Anaphase Onset Research Articles

α-tubulin detyrosination fine-tunes kinetochore-microtubule attachments

Post-translational cycles of α-tubulin detyrosination and tyrosination generate microtubule diversity, the cellular functions of which remain largely unknown. Here we show that α-tubulin detyrosination regulates kinetochore-microtubule attachments to ensure normal chromosome oscillations and timely anaphase onset during mitosis. Remarkably, detyrosinated α-tubulin levels near kinetochore microtubule plus-ends depend on the direction of chromosome motion during metaphase. Proteomic analyses unveil that the KNL-1/MIS12/NDC80 (KMN) network that forms the core microtubule-binding site at kinetochores and the microtubule-rescue protein CLASP2 are enriched on tyrosinated and detyrosinated microtubules during mitosis, respectively. α-tubulin detyrosination enhances CLASP2 binding and NDC80 complex diffusion along the microtubule lattice in vitro. Rescue experiments overexpressing NDC80, including variants with slower microtubule diffusion, suggest a functional interplay with α-tubulin detyrosination for the establishment of a labile kinetochore-microtubule interface. These results offer a mechanistic explanation for how different detyrosinated α-tubulin levels near kinetochore microtubule plus-ends fine-tune load-bearing attachments to both growing and shrinking microtubules.

Aurora B controls anaphase onset and error-free chromosome segregation in trypanosomes.

Kinetochores form the interface between chromosomes and spindle microtubules and are thus under tight control by a complex regulatory circuitry. The Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint. Intriguingly, Aurora B is conserved even in kinetoplastids, a group of early-branching eukaryotes which possess a unique set of kinetochore proteins. It remains unclear how their kinetochores are regulated to ensure faithful chromosome segregation. Here, we show in Trypanosoma brucei that Aurora B activity controls the metaphase-to-anaphase transition through phosphorylation of the divergent Bub1-like protein KKT14. Depletion of KKT14 overrides the metaphase arrest resulting from Aurora B inhibition, while expression of non-phosphorylatable KKT14 delays anaphase onset. Finally, we demonstrate that re-targeting Aurora B to the outer kinetochore suffices to promote mitotic exit but causes extensive chromosome missegregation in anaphase. Our results indicate that Aurora B and KKT14 are involved in an unconventional circuitry controlling cell cycle progression in trypanosomes.

Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20Slp1 under stress

Mitotic anaphase onset is a key cellular process tightly regulated by multiple kinases. The involvement of mitogen-activated protein kinases (MAPKs) in this process has been established in Xenopus egg extracts. However, the detailed regulatory cascade remains elusive, and it is also unknown whether the MAPK-dependent mitotic regulation is evolutionarily conserved in the single-cell eukaryotic organisms such as fission yeast (Schizosaccharomyces pombe). Here, we show that two MAPKs in S. pombe indeed act in concert to restrain anaphase-promoting complex/cyclosome (APC/C) activity upon activation of the spindle assembly checkpoint (SAC). One MAPK, Pmk1, binds to and phosphorylates Slp1Cdc20, the co-activator of APC/C. Phosphorylation of Slp1Cdc20 by Pmk1, but not by Cdk1, promotes its subsequent ubiquitylation and degradation. Intriguingly, Pmk1-mediated phosphorylation event is also required to sustain SAC under environmental stress. Thus, our study establishes a new underlying molecular mechanism of negative regulation of APC/C by MAPK upon stress stimuli, and provides a previously unappreciated framework for regulation of anaphase entry in eukaryotic cells.

Disappearance of Cdc14 from the daughter spindle pole body requires Glc7-Bud14.

The conserved phosphatase Cdc14 facilitates mitotic exit in budding yeast by counteracting mitotic cyclin-dependent kinase activity. Cdc14 is kept in the nucleolus until anaphase onset, when it is released transiently into the nucleoplasm. In late anaphase, Cdc14 is fully released into the cytoplasm upon activation of the mitotic exit network (MEN) to trigger mitotic exit. Cdc14 also localizes to yeast spindle pole bodies (SPBs) in anaphase and dephosphorylates key targets residing on SPBs to allow SPB duplication and prime the MEN. Protein phosphatase 1 (Glc7) with regulatory subunit Bud14 is another phosphatase that plays a key role in the spatiotemporal control of mitotic exit. In this study, we investigated the regulation of Cdc14 localization by Bud14-Glc7. We used fluorescence microscopy to analyze Cdc14 localization in BUD14 wildtype and BUD14 knockout cells (bud14Δ) as well as in cells expressing a mutant allele of BUD14 (bud14-F379A) that cannot bind Glc7. We also utilized a yeast two-hybrid (Y2H) system to examine the interaction of Bud14 with Cdc14. We found that Cdc14 remains at the SPBs longer in bud14Δ and bud14-F379A compared to wildtype cells. This effect is limited to the SPB that has migrated to the daughter cell (dSPB). Cdc14 localizes to both SPBs shortly after anaphase onset. In mid-to-late anaphase, levels of Cdc14 increase at the dSPB in both wildtype and bud14Δ cells. With mitotic exit, Cdc14 disappears from the dSPB in wildtype cells but not in bud14Δ cells. Accordingly, 50% of bud14Δ cells in G1 have Cdc14 at their SPBs. We also found that Cdc14 localization at the dSPB was largely, but not entirely, dependent on Bfa1 in bud14Δ cells. Furthermore, Bud14 interacted with Cdc14 in the Y2H system. Our results suggest that Glc7-Bud14 is part of a mechanism that promotes Cdc14 disappearance from the dSPB.

