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The kinetochore encodes a mechanical switch to disrupt spindle assembly checkpoint signalling

Nature Cell Biology volume 17, pages 868–879 (2015)Cite this article

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Abstract

The spindle assembly checkpoint (SAC) is a unique signalling mechanism that responds to the state of attachment of the kinetochore to spindle microtubules. SAC signalling is activated by unattached kinetochores, and it is silenced after these kinetochores form end-on microtubule attachments. Although the biochemical cascade of SAC signalling is well understood, how kinetochore–microtubule attachment disrupts it remained unknown. Here we show that, in budding yeast, end-on microtubule attachment to the kinetochore physically separates the Mps1 kinase, which probably binds to the calponin homology domain of Ndc80, from the kinetochore substrate of Mps1, Spc105 (KNL1 orthologue). This attachment-mediated separation disrupts the phosphorylation of Spc105, and enables SAC silencing. Additionally, the Dam1 complex may act as a barrier that shields Spc105 from Mps1. Together these data suggest that the protein architecture of the kinetochore encodes a mechanical switch. End-on microtubule attachment to the kinetochore turns this switch off to silence the SAC.

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Figure 1: Cell-cycle effects of anchoring Mps1 to the kinetochore using rapamycin-induced dimerization.
Figure 2: Testing the sensitivity of SAC signalling steps to the attachment status of the kinetochore.
Figure 3: The ability of Mps1 to activate the SAC depends on its position in the kinetochore.
Figure 4: The Dam1 complex defines a boundary for SAC signalling by anchored Mps1.
Figure 5: Phosphorylation of the Spc105 phosphodomain by Mps1 is sufficient to activate the SAC.
Figure 6: Spc105120:329 activates the SAC only when it is anchored in the outer kinetochore.
Figure 7: The proximity between the CH domains of Ndc80 and the phosphodomain of Spc105 within the kinetochore controls SAC signalling.
Figure 8: The mechanical-switch model for attachment-sensitive SAC signalling.

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Acknowledgements

We thank A. Murray (Harvard University, USA), B. Glick (University of Chicago, USA), D. Drubin (University of California, Berkeley, USA), M. Winey (University of Colorado, Boulder, USA) and S. Biggins (Fred Hutchinson Cancer Research Center, Seattle, USA) for sharing reagents, and I. Cheeseman, J. DeLuca, M. Duncan, R. McIntosh, and Y. Yamashita for comments on the manuscript. We also thank L. Humphrey and S. Han for help with imaging and data analysis. A.P.J. is supported by the Career Award at the Scientific Interface from the Burroughs Wellcome Fund. This work was financially supported by R01-GM-105948.

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Authors and Affiliations

  1. Biophysics, University of Michigan, 930 N. University Avenue Ann Arbor, Michigan 48109, USA,

    Pavithra Aravamudhan & Ajit P. Joglekar

  2. Cell and developmental biology, University of Michigan, 109 Zina Pitcher Place, 3067 BSRB Ann Arbor, Michigan 48109, USA,

    Alan A. Goldfarb & Ajit P. Joglekar

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Contributions

A.P.J. and P.A. designed and performed the experiments, and wrote the manuscript; P.A. and A.A.G. performed the rapamycin plating, and cell-cycle kinetics experiments. A.P.J., P.A. and A.A.G. constructed all the yeast strains.

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Correspondence to Ajit P. Joglekar.

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Integrated supplementary information

Supplementary Figure 3 Effects of anchoring key SAC regulators to Mtw1-C on the cell cycle.

