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1.
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
2.
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
3.
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
4.
Prog Mol Subcell Biol ; 56: 3-27, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28840231

RESUMEN

A number of paths have led to the present list of centromere proteins, which is essentially complete for constitutive structural proteins, but still may be only partial if we consider the many other proteins that briefly visit the centromere and kinetochore to fine-tune the chromatin and adjust other functions. Elegant genetics led to the description of the budding yeast point centromere in 1980. In the same year was published the serendipitous discovery of antibodies that stained centromeres of human mitotic chromosomes in antisera from CREST patients. Painstaking biochemical analyses led to the identification of the human centromere antigens several years later, with the first yeast proteins being described 6 years after that. Since those early days, the discovery and cloning of centromere and kinetochore proteins has largely been driven by improvements in technology. These began with expression cloning methods, which allowed antibodies to lead to cDNA clones. Next, functional screens for kinetochore proteins were made possible by the isolation of yeast centromeric DNAs. Ultimately, the completion of genome sequences for humans and model organisms permitted the coupling of biochemical fractionation with protein identification by mass spectrometry. Subsequent improvements in mass spectrometry have led to the current state where virtually all structural components of the kinetochore are known and where a high-resolution map of the entire structure will likely emerge within the next several years.


Asunto(s)
Centrómero , Cinetocoros , Espectrometría de Masas , Centrómero/química , Humanos , Cinetocoros/química , Saccharomycetales
5.
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
6.
Nat Cell Biol ; 4(7): 502-8, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12068306

RESUMEN

Cajal bodies (CBs) are nuclear organelles that contain factors required for splicing, ribosome biogenesis and transcription. Our previous analysis in living cells showed that CBs are dynamic structures. Here, we show that CB mobility is described by anomalous diffusion and that bodies alternate between association with chromatin and diffusion within the interchromatin space. CB mobility increases after ATP depletion and inhibition of transcription, suggesting that the association of CB and chromatin requires ATP and active transcription. This behaviour is fundamentally different from the ATP-dependent mobility observed for chromatin and suggests that a novel mechanism governs CB, and possibly other, nuclear body dynamics.


Asunto(s)
Adenosina Trifosfato/fisiología , Cromatina/fisiología , Cuerpos Enrollados/fisiología , Difusión , Células HeLa , Humanos , Transcripción Genética
7.
J Cell Biol ; 173(4): 469-76, 2006 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-16717124

RESUMEN

Among the earliest events in postmitotic nuclear envelope (NE) assembly are the interactions between chromatin and the membranes that will fuse to form the NE. It has been proposed that interactions between integral NE proteins and chromatin proteins mediate initial membrane recruitment to chromatin. We show that several transmembrane NE proteins bind to DNA directly and that NE membrane proteins as a class are enriched in long, basic domains that potentially bind DNA. Membrane fractions that are essential for NE formation are shown to bind directly to protein-free DNA, and our data suggest that these interactions are critical for early steps in NE assembly.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Animales , Ciclo Celular/fisiología , Cromatina/metabolismo , Citosol/metabolismo , Proteínas de Unión al ADN/química , Femenino , Proteínas de la Membrana/química , Membrana Nuclear/ultraestructura , Oocitos , Estructura Terciaria de Proteína/fisiología , Factores de Tiempo , Vesículas Transportadoras/metabolismo , Xenopus laevis
8.
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
9.
Cells ; 6(2)2017 Mar 25.
Artículo en Inglés | MEDLINE | ID: mdl-28346356

RESUMEN

Lamins form a scaffold lining the nucleus that binds chromatin and contributes to spatial genome organization; however, due to the many other functions of lamins, studies knocking out or altering the lamin polymer cannot clearly distinguish between direct and indirect effects. To overcome this obstacle, we specifically targeted the mapped histone-binding site of A/C lamins by microinjecting antibodies specific to this region predicting that this would make the genome more mobile. No increase in chromatin mobility was observed; however, interestingly, injected cells failed to go through mitosis, while control antibody-injected cells did. This effect was not due to crosslinking of the lamin polymer, as Fab fragments also blocked mitosis. The lack of genome mobility suggested other lamin-chromatin interactions. To determine what these might be, mini-lamin A constructs were expressed with or without the histone-binding site that assembled into independent intranuclear structures. HP1, CenpB and PML proteins accumulated at these structures for both constructs, indicating that other sites supporting chromatin interactions exist on lamin A. Together, these results indicate that lamin A-chromatin interactions are highly redundant and more diverse than generally acknowledged and highlight the importance of trying to experimentally separate their individual functions.

