Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding.
ABSTRACT: Premature anaphase onset is prevented by the mitotic checkpoint through production of a "wait anaphase" inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, we demonstrate that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. We show that purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. Our results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1.
Project description:The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1-Bub3 and BubR1-Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1-Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1-Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment.
Project description: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.
Project description:The mitotic checkpoint acts to maintain chromosome content by generation of a diffusible anaphase inhibitor. Unattached kinetochores catalyze a conformational shift in Mad2, converting an inactive open form into a closed form that can capture Cdc20, the mitotic activator of the APC/C ubiquitin ligase. Mad2 binding is now shown to promote a functional switch in Cdc20, exposing a previously inaccessible site for binding to BubR1's conserved Mad3 homology domain. BubR1, but not Mad2, binding to APC/C(Cdc20) is demonstrated to inhibit ubiquitination of cyclin B. Closed Mad2 is further shown to catalytically amplify production of BubR1-Cdc20 without necessarily being part of the complex. Thus, the mitotic checkpoint is produced by a cascade of two catalytic steps: an initial step acting at unattached kinetochores to produce a diffusible Mad2-Cdc20 intermediate and a diffusible step in which that intermediate amplifies production of BubR1-Cdc20, the inhibitor of cyclin B ubiquitination, by APC/C(Cdc20).
Project description:The mitotic checkpoint monitors kinetochore-microtubule attachment, delays anaphase onset and prevents aneuploidy when unattached or tensionless kinetochores are present in cells. Mitotic arrest deficiency 1 (MAD1) is one of the evolutionarily conserved core mitotic checkpoint proteins. MAD1 forms a cell cycle independent complex with MAD2 through its MAD2 interaction motif (MIM) in the middle region. Such a complex is enriched at unattached kinetochores and functions as an unusual catalyst to promote conformational change of additional MAD2 molecules, constituting a crucial signal amplifying mechanism for the mitotic checkpoint. Only MAD2 in its active conformation can be assembled with BUBR1 and CDC20 to form the Mitotic Checkpoint Complex (MCC), which is a potent inhibitor of anaphase onset. Recent research has shed light on how MAD1 is recruited to unattached kinetochores, and how it carries out its catalytic activity. Here we review these advances and discuss their implications for future research.
Project description:The spindle assembly checkpoint (SAC) delays anaphase until all chromosomes are bioriented on the mitotic spindle. Under current models, unattached kinetochores transduce the SAC by catalyzing the intramitotic production of a diffusible inhibitor of APC/C(Cdc20) (the anaphase-promoting complex/cyclosome and its coactivator Cdc20, a large ubiquitin ligase). Here we show that nuclear pore complexes (NPCs) in interphase cells also function as scaffolds for anaphase-inhibitory signaling. This role is mediated by Mad1-Mad2 complexes tethered to the nuclear basket, which activate soluble Mad2 as a binding partner and inhibitor of Cdc20 in the cytoplasm. Displacing Mad1-Mad2 from nuclear pores accelerated anaphase onset, prevented effective correction of merotelic errors, and increased the threshold of kinetochore-dependent signaling needed to halt mitosis in response to spindle poisons. A heterologous Mad1-NPC tether restored Cdc20 inhibitor production and normal M phase control. We conclude that nuclear pores and kinetochores both emit "wait anaphase" signals that preserve genome integrity.
Project description:To prevent aneuploidy, cells require a mitotic surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC prevents metaphase/anaphase transition by blocking the ubiquitylation and destruction of cyclin B and securin via the Cdc20-activated anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway. This checkpoint involves the kinetochore proteins Mad2, BubR1, and Cdc20. Mad2 and BubR1 are inhibitors of the APC/C, but Cdc20 is an activator. Exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear; in vertebrates, most current models suggest that kinetochore-bound Mad2 is required for initial binding to Cdc20 to form a stable complex that includes BubR1. Here, we show that the Mad2 kinetochore dimerization recruitment mechanism is conserved and that the recruitment of Cdc20 to kinetochores in Drosophila requires BubR1 but not Mad2. BubR1 and Mad2 can bind to Cdc20 independently, and the interactions are enhanced after cells are arrested at mitosis by the depletion of Cdc27 using RNA interference (RNAi) in S2 cells or by MG132 treatment in syncytial embryos. These findings offer an explanation of why BubR1 is more important than Mad2 for SAC function in flies. These findings could lead to a better understanding of vertebrate SAC mechanisms.
Project description:The mitotic checkpoint (also known as the spindle assembly checkpoint) prevents premature anaphase onset through generation of an inhibitor of the E3 ubiquitin ligase APC/C, whose ubiquitination of cyclin B and securin targets them for degradation. Combining in vitro reconstitution and cell-based assays, we now identify dual mechanisms through which Bub3 promotes mitotic checkpoint signaling. Bub3 enhances signaling at unattached kinetochores not only by facilitating binding of BubR1 but also by enhancing Cdc20 recruitment to kinetochores mediated by BubR1's internal Cdc20 binding site. Downstream of kinetochore-produced complexes, Bub3 promotes binding of BubR1's conserved, amino terminal Cdc20 binding domain to a site in Cdc20 that becomes exposed by initial Mad2 binding. This latter Bub3-stimulated event generates the final mitotic checkpoint complex of Bub3-BubR1-Cdc20 that selectively inhibits ubiquitination of securin and cyclin B by APC/C(Cdc20). Thus, Bub3 promotes two distinct BubR1-Cdc20 interactions, involving each of the two Cdc20 binding sites of BubR1 and acting at unattached kinetochores or cytoplasmically, respectively, to facilitate production of the mitotic checkpoint inhibitor.
Project description:The spindle checkpoint senses unattached kinetochores and inhibits the Cdc20-bound anaphase-promoting complex or cyclosome (APC/C), to delay anaphase, thereby preventing aneuploidy. A critical checkpoint inhibitor of APC/C(Cdc20) is the mitotic checkpoint complex (MCC). It is unclear whether MCC suffices to inhibit all cellular APC/C. Here we show that human checkpoint kinase Bub1 not only directly phosphorylates Cdc20, but also scaffolds Plk1-mediated phosphorylation of Cdc20. Phosphorylation of Cdc20 by Bub1-Plk1 inhibits APC/C(Cdc20) in vitro and is required for checkpoint signalling in human cells. Bub1-Plk1-dependent Cdc20 phosphorylation is regulated by upstream checkpoint signals and is dispensable for MCC assembly. A phospho-mimicking Cdc20 mutant restores nocodazole-induced mitotic arrest in cells depleted of Mad2 or BubR1. Thus, Bub1-Plk1-mediated phosphorylation of Cdc20 constitutes an APC/C-inhibitory mechanism that is parallel, but not redundant, to MCC formation. Both mechanisms are required to sustain mitotic arrest in response to spindle defects.
Project description:The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit.
Project description:The mitotic checkpoint monitors kinetochore-microtubule attachment and prevents anaphase until all kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to kinetochores. Unattached, checkpoint-active kinetochores accumulate multiple checkpoint proteins, which are depleted from kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached kinetochores, it is not known what changes at kinetochores are essential for anaphase promoting complex/cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, we show that recruiting the checkpoint protein Mad1 to metaphase kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 or BubR1. Furthermore, Mad2 binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of our checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at kinetochores.