Publications by authors named "Juan F Giménez-Abián"

27 Publications

  • Page 1 of 1

Analyzing Mitotic Chromosome Structural Defects After Topoisomerase II Inhibition or Mutation.

Methods Mol Biol 2018 ;1703:191-215

Department of Genetics, Cell Biology & Development, University of Minnesota, 420 Washington Ave SE, Minneapolis, MN, 55455, USA.

For analyzing chromosome structural defects that result from topoisomerase II (topo II) dysfunction we have adapted classical cell cycle experiments, classical cytological techniques and the use of a potent topo II inhibitor (ICRF-193). In this chapter, we describe in detail the protocols used and we discuss the rational for our choice and for the adaptations applied. We clarify in which cell cycle stages each of the different chromosomal aberrations induced by inhibiting topo II takes place: lack of chromosome segregation, undercondensation, lack of sister chromatid resolution, and lack of chromosome individualization. We also put these observations into the context of the two topo II-dependent cell cycle checkpoints. In addition, we have devised a system to analyze phenotypes that result when topo II is mutated in human cells. This serves as an alternative strategy to the use of topo II inhibitors to perturb topo II function.
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http://dx.doi.org/10.1007/978-1-4939-7459-7_15DOI Listing
July 2018

RepA-WH1 prionoid: Clues from bacteria on factors governing phase transitions in amyloidogenesis.

Prion 2016 ;10(1):41-9

a Department of Cellular & Molecular Biology , Centro de Investigaciones Biológicas - CSIC , Madrid , Spain.

In bacterial plasmids, Rep proteins initiate DNA replication by undergoing a structural transformation coupled to dimer dissociation. Amyloidogenesis of the 'winged-helix' N-terminal domain of RepA (WH1) is triggered in vitro upon binding to plasmid-specific DNA sequences, and occurs at the bacterial nucleoid in vivo. Amyloid fibers are made of distorted RepA-WH1 monomers that assemble as single or double intertwined tubular protofilaments. RepA-WH1 causes in E. coli an amyloid proteinopathy, which is transmissible from mother to daughter cells, but not infectious, and enables conformational imprinting in vitro and in vivo; i.e. RepA-WH1 is a 'prionoid'. Microfluidics allow the assessment of the intracellular dynamics of RepA-WH1: bacterial lineages maintain two types (strains-like) of RepA-WH1 amyloids, either multiple compact cytotoxic particles or a single aggregate with the appearance of a fluidized hydrogel that it is mildly detrimental to growth. The Hsp70 chaperone DnaK governs the phase transition between both types of RepA-WH1 aggregates in vivo, thus modulating the vertical propagation of the prionoid. Engineering chimeras between the Sup35p/[PSI(+)] prion and RepA-WH1 generates [REP-PSI(+)], a synthetic prion exhibiting strong and weak phenotypic variants in yeast. These recent findings on a synthetic, self-contained bacterial prionoid illuminate central issues of protein amyloidogenesis.
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http://dx.doi.org/10.1080/19336896.2015.1129479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981189PMC
January 2017

A novel chromatin tether domain controls topoisomerase IIα dynamics and mitotic chromosome formation.

J Cell Biol 2013 Nov;203(3):471-86

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455.

DNA topoisomerase IIα (Topo IIα) is the target of an important class of anticancer drugs, but tumor cells can become resistant by reducing the association of the enzyme with chromosomes. Here we describe a critical mechanism of chromatin recruitment and exchange that relies on a novel chromatin tether (ChT) domain and mediates interaction with histone H3 and DNA. We show that the ChT domain controls the residence time of Topo IIα on chromatin in mitosis and is necessary for the formation of mitotic chromosomes. Our data suggest that the dynamics of Topo IIα on chromosomes are important for successful mitosis and implicate histone tail posttranslational modifications in regulating Topo IIα.
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http://dx.doi.org/10.1083/jcb.201303045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3824022PMC
November 2013

Cohesin is needed for bipolar mitosis in human cells.

Cell Cycle 2010 May 15;9(9):1764-73. Epub 2010 May 15.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, MN, USA.

Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin.
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http://dx.doi.org/10.4161/cc.9.9.11525DOI Listing
May 2010

Determinants of Rad21 localization at the centrosome in human cells.

Cell Cycle 2010 May 15;9(9):1759-63. Epub 2010 May 15.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, MN, USA.

