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EASTBIO Structure-driven dissection and inhibition of mitotic regulators in the fungal pathogen Cryptococcus neoformans


School of Biological Sciences

Prof K Hardwick , Dr J Arulanandam Wednesday, January 06, 2021 Competition Funded PhD Project (Students Worldwide)

About the Project

Cryptococcus neoformans is an understudied fungus with a fascinating life cycle, forming polyploid Titan cells during infection in a bid to evade the immune system. It kills hundreds of thousands of immune-compromised AIDS patients each year, mainly in Africa.
We study mitosis and chromosome segregation, with a particular focus on the spindle checkpoint and error correction pathways. This checkpoint control monitors interactions between chromosomes and the mitotic spindle and delays mitotic progression until all pairs of sister chromatids are attached appropriately to spindle microtubules. The checkpoint provides time for the error-correction machinery (such as Aurora B and the chromosomal passenger complex) to resolve inappropriate kinetochore-microtubule attachments. Mps1 and Bub1 kinases are central players in the spindle checkpoint, but also have roles in error-correction and are candidate drug targets. You will express, purify, crystallise and solve the structures of important mitotic regulators, such as highly conserved domains of CnBub1, Mps1, Aurora or Sgo1. Structures will be used to screen for and identify small molecule inhibitors of fungal kinases. Lead compounds have already been developed for their human homologues.

Typically, Mps1 activation at unattached kinetochores triggers checkpoint signalling early in mitosis. We have found that Mps1 overexpression is sufficient to activate the Cryptococcus checkpoint, to recruit Bub1 to kinetochores and to arrest cells in metaphase.
You will carry out in vivo structure-function studies on one or more mitotic regulators. CRISPR-mediated genome engineering will be employed to determine the consequences of specific mutations in conserved domains. How do they impact yeast viability, Titan cell formation, polyploidy and aneuploidy? Small molecule inhibitors will be screened for, that produce similar loss of function phenotypes.

Skills developed:
Recombinant protein purification, crystallisation and structure determination
In silico modelling and docking
In vitro validation of inhibitor hits using biophysical binding studies
In vitro kinase assays
Purification of protein complexes from yeast cells, analysis via mass spectrometry
CRISPR-mediated genome engineering and molecular genetics in Cryptococcus neoformans
Quantitative live-cell fluorescent imaging, with microfluidics
Quantitative in vitro titan cell formation assay and in vivo infectivity assays.
Synthetic biology: engineering of a chemically-induced (abscisic acid) cell cycle block.

The School of Biological Sciences is committed to Equality & Diversity: https://www.ed.ac.uk/biology/equality-and-diversity

How to Apply:
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Funding Notes

This 4 year PhD project is part of a competition funded by EASTBIO BBSRC Doctoral Training Partnership View Website. This opportunity is open to UK and International students and provides funding to cover stipend and UK level tuition fees. The fee difference will be covered by the University of Edinburgh for successful international applicants. Please refer to UKRI website (View Website) and Annex B of the UKRI Training Grant Terms and Conditions (View Website) for full eligibility criteria.

References

(1) London, N. and Biggins, S. (2014). Signalling dynamics in the spindle checkpoint response. Nature Reviews Mol Cell Biology, 15, 736-747.

(2) Yuan, I., Leontiou, I., Amin, P., May, K.M., Chafraidh, S.S., Zlamalova, E. and Hardwick, K.G. (2017). Generation of a spindle checkpoint arrest from synthetic signalling assemblies. Current Biology 27, 137-143.

(3) Dambuza, I.M., Drake, T., Chapuis, A., Zhou, X., Correia, J., Taylor-Smith, L., LeGrave, N., Rasmussen, T., Fisher, M.C., Bicanic, T., et al. (2018). The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis. PLoS Pathogens 14, e1006978.
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