejos Multiproteicos/metabolismo , Proteolisis/efectos de los fármacos
7.
J Biol Chem ; 290(35): 21460-72, 2015 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-26175154

RESUMEN

The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼ 32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼ 80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible "dog leash," allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.


Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Microtúbulos/metabolismo , Mitosis , Secuencia de Aminoácidos , Animales , Aurora Quinasa B/metabolismo , Línea Celular , Proliferación Celular , Pollos , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Mutación , Fosforilación , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad
8.
J Biol Chem ; 289(45): 31617-23, 2014 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-25248749

RESUMEN

Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Desoxirribonucleasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Ácidos Indolacéticos/metabolismo , Animales , Anexina A5/metabolismo , Apoptosis , Muerte Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Pollos , Fragmentación del ADN , Activación Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Técnicas de Inactivación de Genes , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/enzimología
9.
J Cell Sci ; 125(Pt 6): 1591-604, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22344259

RESUMEN

In vertebrates, two condensin complexes exist, condensin I and condensin II, which have differing but unresolved roles in organizing mitotic chromosomes. To dissect accurately the role of each complex in mitosis, we have made and studied the first vertebrate conditional knockouts of the genes encoding condensin I subunit CAP-H and condensin II subunit CAP-D3 in chicken DT40 cells. Live-cell imaging reveals highly distinct segregation defects. CAP-D3 (condensin II) knockout results in masses of chromatin-containing anaphase bridges. CAP-H (condensin I)-knockout anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death. Super-resolution microscopy reveals that condensin-I-depleted mitotic chromosomes are wider and shorter, with a diffuse chromosome scaffold, whereas condensin-II-depleted chromosomes retain a more defined scaffold, with chromosomes more stretched and seemingly lacking in axial rigidity. We conclude that condensin II is required primarily to provide rigidity by establishing an initial chromosome axis around which condensin I can arrange loops of chromatin.


Asunto(s)
Adenosina Trifosfatasas/fisiología , Cromosomas/genética , Proteínas de Unión al ADN/fisiología , Mitosis/genética , Complejos Multiproteicos/fisiología , Adenosina Trifosfatasas/deficiencia , Adenosina Trifosfatasas/genética , Animales , Línea Celular Tumoral , Pollos , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Técnicas de Inactivación de Genes/métodos , Complejos Multiproteicos/deficiencia , Complejos Multiproteicos/genética
10.
bioRxiv ; 2024 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-38659940

RESUMEN

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at ~1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

11.
J Biol Chem ; 285(51): 40163-70, 2010 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-20956539

RESUMEN

INCENP, Borealin, Survivin, and Aurora B kinase comprise the chromosomal passenger complex, an essential regulator of mitotic events. INCENP (inner centromere protein) binds and activates Aurora B through a feedback loop involving phosphorylation of a Thr-Ser-Ser (TSS) motif near the INCENP C terminus. Here, we have examined the role of the TSS motif in vertebrate cells using an DT40 INCENP(ON/OFF) conditional knock-out cell line in which mutants are expressed in the absence of wild-type INCENP. Our analysis confirms that regulated phosphorylation of the two serine residues (presumably by Aurora B) is critical for full activation of the kinase and is essential for cell viability. Cells expressing INCENP mutants bearing either phospho-null (TAA) or phospho-mimetic (TEE) mutations exhibit significant levels of Aurora B kinase activity but fail to undergo normal spindle elongation or complete cytokinesis. This work confirms previous suggestions that INCENP can act as a rheostat, with different INCENP mutants promoting differing degrees of kinase activation. Our results also reveal that mitotic progression is accompanied by a requirement for progressively higher levels of Aurora B kinase activity.


Asunto(s)
Proteínas Aviares/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Citocinesis/fisiología , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Secuencias de Aminoácidos , Animales , Aurora Quinasas , Proteínas Aviares/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Pollos , Proteínas Cromosómicas no Histona/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Huso Acromático/genética
12.
Proc Natl Acad Sci U S A ; 105(7): 2457-62, 2008 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-18263736

RESUMEN

We describe a method for the isolation of conditional knockouts of essential multiply spliced genes in which the entire body of the gene downstream of the ATG start codon is left untouched but can be switched off rapidly and completely by adding tetracycline to the culture medium. The approach centers on a "promoter-hijack" strategy in which the gene's promoter is replaced with a minimal promoter responsive to the tetracycline-repressible transactivator (tTA). Elsewhere in the genome, a cloned fragment of the gene's promoter is used to drive expression of a tTA. Thus, the gene is essentially regulated by its own promoter but through the intermediary tTA. Using this strategy, we generated a conditional knockout of chromokinesin KIF4A, an important mitotic effector protein whose mRNA is multiply spliced and whose cDNA is highly toxic when overexpressed in cells. We used chicken DT40 cells, but the same strategy should be applicable to ES cells and, eventually, to mice.