(a) Top: Representative images display the expected localization of SAC proteins tagged with Frb-GFP in untreated cells and one hour after the addition of rapamycin. Bottom: Benomyl sensitivity of indicated strains. (b) Representative transmitted light micrographs of four strains treated with rapamycin for 135 min to anchor Mps1, Ipl1, Mad1, or Glc7, at Mtw1-C. The bar graph displays the percentage of large-budded in each case averaged from two independent experiments (more than 50 cells were scored in each trial. Source data are shown in Supplementary Table 3). (c) Effect of the ATP analog 1-NAPP1 on the localization of the Ip1l substrate Sli15-GFP in cells expressing ipl1-as6, an analog-sensitive allele of the Ipl1 kinase67. Representative pre-anaphase cells expressing Sli15-GFP are shown on the right. Quantification of Sli15-GFP fluorescence on the spindle (mean ± s.d. from a single experiment; number of cells scored are displayed at the top) shown on the left. Consistent with published results67, spindle localization of Sli15-GFP significantly increased following 1-NAPP1 treatment indicating that the analog inhibits ipl1-as6. (d) Cell cycle kinetics following the release of S-phase synchronized cells into media containing 1-NAPP1 and rapamycin. Blocking ipl1-as6 activity did not have any effect on SAC activation induced by Mps1 anchored at Mtw1-C. The experiment was performed once and more than 70 cells were scored for each time point (source data are shown in Supplementary Table 3). (e) Bar graph: Frequency of prometaphase and metaphase cells with kinetochore-localized Mps1 (representative micrographs displayed at the top; 18, 12 and 45 cells were analyzed, from left to right). Spindle length was used to classify cells as prometaphase or metaphase cells. Scatter plot (mean ± 95% confidence interval; n = 21, 46 and 66 kinetochore clusters from left to right) displays the abundance of kinetochore-localized Mps1-Frb-GFP in prometaphase, metaphase-arrested cells (by repressing CDC20), and when it is anchored to Mtw1-C in heterozygous diploid strains. Each experiment was performed once. (f) Quantification of Mps1 localization to kinetochores soon after release from metaphase compared to that in anaphase (n = 10 from one experiment). Micrographs on the right show localization of Mps1 relative to spindle pole bodies over a period of 6 min during the metaphase to anaphase transition.

Supplementary Figure 4 Kinase activity of the kinetochore-anchored Mps1 is sufficient for SAC activation.

(a) Cells expressing the analog-sensitive Mps1 allele65, mps1-as1 or wild type Mps1 were treated as indicated at the top. Bar graph displays the percentage of two-budded cells (which form when a mitotic cell fails to sustain the SAC in the presence of a damaged spindle and produce a new bud by re-entering the cell cycle). Bars indicate the average value based on data from two independent experiments. The total number of cells scored is indicated at the top, and the source data are shown in Supplementary Table 3. (b) Inhibition of the diffusible mps1-as1 does not affect SAC activation by Mps1-Frb anchored at the kinetochore. Heterozygous diploid strains expressing mps1-as1 and Mps1-Frb were synchronized in S-phase and released into 1-NMPP1 for 15 min. Rapamycin was then added to anchor Mps1-Frb at Mtw1-C. The anchored Mps1 arrested the cell cycle robustly, and the cells retained the large-budded morphology for a prolonged period of time. The experiment was performed once, and more than 50 cells scored for each time point (the source data are shown in Supplementary Table 3). (c) Micrographs: Mad1 localization relative to the spindle pole body in haploid cells that have Mps1 anchored to the indicated subunit. Bar graph displays the percentage of cells with visible Mad1 localization in between the spindle poles in each case (number of cells scored in one experiment indicated on top). The corresponding metaphase spindle length in each case is presented in the scatter plot (range: 1.8–1.9 ± 0.3 μm; mean ± 95% confidence interval from n = 15, 14, 25, 23 and 33 cells from left to right).

Supplementary Figure 5 Cell cycle effects of anchoring Mps1, Ipl1 or Mad1 constitutively within the kinetochore.

