scholarly journals PP2A-B56 opposes Mps1 phosphorylation of Knl1 and thereby promotes spindle assembly checkpoint silencing

2014 ◽  
Vol 206 (7) ◽  
pp. 833-842 ◽  
Author(s):  
Antonio Espert ◽  
Pelin Uluocak ◽  
Ricardo Nunes Bastos ◽  
Davinderpreet Mangat ◽  
Philipp Graab ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Feedback Loop ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Eukaryotic Cells ◽  
Aurora B ◽  
Safeguard Mechanism

The spindle assembly checkpoint (SAC) monitors correct attachment of chromosomes to microtubules, an important safeguard mechanism ensuring faithful chromosome segregation in eukaryotic cells. How the SAC signal is turned off once all the chromosomes have successfully attached to the spindle remains an unresolved question. Mps1 phosphorylation of Knl1 results in recruitment of the SAC proteins Bub1, Bub3, and BubR1 to the kinetochore and production of the wait-anaphase signal. SAC silencing is therefore expected to involve a phosphatase opposing Mps1. Here we demonstrate in vivo and in vitro that BubR1-associated PP2A-B56 is a key phosphatase for the removal of the Mps1-mediated Knl1 phosphorylations necessary for Bub1/BubR1 recruitment in mammalian cells. SAC silencing is thus promoted by a negative feedback loop involving the Mps1-dependent recruitment of a phosphatase opposing Mps1. Our findings extend the previously reported role for BubR1-associated PP2A-B56 in opposing Aurora B and suggest that BubR1-bound PP2A-B56 integrates kinetochore surveillance and silencing of the SAC.

scholarly journals RanBP2 and SENP3 Function in a Mitotic SUMO2/3 Conjugation-Deconjugation Cycle on Borealin

2009 ◽  
Vol 20 (1) ◽  
pp. 410-418 ◽  
Author(s):  
Ulf R. Klein ◽  
Markus Haindl ◽  
Erich A. Nigg ◽  
Stefan Muller
Keyword(s):  
Mammalian Cells ◽  
Spindle Assembly Checkpoint ◽  
Critical Role ◽  
Specific Interaction ◽  
Regulatory Function ◽  
Mitotic Progression ◽  
Chromosome Congression ◽  
Chromosomal Passenger

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation–deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.

scholarly journals Centromere-localized Aurora B kinase is required for the fidelity of chromosome segregation

2019 ◽  
Vol 219 (2) ◽  
Author(s):  
Cai Liang ◽  
Zhenlei Zhang ◽  
Qinfu Chen ◽  
Haiyan Yan ◽  
Miao Zhang ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Essential Role ◽  
Spindle Assembly Checkpoint ◽  
Histone H3 ◽  
Spindle Assembly ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Chromosome Missegregation ◽  
Proper Timing ◽  
Segregation Of Chromosomes

Aurora B kinase plays an essential role in chromosome bi-orientation, which is a prerequisite for equal segregation of chromosomes during mitosis. However, it remains largely unclear whether centromere-localized Aurora B is required for faithful chromosome segregation. Here we show that histone H3 Thr-3 phosphorylation (H3pT3) and H2A Thr-120 phosphorylation (H2ApT120) can independently recruit Aurora B. Disrupting H3pT3-mediated localization of Aurora B at the inner centromere impedes the decline in H2ApT120 during metaphase and causes H2ApT120-dependent accumulation of Aurora B at the kinetochore-proximal centromere. Consequently, silencing of the spindle assembly checkpoint (SAC) is delayed, whereas the fidelity of chromosome segregation is negligibly affected. Further eliminating an H2ApT120-dependent pool of Aurora B restores proper timing for SAC silencing but increases chromosome missegregation. Our data indicate that H2ApT120-mediated localization of Aurora B compensates for the loss of an H3pT3-dependent pool of Aurora B to correct improper kinetochore–microtubule attachments. This study provides important insights into how centromeric Aurora B regulates SAC and kinetochore attachment to microtubules to ensure error-free chromosome segregation.

scholarly journals Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

1996 ◽  
Vol 16 (7) ◽  
pp. 3576-3586 ◽  
Author(s):  
C H Yang ◽  
J Tomkiel ◽  
H Saitoh ◽  
D H Johnson ◽  
W C Earnshaw
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
In Vitro Assays ◽  
Centromeric Heterochromatin ◽  
Structural Protein ◽  
Chromosomal Site

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

scholarly journals CSC-1

2003 ◽  
Vol 161 (2) ◽  
pp. 229-236 ◽  
Author(s):  
Alper Romano ◽  
Annika Guse ◽  
Ivica Krascenicova ◽  
Heinke Schnabel ◽  
Ralf Schnabel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Genetic Analysis ◽  
Chromosome Segregation ◽  
Kinase Activity ◽  
Aurora B ◽  
Aurora B Kinase ◽  
C Elegans ◽  
The Individual

The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, we have identified a new subunit of the Aurora B kinase complex, CSC-1. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase.

scholarly journals Aurora B Tension Sensing Mechanisms in the Kinetochore Ensure Accurate Chromosome Segregation

2021 ◽  
Vol 22 (16) ◽  
pp. 8818
Author(s):  
Shelby L. McVey ◽  
Jenna K. Cosby ◽  
Natalie J. Nannas
Keyword(s):  
Chromosome Segregation ◽  
Birth Defects ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Aurora B ◽  
Sister Chromatids ◽  
Congenital Birth Defects ◽  
Biochemical Signals ◽  
Segregation Of Chromosomes