Cohesin proteins help maintain the physical associations between sister chromatids that arise in S-phase and are removed in anaphase. Recent studies found that cohesins also localize to the centrosomes, the organelles that organize the mitotic bipolar spindle. We find that the cohesin protein Rad21 localizes to centrosomes in a manner that is dependent upon known regulators of sister chromatid cohesion as well as regulators of centrosome function. These data suggest that Rad21 functions at the centrosome and that the regulators of Rad21 coordinate the centrosome and chromosomal functions of cohesin.
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http://dx.doi.org/10.4161/cc.9.9.11523DOI Listing
May 2010

Rad21 is required for centrosome integrity in human cells independently of its role in chromosome cohesion.

Cell Cycle 2010 May 15;9(9):1774-80. Epub 2010 May 15.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, MN, USA.

Classically, chromosomal functions in DNA repair and sister chromatid association have been assigned to the cohesin proteins. More recent studies have provided evidence that cohesins also localize to the centrosomes, which organize the bipolar spindle during mitosis. Depletion of cohesin proteins is associated with multi-polar mitosis in which spindle pole integrity is compromised. However, the spindle pole defects after cohesin depletion could be an indirect consequence of a chromosomal cohesion defect which might impact centrosome integrity via alterations to the spindle microtubule network. Here we show that the cohesin Rad21 is required for centrosome integrity independently of its role as a chromosomal cohesin. Thus, Rad21 may promote accurate chromosome transmission not only by virtue of its function as a chromosomal cohesin, but also because it is required for centrosome function.
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http://dx.doi.org/10.4161/cc.9.9.11524DOI Listing
May 2010

Cytological analysis of chromosome structural defects that result from topoisomerase II dysfunction.

Methods Mol Biol 2009 ;582:189-207

Reproducción Celular y Animal, CIB, CSIC, Madrid, Spain.

For analyzing chromosome structural defects that result from topoisomerase II (topo II) dysfunction, we have adapted classical cell cycle experiments, classical cytological techniques, and the use of a potent topo II inhibitor (ICRF-193). In this chapter, we describe in detail the protocols used and we discuss the rationale for our choice and for the adaptations applied. We clarify in which cell cycle stages each of the different chromosomal aberrations induced by inhibiting topo II take place: lack of chromosome segregation, undercondensation, lack of sister chromatid resolution, and lack of chromosome individualization. We also put these observations into the context of the two topo II-dependent cell cycle checkpoints.
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http://dx.doi.org/10.1007/978-1-60761-340-4_15DOI Listing
January 2010

Chromosome cohesion - rings, knots, orcs and fellowship.

J Cell Sci 2008 Jul;121(Pt 13):2107-14

Department of Pharmacology, UT-Southwestern Medical Center, 6001 Forest Park Rd, Dallas, TX75390, USA.

Sister-chromatid cohesion is essential for accurate chromosome segregation. A key discovery towards our understanding of sister-chromatid cohesion was made 10 years ago with the identification of cohesins. Since then, cohesins have been shown to be involved in cohesion in numerous organisms, from yeast to mammals. Studies of the composition, regulation and structure of the cohesin complex led to a model in which cohesin loading during S-phase establishes cohesion, and cohesin cleavage at the onset of anaphase allows sister-chromatid separation. However, recent studies have revealed activities that provide cohesion in the absence of cohesin. Here we review these advances and propose an integrative model in which chromatid cohesion is a result of the combined activities of multiple cohesion mechanisms.
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http://dx.doi.org/10.1242/jcs.029132DOI Listing
July 2008

Cohesin is dispensable for centromere cohesion in human cells.

PLoS One 2007 Mar 28;2(3):e318. Epub 2007 Mar 28.

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

Background: Proper regulation of the cohesion at the centromeres of human chromosomes is essential for accurate genome transmission. Exactly how cohesion is maintained and is then dissolved in anaphase is not understood.

Principal Findings: We have investigated the role of the cohesin complex at centromeres in human cells both by depleting cohesin subunits using RNA interference and also by expressing a non-cleavable version of the Rad21 cohesin protein. Rad21 depletion results in aberrant anaphase, during which the sister chromatids separate and segregate in an asynchronous fashion. However, centromere cohesion was maintained before anaphase in Rad21-depleted cells, and the primary constrictions at centromeres were indistinguishable from those in control cells. Expression of non-cleavable Rad21 (NC-Rad21), in which the sites normally cleaved by separase are mutated, resulted in delayed sister chromatid resolution in prophase and prometaphase, and a blockage of chromosome arm separation in anaphase, but did not impede centromere separation.