10.
FEBS Lett ; 580(27): 6435-41, 2006 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-17097643

RESUMEN

The inner nuclear membrane (INM) of eukaryotic cells is characterized by a unique set of transmembrane proteins which interact with chromatin and/or the nuclear lamina. The number of identified INM proteins is steadily increasing, mainly as a result of proteomic and computational approaches. However, despite a link between mutation of several of these proteins and disease, the function of most transmembrane proteins of the INM remains unknown and depletion of many of these proteins from a variety of systems did not produce an obvious phenotype in the affected cells. Here, we report that depletion of the conserved INM protein Lem2 from human cell lines leads to abnormally shaped nuclei and severely reduces cell survival. We suggest that interactions of Lem2 with lamins or chromatin are critical for maintaining the integrity of the nuclear envelope.


Asunto(s)
Cromatina/metabolismo , Laminas/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Supervivencia Celular/genética , Cromatina/genética , Eliminación de Gen , Células HeLa , Humanos , Laminas/genética , Proteínas de la Membrana/genética , Mutación , Membrana Nuclear/genética , Proteínas Nucleares/genética
11.
J Cell Biol ; 210(1): 45-62, 2015 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-26124292

RESUMEN

Coordination of cell growth and proliferation in response to nutrient supply is mediated by mammalian target of rapamycin (mTOR) signaling. In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles. Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects. Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression.


Asunto(s)
Aurora Quinasa A/fisiología , Proteínas de Ciclo Celular/metabolismo , Centrosoma/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Secuencia de Aminoácidos , Activación Enzimática , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Cinesinas/metabolismo , Mitosis , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Huso Acromático/metabolismo , Quinasa Tipo Polo 1
14.
Mol Biol Cell ; 20(24): 5260-75, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19864462

RESUMEN

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere.


Asunto(s)
Cromosomas Humanos/metabolismo , Mitosis , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Anafase/efectos de los fármacos , Aurora Quinasa B , Aurora Quinasas , Segregación Cromosómica/efectos de los fármacos , Cromosomas Humanos/efectos de los fármacos , Cromosomas Humanos/ultraestructura , Citocinesis/efectos de los fármacos , Células HeLa , Humanos , Cinesinas/metabolismo , Cinetocoros/efectos de los fármacos , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Mitosis/efectos de los fármacos , Nocodazol/farmacología , Paclitaxel/farmacología , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Pirimidinas/metabolismo , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Especificidad por Sustrato/efectos de los fármacos , Tionas/metabolismo
15.
Mol Biol Cell ; 19(4): 1753-62, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18256286

RESUMEN

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly.


Asunto(s)
Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Femenino , Humanos , Técnicas In Vitro , Fusión de Membrana , Mutación , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Oocitos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/genética
16.
Cell Struct Funct ; 27(5): 335-41, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12502887

RESUMEN

The use of fluorescence imaging methods, most recently based on fluorescent protein technology, and the availability of high quality fluorescence imaging systems have driven a revolution in cell and molecular biology. Live cell imaging, especially using fluorescence, is now used in a wide variety of assays in academic and commercial laboratories. The use of this technology requires particular attention to be paid to cell engineering, the design of the image acquisition system, the imaging protocol, and subsequent processing and analytic methods. In this review, we discuss each of these steps, highlighting practical techniques developed by us and others.


Asunto(s)
Células Eucariotas/citología , Microscopía Fluorescente , Algoritmos , Animales , Técnicas de Cultivo de Célula , Cámaras de Difusión de Cultivos , Células Eucariotas/metabolismo , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes , Humanos , Procesamiento de Imagen Asistido por Computador , Proteínas Luminiscentes , Procesamiento de Señales Asistido por Computador
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