Spatiotemporal regulation of MELK during mitosis.

Maternal Embryonic Leucine Zipper Kinase (MELK) has been studied intensively in recent years due to its overexpression in multiple cancers. However, the cell biology of MELK remains less characterized despite its well-documented association with mitosis. Here we report a distinctive pattern of human MELK that translocates from the cytoplasm to cell cortex within 3min of anaphase onset. The cortex association lasts about 30min till telophase. The spatiotemporal specific localization of MELK depends on the interaction between its Threonine-Proline (TP) rich domain and kinase associated 1 (KA1) domain, which is regulated by CDK1 kinase and PP4 protein phosphatase. KA1 domains are known to regulate kinase activities through various intramolecular interactions. Our results revealed a new role for KA1 domain to control subcellular localization of a protein kinase.

Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses.

Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase ∼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is ∼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an ∼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.

Measuring and modeling the dynamics of mitotic error correction

Error correction is central to many biological systems and is critical for protein function and cell health. During mitosis, error correction is required for the faithful inheritance of genetic material. When functioning properly, the mitotic spindle segregates an equal number of chromosomes to daughter cells with high fidelity. Over the course of spindle assembly, many initially erroneous attachments between kinetochores and microtubules are fixed through the process of error correction. Despite the importance of chromosome segregation errors in cancer and other diseases, there is a lack of methods to characterize the dynamics of error correction and how it can go wrong. Here, we present an experimental method and analysis framework to quantify chromosome segregation error correction in human tissue culture cells with live cell confocal imaging, timed premature anaphase, and automated counting of kinetochores after cell division. We find that errors decrease exponentially over time during spindle assembly. A coarse-grained model, in which errors are corrected in a chromosome-autonomous manner at a constant rate, can quantitatively explain both the measured error correction dynamics and the distribution of anaphase onset times. We further validated our model using perturbations that destabilized microtubules and changed the initial configuration of chromosomal attachments. Taken together, this work provides a quantitative framework for understanding the dynamics of mitotic error correction.

Microtubule end-on attachment maturation regulates Mps1 association with its kinetochore receptor

Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.

A communication hub for phosphoregulation of kinetochore-microtubule attachment

The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical spindle attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is a 600-Å-long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16-17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long C-terminal extension of Dam1. Results reported here show, both by invitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1. It also binds, less tightly, a conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset.

A non-canonical role of the inner kinetochore in regulating sister-chromatid cohesion at centromeres

The 16-subunit Constitutive Centromere-associated Network (CCAN)-based inner kinetochore is well-known for connecting centromeric chromatin to the spindle-binding outer kinetochore. Here, we report a non-canonical role for the inner kinetochore in directly regulating sister-chromatid cohesion at centromeres. We provide biochemical, X-ray crystal structure, and intracellular ectopic localization evidence that the inner kinetochore directly binds cohesin, a ring-shaped multi-subunit complex that holds sister chromatids together from S-phase until anaphase onset. This interaction is mediated by binding of the 5-subunit CENP-OPQUR sub-complex of CCAN to the Scc1-SA2 sub-complex of cohesin. Mutation in the CENP-U subunit of the CENP-OPQUR complex that abolishes its binding to the composite interface between Scc1 and SA2 weakens centromeric cohesion, leading to premature separation of sister chromatids during delayed metaphase. We further show that CENP-U competes with the cohesin release factor Wapl for binding the interface of Scc1-SA2, and that the cohesion-protecting role for CENP-U can be bypassed by depleting Wapl. Taken together, this study reveals an inner kinetochore-bound pool of cohesin, which strengthens centromeric sister-chromatid cohesion to resist metaphase spindle pulling forces.

Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric FRET-Based Molecular Sensor upon Cell Cycle Progression.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Simvastatin activates the spindle assembly checkpoint and causes abnormal cell division by modifying small GTPases

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of the cholesterol synthesis pathway. It has been used clinically as a lipid-lowering agent to reduce low-density lipoprotein (LDL) cholesterol levels. In addition, antitumor activity has been demonstrated. Although simvastatin attenuates the prenylation of small GTPases, its effects on cell division in which small GTPases play an important role, have not been examined as a mechanism underlying its cytostatic effects. In this study, we determined its effect on cell division. Cell cycle synchronization experiments revealed a delay in mitotic progression in simvastatin-treated cells at concentrations lower than the IC50. Time-lapse imaging analysis indicated that the duration of mitosis, especially from mitotic entry to anaphase onset, was prolonged. In addition, simvastatin increased the number of cells exhibiting misoriented anaphase/telophase and bleb formation. Inhibition of the spindle assembly checkpoint (SAC) kinase Mps1 canceled the mitotic delay. Additionally, the number of cells exhibiting kinetochore localization of BubR1, an essential component of SAC, was increased, suggesting an involvement of SAC in the mitotic delay. Enhancement of F-actin formation and cell rounding at mitotic entry indicates that cortical actin dynamics were affected by simvastatin. The cholesterol removal agent methyl-β-cyclodextrin (MβCD) accelerated mitotic progression differently from simvastatin, suggesting that cholesterol loss from the plasma membrane is not involved in the mitotic delay. Of note, the small GTPase RhoA, which is a critical factor for cortical actin dynamics, exhibited upregulated expression. In addition, Rap1 was likely not geranylgeranylated. Our results demonstrate that simvastatin affects actin dynamics by modifying small GTPases, thereby activating the spindle assembly checkpoint and causing abnormal cell division.

Dynamic localization of the chromosomal passenger complex in trypanosomes is controlled by the orphan kinesins KIN-A and KIN-B.

The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in Trypanosoma brucei, the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.

Discovery of novel, potent and orally available benzoazipinone derivatives that elicit MKLP2-inhibitory phenotypes

Mitotic kinesin-like protein 2 (MKLP2/KIF20A) is a key mitotic regulator frequently overexpressed in human malignancies and its abundance is positively correlated with poor outcomes of the disease. Despite extensive research on MKLP2 as a potential target for oncology, the development of small-molecule inhibitors specific to MKLP2 remains limited. We have previously identified a benzoazipinone compound, HJ81 as a potent disruptor of Aurora kinase B (AURKB) localization during late mitosis. This study reveals that such disruption results from a failure of AURKB relocation at the onset of anaphase and this phenomenon can be specifically attributed to the disablement of MKLP2, a recognized facilitator of the relocation process. Further optimization of HJ81 leads to identifying compounds such as 12a as promising lead inhibitors of MKLP2-mediated processes, with improved pharmacokinetic properties. 12a inhibits the microtubule-stimulated ATPase activity of the recombinant MKLP2 in vitro. Significant suppression of tumor growth was observed in mice bearing the Calu-6 lung cancer cell line when treated with 12a at a well-tolerated dose. Overall, our findings suggest that benzoazipinone derivatives represent a novel chemical scaffold with the potential to be developed to mimic MKLP2 inhibition for cancer treatment.

Securin acetylation prevents precocious separase activation and premature sister chromatid separation

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/CCDC20-mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability.

Reconstitution of nuclear envelope subdomain formation on mitotic chromosomes in semi-intact cells.

In metazoans, the nuclear envelope (NE) disassembles during the prophase and reassembles around segregated chromatids during the telophase. The process of NE formation has been extensively studied using live-cell imaging. At the early step of NE reassembly in human cells, specific pattern-like localization of inner nuclear membrane (INM) proteins, connected to the nuclear pore complex (NPC), was observed in the so-called "core" region and "noncore" region on telophase chromosomes, which corresponded to the "pore-free" region and the "pore-rich" region, respectively, in the early G1 interphase nucleus. We refer to these phenomena as NE subdomain formation. To biochemically investigate this process, we aimed to develop an in vitro NE reconstitution system using digitonin-permeabilized semi-intact mitotic human cells coexpressing two INM proteins, emerin and lamin B receptor, which were labeled with fluorescent proteins. The targeting and accumulation of INM proteins to chromosomes before and after anaphase onset in semi-intact cells were observed using time-lapse imaging. Our in vitro NE reconstitution system recapitulated the formation of the NE subdomain, as in living cells, although chromosome segregation and cytokinesis were not observed. This in vitro NE reconstitution required the addition of a mitotic cytosolic fraction supplemented with a cyclin-dependent kinase inhibitor and energy sources. The cytoplasmic soluble factor(s) dependency of INM protein targeting differed among the segregation states of chromosomes. Furthermore, the NE reconstituted on segregated chromosomes exhibited active nucleocytoplasmic transport competency. These results indicate that the chromosome status changes after anaphase onset for recruiting NPC components.

