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MPS1 localizes to microtubule-attached kinetochores and actively promotes microtubule release

Daniel Hayward, Emile Roberts, Ulrike Gruneberg

Preprint posted on 22 May 2022 https://www.biorxiv.org/content/10.1101/2022.05.23.493048v1

What releases the mitotic kinase, MPS1, from the environment of outer kinetochores? This work challenges previous models and revises our current understanding of the regulatory mechanisms that determine MPS1 localisation patterns in mitotic cells.

Selected by Saanjbati Adhikari

Background

Accurate DNA segregation relies on proper attachments between chromosomes and microtubules. These attachments are mediated by the kinetochore, a macromolecular structure that assembles on the outer centromeric region of chromosomes (reviewed in 1). Incorrect or defective kinetochore-microtubule attachments may lead to unequal division of chromosomes between daughter cells, a phenomenon called aneuploidy, which is a hallmark of several cancers (2, 3). To preserve genomic integrity, incorrectly attached/unattached kinetochores cause activation of a spindle assembly checkpoint (SAC) – an active signalling pathway – that prevents cell cycle progression until all chromosomes are correctly bound (reviewed in 1). SAC orchestrates inhibition of the anaphase promoting complex/cyclosome (APC/C) via several downstream effectors, including the monopolar spindle 1 kinase (MPS1). The current model of error correction mechanisms involves MPS1 recruitment to unattached kinetochores generated by the serine/threonine master kinase, Aurora B (4, 5). MPS1 catalyses formation of the mitotic checkpoint complex (MCC), which directly inhibits the APC/C and stalls mitotic progression until all chromosomes are correctly attached (6, Figure 1). Once end-on attachments are established, microtubules physically compete with MPS1 for the same binding sites on the outer kinetochore, releasing MPS1 from the outer kinetochores (6, 7). Recent studies have shown checkpoint silencing to occur at kinetochores with partial microtubule occupancy (8, 9); therefore, a competition model can not fully explain the release of MPS1 from the environment of attached kinetochores.

Figure 1. Sequential multi-target phosphorylation by MPS1 activates the formation of the mitotic checkpoint complex (Adapted from 5). A schematic of paired sister chromatids has been depicted on the left with a magnified view of one of the unattached kinetochores to highlight MPS1 activity. MPS1 phosphorylates an essential protein of the outer kinetochore complex, Knl1, at multiple methionine-glutamate-leucine-threonine (MELT) motifs (a). This leads to recruitment of the budding uninhibited by benomyl 1–3 complex (Bub1–Bub3) at the outer kinetochore. MPS1 further phosphorylates Cdk1-phosphorylated Bub1 (b), which then recruits the Mad1-Mad2 complex. MPS1 then phosphorylates Mad1 (c), enabling the formation of the Mad1-Cdc20 complex (5) This bipartite complex can bind to BubR1 (regulator of Bub1), forming the mitotic checkpoint complex (MCC), which directly binds to the APC/C for inhibition of substrate recognition (6). 

In this work, Hayward and colleagues revise our current understanding of the regulatory mechanisms that determine MPS1 localisation at kinetochores of mitotic cells. Contrary to previous models that emphasise upon competition between MPS1 and microtubules for the same binding sites on the outer kinetochore, data from this study indicate that MPS1 localisation at the outer kinetochore is modulated by kinase-phosphatase counter activities within an ‘incoherent’ feed-forward loop. 