The accurate segregation of chromosomes is essential for the survival of organisms and cells. Mistakes can lead to aneuploidy, tumorigenesis and congenital birth defects. The spindle assembly checkpoint ensures that chromosomes properly align on the spindle, with sister chromatids attached to microtubules from opposite poles. Here, we review how tension is used to identify and selectively destabilize incorrect attachments, and thus serves as a trigger of the spindle assembly checkpoint to ensure fidelity in chromosome segregation. Tension is generated on properly attached chromosomes as sister chromatids are pulled in opposing directions but resisted by centromeric cohesin. We discuss the role of the Aurora B kinase in tension-sensing and explore the current models for translating mechanical force into Aurora B-mediated biochemical signals that regulate correction of chromosome attachments to the spindle.

scholarly journals SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation

2021 ◽  
Author(s):  
Manjuan Zhang ◽  
Fengrui Yang ◽  
Wenwen Wang ◽  
Xiwei Wang ◽  
Dongmei Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Chromosome Segregation ◽  
Aurora B ◽  
Spindle Microtubule ◽  
Dynamic Interactions ◽  
Chromosome Alignment ◽  
Spindle Microtubules ◽  
And Behavior

Abstract Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP–Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore–microtubule attachments to achieve faithful cell division.

scholarly journals Inhibitors of the Bub1 spindle assembly checkpoint kinase: Synthesis of BAY-320 and comparison with 2OH-BNPP1

2020 ◽  
Author(s):  
Ilma Amalina ◽  
Ailsa Bennett ◽  
Helen Whalley ◽  
David Perera ◽  
Joanne C. McGrail ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Minor Role ◽  
Chromosome Alignment ◽  
High Concentrations ◽  
A Minor ◽  
Target Effects

SummaryBub1 is a serine/threonine kinase proposed to function centrally in both mitotic chromosome alignment and the spindle assembly checkpoint (SAC), however its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors can be invaluable tools for investigation of kinase function, we decided to evaluate the relative potential of two agents (2OH-BNPPI and BAY-320) as Bub1 inhibitors. After confirming that both agents inhibit Bub1 in vitro, we developed a cell based-assay to specifically measure Bub1 inhibition in vivo. For this assay we overexpressed a fusion of Histone 2B and the Bub1 kinase region (Bub1C) tethering it in close proximity to H2A, which generated a strong ectopic H2ApT120 signal along chromosome arms. The ectopic signal generated from Bub1C activity was effectively inhibited by BAY-320, but not 2OH-BNPP1. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localisation. Preliminary experiments using BAY-320 suggested a minor role for Bub1 kinase activity in chromosome alignment and the SAC, however results suggest that BAY-320 may exhibit off-target effects at the concentration required to demonstrate these outcomes. In conclusion, 2OH-BNPP1 may not be an effective Bub1 inhibitor in vivo, and while BAY-320 is able to inhibit Bub1 in vivo, the high concentrations required and potential for off-target effects highlight the ongoing need for improved Bub1 inhibitors.

scholarly journals Whole chromosome loss in tetraploid cells confers tumorigenic potential in a mouse allograft model

2017 ◽  
Author(s):  
Rozario Thomas ◽  
Daniel H Marks ◽  
Yvette Chin ◽  
Robert Benezra
Keyword(s):  
Mammalian Cells ◽  
Spindle Assembly Checkpoint ◽  
Chromosome Loss ◽  
Causative Role ◽  
Embryonic Fibroblasts ◽  
Tumorigenic Potential ◽  
Immunocompromised Mice ◽  
Shed Light

AbstractWhole chromosome gains or losses (aneuploidy) are a hallmark of ~70% of human tumors. Modeling the consequences of aneuploidy has relied on perturbing spindle assembly checkpoint (SAC) components but interpretations of these experiments are clouded by the multiple functions of these proteins. Here we used a Cre recombinase-mediated chromosome loss strategy to individually delete mouse chromosomes 9, 10, 12 or 14 in tetraploid immortalized murine embryonic fibroblasts. While the aneuploid cells generally display a growth disadvantage in vitro, they grow significantly better in low adherence sphere-forming conditions and 3 of the 4 lines are transformed in vivo, forming large and invasive tumors in immunocompromised mice. The aneuploid cells display increased chromosomal instability and DNA damage, a mutator phenotype associated with tumorigenesis in vivo. Thus, these studies demonstrate a causative role for whole chromosome loss in tumorigenesis and may shed light on the early consequences of aneuploidy in mammalian cells.

scholarly journals Structure-Specific Recognition Protein 1 Facilitates Microtubule Growth and Bundling Required for Mitosis

2009 ◽  
Vol 30 (4) ◽  
pp. 935-947 ◽  
Author(s):  
Shelya X. Zeng ◽  
Yanping Li ◽  
Yetao Jin ◽  
Qi Zhang ◽  
David M. Keller ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Spindle Assembly ◽  
Tubulin Polymerization ◽  
Chromosome Movement ◽  
Specific Recognition ◽  
Recognition Protein ◽  
Microtubule Growth

ABSTRACT Tight regulation of microtubule (MT) dynamics is essential for proper chromosome movement during mitosis. Here we show, using mammalian cells, that structure-specific recognition protein 1 (SSRP1) is a novel regulator of MT dynamics. SSRP1 colocalizes with the spindle and midbody MTs, and associates with MTs both in vitro and in vivo. Purified SSRP1 facilitates tubulin polymerization and MT bundling in vitro. Knockdown of SSRP1 inhibits the growth of MTs and leads to disorganized spindle structures, reduction of K-fibers and midbody fibers, disrupted chromosome movement, and attenuated cytokinesis in vivo. These results demonstrate that SSRP1 is crucial for MT growth and spindle assembly during mitosis.

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