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Basis of catalytic assembly of the mitotic checkpoint complex

Nature volume 542, pages 498–502 (2017)Cite this article

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Abstract

In mitosis, for each daughter cell to inherit an accurate copy of the genome from the mother cell, sister chromatids in the mother cell must attach to microtubules emanating from opposite poles of the mitotic spindle, a process known as bi-orientation. A surveillance mechanism, termed the spindle assembly checkpoint (SAC), monitors the microtubule attachment process and can temporarily halt the separation of sister chromatids and the completion of mitosis until bi-orientation is complete1. SAC failure results in abnormal chromosome numbers, termed aneuploidy, in the daughter cells, a hallmark of many tumours. The HORMA-domain-containing protein mitotic arrest deficient 2 (MAD2) is a subunit of the SAC effector mitotic checkpoint complex (MCC). Structural conversion from the open to the closed conformation of MAD2 is required for MAD2 to be incorporated into the MCC1. In vitro, MAD2 conversion and MCC assembly take several hours2,3,4, but in cells the SAC response is established in a few minutes5,6,7. Here, to address this discrepancy, we reconstituted a near-complete SAC signalling system with purified components and monitored assembly of the MCC in real time. A marked acceleration in MAD2 conversion and MCC assembly was observed when monopolar spindle 1 (MPS1) kinase phosphorylated the MAD1–MAD2 complex, triggering it to act as the template for MAD2 conversion and therefore contributing to the establishment of a physical platform for MCC assembly. Thus, catalytic activation of the SAC depends on regulated protein–protein interactions that accelerate the spontaneous but rate-limiting conversion of MAD2 required for MCC assembly.

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Figure 1: Stability of MCC.
Figure 2: Catalytic assembly of MCC.
Figure 3: Molecular requirements of catalytic MCC assembly.
Figure 4: MPS1 activates MAD1.
Figure 5: Role of catalysis in MAD2 activation dynamics.

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Acknowledgements

We thank T. Kapoor and G. Siemeister for sharing reagents; the J.-M. Peters laboratory for the CDC20 expression vector; H. Ploegh for the Sortase expression vector; Y. Wu for help with construction of FRET probes; O. Durczak for technical assistance; A. Ciliberto for suggestions and comments; and G. Vader and the A.M. laboratory for discussions and reading of the manuscript. A.C.F. acknowledges support by an EMBO long-term fellowship (ALTF 1096-2012) and a Marie Curie Intra-European Fellowship (IEF). A.M. acknowledges funding by the Framework Program 7 Integrated Project MitoSys, the Horizon 2020 ERC agreement RECEPIANCE, and the DFG’s Collaborative Research Centre (CRC) 1093.

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Authors and Affiliations

  1. Department of Mechanistic Cell Biology, Max-Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, Dortmund, 44227, Germany

    Alex C. Faesen, Maria Thanasoula, Stefano Maffini, Claudia Breit, Franziska Müller, Suzan van Gerwen, Tanja Bange & Andrea Musacchio

  2. Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstraße, Essen, 45141, Germany

    Andrea Musacchio

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Contributions

A.C.F. and A.M. designed experiments and analysed results. A.C.F., M.T., C.B. and S.v.G. set up recombinant expression systems and purified proteins. A.C.F. performed in vitro experiments. M.T. performed solid-phase binding assays. S.M. and S.v.G. performed cellular SAC assays. F.M. and T.B. performed mass-spectrometry measurements. A.M. supervised the project. A.C.F. and A.M. wrote the manuscript.

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Correspondence to Alex C. Faesen or Andrea Musacchio.

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The authors declare no competing financial interests.

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Reviewer Information

Nature thanks T. Kapoor, J. Pines and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 MCC constituents and MAD2-template model.

a, Schematic of MCC constituents and their domain structure. b, Cartoon model of the crystal structure of the Schizosaccharomyces pombe MCC complex11 (Protein Data Bank accession number: 4AEZ). CDC20 consists mainly of WD40 β-propeller domains, where the N-terminal extension interacts with MAD2 (at the MIM). Mad3 is the yeast orthologue of BUBR1. BUBR1, which is constitutively bound to BUB3, contains many functional motifs and structural domains, a few of which are highlighted in a. c, Cartoon models of the crystal structures of O-MAD2 and C-MAD2. The HORMA domain of MAD2 exists in two distinct topologies: O-MAD2, when unbound by ligand; and C-MAD2, when bound to the MIMs of MAD1 or CDC20 (refs 15,16). The change in topology is due to relocation of mobile elements of the structure, indicated in grey. d, Schematic representation of MAD1 and MAD1-deletion mutants used in this study. e, Schematic representation of the MAD2 template model.