Main findings:

  1. Firstly, the authors asked if MPS1 recruitment to kinetochores is regulated through kinase and phosphatase activities, instead of direct competition with microtubules for identical binding sites on the outer kinetochore. They found that kinetochores of prometaphase cells can be bound to both MPS1 and microtubules at the same time. These transient interactions could be stabilised by treatment of cells at reduced temperature. To compare MPS1 localisation at attached versus unattached kinetochores, they treated HeLa cells with STLC to create monopolar spindles, i.e. sister kinetochores with one attached and one unattached kinetochore. MPS1 was shown to localise equally on both kinetochores in such cells, when treated at a reduced temperature. Based on these observations, they suggest that microtubules do not compete with MPS1 for binding to the outer kinetochore protein, rather there exists a transient state where both MPS1 and microtubules are simultaneously attached to kinetochores. 
  2. BUBR1 directly interacts with and recruits PP2A-B56 to outer kinetochores (10). PP2A-B56 also modulates MPS1 activity at unattached kinetochores (11). In this work, inhibition of PP1 and PP2 phosphatases significantly increased endogenous MPS1 localisation at bioriented kinetochores (chromosomes attached to microtubules from opposite poles). Furthermore, abrogation of PP2A-B56 interaction with BUBR1 led to MPS1 localisation at both attached and unattached kinetochores of monopolar spindles. This was in contrast to cells with endogenous intact BUBR1, where MPS1 was selectively recruited to unattached kinetochores. Together, these results indicate that a specific pool of BUBR1-bound PP2A-B56 might be responsible for making MPS1 sensitive to microtubule attachments.
  3. To understand whether autophosphorylation of MPS1 affects its kinetochore localisation, all known autophosphorylation sites in the N-terminal region of MPS1 were silenced by Alanine-substitution. This significantly increased endogenous MPS1 levels at unattached kinetochores. However, bioriented kinetochores of metaphase cells did not show increased localisation of MPS1 mutants lacking autophosphorylation sites. Thus, while autophosphorylation regulates MPS1 localisation at unattached kinetochores, it is not responsible for release of MPS1 from microtubule-attached kinetochores.
  4. To explore whether Aurora B regulates MPS1 recruitment to attached kinetochores, a dimerisation module was designed (12). This module/cassette allowed Rapamycin-inducible delivery of Aurora B kinase to the outer kinetochore protein, Mis12, in HeLa cells endogenously expressing GFP-MPS1. Targeting Aurora B to bioriented kinetochores caused rapid recruitment of MPS1 to the outer kinetochore, comparable to MPS1 levels at unattached kinetochores. This suggests that high Aurora B levels at the outer kinetochore are sufficient for MPS1 localisation and maintenance, even when chromosomes are bioriented.
  5. To describe the role of MPS1 in error correction, the authors inhibited MPS1 in bioriented cells with rapamycin-inducible targeted delivery of Aurora B at the outer kinetochore. A stark delay in the generation of unattached kinetochores was observed, suggesting the necessity of MPS1 in the resolution of incorrect microtubule-kinetochore attachments. Then, they describe through co-depletion studies that MPS1 can rescue PP2A inhibition more effectively than Aurora B inhibition. This adds further supportive evidence to a model where MPS1 and PP2A-B56 act antagonistically in the regulation and maintenance of kinetochore-microtubule attachments. Overall, the authors propose a model, wherein MPS1 constitutes a novel feed forward loop: MPS1 is recruited to incorrectly attached kinetochores in an Aurora B-dependent manner, where MPS1 catalyses the checkpoint complex and recruits the BUBR1-bound PP2A-B56. Biorientation and fulfilment of the spindle activation checkpoint attenuates AuroraB levels at the kinetochores, which further leads to counteraction of MPS1 activity by the BUBR1-recruited pool of PP2A-B56. This eventually releases MPS1 from the environment of attached kinetochores, allowing stabilisation of attached kinetochores and mitotic progression.