Conclusions: These data indicate that cohesin complexes are dispensable for sister cohesion in early mitosis, yet play an important part in the fidelity of sister separation and segregation during anaphase. Cleavage at the separase-sensitive sites of Rad21 is important for arm separation, but not for centromere separation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000318PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820851PMC
March 2007

Regulation of centromeric cohesion by sororin independently of the APC/C.

Cell Cycle 2007 Mar 1;6(6):714-24. Epub 2007 Mar 1.

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Regulated separation of sister chromatids is the key event of mitosis. Sister chromatids remain cohered from the moment of DNA duplication until anaphase. Two known factors account for cohesion: DNA catenations and cohesin complexes. Premature loss of centromeric cohesion is prevented by the spindle checkpoint. Here we show that sororin, a protein implicated in promoting cohesion through effects on cohesin complexes, is involved in maintenance of cohesion in response to the spindle checkpoint. Sororin-depleted cells reach prometaphase with cohered sister chromatids and are able to form metaphase plates. However, loss of cohesion in anaphase is asynchronous and cells are unresponsive to the spindle checkpoint, accumulating with separated sisters scattered throughout the cytoplasm. These phenotypes are similar to those seen after Shugoshin depletion, suggesting that sororin and Shugoshin might act in concert. Furthermore, sororin-depleted and Shugoshin-depleted cells lose cohesion independently of the APC/C. Therefore, sororin and Shugoshin protect centromeric cohesion in response to the spindle checkpoint, but prevent the removal of cohesion by a mechanism independent of the APC/C.
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http://dx.doi.org/10.4161/cc.6.6.3935DOI Listing
March 2007

PIASgamma is required for faithful chromosome segregation in human cells.

PLoS One 2006 Dec 20;1:e53. Epub 2006 Dec 20.

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America.

Background: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood.

Principal Findings: We identify PIASgamma as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASgamma, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASgamma-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASgamma and Topoisomerase II.

Conclusions: PIASgamma directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASgamma in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0000053PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1762334PMC
December 2006

Topoisomerase II checkpoints: universal mechanisms that regulate mitosis.

Cell Cycle 2006 Sep 1;5(17):1925-8. Epub 2006 Sep 1.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Checkpoint controls confer order to the cell cycle and help prevent genome instability. Here we discuss the Topoisomerase II (Decatenation) Checkpoint which functions to regulate mitotic progression so that chromosomes can be efficiently condensed in prophase and can be segregated with high fidelity in anaphase.
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http://dx.doi.org/10.4161/cc.5.17.3200DOI Listing
September 2006

A mitotic topoisomerase II checkpoint in budding yeast is required for genome stability but acts independently of Pds1/securin.

Genes Dev 2006 May;20(9):1162-74

Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Topoisomerase II (Topo II) performs topological modifications on double-stranded DNA molecules that are essential for chromosome condensation, resolution, and segregation. In mammals, G2 and metaphase cell cycle delays induced by Topo II poisons have been proposed to be the result of checkpoint activation in response to the catenation state of DNA. However, the apparent lack of such controls in model organisms has excluded genetic proof that Topo II checkpoints exist and are separable from the conventional DNA damage checkpoint controls. But here, we define a Topo II-dependent G2/M checkpoint in a genetically amenable eukaryote, budding yeast, and demonstrate that this checkpoint enhances cell survival. Conversely, a lack of the checkpoint results in aneuploidy. Neither DNA damage-responsive pathways nor Pds1/securin are needed for this checkpoint. Unusually, spindle assembly checkpoint components are required for the Topo II checkpoint, but checkpoint activation is not the result of failed chromosome biorientation or a lack of spindle tension. Thus, compromised Topo II function activates a yeast checkpoint system that operates by a novel mechanism.
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http://dx.doi.org/10.1101/gad.1367206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1472475PMC
May 2006

Evidence that the yeast spindle assembly checkpoint has a target other than the anaphase promoting complex.

Cell Cycle 2005 Nov 5;4(11):1555-7. Epub 2005 Nov 5.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

The spindle assembly checkpoint monitors biorientation of chromosomes on the metaphase spindle and inhibits the Anaphase Promoting Complex (APC) specificity factor Cdc20. If APC-Cdc20 is the sole target of the spindle checkpoint, then cells lacking APC and its targets, B-type cyclin and securin, would lack spindle checkpoint function. We tested this hypothesis in yeast cells that are APC-null. Surprisingly, we find that such yeast cells are able to activate the spindle assembly checkpoint, delaying cell cycle progression in G2/M phase. These data suggest that the spindle checkpoint has a non-APC target that can restrain anaphase onset.
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http://dx.doi.org/10.4161/cc.4.11.2144DOI Listing
November 2005

Anaphase promoting complex or cyclosome?