Extended Data Figure 2 Characterizing the MCC using FRET sensors.

a, Coomassie-stained SDS–PAGE gel of recombinant proteins used in this study. b, Fluorescence emission spectrum of MCCS1 excited at 430 nm. The concentration of all proteins is 100 nM, expect for the CDC20 peptide, which was used in large excess (5 μM) in competition reactions. Signals were normalized to peak donor emission at 470 nm. No change in emission was observed in presence of only O-MAD2–TAMRA, or when CFP–CDC20 was measured in isolation (black) or with a TAMRA-labelled peptide (green). The excess of CDC20 peptide competed for MAD2 binding and no FRET was observed (brown). c, In an additional control for MCCS1, CFP–CDC20 was tested against TAMRA-labelled ‘loopless’ (O-MAD2-LL–TAMRA), a MAD2 mutant that is locked in the O-MAD2 conformation and cannot bind CDC20 (ref. 18). Assay conditions were as described in b. d, MCC formation relies on the presence of CDC20. Fluorescence emission spectra of MCCS2 (or parts thereof) excited at 430 nm. No change in emission was observed in the presence of only O-MAD2–TAMRA (no CFP–BUBR1–BUB3, blue) or when CFP–BUBR1–BUB3 was measured in isolation (black), in the presence of O-MAD2–TAMRA (without CDC20, purple), or in the presence of CDC20 and a TAMRA-labelled peptide not conjugated to MAD2 (green). The only condition that led to changes in donor and acceptor emission spectra was when CFP–BUBR1–BUB3, TAMRA–MAD2 and CDC20 were present at the same time (red). FRET efficiency upon complex formation at equilibrium was 35%. The concentration of all proteins was 100 nM. Signals were normalized to peak donor emission at 470 nm. e, In an additional control for MCCS2, CFP–BUBR1 was tested in the presence of CDC20 against O-MAD2-LL–TAMRA. Assay conditions were as described in d. f, Recombinant O-MAD2–TAMRA, CDC20 and CFP–BUBR1–BUB3 form the MCC complex. Size-exclusion chromatography elution profiles of O-MAD2–TAMRA (dark blue trace), CDC20 (green trace), CFP–BUBR1–BUB3 (light blue trace) or all mixed to form the MCC complex (orange trace). The shift in the elution profile indicates complex formation. g, BUB3 does not affect MCCcore stability. A titration experiment determining the binding isotherms of the MCC complex, using MCCS2 in the presence (red) or absence (blue) of BUB3, showed indistinguishable apparent Kd values. Data are mean ± s.e.m. of three independent technical replicates of the experiments.

Extended Data Figure 3 Microinjection of recombinant fluorescent MCC proteins.

a, b, Recombinant fluorescent MCC proteins injected into mitotic cells localize to kinetochores. HeLa cells constitutively expressing LAP–BUB1 (a) or transiently expressing mCherry–CENP-A (b) were synchronized in the G2 phase of the cell cycle by treatment with the CDK1 inhibitor RO3306 (ref. 42) and released into mitosis in the presence of nocodazole. Shortly after release, cells were injected with either TAMRA–MAD2 or TAMRA (a), or with CFP–BUBR1–BUB3 (b). Cells were live-imaged both before (Pre) and after (Post) microinjection. Scale bars, 2 μm. Number of injected cells, n, for TAMRA, n = 2; for TAMRA–MAD2, n = 9; for mTurquoise–BUBR1–BUB3, n = 8.

