Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Cryptococcus Neoformans

TISSUE(S): Cell Culture

SUBMITTER: Kevin Hardwick

LAB HEAD: Professor Kevin Hardwick

PROVIDER: PXD052327 | Pride | 2024-05-27

REPOSITORIES: Pride

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			Action	DRS
	Aktar_etal_2024_GFPMad1_arr_vs_H99_vs_GFPMad1_cycl.7z	Other
	Aktar_etal_2024_GFP_Mad1_arr_Rep1.raw	Raw
	Aktar_etal_2024_GFP_Mad1_arr_Rep2.raw	Raw
	Aktar_etal_2024_GFP_Mad1_arr_Rep3.raw	Raw
	Aktar_etal_2024_GFP_Mad1_cycl_Rep1.raw	Raw

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Project description:Ibrahim2008 - Mitotic Spindle Assembly Checkpoint - Convey variant The Mitotic Spindle Assembly Checkpoint ((M)SAC) is an evolutionary conserved mechanism. This model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. MCC:APC dissociation is described by two alternatives models, namely the "Dissociation" and the "Convey" model variants. Both these model are available in BioModels Database. This model corresponds to the "Convey" variant. This model is described in the article: In-silico modeling of the mitotic spindle assembly checkpoint. Ibrahim B, Diekmann S, Schmitt E, Dittrich P PLoS One. 2008 Feb 6;3(2):e1555. Abstract: BACKGROUND: The Mitotic Spindle Assembly Checkpoint ((M)SAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer. PRINCIPLE FINDINGS: We have constructed and validated for the human (M)SAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the (M)SAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the "Dissociation" and the "Convey" model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments. CONCLUSION: Only in the controlled case, our models show (M)SAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for (M)SAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding. This model describes the controlled dissociation variant of the mitotic spindle assembly checkpoint. If the tool you use has problems with events, you can uncomment the assignment rules for u and u_prime and comment out the list of events. In accordance with the authors due to typos in the original publication some initial conditions and parameters were slightly changed in the model: article model [O-Mad2] 1.5e-7 M 1.3e-7 M [BubR1:Bub3] 1.30e-7 M 1.27e-7 M k -4 0.01 M -1 s -1 0.02 M -1 s -1 k -5 0.1 M -1 s -1 0.2 M -1 s -1 This model is hosted on BioModels Database and identified by: MODEL6655578762 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Ibrahim2008 - Mitotic Spindle Assembly Checkpoint - Dissociation variant The Mitotic Spindle Assembly Checkpoint ((M)SAC) is an evolutionary conserved mechanism. This model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. MCC:APC dissociation is described by two alternatives models, namely the "Dissociation" and the "Convey" model variants. Both these model are available in BioModels Database. This model corresponds to the "Dissociation" variant. This model is described in the article: In-silico modeling of the mitotic spindle assembly checkpoint. Ibrahim B, Diekmann S, Schmitt E, Dittrich P PLoS One. 2008 Feb 6;3(2):e1555. Abstract: BACKGROUND: The Mitotic Spindle Assembly Checkpoint ((M)SAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer. PRINCIPLE FINDINGS: We have constructed and validated for the human (M)SAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the (M)SAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the "Dissociation" and the "Convey" model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments. CONCLUSION: Only in the controlled case, our models show (M)SAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for (M)SAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding. This model describes the controlled dissociation variant of the mitotic spindle assembly checkpoint. If the tool you use has problems with events, you can uncomment the assignment rules for u and u_prime and comment out the list of events. In accordance with the authors due to typos in the original publication some initial conditions and parameters were slightly changed in the model: article model [O-Mad2] 1.5e-7 M 1.3e-7 M [BubR1:Bub3] 1.30e-7 M 1.27e-7 M k -4 0.01 M -1 s -1 0.02 M -1 s -1 k -5 0.1 M -1 s -1 0.2 M -1 s -1 This model is hosted on BioModels Database and identified by: MODEL6655615431 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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

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