Cell Cycle 2005 Nov 26;4(11):1585-92. Epub 2005 Nov 26.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

The anaphase promoting complex/cyclosome (APC/C) was initially described as a multi-subunit protein complex that ubiquitinates anaphase inhibitors thus targeting them for destruction by proteasomes to initiate loss of sister chromatid cohesion. However, recent studies have identified important new functions of the APC/C. Moreover, sister centromere separation can occur in the absence of APC/C activity in mammals, indicating that anaphase onset might be triggered by multiple factors. Here we discuss whether the APC/C functions primarily as the anaphase trigger, or whether it has more general properties, relevant for cell cycle control at multiple developmental and cell cycle stages. Additionally, we discuss the validity of the APC-dependent model for sister segregation in mammals.
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http://dx.doi.org/10.4161/cc.4.11.2143DOI Listing
November 2005

Proteasome activity is required for centromere separation independently of securin degradation in human cells.

Cell Cycle 2005 Nov 14;4(11):1558-60. Epub 2005 Nov 14.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Loss of centromere cohesion during anaphase in human cells is regulated by the spindle assembly checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). APC-Cdc20 adds ubiquitin chains to securin inducing its destruction by the proteasome and these events correlate with the loss of sister chromatid cohesion and the onset of anaphase. But whether securin destruction is necessary and sufficient for anaphase initiation is not clear. Therefore, we asked if proteasome activity is needed for anaphase onset in human cells that lack securin. We find that even in the absence of securin, a metaphase block with cohered sister centromeres can be enforced in the absence of proteasome activity. Therefore, other targets of the proteasome must be degraded to allow anaphase onset.
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http://dx.doi.org/10.4161/cc.4.11.2145DOI Listing
November 2005

Regulated separation of sister centromeres depends on the spindle assembly checkpoint but not on the anaphase promoting complex/cyclosome.

Cell Cycle 2005 Nov 7;4(11):1561-75. Epub 2005 Nov 7.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

Key to faithful genetic inheritance is the cohesion between sister centromeres that physically links replicated sister chromatids and is then abruptly lost at the onset of anaphase. Misregulated cohesion causes aneuploidy, birth defects and perhaps initiates cancers. Loss of centromere cohesion is controlled by the spindle checkpoint and is thought to depend on a ubiquitin ligase, the Anaphase Promoting Complex/Cyclosome (APC). But here we present evidence that the APC pathway is dispensable for centromere separation at anaphase in mammals, and that anaphase proceeds in the presence of cyclin B and securin. Arm separation is perturbed in the absence of APC, compromising the fidelity of segregation, but full sister chromatid separation is achieved after a delayed anaphase. Thereafter, cells arrest terminally in telophase with high levels of cyclin B. Extending these findings we provide evidence that the spindle checkpoint regulates centromere cohesion through an APC-independent pathway. We propose that this Centromere Linkage Pathway (CLiP) is a second branch that stems from the spindle checkpoint to regulate cohesion preferentially at the centromeres and that Sgo1 is one of its components.
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http://dx.doi.org/10.4161/cc.4.11.2146DOI Listing
November 2005

Separase is required at multiple pre-anaphase cell cycle stages in human cells.

Cell Cycle 2005 Nov 7;4(11):1576-84. Epub 2005 Nov 7.

Department of Genetics, Cell Biology & Development, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.

The yeast separase proteins Esp1 and Cut1 are required for loss of sister chromatid cohesion that occurs at the moment of anaphase onset. Circumstantial evidence has linked human separase to centromere separation at anaphase, but a direct test that the role of this enzyme is functionally conserved with the yeast proteins is lacking. Here we describe the effects of separase depletion from human cells using RNA interference. Surprisingly, HeLa cells lacking separase are delayed or arrest at the G2-M phase transition. This arrest is not likely due to the activation of a known checkpoint control, but may be a result of a failure to construct a mitotic chromosome. Without separase, cells also have a prolonged prometaphase, perhaps resulting from defects in spindle assembly or dynamics. In cells that reach mitosis, sister arm resolution and separation are perturbed, whereas in anaphase cells sister centromeres do appear to separate. These data indicate that separase function is not restricted to anaphase initiation and that its role in promoting loss of sister chromatid cohesion might be preferentially at arms but not centromeres.
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http://dx.doi.org/10.4161/cc.4.11.2147DOI Listing
November 2005

Roles of polo-like kinase 1 in the assembly of functional mitotic spindles.