Extended Data Figure 4 MCC assembly kinetics.

a, The CDC20–MAD2 complex forms slowly. The time-dependent change of acceptor (left) and donor (right) fluorescence (normalized to values at equilibrium) with 10 nM CFP–BUBR1–BUB3 (see Supplementary Information Section G for details of the effects of BUBR1 concentration on reaction rate of MCCS2) and 500 nM CDC20 with varying concentrations of O-MAD2–TAMRA. Signal changes were fitted to single exponential curves. b, After the single-exponential fitting of the curves in a, the apparent first order rate constants (kobs) were plotted as a function of MAD2 concentration, with kon being the slope of the resulting curve. These kon values depend on the concentration of BUBR1 (see c and Supplementary Information Section G). c, MCC assembly assay performed with MCCS2 with 100 nM O-MAD2–TAMRA, 500 nM CDC20 and the indicated concentrations of CFP–BUBR1–BUB3. d, BUBR1 does not influence the assembly kinetics of the MCC. Monitoring the assembly of CDC20–MAD2 (MCCS1; blue), CDC20–MAD2 with dark BUBR1–BUB3 (MCCS1; red) and BUBR1–MAD2 with dark CDC20 (green) shows indistinguishable rates. e, Catalysis rates scale linearly with catalyst concentration. After pre-incubation of the catalyst proteins, MCC assembly was monitored with MCCS2 (sensor concentrations were 100 nM, except for CDC20, which was 500 nM) at varying catalyst concentrations. Initial velocity (Vi) signal changes were plotted against catalyst concentration, revealing a linear dependency. f, Catalysis of MCC formation could be observed with both FRET sensors. After pre-incubation of MAD1–C-MAD2, BUB1–BUB3 and MPS1 at 1 μM for 30 min, similar catalysis rates were observed with either MCCS1 (blue) or MCCS2 (red). The assay was performed as described in Fig. 2b, with all proteins at 100 nM. Data are mean ± s.e.m. of three independent technical replicates of the experiments (b, e).

Extended Data Figure 5 Molecular requirements of catalytic MCC assembly.

a, Catalytic MCC assembly requires MAD1–C-MAD2, MPS1, ATP and BUB1–BUB3. MCC assembly was monitored with MCCS2, as described in Fig. 2b, using 100 nM catalysts. Individual components were omitted as indicated. The same control profiles (black and red curves) are shown here and in Fig. 2. b, MAD1 with the N-terminal 419 residues deleted (MAD1420-C; red) is a minimal construct capable of full catalysis. A reduction in the catalytic rate was observed with MAD1 with the N-terminal 484 residues deleted (MAD1485-C; purple) compared to full-length MAD1 (MAD1FL; yellow) or MAD1420-C. The assay was performed with MCCS2 as described in Fig. 2b, using 100 nM catalysts. Catalytic activation is salt-sensitive, probably because high salt inhibits phosphorylation-mediated polar interactions (see c, d). c, d, Catalysis is sensitive to salt concentration. MCC assembly was monitored with MCCS2, using 75 mM (red), 150 mM (blue), 300 mM (green) or 500 mM NaCl (brown), in the absence (c) or presence (d) of catalysts. The assay was performed with MCCS2, as described in Fig. 2b.

Extended Data Figure 6 Inhibiting catalysis.

a, Inhibiting MPS1 and BUB1 during pre-incubation strongly reduces catalysis. Adding both Reversine and BAY-320 to the pre-incubation solution of catalysts strongly reduced the catalysis of MCC formation. Adding the inhibitors after pre-incubation but before addition to the MCCS2 components did not affect catalysis. Final concentrations of inhibitors were 50 μM during pre-incubation and 5 μM in the assay. b, As in Fig. 4a, but with the BUB1 inhibitor BAY-320 rather than Reversine. BUB1 without functional kinase activity (BUB1KD) was used as a control. c, Catalysis rates remained unchanged when the pre-incubation of catalyst proteins was ‘split’ into two reactions (MAD1–C-MAD2 together with MPS1 or BUB1 alone; compare green to red). Assay performed with MCCS2, as described in Fig. 2b, using 100 nM catalysts. d, MAD1–C-MAD2 is phosphorylated by MPS1. Catalysis rates remained unchanged when the pre-incubation of catalyst proteins was split into two reactions (MAD1–C-MAD2 together with MPS1 or BUB1 alone; compare green to red). Adding the kinase inhibitors Reversine (targeting MPS1) and BAY-320 (BUB1) to the pre-incubation reaction strongly reduced the rates of catalysis (orange). However, inverting the inhibitors had no effect on the catalysis rates (blue). Assay performed with MCCS2, as described in Fig. 2b, using 100 nM catalysts. Final concentrations of inhibitors are 5 μM in assay (50 μM during pre-incubation).