Asunto(s)
Genes cdc , Regiones Promotoras Genéticas/genética , Empalme del ARN/genética , Animales , Línea Celular , Pollos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Esenciales/genética , Genoma/genética , Cinesinas/genética , Cinesinas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transgenes/genética
13.
Biochem Soc Trans ; 38(6): 1655-9, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21118143

RESUMEN

The CPC [chromosomal passenger complex; INCENP (inner centromere protein), Aurora B kinase, survivin and borealin] is implicated in many mitotic processes. In the present paper we describe how we generated DT40 conditional-knockout cell lines for incenp1 and survivin1 to better understand the role of these CPC subunits in the control of Aurora B kinase activity. These lines enabled us to reassess current knowledge of survivin function and to show that INCENP acts as a rheostat for Aurora B activity.


Asunto(s)
Línea Celular , Proteínas Cromosómicas no Histona/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Apoptosis , Aurora Quinasa B , Aurora Quinasas , Pollos , Proteínas Cromosómicas no Histona/genética , Cromosomas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Mitosis
14.
Cell Rep ; 32(12): 108177, 2020 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-32966795

RESUMEN

Cells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific manner after replication stress, marking regions of instability in cancer. Despite such a distinct defect, no model fully provides a molecular explanation for CFSs. We show that CFSs are characterized by impaired chromatin folding, manifesting as disrupted mitotic structures visible with molecular fluorescence in situ hybridization (FISH) probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Cytogenetic and molecular lesions are marked by faulty condensin loading at CFSs, a defect in condensin-I-mediated compaction, and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that, in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS expression, concomitantly providing an environment for MIDAS, which, if not resolved, results in chromosome instability.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Sitios Frágiles del Cromosoma , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Estrés Fisiológico , Afidicolina/farmacología , Cromatina/metabolismo , ADN/biosíntesis , Replicación del ADN/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Femenino , Fase G2/efectos de los fármacos , Células HCT116 , Humanos , Masculino , Mitosis/efectos de los fármacos , Modelos Biológicos , Estrés Fisiológico/efectos de los fármacos
15.
Science ; 359(6376)2018 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-29348367

RESUMEN

Mitotic chromosomes fold as compact arrays of chromatin loops. To identify the pathway of mitotic chromosome formation, we combined imaging and Hi-C analysis of synchronous DT40 cell cultures with polymer simulations. Here we show that in prophase, the interphase organization is rapidly lost in a condensin-dependent manner, and arrays of consecutive 60-kilobase (kb) loops are formed. During prometaphase, ~80-kb inner loops are nested within ~400-kb outer loops. The loop array acquires a helical arrangement with consecutive loops emanating from a central "spiral staircase" condensin scaffold. The size of helical turns progressively increases to ~12 megabases during prometaphase. Acute depletion of condensin I or II shows that nested loops form by differential action of the two condensins, whereas condensin II is required for helical winding.


Asunto(s)
Cromosomas/química , Cromosomas/genética , Mitosis , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Biología Computacional , Proteínas de Unión al ADN/metabolismo , Genómica , Interfase , Complejos Multiproteicos/metabolismo , Prometafase , Profase , Xenopus laevis
16.
Curr Biol ; 28(23): 3824-3832.e6, 2018 12 03.
Artículo en Inglés | MEDLINE | ID: mdl-30449668

RESUMEN

Distinct protein phosphorylation levels in interphase and M phase require tight regulation of Cdk1 activity [1, 2]. A bistable switch, based on positive feedback in the Cdk1 activation loop, has been proposed to generate different thresholds for transitions between these cell-cycle states [3-5]. Recently, the activity of the major Cdk1-counteracting phosphatase, PP2A:B55, has also been found to be bistable due to Greatwall kinase-dependent regulation [6]. However, the interplay of the regulation of Cdk1 and PP2A:B55 in vivo remains unexplored. Here, we combine quantitative cell biology assays with mathematical modeling to explore the interplay of mitotic kinase activation and phosphatase inactivation in human cells. By measuring mitotic entry and exit thresholds using ATP-analog-sensitive Cdk1 mutants, we find evidence that the mitotic switch displays hysteresis and bistability, responding differentially to Cdk1 inhibition in the mitotic and interphase states. Cdk1 activation by Wee1/Cdc25 feedback loops and PP2A:B55 inactivation by Greatwall independently contributes to this hysteretic switch system. However, elimination of both Cdk1 and PP2A:B55 inactivation fully abrogates bistability, suggesting that hysteresis is an emergent property of mutual inhibition between the Cdk1 and PP2A:B55 feedback loops. Our model of the two interlinked feedback systems predicts an intermediate but hidden steady state between interphase and M phase. This could be verified experimentally by Cdk1 inhibition during mitotic entry, supporting the predictive value of our model. Furthermore, we demonstrate that dual inhibition of Wee1 and Gwl kinases causes loss of cell-cycle memory and synthetic lethality, which could be further exploited therapeutically.


