Opportunities to join the lab.
Funded by the Wellcome Trust
Our lab, in collaboration with JP's lab and Kaustuv Sanyal's lab (Bengalaru), was recently awarded an 8 year Wellcome Trust Discovery Award to study mechanisms of cell division and aneuploidy in Cryptococcus. Several positions are currently available. Please contact us by email if interested.
PhD studentships
Cryptostasis: dissecting roles of the Cryptococcus neoformans proteasome through structure informed analysis
Supervisors:
Kevin Hardwick, Institute of Cell Biology, University of Edinburgh
Manu De Rycker, Drug Discovery Unit, University of Dundee
Summary:
Cryptococcus neoformans is an important human fungal pathogen 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, chromosome segregation and aneuploidy. Mitotic errors can lead to an incorrect number of chromosomes (aneuploidy) and this is typically deleterious to cells as it induces proteotoxic stress. However, in certain situations it can lead to drug resistance and thus is relevant to treatment of Cryptococcus in the clinic. We are interested in how Cryptococcus deals with proteotoxic stress, along with its resistance mechanisms to other stresses encountered during infections (high body temperature, oxidative stress, hypoxia). A key player in cellular proteostasis is the 26S proteasome, which consists of a 20S core particle and a 19S regulatory particle. The catalytic activities of the core particle are found in chymotrypsin-like (beta5), trypsin-like (beta2) and caspase-like (beta1) sub-units.
The project offers a unique opportunity to engage with a variety of essential lab techniques. You will tag multiple sub-units of the Cryptococcus proteasome with fluorescent proteins and analyse its localisation in live yeast, and the clinically relevant titan and titanide cells. You will purify the proteasome from Cryptococcus cells, and employ cryo-electron microscopy to study its structure. Mass spectrometry will be used to identify all the protein sub-units present. CRISPR-mediated genome engineering will be employed to determine the consequences of specific mutations in conserved domains of each type of beta sub-unit. How do these mutations impact stress sensitivity, viability, titan cell formation and virulence? In parallel, in silico modelling and docking studies will be used to characterise the mode of action of known proteasome inhibitors. Importantly, your Cryptococcus proteasome structure and active-site characterisation will be used to inform screens for novel proteasome inhibitors. Hence, you will directly contribute to providing key tools in the lab which could, in the longer term, be tested in combinatorial cryptococcal therapies.
The project is ideal for a student looking to understand fundamental cellular machinery in a critically understudied, human fungal pathogen.
Skills that you will develop include:
Proteasome purification from Cryptococcus neoformans
Mass spectrometry
Cryo-EM structure determination
In silico modelling and small molecule docking
In vitro proteasome activity assays
CRISPR-mediated genome engineering in Cryptococcus neoformans
Quantitative live-cell fluorescent imaging with microfluidics
Quantitative in vitro titan cell formation assays
References:
The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis. Dambuza IM, Drake T, Chapuis A, Zhou X, Correia J, Taylor-Smith L, LeGrave N, Rasmussen T, Fisher MC, Bicanic T et al (2018). PLoS Pathogens, 14: e1006978
Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint. Aktar K, Davies T, Leontiou I, Clark I, Spanos C, Wallace E, Tuck L, Jeyaprakash AA, and Hardwick KG (2024). PLoS Genetics, 20: e1011302
Pharmacological and structural understanding of the Trypanosoma cruzi proteasome provides key insights for developing site-specific inhibitors. Eadsforth TC, Torrie LS, Rowland P, Edgar EV, MacLean LM, Paterson C, Robinson DA, Shepherd SM, Thomas J, Thomas MG, Gray DW, Postis VLG, De Rycker M (2025). J Biol Chem. 301(1):108049. doi: 10.1016/j.jbc.2024.108049.
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