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Specialisation of meiotic kinetochores revealed through a synthetic spindle assembly checkpoint strategy.

ABSTRACT: Meiosis creates haploid gametes through two sequential M phases. While many studies have focused on meiosis I, the molecular events which drive and define meiosis II are largely unknown. Here, we report a novel cell synchronization strategy which allows for collection of yeast cells arrested at metaphase I or metaphase II, enabling better characterisation of meiosis II events. The method relies on chemically-inducible dimerization of ectopic copies of spindle assembly checkpoint (SAC) proteins Mps1 and Spc105. Using this synthetic SAC (SynSAC) approach, we found that the SAC response is weaker in metaphase I compared to metaphase II and that the PP1 binding site within Spc105 contributes to restraining the MI SAC response. Furthermore, we demonstrate the utility of the SynSAC approach by analysing the composition and phosphorylation of kinetochores from metaphase I and metaphase II. This revealed an increase in the abundance of outer kinetochore proteins in meiotic metaphase I and reduced phosphorylation on metaphase II kinetochore proteins. Overall, we present the SynSAC method as a valuable tool for analysis of both meiotic metaphases.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Adele Marston

LAB HEAD: Adele Marston

PROVIDER: PXD067911 | Pride | 2026-03-04

REPOSITORIES: Pride

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			Action	DRS
	DIANN_Koch_etal_2025_51K_51N_N_PE_A_F_Final_Analysis_report.pr_matrix.7z	Other
	Koch_etal_2025_Metaphase1_51M_N_A_Dsn1-His-FLAG_Rep1.raw	Raw
	Koch_etal_2025_Metaphase1_51M_N_B_Dsn1-His-FLAG_Rep2.raw	Raw
	Koch_etal_2025_Metaphase1_51M_N_C_Dsn1-His-FLAG_Rep3.raw	Raw
	Koch_etal_2025_Metaphase1_51M_N_D_NoTag_Rep1.raw	Raw

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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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Specialisation of meiotic kinetochores revealed through a synthetic spindle assembly checkpoint strategy.

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