Chromatin compaction by condensin I, intra-kinetochore stretch and tension, and anaphase onset, in collective spindle assembly checkpoint interaction

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

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Interactions between N-Terminal Modules in MPS1 Enable Spindle Checkpoint Silencing

10.1101/438903 ◽

2018 ◽

Author(s):

Spyridon T. Pachis ◽

Yoshitaka Hiruma ◽

Anastassis Perrakis ◽

Geert J.P.L. Kops

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Intramolecular Interactions ◽

Mitotic Progression ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Regulatory Input ◽

Tpr Domain

ABSTRACTFaithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until all chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to unattached kinetochores to initiate SAC signaling, and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here we show that a helical fragment within the kinetochore-targeting NTE module of MPS1 is required for interactions with kinetochores, and also forms intramolecular interactions with its adjacent TPR domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, ineffecient MPS1 delocalization from kinetochores upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MSP1-NDC80-C interactions.

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The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness the Spindle Assembly Checkpoint

10.1101/2020.01.07.897876 ◽

2020 ◽

Cited By ~ 1

Author(s):

Babhrubahan Roy ◽

Simon JY Han ◽

Adrienne N. Fontan ◽

Ajit P. Joglekar

Keyword(s):

Chromosome Segregation ◽

Copy Number ◽

Genome Stability ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Evolutionary Trend ◽

Binding Affinities ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Maintain Genome Stability

SummaryThe Spindle Assembly Checkpoint (SAC) maintains genome stability while enabling timely anaphase onset. To maintain genome stability, the SAC must be strong so that it delays cell division even if one chromosome is unattached, but for timely anaphase onset, it must be responsive to silencing mechanisms. How it meets these potentially antagonistic requirements is unclear. Here we show that the balance between SAC strength and responsiveness is determined by the number of ‘MELT’ motifs in the kinetochore protein Spc105/KNL1 and their Bub3-Bub1 binding affinities. Spc105/KNL1 must contain many strong MELT motifs to prevent chromosome missegregation, but not too many, because this delays SAC silencing and anaphase onset. We demonstrate this by constructing a Spc105 variant that trades SAC responsiveness for significantly improved chromosome segregation accuracy. We propose that the necessity of balancing SAC strength with responsiveness drives the evolutionary trend of MELT motif number amplification and degeneration of their functionally optimal amino acid sequence.

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PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores

The Journal of Cell Biology ◽

10.1083/jcb.200703133 ◽

2007 ◽

Vol 179 (4) ◽

pp. 601-609 ◽

Cited By ~ 45

Author(s):

Emilie Montembault ◽

Stéphanie Dutertre ◽

Claude Prigent ◽

Régis Giet

Keyword(s):

Rna Processing ◽

Mitotic Spindle ◽

Messenger Rna ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Checkpoint Proteins ◽

Checkpoint Protein ◽

Anaphase Onset ◽

Spindle Microtubules ◽

Spindle Assembly Checkpoint Protein

The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre–messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) failure. Thus, we find that PRP4 is necessary for recruitment or maintenance of the checkpoint proteins MPS1, MAD1, and MAD2 at the kinetochores. Our data clearly identify PRP4 as a previously unrecognized kinetochore component that is necessary to establish a functional SAC.

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BubR1 recruitment to the kinetochore via Bub1 enhances Spindle Assembly Checkpoint signaling

10.1101/2021.08.12.456107 ◽

2021 ◽

Author(s):

Anand Banerjee ◽

Chu Chen ◽

Lauren Humphrey ◽

John J. Tyson ◽

Ajit Joglekar

Keyword(s):

Mathematical Modeling ◽

Dose Response ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Checkpoint ◽

Local Concentration ◽

Checkpoint Signaling ◽

Response Characteristics ◽

Anaphase Onset ◽

Mitotic Checkpoint Complex

During mitosis, unattached kinetochores in a dividing cell generate the anaphase-inhibitory Mitotic Checkpoint Complex (MCC) to activate the Spindle Assembly Checkpoint (SAC) and delay anaphase onset. To generate MCC, these kinetochores recruit MCC constituent proteins including the protein BubR1. The increased local concentration of BubR1 resulting from this recruitment should enhance MCC generation, but prior studies found this not to be the case. We analyzed the contribution of two BubR1 recruitment pathways to MCC generation in human kinetochores. For these analyses, we isolated a subset of the MCC generation reactions to the cytosol using ectopic SAC activation systems. These analyses and mathematical modeling show that BubR1 binding to the SAC protein Bub1, but not to the 'KI' motifs in the kinetochore protein Knl1, significantly enhances the rate of Bub1-mediated MCC generation in the kinetochore. Our work also suggests that Bub1-BubR1 stoichiometry will strongly influence the dose-response characteristics of SAC signaling.

