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Mad1 promotes chromosome congression by anchoring a kinesin motor to the kinetochore

Nature Cell Biology volume 17, pages 1124–1133 (2015)Cite this article

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Abstract

For proper partitioning of genomes in mitosis, all chromosomes must be aligned at the spindle equator before the onset of anaphase. The spindle assembly checkpoint (SAC) monitors this process, generating a ‘wait anaphase’ signal at unattached kinetochores of misaligned chromosomes. However, the link between SAC activation and chromosome alignment is poorly understood. Here we show that Mad1, a core SAC component, plays a hitherto concealed role in chromosome alignment. Protein–protein interaction screening revealed that fission yeast Mad1 binds the plus-end-directed kinesin-5 motor protein Cut7 (Eg5 homologue), which is generally thought to promote spindle bipolarity. We demonstrate that Mad1 recruits Cut7 to kinetochores of misaligned chromosomes and promotes chromosome gliding towards the spindle equator. Similarly, human Mad1 recruits another kinetochore motor CENP-E, revealing that Mad1 is the conserved dual-function protein acting in SAC activation and chromosome gliding. Our results suggest that the mitotic checkpoint has co-evolved with a mechanism to drive chromosome congression.

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Figure 1: Identification of a Mad1-interacting protein.
Figure 2: Mad1 targets Cut7 to unattached kinetochores.
Figure 3: The Mad1-KAKA mutant unable to recruit Cut7 is defective in chromosome bi-orientation but intact in SAC activation.
Figure 4: Cut7 dimer at kinetochores promotes chromosome bi-orientation.
Figure 5: Cut7 facilitates chromosome alignment by promoting chromosome gliding towards the spindle equator.
Figure 6: Human Mad1 is required for chromosome alignment.
Figure 7: Human Mad1 targets CENP-E/kinesin-7 to unattached kinetochores.
Figure 8: Schematic depiction of Mad1–kinesin interplay in S. pombe and human cells.

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Acknowledgements

We thank the Yeast Genetic Resource Center (YGRC) for yeast strains, S. Hauf for critically reading the manuscript, J. Pines (Gurdon Institute in UK) for hMad1 RNAi information and all members of the Watanabe laboratory for their support and discussion. This work was supported in part by a JSPS Research Fellowship (to T.A.) and MEXT KAKENHI Grant Number 25000014 (to Y.W.).

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Authors and Affiliations

  1. Laboratory of Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Tokyo 113-0032, Japan

    Takashi Akera, Yuhei Goto & Yoshinori Watanabe

  2. Graduate Program in Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Yayoi, Tokyo 113-0032, Japan

    Takashi Akera, Yuhei Goto, Masamitsu Sato, Masayuki Yamamoto & Yoshinori Watanabe

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  1. Takashi Akera
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Contributions

T.A. designed and performed most experiments. Y.G. isolated Cut7 as a Mad1 interactor. M.S., M.Y. and Y.W. supervised the project. T.A. and Y.W. wrote the manuscript.

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Correspondence to Yoshinori Watanabe.

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Integrated supplementary information

Supplementary Figure 1 Mad1 directly associates with Cut7.

(a) Amino acid sequence of the N-terminal region of fission yeast Mad1. Amino acids sequence highlighted in blue represents the region required for Cut7 binding (Top). Yeast two-hybrid assay for mapping the minimal Cut7 interaction domain in Mad1 N-terminus (Bottom). (b) Yeast two-hybrid assay showing KAKA mutation abolished the interaction between Mad1 N-terminal region and Cut7. (c) Yeast two-hybrid assay showing mad1-4A mutation (R510A, V511A, L512A, Q513A) abolished the interaction between Mad1 and Mad2.

Supplementary Figure 2 Cut7 motor activity is essential for its kinetochore function.

