Negative feedback at kinetochores underlies a responsive spindle checkpoint signal

Accurate chromosome segregation requires coordination between microtubule attachment and spindle checkpoint signaling at the kinetochore. The kinetochore-localized KMN (KNL-1/Mis12 complex/Ndc80 complex) network, which mediates microtubule attachment and scaffolds checkpoint signaling, harbors two distinct microtubule-binding activities: the load-bearing activity of the Ndc80 complex and a less well-understood activity in KNL-1. In this paper, we show that KNL-1 microtubule-binding and -bundling activity resides in its extreme N terminus. Selective perturbation of KNL-1 microtubule binding in Caenorhabditis elegans embryos revealed that this activity is dispensable for both load-bearing attachment formation and checkpoint activation but plays a role in checkpoint silencing at the kinetochore. Perturbation of both microtubule binding and protein phosphatase 1 docking at the KNL-1 N terminus additively affected checkpoint silencing, indicating that, despite their proximity in KNL-1, these two activities make independent contributions. We propose that microtubule binding by KNL-1 functions in checkpoint silencing by sensing microtubules attached to kinetochores and relaying their presence to eliminate generation of the checkpoint signal.

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CENP-I and Aurora B act as a molecular switch that ties RZZ/Mad1 recruitment to kinetochore attachment status

The Journal of Cell Biology ◽

10.1083/jcb.201307137 ◽

2014 ◽

Vol 205 (4) ◽

pp. 541-554 ◽

Cited By ~ 31

Author(s):

Daniel R. Matson ◽

P. Todd Stukenberg

Keyword(s):

Kinase Activity ◽

Spindle Checkpoint ◽

Molecular Switch ◽

Aurora B ◽

Aurora B Kinase ◽

Dynamic Regulation ◽

Checkpoint Proteins ◽

Centromere Protein ◽

Stable Association ◽

Checkpoint Signal

The RZZ (Rod, ZW10, and Zwilch) complex and Mad1 proteins tightly associate with kinetochores to generate the spindle checkpoint signal, but they are released when a kinetochore forms mature microtubule attachments. Here we demonstrate that the centromere protein CENP-I is required to generate a stable association of RZZ and Mad1 with kinetochores. CENP-I also inhibits their removal by dynein stripping. This regulation of Mad1 and RZZ dissociation functions independently of Aurora B, which regulates their association. We show that the microtubule status of each kinetochore independently dictates the recruitment of Aurora B kinase, kinase activity on a kinetochore substrate, and loading of spindle checkpoint proteins. This dynamic regulation of Mad1 association by Aurora B is only uncovered when CENP-I is depleted, consistent with our finding that CENP-I inhibits the dissociation of Mad1. We conclude that the dual activities of Aurora B and CENP-I generate a molecular switch that maintains a robust spindle checkpoint signal at prometaphase kinetochores until they attain mature attachments to microtubules.

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BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1

The Journal of Cell Biology ◽

10.1083/jcb.200204048 ◽

2002 ◽

Vol 158 (3) ◽

pp. 487-496 ◽

Cited By ~ 109

Author(s):

Rey-Huei Chen

Keyword(s):

Spindle Checkpoint ◽

Kinase Domain ◽

Wild Type ◽

Checkpoint Proteins ◽

Anaphase Onset ◽

Egg Extracts ◽

Xenopus Egg ◽

Spindle Microtubules ◽

Xenopus Egg Extracts ◽

Checkpoint Signal

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.

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Spindle Checkpoint Protein Bub1 Is Required for Kinetochore Localization of Mad1, Mad2, Bub3, and Cenp-E, Independently of Its Kinase Activity

The Journal of Cell Biology ◽

10.1083/jcb.153.6.1239 ◽

2001 ◽

Vol 153 (6) ◽

pp. 1239-1250 ◽

Cited By ~ 141

Author(s):

Hilary Sharp-Baker ◽

Rey-Huei Chen

Keyword(s):

Mitotic Spindle ◽

Kinase Activity ◽

Spindle Checkpoint ◽

Wild Type ◽

Checkpoint Proteins ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Checkpoint Signal

The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1–Mad2. However, Bub1 differs from Mad1–Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

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