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Kinetochore protein Cnn1 promotes sister chromatid bi-orientation by modulating intraKMN affinities in a biphasic, phospho-regulated manner.

ABSTRACT: Kinetochores are large proteinaceous structures that assemble on centromeres to align sister chromatids to the mitotic spindle and orchestrate their segregation to the daughter cells. At the core of kinetochores lies a network of proteins known as KMN, whose microtubule-binding activity is regulated by the Ipl1/Aurora B and Mps1 kinases. Here, we have characterized a novel structural yeast kinetochore protein named Cnn1 and show that it regulates KMN-spindle activity by modulating the affinity between the KMN subcomplexes at their basis. Cnn1 supports KMN from S phase to anaphase, and is controlled by the Cdc28, Mps1 and Ipl1 kinases. Cnn1 has evolved to associate the KMN complexes into a coherent network activity to ensure efficient

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE31567 | GEO | 2012/05/06

SECONDARY ACCESSION(S): PRJNA145699

REPOSITORIES: GEO

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Project description:Cryptococcus neoformans is 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 checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1D or mad2D strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. 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 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 arrest Cryptococcus cells 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 effective in vitro substrate 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 the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

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Kinetochore protein Cnn1 promotes sister chromatid bi-orientation by modulating intraKMN affinities in a biphasic, phospho-regulated manner.

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