Curr Biol 2004 Oct;14(19):1712-22

Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

Background: The stable association of chromosomes with both poles of the mitotic spindle (biorientation) depends on spindle pulling forces. These forces create tension across sister kinetochores and are thought to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint. Polo-like kinase 1 (Plk1) has been implicated in regulating centrosome maturation, mitotic entry, sister chromatid cohesion, the anaphase-promoting complex/cyclosome (APC/C), and cytokinesis, but it is unknown if Plk1 controls chromosome biorientation.

Results: We have analyzed Plk1 functions in synchronized mammalian cells by RNA interference (RNAi). Plk1-depleted cells enter mitosis after a short delay, accumulate in a preanaphase state, and subsequently often die by apoptosis. Spindles in Plk1-depleted cells lack focused poles and are not associated with centrosomes. Chromosomes attach to these spindles, but the checkpoint proteins Mad2, BubR1, and CENP-E are enriched at many kinetochores. When Plk1-depleted cells are treated with the Aurora B inhibitor Hesperadin, which silences the spindle checkpoint by stabilizing microtubule-kinetochore interactions, cells degrade APC/C substrates and exit mitosis without chromosome segregation and cytokinesis. Experiments with monopolar spindles that are induced by the kinesin inhibitor Monastrol indicate that Plk1 is required for the assembly of spindles that are able to generate poleward pulling forces.

Conclusions: Our results imply that Plk1 is not essential for mitotic entry and APC/C activation but is required for proper spindle assembly and function. In Plk1-depleted cells spindles may not be able to create enough tension across sister kinetochores to stabilize microtubule-kinetochore interactions and to silence the spindle checkpoint.
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http://dx.doi.org/10.1016/j.cub.2004.09.049DOI Listing
October 2004

Regulation of sister chromatid cohesion between chromosome arms.

Curr Biol 2004 Jul;14(13):1187-93

Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030 Vienna, Austria.

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.
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http://dx.doi.org/10.1016/j.cub.2004.06.052DOI Listing
July 2004

Loss of the anaphase-promoting complex in quiescent cells causes unscheduled hepatocyte proliferation.

Genes Dev 2004 Jan;18(1):88-98

Research Institute of Molecular Pathology (IMP), A-1030 Vienna, Austria.

The anaphase-promoting complex or cyclosome (APC/C) is an ubiquitin protein ligase that together with Cdc20 and Cdh1 targets mitotic proteins for degradation by the proteosome. APC-Cdc20 activity during mitosis triggers anaphase by destroying securin and cyclins. APC-Cdh1 promotes degradation of cyclins and other proteins during G(1). We show that loss of APC/C during embryogenesis is early lethal before embryonic day E6.5 (E6.5). To investigate the role of APC/C in quiescent cells, we conditionally inactivated the subunit Apc2 in mice. Deletion of Apc2 in quiescent hepatocytes caused re-entry into the cell cycle and arrest in metaphase, resulting in liver failure. Re-entry into the cell cycle either occurred without any proliferative stimulus or could be easily induced. We demonstrate that the APC has an additional function to prevent hepatocytes from unscheduled re-entry into the cell cycle.
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http://dx.doi.org/10.1101/gad.285404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC314282PMC
January 2004

p53-independent checkpoint controls in a plant cell model.

Biol Res 2003 ;36(3-4):381-8

Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9. E-28040-Madrid, Spain.

Allium cepa L. meristems were used as a plant model to study the p53-independent control of S and G2 phases by checkpoint pathways, in eukaryotic cells. Checkpoint blocks were induced at early and mid S by hydroxyurea. After their spontaneous override, cells became accumulated in G2-prophase, giving rise later on to a delayed mitotic wave. Cell growth was maintained during the checkpoint blocks, as the delayed mitoses were larger in size than the control ones. Under continuous hydroxyurea treatment, the delayed mitotic was formed by two subpopulations: normal mitoses corresponding to cells having properly recovered from the checkpoint block, and abnormal ones resulting from checkpoint adaptation. These latter cells displayed broken chromatids as they had unduly overriden the G2 checkpoint block, without completing DNA repair. The frequency of the checkpoint-adapted mitoses increased with the hydroxyurea concentration from 0.25 to 1.0 mM. However, from 1 mM hydroxyurea upwards, some of the cells lost their competence for checkpoint adaptation. Therefore, the dose of a genotoxic agent that still allows G2 checkpoint adaptation should always be applied in order to get rid of uncontrolled proliferating cells. This is specially suitable for cells lacking a functional p53 protein.
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http://dx.doi.org/10.4067/s0716-97602003000300009DOI Listing
March 2004

Replication-coupled topoisomerase II templates the mitotic chromosome scaffold?

