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Using lattice light sheet microscopy to determine how the Spindle Assembly Checkpoint turns off


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  Prof J Pines, Mr Ben Atkinson  No more applications being accepted  Funded PhD Project (Students Worldwide)

London United Kingdom Biochemistry Biophysics Cancer Biology Cell Biology Optical Physics Biological Sciences

About the Project

Project Description 

To maintain genomic stability the mechanisms of mitosis divide the genome such that the two daughter cells receive an equal and identical copy of the genome. To ensure this, the Spindle Assembly Checkpoint (SAC) prevents sister chromatid separation should any kinetochore fail to attach correctly to the spindle. (Reviewed in Lara-Gonzales et al., 2021.) Unattached kinetochores generate an anaphase inhibitor composed of the MAD2, BUB3 and BUBR1 Spindle Assembly Checkpoint proteins. Kinetochores that stably attach to microtubules no longer generate the inhibitor but we do not understand how microtubule attachment does this. Is one attached microtubule sufficient, or are several required? Is the ability of microtubules to ‘strip’ kinetochores of the inhibitor proteins fast enough? To answer these questions we need to image microtubule attachment to kinetochores in real-time and quantify the changes in MAD2 and BUBR1 at kinetochores. We can now do this using lattice-lightsheet microscopy and endogenously tagged proteins. The specific aims at the start of the project are as follows:

  • Generate cells with endogenously tagged kinetochore and microtubule markers by CRISPR/Cas9 gene editing
  • Optimise lattice light-sheet imaging to resolve kinetochore-microtubule attachment in living cells
  • Measure the kinetics of MAD1, MAD2 and BUBR1 localisation to unattached kinetochores using existing endogenously tagged cell lines and lattice-lightsheet imaging and FRAP
  • Determine the effect of microtubule attachment on MAD2 and BUBR1 residence at kinetochores
  • Perturb known effectors of the microtubule attachment and the Spindle Assembly Checkpoint to determine how MAD2 and BUBR1 are removed from kinetochores upon microtubule attachment.

These experiments will form the basis for hypotheses to explain how microtubule attachment turns off the SAC. Our quantitative experiments will determine whether there is a simple inverse relationship between number of microtubules attached and number of SAC proteins at a kinetochore (unlikely) or whether SAC proteins exhibit switch-like behaviour, as previously reported for exogenous markers (Kuhn and Dumont, 2019). If SAC proteins do have a switch-like response we will determine whether this is controlled by Spindly and dynein-stripping of MAD1 and/or whether post-translational modifications of MAD1 and MAD2 are involved by introducing mutants of known phosphorylation sites.

Keywords

  • Quantitative biology
  • Cell Biology
  • Mitosis

Candidate profile

Candidates must have a First class or Upper Second class BSc Honours/MSc in biology, biochemistry, physics or, biophysics. An interest in super-resolution microscopy would be an advantage.

How to apply

To view the full project proposal and details on how to apply using our online recruitment portal, please go to icr.ac.uk/phds. Please ensure that you read and follow the application instructions very carefully.

Please note we only accept applications via the online application system apply.icr.ac.uk.

Applications close at 11:55pm UK time on 14 November 2021.


Funding Notes

Students receive an annual stipend, currently £21,000 per annum, as well as having tuition fees (both UK and EU/overseas) and project costs paid for the four-year duration. We are open to applications from any eligible candidates and are committed to attracting and developing the best minds in the world. We particularly welcome applicants from British Black and ethnic minority backgrounds, as they are under-represented at PhD level within the ICR and nationwide.

References

- Kuhn, J and Dumont, S., Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner, J. Cell Biol., (2019) https://doi.org/10.1083/jcb.201902105
- Lara-Gonzales, P., Pines, J., and Desai, A., Spindle assembly checkpoint activation and silencing at kinetochores, Seminars in Cell and Developmental Biology, (2021) https://doi.org/10.1016/j.semcdb.2021.06.009
- Chen, B., et al., Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution, Science, (2014), DOI: 10.1126/science.1257998
- Gassmann, R., Holland, A.J., Varma, D., ,Wan, X., ,Çivril, F., Cleveland, D.W, Oegema, K., Salmon, E.D, Desai, A., Removal of Spindly from microtubule-attached kinetochores controls spindle checkpoint silencing in human cells Genes Dev., http://www.genesdev.org/cgi/doi/10.1101/gad.1886810. (2010).
- Eric R. Griffis,E.R., Stuurman,N. and Vale, R.D, Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore J Cell Biol. (2007) https://doi.org/10.1083/jcb.200702062
- B.J. Howell, B.F. McEwen, J.C. Canman, D.B. Hoffman, E.M. Farrar, C.L. Rieder, E.D. Salmon, Cytoplasmic dynein/dynactin drives kinetochore protein transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation J. Cell Biol. (2001) https://doi.org/10.1083/jcb.200105093
- Jackman, MR, Marcozzi, C., Barbiero, M., Pardo, M., Yu, L., Tyson, A.L., Choudhary, JS., Pines, J. (2020) Cyclin B1-Cdk1 facilitates MAD1 release from the nuclear pore to ensure a robust spindle checkpoint J. Cell Biol., doi.org/10.1083/jcb.201907082
- Kuhn, J and Dumont, S., Mammalian kinetochores count attached microtubules in a sensitive and switch-like manner, J. Cell Biol., (2019) https://doi.org/10.1083/jcb.201902105
- Lara-Gonzales, P., Pines, J., and Desai, A., Spindle assembly checkpoint activation and silencing at kinetochores, Seminars in Cell and Developmental Biology, (2021) https://doi.org/10.1016/j.semcdb.2021.06.009