mps1andmadmutations reduceCryptococcus neoformanstitan cell viability

Abstract

AbstractCryptococcus neoformansis an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle assembly checkpoint (SAC) which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate thatCryptococcus mad1Δormad2Δstrains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die rapidly as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates thatmadmutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved inCryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrestCryptococcuscells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effectivein vitrosubstrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of theGAL-MPS1arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step inCryptococcusspindle checkpoint signalling. Finally, we analyse the phenotype ofmadandmps1mutants during titan cell generation: quantitating viability of titan cells and their daughters generated during the ensuing reductive division. Themad1Δ, mad2ΔandmpsΔmutants show significantly reduced viability: many titans are dead and others produce slow growing colonies. We propose that theseCryptococcus neoformanscheckpoint proteins have important roles in ensuring high fidelity chromosome segregation during stressful conditions, such that those heightened during its polyploid infection cycle.