(a) Cell cycle kinetics of asynchronous cultures where Mps1 is anchored at the C-termini of indicated kinetochore subunits in wild-type or SAC null strains (mad2Δ). The experiment was performed once. More than 50 cells were scored for each time point. The source data are shown in Supplementary Table 3. (b) Quantification of Mps1-Frb-GFP (mean ± 95% confidence interval; n = 35, 36, 47, 43, 27, 26, 33, 31, 66, 35 and 46 kinetochore clusters from left to right) anchored at indicated kinetochore subunits measured 45 min after rapamycin treatment and normalized relative to endogenous Mps1 in metaphase-arrested cells. Note that the recruitment of Mps1 at Dad4-C and Ctf19-C that activates the SAC is comparable to that at Ask1-C which does not activate the SAC (see Supplementary Fig. 2c). (c) Reducing the length of the flexible linker connecting Mps1 and Frb (from 24 to 7 amino acids) did not change the effect of anchoring Mps1 on colony growth. Bars represent the average values calculated from two independent experiments. Cumulative number of colonies counted is displayed at the bottom. The source data are shown in Supplementary Table 3. (d,e) The number of colonies formed on rapamycin-containing plates relative to control plates, when Ipl1 (in d) or Mad1 (in e) is constitutively anchored at the indicated positions. Bars represent the average values calculated from 3 or 4 independent experiments. The source data are shown in Supplementary Table 3. The number of colonies on rapamycin-containing plates exceeding that on the control is likely due to pipetting errors. Reduced number of colonies upon anchoring Ipl1-Frb-GFP at Ndc80-C and N-Ndc80 may be due to attachment defects caused by Ipl1-mediated phosphorylation of kinetochore proteins13. The loss of colony formation upon Mad1 anchoring at N-Ndc80 is also likely due to attachment defects (see f,g). (f) Mad1 anchored at N-Ndc80 generates unattached kinetochores (arrowheads) in a large fraction of cells (60 out of 108 cells had visible defects in kinetochore cluster morphology). (g) Graph presents the fraction of cells expressing Spc105-6A or Spc105 that arrested with large-buds when Mad1 was anchored at N-Ndc80 (rapamycin treatment for 4 h). The experiment was performed once. The number of cells scored is indicated on top of bars.

Supplementary Figure 6 Anchoring Mps1 to Dam1 subunits leads to different phenotypes.

(a) Subunit organization of the Dam1 complex defined in ref. 27 is color-coded according to the scheme displayed on the right. The untested subunits are Duo1 and Hsk4. Bar graph displays the number of colonies formed on rapamycin relative to the control plates. Bars represent averages from two independent experiments. The total numbers of colonies scored are displayed at the bottom. The source data are also shown in Supplementary Table 3. (b) Cell cycle kinetics of rapamycin treated (to anchor Mps1 at indicated subunits) or untreated (control) cells. The experiment was performed once and more than 60 cells were scored for each time point; the source data are shown in Supplementary Table 3. Since Mps1 anchored at Dad2-C, Dad4-C or Spc34-C transiently activated the SAC, we tested if shortening the linker connecting Mps1 and Frb (from 24 to 7 amino acids, also See Supplementary Fig. 3c) eliminates this transient SAC activation by restricting access to N-Spc105. However, anchored Mps1 activated the SAC in spite of the shorter linker.

Supplementary Figure 7 SAC signaling induced by rapamycin-induced dimerization of Spc105120: 329 and Mps1 does not require functional kinetochores.

(a) Representative images show Spc105120: 329 anchored to Mps1 (rapamycin treatment for 45 min) localizing to the kinetochores. Mad1 also co-localizes with these kinetochore clusters. (b) Cells carrying the temperature-sensitive ndc10-1 allele and expressing Spc105120: 329 and Mps1-Fkbp12 were treated as indicated at the top. When released at the restrictive temperature from G1 arrest, these cells go through the cell cycle without assembling functional kinetochores and fail in cytokinesis66, and give rise to cells with two buds (black bars; also see transmitted light micrograph top-right). However, when the same experiment was conducted in rapamycin containing media, the emergence of two budded cells was delayed by an hour (light gray bars). We attribute this delay to SAC activation, which is also observed when Spc105120: 329 is anchored to Mps1 in NDC10 cells at 37 °C (dark gray bars). Bars represent averages from 2 independent experiments. The source data are shown in Supplementary Table 3.

Supplementary Figure 8 SAC signaling induced by Mps1 anchored at N-Ndc80 depends on the attachment-state of the kinetochore.

