Molecular dissection of the spindle assembly checkpoint signalling in Cryptococcus neoformans

Abstract

Cryptococcosis is a severe fungal infection caused by an opportunistic fungal pathogen, Cryptococcus neoformans which has been medically significant for more than half of the last century. This yeast displays noticeable ploidy shifts during in vivo pulmonary infection. These polyploid cells often generate aneuploid progeny which has been repeatedly reported as one of the main virulence factors for disease progression. Given this capability of escaping equal chromosome segregation during mitosis, they are possibly escaping several cell cycle controls including the spindle assembly checkpoint. The spindle assembly checkpoint is undescribed in this fungal pathogen. Therefore, I aimed to understand how this checkpoint signalling contribute to cell division in C. neoformans. My current aim is to study one of the critical spindle assembly checkpoint proteins, Mad1, which remains undescribed in this fungal pathogen. Deletion of mad1 and mad2 in Cryptococcus showed sensitivity to anti-microtubules drugs. Microscopy and microfluidics data revealed that the mad1 and mad2 mutants were unable to maintain mitotic arrest in response to such drugs. Both proteins were also found to be important for Titan cell viability. Mad1 showed localisation to unattached kinetochores of arrested cells. Purified Mad1 complexes showed interactions with other checkpoint proteins Bub1, Mad2, Cdc20 and Mps1, by co-immunoprecipitation and mass spectrometry. I believe that several of these interactions are driven by phosphorylation. I found Mad1 to be phosphorylated by recombinant Mps1 kinase. I have generated several Mad1 phospho-mutants and some show defects in checkpoint signalling. Thus, Mad1 protein-protein interactions could be regulated by kinases such as Mps1, Cdk, Plk1 or Bub1 kinase and this may affect Mad1 interaction with Cdc20 (the APC/C co-activator). This study leads to a plausible molecular explanation of Mad1 contribution in MCC assembly (Mitotic checkpoint complex). The precise in vivo functions of Mad1 and more details of the underlying molecular mechanisms of spindle assembly checkpoint signalling in this understudied pathogenic fungus will be discussed.