Conserved signalling functions for Mps1, Mad1 and Mad2 in the Cryptococcus neoformans spindle checkpoint.

Autor: Aktar K; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom., Davies T; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom., Leontiou I; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom., Clark I; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom., Spanos C; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom., Wallace E; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom., Tuck L; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom., Jeyaprakash AA; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.; Gene Center, Department of Biochemistry, Ludwig Maximilians Universitat, Munich, Germany., Hardwick KG; Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.
Jazyk: angličtina
Zdroj: PLoS genetics [PLoS Genet] 2024 Jun 03; Vol. 20 (6), pp. e1011302. Date of Electronic Publication: 2024 Jun 03 (Print Publication: 2024).
DOI: 10.1371/journal.pgen.1011302
Abstrakt: 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 mad1Δ or mad2Δ 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.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2024 Aktar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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