Extended Data Figure 7 MPS1 phosphorylation of MAD1.

a, Phosphorylation sites of MAD1 by MPS1. The peptide sequence with the phosphorylated residue in bold, the amino acid position within the protein, the P value of the posterior error probability for the identified peptide (PEP) and the Andromeda search engine score (score) are shown. Residue numbers in bold indicate phosphorylation sites found in at least two experiments. b–d, In b, HeLa cells were transfected with mCherry (Control), mCherry–MIS12–MAD1WT (WT), mCherry–MIS12–MAD1S428A (S428A), mCherry–MIS12–MAD1RWD-A (RWD-A) or mCherry–MIS12–MAD1S428A/RWD-A (S428A, RWD-A) as described and quantified in the legend of Fig. 4c. Shown are mitotic cells, representative of the mitotic population in each cohort (mCherry control, 59 cells; mCherry–MIS12–MAD1WT, 247 cells; mCherry–MIS12–MAD1S428A, 203 cells; mCherry–MIS12–MAD1RWD-A, 83 cells; mCherry–MIS12–MAD1S428A/RWD-A, 91 cells). Following a 30-h transfection with the indicated constructs, cells were fixed and processed for western blotting (c) or immunofluorescence (d and Fig. 4c). Western blot analysis showed that expression levels of mCherry–MIS12–MAD1 fusions were lower than endogenous MAD1 levels (c). Scale bars, 5 μm. Quantification of the kinetochore signal was performed on unmodified Z series images. Following background subtraction, a ratio for mCherry–MIS12–MAD1/CREST intensity signals was calculated. All ratios were normalized to the mean of mCherry–MIS12–MAD1WT ratio. Quantifications are based on two independent biological replicates of the experiment, for a total of five cells for each condition, where 254 (mCherry–MIS12–MAD1WT), 143 (mCherry–MIS12–MAD1S428A), 207 (mCherry–MIS12–MAD1RWD-A) or 188 (mCherry–MIS12–MAD1S428A/RWD-A) kinetochores were analysed. Shown is a box-and-whiskers graph, indicating the median, a box with the 25–75th percentile and hinges indicating the upper and lower limits of the data points.

Extended Data Figure 8 MAD1 and BUB1 interact to combine O-MAD2 and CDC20.

a, MAD1–C-MAD2 and BUB1–BUB3 together form the MCC enzyme, while MPS1 suffices in sub-stoichiometric amounts. Lowering the concentration of all catalysts increased the half-life (halftime) tenfold (compare conditions 1 and 8). Lowering individual components reduces rates to intermediate levels for MAD1–C-MAD2 (condition 2) and BUB1–BUB3 (condition 3), but not MPS1 (condition 4). Lowering both MAD1–C-MAD2 and BUB1–BUB3 (condition 5) mimics the reduction of all components (condition 8), whereas reducing MAD1–C-MAD2 or BUB1–BUB3 in combination with MPS1 (conditions 6 and 7, respectively) only resulted in intermediate rates. Assays were performed with MCCS2 as described in Fig. 2b, using either 100 nM (1×) or 10 nM (0.1×) catalysts. b, Excluding BUBR1 does not affect catalytic rates (green and blue). Assays were performed using MCCS1, all proteins were present at 100 nM. c, The interaction between BUB1 and CDC20 enhances binding with MAD2. A BUB1 construct that does not bind CDC20 (the KEN1-ABBA mutant (BUB1KEN1-ABBAmut); purple) yields similar rates as in the absence of BUB1 (grey). Assay performed with MCCS2 and as described in Fig. 2b, using 100 nM catalysts. d, MAD1–C-MAD2 and BUB1–BUB3 show an ATP-dependent interaction in the presence of MPS1. Pull-down experiment using MBP–MAD1–C-MAD2 as bait. Assay was performed with 1 μM MAD1–C-MAD2, 2 μM BUB1–BUB3 and 400 nM MPS1. e, Values of FRET from MCCS2 (1 nM CFP–BUBR1 and 500 nM CDC20) after equilibration with or without catalysts (25 nM catalyst concentration). Data are mean ± s.e.m. of three independent technical replicates of the experiment (b, e).

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Faesen, A., Thanasoula, M., Maffini, S. et al. Basis of catalytic assembly of the mitotic checkpoint complex. Nature 542, 498–502 (2017). https://doi.org/10.1038/nature21384

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