What I like about this work:

My initial enthusiasm regarding MPS1 stemmed from the various controversies around localisation patterns of this master kinase at metaphase kinetochores and its role in error correction mechanisms of kinetochore-microtubule attachments. My doctoral research project focuses on a microtubule and kinetochore-associated protein, Astrin, which has been implicated in stabilising chromosome-microtubule attachments via its spatio-temporally regulated interaction with protein phosphatase 1 (PP1). When I read the abstract of the current work by Hayward et al., 2022, I could appreciate the kinase-phosphatase interplay since Astrin-PP1 interaction also regulates a finely tuned feedback loop (13). MPS1 is a core component of the spindle assembly checkpoint, therefore, it was critical to unveil its dynamics at the crucial stages of mitosis. In my opinion, MPS1’s direct involvement in the resolution of incorrect kinetochore-microtubule attachments can potentially have novel implications in cancer and cell biology research, since MPS1 is an important prognostic biomarker for several cancers (14) .

My questions to authors:

  1. Plk1 and MPS1 were shown to cooperatively regulate the establishment and maintenance of the SAC (15). Same study showed that Plk1 directly phosphorylates MPS1, which increases MPS1’s activity and turnover rate at the kinetochores during the G2/M phase transition and through early mitosis. I am curious to know whether you think Plk1 might have an effect on the PP2A-B56-dependent regulation of MPS1 at attached/bioriented kinetochores, or not? 
  2. It is also known that MPS1 and Plk1 synergistically phosphorylate KNL1, an outer kinetochore protein essential for recruitment of SAC signalling components to the kinetochore (15). Do you think including KNL1 in the existing model might draw a more comprehensive picture of the kinetochore status during the ‘transient’ state (when MPS1 can be found at microtubule-attached kinetochores)?

References

  1. Cheeseman, I. M. & Desai, A. Molecular architecture of the kinetochore–microtubule interface. Nat Rev Mol Cell Biol 9, 33–46 (2008).
  2. Santaguida, S. & Amon, A. Short- and long-term effects of chromosome mis-segregation and aneuploidy. Nat Rev Mol Cell Biol 16, 473–485 (2015).
  3. Gui, P. et al. Mps1 dimerization and multisite interactions with Ndc80 complex enable responsive spindle assembly checkpoint signaling. Journal of Molecular Cell Biology 12, 486–498 (2020).
  4. Sudakin, V., Chan, G. K. T. & Yen, T. J. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. Journal of Cell Biology 154, 925–936 (2001).
  5. Ji, Z., Gao, H., Jia, L., Li, B. & Yu, H. A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling. eLife 6, e22513 (2017).
  6. Ji, Z., Gao, H. & Yu, H. Kinetochore attachment sensed by competitive Mps1 and microtubule binding to Ndc80C. Science 348, 1260–1264 (2015).
  7. Hiruma, Y. et al. Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling. Science 348, 1264–1267 (2015).
  8. Dudka, D. et al. Complete microtubule–kinetochore occupancy favours the segregation of merotelic attachments. Nat Commun 9, 2042 (2018).
  9. Kuhn, J. & Dumont, S. Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner. Journal of Cell Biology 218, 3583–3596 (2019).
  10. Kruse, T. et al. Direct binding between BubR1 and B56–PP2A phosphatase complexes regulate mitotic progression. Journal of Cell Science 126, 1086–1092 (2013).
  11. Hayward, D. et al. Checkpoint signaling and error correction require regulation of the MPS1 T-loop by PP2A-B56. Journal of Cell Biology 218, 3188–3199 (2019).
  12. Ballister, E. R., Riegman, M. & Lampson, M. A. Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint. Journal of Cell Biology 204, 901–908 (2014).
  13. Song, X., Conti, D., Shrestha, R. L., Braun, D. & Draviam, V. M. Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation. Nat Commun 12, 7010 (2021).
  14. Xie, Yuan et al. Mps1/TTK: a novel target and biomarker for cancer. Journal of drug targeting vol. 25, 112-118 (2017). 
  15. von Schubert, C. et al. Plk1 and Mps1 Cooperatively Regulate the Spindle Assembly Checkpoint in Human Cells. Cell Reports 12, 66–78 (2015).

 

Posted on: 17 August 2022

doi: https://doi.org/10.1242/prelights.32508

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