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Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

eLife ◽

10.7554/elife.25366 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 34

Author(s):

Margarida Moura ◽

Mariana Osswald ◽

Nelson Leça ◽

João Barbosa ◽

António J Pereira ◽

...

Keyword(s):

Protein Phosphatase ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Mitotic Exit ◽

Protein Phosphatase 1 ◽

Anaphase Onset ◽

Phosphorylation Cascade ◽

Spindle Microtubules ◽

Early Mitosis

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

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Coupling of Cdc20 inhibition and activation by BubR1

10.1101/2020.12.18.423461 ◽

2020 ◽

Author(s):

Jamin Hein ◽

Dimitriya H Garvanska ◽

Isha Nasa ◽

Arminja Kettenbach ◽

Jakob Nilsson

Keyword(s):

Ubiquitin Ligase ◽

Cross Talk ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cyclin B1 ◽

Mitotic Checkpoint ◽

Anaphase Onset ◽

Mitotic Checkpoint Complex

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by Cyclin B1-Cdk1 independently inhibits APC/C-Cdc20 activation. This creates a conundrum for how Cdc20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively our work reveals how Cdc20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.

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Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore

The Journal of Cell Biology ◽

10.1083/jcb.200702062 ◽

2007 ◽

Vol 177 (6) ◽

pp. 1005-1015 ◽

Cited By ~ 144

Author(s):

Eric R. Griffis ◽

Nico Stuurman ◽

Ronald D. Vale

Keyword(s):

Spindle Assembly Checkpoint ◽

Metaphase Plate ◽

Spindle Assembly ◽

Cytoplasmic Dynein ◽

Checkpoint Proteins ◽

Drosophila Melanogaster S2 Cells ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Cell Biol ◽

Novel Protein

The eukaryotic spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores and prevents anaphase onset until all kinetochores are aligned on the metaphase plate. In higher eukaryotes, cytoplasmic dynein is involved in silencing the SAC by removing the checkpoint proteins Mad2 and the Rod–Zw10–Zwilch complex (RZZ) from aligned kinetochores (Howell, B.J., B.F. McEwen, J.C. Canman, D.B. Hoffman, E.M. Farrar, C.L. Rieder, and E.D. Salmon. 2001. J. Cell Biol. 155:1159–1172; Wojcik, E., R. Basto, M. Serr, F. Scaerou, R. Karess, and T. Hays. 2001. Nat. Cell Biol. 3:1001–1007). Using a high throughput RNA interference screen in Drosophila melanogaster S2 cells, we have identified a new protein (Spindly) that accumulates on unattached kinetochores and is required for silencing the SAC. After the depletion of Spindly, dynein cannot target to kinetochores, and, as a result, cells arrest in metaphase with high levels of kinetochore-bound Mad2 and RZZ. We also identified a human homologue of Spindly that serves a similar function. However, dynein's nonkinetochore functions are unaffected by Spindly depletion. Our findings indicate that Spindly is a novel regulator of mitotic dynein, functioning specifically to target dynein to kinetochores.

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Chromosome biorientation and APC activity remain uncoupled in oocytes with reduced volume

The Journal of Cell Biology ◽

10.1083/jcb.201606134 ◽

2017 ◽

Vol 216 (12) ◽

pp. 3949-3957 ◽

Cited By ~ 13

Author(s):

Simon I.R. Lane ◽

Keith T. Jones

Keyword(s):

Cell Volume ◽

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Anaphase Promoting Complex ◽

Chromosome Missegregation ◽

Anaphase Onset ◽

Reduced Volume ◽

Chromosome Attachment ◽

Meiosis I

The spindle assembly checkpoint (SAC) prevents chromosome missegregation by coupling anaphase onset with correct chromosome attachment and tension to microtubules. It does this by generating a diffusible signal from free kinetochores into the cytoplasm, inhibiting the anaphase-promoting complex (APC). The volume in which this signal remains effective is unknown. This raises the possibility that cell volume may be the reason the SAC is weak, and chromosome segregation error-prone, in mammalian oocytes. Here, by a process of serial bisection, we analyzed the influence of oocyte volume on the ability of the SAC to inhibit bivalent segregation in meiosis I. We were able to generate oocytes with cytoplasmic volumes reduced by 86% and observed changes in APC activity consistent with increased SAC control. However, bivalent biorientation remained uncoupled from APC activity, leading to error-prone chromosome segregation. We conclude that volume is one factor contributing to SAC weakness in oocytes. However, additional factors likely uncouple chromosome biorientation with APC activity.

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