Close

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

Updated 17 January 2023 with a postLight by Saanjbati Adhikari

The preprint by Hayward et al., 2022 was discussed on this platform in August, 2022. The published version of this work is now available in Current Biology: https://doi.org/10.1016/j.cub.2022.10.047

There are no major revisions in the experiments discussed, however, there are important modifications that have added clarity to the final piece of published work.

The authors have redesigned the schematic of their hypothesised model in Figure 1A. The preprint included an in-depth schematic at the beginning, discussing “stable” to “transient” kinetochore status, and possible roles of Aurora B kinase and the spindle assembly checkpoint (SAC). This, in my opinion, was a big giveaway and didn’t allow readers to be slowly introduced to the interesting results. The current version maintains suspense of the story and allows readers to appreciate the eventual addition of layers of complexity.

Another important modification is the addition of subpanels J and K in Figure 2, which were in the supplementary material (extended data figure 4) of the preprint. Here, the authors show that lack of N-terminal autophosphorylation in MPS1 continues to cause detachment of MPS1 from kinetochores, similar to the wild type. This proves that autophosphorylation of MPS1 is not the factor that confers microtubule sensitivity to it. This, in my opinion, is an important discovery that indeed needs to be discussed in the main figures. Furthermore, the authors have conducted experiments to show fluorescence recovery after photobleaching (FRAP) of cells to also be independent of N-terminal autophosphorylation sites on MPS1 (Figure 2I). These results provide orthogonal evidence to confirm that MPS1 removal from kinetochores is not affected by its N-terminal autophosphorylation sites. Also, supplementary figures S2P-S confirm that kinase-dead MPS1 can not recruit the cellular phosphatases, PP1 and PP2A, to kinetochores. In the absence of these phosphatases, recruitment of Aurora B-dependent MPS1 is not counteracted, therefore inactive MPS1 remains at the metaphase plate. Overall, I think, these added experiments provide a holistic story and help readers to better understand how a kinase-phosphatase counterbalance modulates MPS1 recruitment at the kinetochores.

The last touch-up I would like to discuss is the final model presented in Figure 4E. It provides a well-rounded finishing touch, since it fills in the gaps present in Figure 1A, without being repetitive. Although the schematic was included in the preprint as well, I prefer the modified version since it looks more aesthetically pleasing and is clearer to the eye.

 

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 , updated on: 17 January 2023

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

Read preprint (No Ratings Yet)

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

preLights peer support – preprints of interest

This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.

 



List by preLights peer support

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.

 



List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.

 



List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Dey Lab, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Dey Lab, Amanda Haage

Also in the molecular biology category:

‘In preprints’ from Development 2022-2023

A list of the preprints featured in Development's 'In preprints' articles between 2022-2023

 



List by Alex Eve, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23

 



List by Alex Eve

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.

 



List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.

 



List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.

 



List by Nadja Hümpfer et al.

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

MitoList

This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.

 



List by Sandra Franco Iborra
Close