(a) The signals of CFP−Cnp3C, Cut7d-CFP−Cnp3C and Cut7*d-CFP−Cnp3C were observed in the indicated cells. The centromeres were visualized by cen2-GFP. (b) Serial dilution assay showing that motor-dead version of Cut7d (Cut7*d) cannot suppress the TBZ hypersensitivity of mad1-KAKA cells (TBZ 15 μg ml−1). (c) The indicated cells were monitored for cen2-GFP segregation in bi-nucleate cells as in Fig. 1b. Over 500 cells per strain were analysed. Error bars, s.e.m. for n = 3 independent experiments. (d) Serial dilution assay showing that cut7d−cnp3C suppresses the TBZ hypersensitivity of mad1Δ cells (10 μg ml−1). (e) The indicated cells were monitored for cen2-GFP segregation in bi-nucleate cells as in Fig. 1b (over 500 cells per strain were analysed). Error bars represent s.e.m. for n = 3 independent experiments. (f) Serial dilution assays showing that centromere targeting of Cut7 can suppress the TBZ hypersensitivity of bub1Δ, bub3Δ, and mph1Δ cells. Scale bars, 4 μm.

Supplementary Figure 3 Deletion of counteracting kinesins enhances Cut7-mediated chromosome gliding.

Chromosome gliding assays were performed as in Fig. 5c–e in the absence of minus-end directed kinesin-14 motors (Pkl1 and Klp2 in fission yeast). Kinesin-14 motors were implicated in chromosome movement toward the spindle poles in several organisms including fission yeast37. Note that greater chromosome gliding observed in Kinesin-14 deleted cells, supporting the idea that Cut7 promotes plus-end directed gliding. Around 20 cells per strain were analysed. Scale bars, 2 μm.

Supplementary Figure 4 CENP-E is targeted to the kinetochores by hMad1.

(a) HeLa cells treated with the indicated siRNAs were examined by immunoblot using the indicated antibodies. Note that hMad1-1 siRNA is less effective than hMad1 siRNA, which is used in Figs 6 and 7. (b) HeLa cells treated with control siRNA or hMad1-1 siRNA were arrested in metaphase by MG132 and examined for chromosome alignment as in Fig. 6a. Error bars, s.e.m. for n = 3 independent experiments. Statistical significances (t-test, two-tailed) were assessed (*P < 0.05). (c) HeLa cells treated with control siRNA or hMad1-1 siRNA arrested in mitosis by nocodazole and MG132 were examined by immunostaining (left). Localization was quantified as the ratio of fluorescence intensity of CENP-E to the value of CENP-C in 10 kinetochores from each cell (right). Error bars, s.e.m. for n = 9 cells for each. Statistical significances (t-test, two-tailed) were assessed (*P < 0.05). (d) HeLa cells treated with indicated siRNAs were arrested by Monastrol and released into MG132 after the washout of Monastrol. To prevent premature mitotic exits in hMad1-depleted cells treated with Monastrol, MG132 was added to the culture 1 h after the Monastrol addition. In this synchronous mitotic culture, alignment defects in hMad1-depleted cells appeared transiently only after 1 h after release but not anymore at 2 h. This contrasts to the results in CENP-E-depleted cells, in which alignment defects were persistent. Because CENP-E is only partly displaced from centromeres in hMad1-depleted cells (Fig. 7a), this residual CENP-E might finally complete the alignment. Over 30 cells were analysed for each. (e) HeLa cells expressing hMad1-3Flag-HA or hMad1-5A-3Flag-HA arrested in mitosis by nocodazole were examined by immunostaining (left). Localization was quantified as the ratio of fluorescence intensity of CENP-E to the value of CENP-C in 10 kinetochores from each cell (right). Error bars, s.e.m. for n = 7 cells for each. Statistical significances (t-test, two-tailed) were assessed (**P < 0.005). hMad1 overexpression enhanced the CENP-E accumulation at kinetochores in N-terminal motif-dependent manner. (f) hMad1-depleted HeLa cells expressing RNAi-resistant hMad1-3Flag-HA or hMad1-5A-3Flag-HA were examined for its nuclear envelope localization by immunostaining. Single sections of the images are shown. Uncropped images of blots are shown in Supplementary Fig. 8. Scale bars, 4 μm.

Supplementary Figure 5 Eg5 does not localize at kinetochores.

Non-treated or nocodazole-treated HeLa cells were examined for Eg5 localization by immunostaining. Note that Eg5 was not detected at the kinetochores even in the absence of attachment (+ Nocodazole). Scale bars, 2 μm.

