*EASTBIO* Spindle checkpoint signalling in a human fungal pathogen

Background: We study mitosis and chromosome segregation in yeast, with a particular focus on the spindle checkpoint. This cell cycle 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 (1). We recently started to employ synthetic biology approaches to analyse this signalling pathway, and have now successfully generated a ‘wait anaphase’ signal entirely independently of kinetochores in fission yeast. This requires tethering of Mps1 kinase to one of its key substrates (2), thereby producing an active signalling scaffold that is sufficient to generate a prolonged metaphase arrest.

Cryptococcus neoformans is a basidiomycete fungal pathogen responsible for ~1 million new infections each year in immunocompromised patients, with many infections leading to meningitis and death. You will study how this budding yeast deals with prolonged mitotic arrests, such as those imposed by anti-microtubule drugs and our synthetic checkpoint signalling platform. The molecular components of the spindle checkpoint are somewhat simplified in this yeast, with very interesting implications for the mechanistic generation of spindle checkpoint signals.

Project:
a) You will generate and analyse spindle checkpoint mutants in Cryptococcus, and employ live-cell imaging to localise the Mad and Bub proteins throughout its cell cycle.
b) You will employ Gibson assembly cloning to build novel, synthetic signalling assemblies, in both fission yeast and in Cryptococcus. The active scaffolds will then be purified biochemically from yeast cells and analysed using phospho-proteomics to identify associated proteins and their post-translational modifications. Mutations will then be employed to disable and hyper-activate these signalling scaffolds.
c) Prolonged mitotic arrest in fission yeast frequently leads to chromosome mis-segregation, aneuploidy and cell death. You will employ live-cell imaging (3) to study how Cryptococcus cells behave during and after prolonged mitotic arrests.

This project will improve our mechanistic understanding of the spindle checkpoint, and aims to improve strategies for killing Cryptococcus neoformans during infection. A deeper understanding of the cell cycle controls employed by this pathogen will be critical for successful strategies to be developed. This project also provides for an excellent training opportunity, using a broad range of techniques to study cell cycle regulation and chromosome segregation in both a model organism and an important human pathogen.