Asunto(s)
Ciclo Celular , Mitosis , Ciclo Celular/genética , Células HeLa , Humanos , Interfase/genética , Mitosis/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación
17.
Science ; 361(6404): 806-810, 2018 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-30139873

RESUMEN

The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)-directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.


Asunto(s)
Fase G2/genética , Mitosis/genética , Fase S/genética , Antígenos de Superficie/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Ciclina B1/antagonistas & inhibidores , Ciclina B1/metabolismo , Daño del ADN/genética , Replicación del ADN/genética , Proteína Forkhead Box M1/metabolismo , Redes Reguladoras de Genes , Células HCT116 , Humanos , Fosforilación , Telomerasa
18.
Subcell Biochem ; 40: 75-90, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17623901

RESUMEN

Chicken DT40 cells are a very favourable system to use for the study of apoptosis. These cells undergo apoptosis readily in response to a variety of physiological and experimental stimuli. Their response to chemotherapeutic agents such as etoposide is rapid and efficient, facilitating biochemical analysis of apoptotic execution. Because many of the genes involved in apoptotic execution are not essential for cellular life, DT40 cells also provide a ready system for the genetic analysis of apoptosis, in which true null mutations may be isolated. Here we describe standard procedures for the induction and analysis of apoptosis in DT40 cells. We also describe a few of the conclusions from our studies in which several components of the apoptotic execution pathway have been knocked out.


Asunto(s)
Apoptosis/genética , Animales , Linfocitos B/citología , Línea Celular , Pollos
19.
Open Biol ; 6(1): 150230, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26791246

RESUMEN

Most studies using knockout technologies to examine protein function have relied either on shutting off transcription (conventional conditional knockouts with tetracycline-regulated gene expression or gene disruption) or destroying the mature mRNA (RNAi technology). In both cases, the target protein is lost at a rate determined by its intrinsic half-life. Thus, protein levels typically fall over at least 1-3 days, and cells continue to cycle while exposed to a decreasing concentration of the protein. Here we characterise the kinetochore proteome of mitotic chromosomes isolated from a cell line in which the essential kinetochore protein CENP-T is present as an auxin-inducible degron (AID) fusion protein that is fully functional and able to support the viability of the cells. Stripping of the protein from chromosomes in early mitosis via targeted proteasomal degradation reveals the dependency of other proteins on CENP-T for their maintenance in kinetochores. We compare these results with the kinetochore proteome of conventional CENP-T/W knockouts. As the cell cycle is mostly formed from G1, S and G2 phases a gradual loss of CENP-T/W levels is more likely to reflect dependencies associated with kinetochore assembly pre-mitosis and upon entry into mitosis. Interestingly, a putative super-complex involving Rod-Zw10-zwilch (RZZ complex), Spindly, Mad1/Mad2 and CENP-E requires the function of CENP-T/W during kinetochore assembly for its stable association with the outer kinetochore, but once assembled remains associated with chromosomes after stripping of CENP-T during mitosis. This study highlights the different roles core kinetochore components may play in the assembly of kinetochores (upon entry into mitosis) versus the maintenance of specific components (during mitosis).


Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Técnicas de Inactivación de Genes , Ácidos Indolacéticos/metabolismo , Cinetocoros/metabolismo , Mitosis , Animales , Muerte Celular , Línea Celular , Supervivencia Celular , Pollos , Metafase , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteolisis , Proteómica
20.
Mol Cell Biol ; 35(20): 3566-78, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26240282

RESUMEN

Aurora kinase B, one of the three members of the mammalian Aurora kinase family, is the catalytic component of the chromosomal passenger complex, an essential regulator of chromosome segregation in mitosis. Aurora B is overexpressed in human tumors although whether this kinase may function as an oncogene in vivo is not established. Here, we report a new mouse model in which expression of the endogenous Aurkb locus can be induced in vitro and in vivo. Overexpression of Aurora B in cultured cells induces defective chromosome segregation and aneuploidy. Long-term overexpression of Aurora B in vivo results in aneuploidy and the development of multiple spontaneous tumors in adult mice, including a high incidence of lymphomas. Overexpression of Aurora B also results in a reduced DNA damage response and decreased levels of the p53 target p21(Cip1) in vitro and in vivo, in line with an inverse correlation between Aurora B and p21(Cip1) expression in human leukemias. Thus, overexpression of Aurora B may contribute to tumor formation not only by inducing chromosomal instability but also by suppressing the function of the cell cycle inhibitor p21(Cip1).


Asunto(s)
Aneuploidia , Aurora Quinasa B/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/metabolismo , Expresión Génica , Silenciador del Gen , Ratones Endogámicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética
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