Supplementary Figure 6 Fission yeast Mad1 uses the N-terminal conserved motif for Cut7 binding.

(a) His-Cut7 was pulled down by GST-Mad1. Cut7 was efficiently pulled down by wild-type Mad1, whereas it was pulled down to a lesser extent by Mad1-5A. (b) The indicated cells carrying the nda3-KM311 mutation were cultured at the restrictive temperature and scored for Plo1-GFP-positive cells. Note that Mad1-5A can efficiently activate the SAC. Over 100 cells per strain were analysed. Error bars, s.e.m. for n = 3 independent experiments. (c) Serial dilution assay showing that mad1-5A mutant shows hypersensitivity to TBZ as mad1Δ and mad1-KAKA cells (TBZ 15 μg ml−1). (d) The single section of interphase cells expressing Mad1-GFP, Mad1-KAKA-GFP or Mad1-5A-GFP. Note that both Mad1-KAKA-GFP and Mad1-5A-GFP properly localize to the nuclear envelope. Uncropped images of blots are shown in Supplementary Fig. 8. Scale bar, 3 μm.

Supplementary Figure 7 Potential Cdk1 phosphorylation site T1011 in Cut7 is not involved in Cut7-Mad1 interaction.

(a) Alignment of conserved BimC motif in Cut7 family motor proteins. Cdk1 phosphorylation site in human46 is highlighted in red. Identical or similar residues are highlighted in blue. Alignments of 997-1019 from S. pombe Cut7[gene bank number x57513], 912-934 from H.sapiense Eg5[x85137], aa.923-945 from X.laevis Eg5[x71864], 992-1014 A.nidulans BimC [M32075]. (b) A yeast two-hybrid assay shows that the potential Cdk1 phosphorylation sites (T1011) in Cut7 is not important for Mad1 interaction. (c) The GFP signals were measured in the indicated mitotic (nda3-KM311) cells expressing Cut7-GFP or Cut7-T1011A-GFP, Mis6-2mCherry (kinetochore) and Sfi1-CFP (SPB). Note that kinetochore localization of Cut7-T1011A mutant was intact. Error bars, s.e.m. for n = 30 kinetochores detached from SPB from 10 cells. (d) Serial dilution assay (28 °C, 34 °C, TBZ 15 μg ml−1 at 28 °C). Note that cut7-T1011A mutant cells show temperature sensitivity at 34 °C, and does not show TBZ sensitivity unlike mad1-KAKA. Scale bar, 3 μm.

Supplementary Figure 8 uncropped images of blottings and gels.

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Live imaging of mitotic HeLa cell expressing H2B-mCherry.

Original Live-cell movie of HeLa cell shown in Fig. 6b (Control). Exposures of 0.05 s with a 2 × 2 bin were acquired every 3 min. (MOV 291 kb)

Live imaging of CENP-E-depleted-mitotic HeLa cell expressing H2B-mCherry.

Original Live-cell movie of CENP-E-depleted HeLa cell shown in Fig. 6b (CENP-E RNAi). Exposures of 0.05 s with a 2 × 2 bin were acquired every 3 min. (MOV 525 kb)

Live imaging of hMad1-depleted-mitotic HeLa cell expressing H2B-mCherry.

Original Live-cell movie of hMad1-depleted HeLa cell shown in Fig. 6b (hMad1 RNAi example 1). Note that cell exited from mitosis prematurely. Exposures of 0.05 s with a 2 × 2 bin were acquired every 3 min. (MOV 84 kb)

Live imaging of hMad1-depleted-mitotic HeLa cell expressing H2B-mCherry.

Original Live-cell movie of hMad1-depleted HeLa cell shown in Fig. 6b (hMad1 RNAi example 2). Note that although in the presence of unaligned chromosomes, cell entered anaphase due to the defective SAC. Exposures of 0.05 s with a 2 × 2 bin were acquired every 3 min. (MOV 169 kb)

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Akera, T., Goto, Y., Sato, M. et al. Mad1 promotes chromosome congression by anchoring a kinesin motor to the kinetochore. Nat Cell Biol 17, 1124–1133 (2015). https://doi.org/10.1038/ncb3219

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