An Attachment-Independent Biochemical Timer of the Spindle Assembly Checkpoint

Qian, Junbin; García-Gimeno, Maria Adelaida; Beullens, Monique; Manzione, Maria Giulia; Hoeven, Gerd Van der; Igual, J. Carlos; Heredia, Miguel López de; Sanz, Pascual; Gelens, Lendert; Bollen, Mathieu

doi:10.1016/j.molcel.2017.10.011

Cited by 65 publications

(116 citation statements)

References 51 publications

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“…Our finding that the expanded domain can be fully detached from the underlying kinetochore, either by acute CDK1 inhibition or by combining GFP::ROD over-expression with Knl1 depletion, suggests that RZZ-dependent expansion results in a distinct multiprotein assembly that is at least transiently stable without the KMN network. Consistent with recent studies [13,17], our findings imply that kinetochores contain two distinct pools of Mad1-Mad2: one pool recruited by the KMN network through a direct interaction with Bub1 [7][8][9], and another pool localized to the expanded outer domain, most likely recruited through a direct interaction with RZZ. The presence of BUBR1 and BUB3 on detached crescents may indicate that the expanded domain is able to generate MCC independently of BUB1.…”

Section: Discussionsupporting

confidence: 91%

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Gassmann

Pereira

Reis

et al. 2018

Preprint

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SUMMARYThe kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signalling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, while dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into µm-scale filaments, and we identify the ROD-1 β-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZ’s capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

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Section: Discussionsupporting

confidence: 91%

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Gassmann

Pereira

Reis

et al. 2018

Preprint

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“…An initial high level of Bub1 phosphorylation might ensure that a large fraction of Mad1 is bound to Bub1 at early stages of mitosis to ensure a high level of MCC production to establish the checkpoint (Qian et al, 2017). The amount of Mad1-Bub1 complex likely changes over time as the amount of MCC that needs to be produced for establishing and maintaining the SAC have different thresholds during mitosis.…”

Section: Discussionmentioning

confidence: 99%

“…It is clear that phosphorylation of Met-Glu-Leu-Thr (MELT) repeats in the outer kinetochore protein KNL1 by the checkpoint kinase Mps1 generates binding sites for the checkpoint complexes Bub1/Bub3 and BubR1/Bub3 (London et al, 2012;Shepperd et al, 2012;Yamagishi et al, 2012;Vleugel et al, 2013Vleugel et al, , 2015bZhang et al, 2014Zhang et al, , 2016. Subsequent phosphorylation of Bub1 by Mps1 then facilitates an interaction between Mad1 and Bub1 a mechanism conserved from yeast to man (London & Biggins, 2014;Mora-Santos et al, 2016;Faesen et al, 2017;Ji et al, 2017;Qian et al, 2017;Zhang et al, 2017). Mad1 is in a stable complex with Mad2 and the recruitment of Mad1/Mad2 to kinetochores is essential because this complex catalyzes the first step in MCC formation by loading Mad2 onto Cdc20 (De Antoni et al, 2005;Faesen et al, 2017;Ji et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

et al. 2019

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Kinetochore localized Mad1 is essential for generating a “wait anaphase” signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod‐Zw10‐Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1‐Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1‐Bub1 complex.

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“…Current models suggest that an unattached kinetochore activates the SAC by allowing Mps1 kinase to phosphorylate KNL1 at sites known as ‘MELT motifs’ due to their consensus sequence (Figure 1B-C) [7-10]. This event is followed by the sequential recruitment of Bub3-Bub1 and Mad1-Mad2, along with Bub3-BubR1 and Cdc20 to the kinetochore, with Mps1 phosphorylation playing a licensing role for each step (Figure 1B) [2, 11-19]. We refer to this biochemical cascade as the ‘core SAC signaling cascade’ (dashed gray box in Figure 1B).…”

Section: Introductionmentioning

confidence: 99%

Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design

et al. 2019

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Summary Switch-like activation of the Spindle Assembly Checkpoint (SAC) is critical for accurate chromosomes segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the “eSAC”. The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins, and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior.

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An Attachment-Independent Biochemical Timer of the Spindle Assembly Checkpoint

Cited by 65 publications

References 51 publications

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design

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