S-phase synchronized cells were treated as indicated in the schematic at the top and the percentage of large-budded cells formed after 100 min was measured as an indicator of cell cycle arrest Mps1 anchored at Mtw1-C constitutively activated the SAC in the presence of attachments and in nocodazole. However, Mps1 anchored at N-Ndc80 allowed normal cell cycle progression and caused cell cycle arrest only in the presence of unattached kinetochores in nocodazole. Bars represent the average from 2 independent experiments. More than 50 cells were scored for each condition in each experiment. The source data are shown in Supplementary Table 3.

Supplementary Figure 9 Effect of spindle disruption on SAC protein recruitment and kinetochore architecture.

(a) Spindle disruption with nocodazole generates two or three kinetochore clusters within the nuclei of most budding yeast cells as reported previously64. The cluster that contained majority of the kinetochores (large, asterisks) localized proximal to the collapsed spindle pole bodies (visualized by Spc97-GFP). One or two smaller kinetochore clusters (small, arrowheads) were found distal to the spindle pole bodies. Bar graph displays the percentage of large or small clusters that are proximal to the spindle pole body. The cumulative numbers of clusters scored in 2 independent experiments are indicated at the bottom. (b) Consistent with Gillett et al. 2004, the smaller kinetochore clusters (arrowheads) in nocodazole recruit significantly higher levels of Mps1 and Bub1 than the large cluster. Mad1 was undetectable at the large clusters (bars indicate average from 2 independent experiments). 76 out of 82 smaller clusters recruited Mad1, but only 3 out of 65 large clusters had detectable Mad1. Therefore, we classify the smaller clusters as SAC-active and the larger clusters as SAC-inactive. (c) Dam1 complex (visualized with Ask1-mCherry) is retained at the SAC-inactive cluster, whereas it is significantly reduced at the SAC-active clusters in nocodazole. Quantification of Ask1-mCherry fluorescence measured relative to Spc24-mCherry fluorescence is displayed on the right. The experiment was performed once and horizontal bars represent mean ± 95% confidence intervals, n = 33, 72, 45 and 81 kinetochore clusters (left to right). Since microtubule attachment is necessary for Dam1 recruitment to the kinetochore42, this observation suggests that the kinetochores located proximal to the collapsed spindle pole bodies retain microtubule attachment. (d) Measurement of FRET between GFP-Spc105 and either mCherry-Nuf2 or mCherry-Ndc80 in SAC-active and SAC- inactive kinetochore clusters. The horizontal bars represent mean ± 95% confidence interval; data pooled from more than two independent experiments. P-values were computed using non-parametric Mann–Whitney test; n = 121, 39, 110, 87, 101, 49, 90 and 47 kinetochore clusters from left to right). FRET between mCherry-Nuf2 (or mCherry-Ndc80) and GFP-Spc105 in the SAC-inactive kinetochore cluster is higher than metaphase FRET value, and significantly lower than the FRET observed in the SAC-active cluster (∼50%, P-value ∼ 0.04). Note that the significantly higher FRET in anaphase compared to metaphase clusters is consistent with the previously reported reduction in the length of Ndc80 complex in anaphase20.

Supplementary Figure 10 Spc105120: 329 restores the SAC when it is anchored to unattached kinetochores in SAC-null strains.

Top: Experimental scheme. Bar graph: Fraction of nocodazole-treated cells with two buds in the presence and absence of rapamycin in cells expressing spc105-6A (see micrographs on the left). Note the diffuse nuclear localization of Spc105120: 329 in the absence of rapamycin. When Spc105120: 329 was anchored either at N-Ndc80 or at Spc24-C, it restored the SAC. The cells arrested with large buds (transmitted light micrograph on the right). In this condition, Spc105120: 329 is visible as multiple puncta corresponding to kinetochore clusters that form when budding yeast cells are treated with nocodazole (see Supplementary Fig. 7). Data represent mean from 2 independent trials. More than 100 cells were scored for each treatment. The source data are shown in Supplementary Table 3.

Supplementary Table 1 Strains used in this study.
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Supplementary Table 2 Plasmids used in this study.
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Aravamudhan, P., Goldfarb, A. & Joglekar, A. The kinetochore encodes a mechanical switch to disrupt spindle assembly checkpoint signalling. Nat Cell Biol 17, 868–879 (2015). https://doi.org/10.1038/ncb3179

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