Cell Cycle 2003 May-Jun;2(3):230-2

Institute for Molecular Pathology, Vienna, Austria.

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September 2003

A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed by ectopic expression of mitotic cyclin B2.

Cell Cycle 2002 May-Jun;1(3):187-92

Centro de Investigaciones Biológicas, CSIC, Velázquez 144, 28006 Madrid, Spain.

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.
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December 2002

DNA-damage-independent checkpoints: yeast and higher eukaryotes.

Cell Cycle 2002 Jan;1(1):16-33

The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

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January 2002

Regulation of human separase by securin binding and autocleavage.

Curr Biol 2002 Aug;12(16):1368-78

Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, 1030, Vienna, Austria.

Background: Sister chromatid separation is initiated by separase, a protease that cleaves cohesin and thereby dissolves sister chromatid cohesion. Separase is activated by the degradation of its inhibitor securin and by the removal of inhibitory phosphates. In human cells, separase activation also coincides with the cleavage of separase, but it is not known if this reaction activates separase, which protease cleaves separase, and how separase cleavage is regulated.

Results: Inhibition of separase expression in human cells by RNA interference causes the formation of polyploid cells with large lobed nuclei. In mitosis, many of these cells contain abnormal chromosome plates with unseparated sister chromatids. Inhibitor binding experiments in vitro reveal that securin prevents the access of substrate analogs to the active site of separase. Upon securin degradation, the active site of full-length separase becomes accessible, allowing rapid autocatalytic cleavage of separase at one of three sites. The resulting N- and C-terminal fragments remain associated and can be reinhibited by securin. A noncleavable separase mutant retains its ability to cleave cohesin in vitro.

Conclusions: Our results suggest that separase is required for sister chromatid separation during mitosis in human cells. Our data further indicate that securin inhibits separase by blocking the access of substrates to the active site of separase. Securin proteolysis allows autocatalytic processing of separase into a cleaved form, but separase cleavage is not essential for separase activation.
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http://dx.doi.org/10.1016/s0960-9822(02)01073-4DOI Listing
August 2002

DNA catenations that link sister chromatids until the onset of anaphase are maintained by a checkpoint mechanism.

Eur J Cell Biol 2002 Jan;81(1):9-16

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

Treatment of Allium cepa meristematic cells in metaphase with the topoisomerase II inhibitor ICRF-193, results in bridging of the sister chromatids at anaphase. Separation of the sisters in experimentally generated acentric chromosomal fragments was also inhibited by ICRF-193, indicating that some non-centromeric catenations also persist in metaphase chromosomes. Thus, catenations must be resolved by DNA topoisomerase II at the metaphase-to-anaphase transition to allow segregation of sisters. A passive mechanism could maintain catenations holding sisters until the onset of anaphase. At this point the opposite tension exerted on sister chromatids could render the decatenation reaction physically more favorable than catenation. But this possibility was dismissed as acentric chromosome fragments were able to separate their sister chromatids at anaphase. A timing mechanism (a common trigger for two processes taking different times to be completed) could passively couple the resolution of the last remaining catenations to the moment of anaphase onset. This possibility was also discarded as cells arrested in metaphase with microtubule-destabilising drugs still displayed anaphase bridges when released in the presence of ICRF-193. It is possible that a checkpoint mechanism prevents the release of the last catenations linking sisters until the onset of anaphase. To test whether cells are competent to fully resolve catenations before anaphase onset, we generated multinucleate plant cells. In this system, the nuclei within a single multinucleate cell displayed differences in chromosome condensation at metaphase, but initiated anaphase synchronously. When multinucleates were treated with ICRF-193 at the metaphase-toanaphase transition, tangled and untangled anaphases were observed within the same cell. This can only occur if cells are competent to disentangle sister chromatids before the onset of anaphase, but are prevented from doing so by a checkpoint mechanism.
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http://dx.doi.org/10.1078/0171-9335-00226DOI Listing
January 2002