CYR61 Acts as an Intracellular Microtubule-Associated Protein and Coordinates Mitotic Progression via PLK1-FBW7 Pathway.

Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.

Overexpression of JNK-associated leucine zipper protein induces chromosomal instability through interaction with dynein light intermediate chain 1.

The c-Jun N-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein of mitogen-activated protein kinase signaling pathways, is a multifunctional protein involved in a variety of cellular processes. It has been reported that JLP is overexpressed in various types of cancer and is expected to be a potential therapeutic target. However, whether and how JLP overexpression affects non-transformed cells remain unknown. Here, we aimed to investigate the effect of JLP overexpression on chromosomal stability in human non-transformed cells and the mechanisms involved. We found that aneuploidy was induced in JLP-overexpressed cells. Moreover, we established JLP-inducible cell lines and observed an increased frequency of chromosome missegregation, reduced time from nuclear envelope breakdown to anaphase onset, and decreased levels of the spindle assembly checkpoint (SAC) components at the prometaphase kinetochore in cells overexpressing the wild-type JLP. In contrast, we observed that a point mutant JLP lacking the ability to interact with dynein light intermediate chain 1 (DLIC1) failed to induce chromosomal instability. Our results suggest that overexpression of the wild-type JLP facilitates premature SAC silencing through interaction with DLIC1, leading to aneuploidy. This study provides a novel insight into the mechanism through which JLP overexpression is associated with cancer development and progression.

The Aurora kinase B relocation blocker LXY18 triggers mitotic catastrophe selectively in malignant cells.

The mitotic regulator, Aurora kinase B (AURKB), is frequently overexpressed in malignancy and is a target for therapeutic intervention. The compound, LXY18, is a potent, orally available small molecule that inhibits the proper localization of AURKB during late mitosis, without affecting its kinase activity. In this study, we demonstrate that LXY18 elicits apoptosis in cancer cells derived from various indications, but not in non-transformed cell lines. The apoptosis is p53-independent, triggered by a prolonged mitotic arrest and occurs predominantly in mitosis. Some additional cells succumb post-mitotic slippage. We also demonstrate that cancer cell lines refractory to AURKB kinase inhibitors are sensitive to LXY18. The mitotic proteins MKLP2, NEK6, NEK7 and NEK9 are known regulators of AURKB localization during the onset of anaphase. LXY18 fails to inhibit the catalytic activity of these AURKB localization factors. Overall, our findings suggest a novel activity for LXY18 that produces a prolonged mitotic arrest and lethality in cancer cells, leaving non-transformed cells healthy. This new activity suggests that the compound may be a promising drug candidate for cancer treatment and that it can also be used as a tool compound to further dissect the regulatory network controlling AURKB localization.

Signaling protein abundance modulates the strength of the spindle assembly checkpoint

During mitosis, unattached kinetochores in a dividing cell signal to the spindle assembly checkpoint (SAC) to delay anaphase onset and prevent chromosome missegregation.1,2,3,4 The signaling activity of these kinetochores and the likelihood of chromosome missegregation depend on the amount of SAC signaling proteins each kinetochore recruits.5,6,7,8 Therefore, factors that control SAC protein recruitment must be thoroughly understood. Phosphoregulation of kinetochore and SAC signaling proteins due to the concerted action of many kinases and phosphatases is a significant determinant of the SAC protein recruitment to signaling kinetochores.9 Whether the abundance of SAC proteins also influences the recruitment and signaling activity of human kinetochores has not been studied.8,10 Here, we reveal that the low cellular abundance of the SAC signaling protein Bub1 limits its own recruitment and that of BubR1 and restricts the SAC signaling activity of the kinetochore. Conversely, Bub1 overexpression results in higher recruitment of SAC proteins, producing longer delays in anaphase onset. We also find that the number of SAC proteins recruited by a signaling kinetochore is inversely correlated with the total number of signaling kinetochores in the cell. This correlation likely arises from the competition among the signaling kinetochores to recruit from a limited pool of signaling proteins, including Bub1. The inverse correlation may allow the dividing cell to prevent a large number of signaling kinetochores in early prophase from generating an overly large signal while enabling the last unaligned kinetochore in late prometaphase to signal at the maximum strength.