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An Extended, Boolean Model of the Septation Initiation Network in S.Pombe Provides Insights into Its Regulation.

Chasapi A, Wachowicz P, Niknejad A, Collin P, Krapp A, Cano E, Simanis V, Xenarios I - PLoS ONE (2015)

Bottom Line: In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach.The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set.Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.

View Article: PubMed Central - PubMed

Affiliation: Vital-IT Group, Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland.

ABSTRACT
Cytokinesis in fission yeast is controlled by the Septation Initiation Network (SIN), a protein kinase signaling network using the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this paper, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.

No MeSH data available.


Related in: MedlinePlus

Fin1p over-expression results in Cdc16p disassociation from the SPB.(A) Cells expressing the labeled tubulin marker leu1::m-Cherry-atb2 and cdc16-GFP were induced to express fin1 from the medium strength nmt41 promoter [80]. Cells transformed with empty vector served as control. Cells were grown in medium without thiamine for 27h at 25°C. Cells were imaged and the intensity of SPB associated cdc16-GFP signal was analyzed as described in [12]. The panel shows m-Cherry-atb2 leu1::cdc16-GFP(ura4+) cells bearing REP41 or REP41-fin1. The scale bar is 10 μm. (B) The SPB associated signal was determined in interphase cells in each strain. Since REP41-fin1 eventually leads to a mitotic arrest [87] interphase cells were identified by the presence of an interphasic microtubule array. The box shows 25%-75% range for the population, the line indicates the median. The bars indicate 10% and 90% range for the population, and dots indicate more extreme individual values. The y-axis shows fluorescence intensity on an arbitrary scale. (C) Cells bearing the leu1::cdc16-HA allele were induced to express fin1 (ON) by growing them in defined minimal medium [81] in the presence (OFF) or absence (ON) of 2mM thiamine. Protein extracts were prepared 27h after induction and analyzed by western blotting using monoclonal antibody 12CA5. The anti-α-tubulin monoclonal antibody TAT-1 [90] was used as a control.
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pone.0134214.g005: Fin1p over-expression results in Cdc16p disassociation from the SPB.(A) Cells expressing the labeled tubulin marker leu1::m-Cherry-atb2 and cdc16-GFP were induced to express fin1 from the medium strength nmt41 promoter [80]. Cells transformed with empty vector served as control. Cells were grown in medium without thiamine for 27h at 25°C. Cells were imaged and the intensity of SPB associated cdc16-GFP signal was analyzed as described in [12]. The panel shows m-Cherry-atb2 leu1::cdc16-GFP(ura4+) cells bearing REP41 or REP41-fin1. The scale bar is 10 μm. (B) The SPB associated signal was determined in interphase cells in each strain. Since REP41-fin1 eventually leads to a mitotic arrest [87] interphase cells were identified by the presence of an interphasic microtubule array. The box shows 25%-75% range for the population, the line indicates the median. The bars indicate 10% and 90% range for the population, and dots indicate more extreme individual values. The y-axis shows fluorescence intensity on an arbitrary scale. (C) Cells bearing the leu1::cdc16-HA allele were induced to express fin1 (ON) by growing them in defined minimal medium [81] in the presence (OFF) or absence (ON) of 2mM thiamine. Protein extracts were prepared 27h after induction and analyzed by western blotting using monoclonal antibody 12CA5. The anti-α-tubulin monoclonal antibody TAT-1 [90] was used as a control.

Mentions: In the PKN of the model there were no negative regulators targeting GAP components during early mitosis, which resulted in suboptimal outcomes during the simulations of early mitosis; i.e. the simulation would produce a steady state where the GAP remained active in early mitosis. Since removal of the SIN GAP from the SPB is an early step in the activation of the SIN after entry into mitosis [12,74,89], we modeled whether GAP components could be regulated by fin1. Since Cdc16p contains several sites matching the established consensus for mammalian Nek2 (one of the orthologues of nimA), the effect of increased expression of fin1 on Cdc16p localization was investigated by in vivo experiments. Expression of fin1 from the medium strength nmt-41 promoter [80] resulted in partial displacement of Cdc16p-GFP from SPBs in interphase cells (Fig 5A). Quantification of the SPB-associated signal of Cdc16p-GFP in interphase cells revealed that it was significantly decreased upon expression of fin1 (Fig 5B). This was not due to a significant alteration of the steady state level of Cdc16p (Fig 5C).


An Extended, Boolean Model of the Septation Initiation Network in S.Pombe Provides Insights into Its Regulation.

Chasapi A, Wachowicz P, Niknejad A, Collin P, Krapp A, Cano E, Simanis V, Xenarios I - PLoS ONE (2015)

Fin1p over-expression results in Cdc16p disassociation from the SPB.(A) Cells expressing the labeled tubulin marker leu1::m-Cherry-atb2 and cdc16-GFP were induced to express fin1 from the medium strength nmt41 promoter [80]. Cells transformed with empty vector served as control. Cells were grown in medium without thiamine for 27h at 25°C. Cells were imaged and the intensity of SPB associated cdc16-GFP signal was analyzed as described in [12]. The panel shows m-Cherry-atb2 leu1::cdc16-GFP(ura4+) cells bearing REP41 or REP41-fin1. The scale bar is 10 μm. (B) The SPB associated signal was determined in interphase cells in each strain. Since REP41-fin1 eventually leads to a mitotic arrest [87] interphase cells were identified by the presence of an interphasic microtubule array. The box shows 25%-75% range for the population, the line indicates the median. The bars indicate 10% and 90% range for the population, and dots indicate more extreme individual values. The y-axis shows fluorescence intensity on an arbitrary scale. (C) Cells bearing the leu1::cdc16-HA allele were induced to express fin1 (ON) by growing them in defined minimal medium [81] in the presence (OFF) or absence (ON) of 2mM thiamine. Protein extracts were prepared 27h after induction and analyzed by western blotting using monoclonal antibody 12CA5. The anti-α-tubulin monoclonal antibody TAT-1 [90] was used as a control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526654&req=5

pone.0134214.g005: Fin1p over-expression results in Cdc16p disassociation from the SPB.(A) Cells expressing the labeled tubulin marker leu1::m-Cherry-atb2 and cdc16-GFP were induced to express fin1 from the medium strength nmt41 promoter [80]. Cells transformed with empty vector served as control. Cells were grown in medium without thiamine for 27h at 25°C. Cells were imaged and the intensity of SPB associated cdc16-GFP signal was analyzed as described in [12]. The panel shows m-Cherry-atb2 leu1::cdc16-GFP(ura4+) cells bearing REP41 or REP41-fin1. The scale bar is 10 μm. (B) The SPB associated signal was determined in interphase cells in each strain. Since REP41-fin1 eventually leads to a mitotic arrest [87] interphase cells were identified by the presence of an interphasic microtubule array. The box shows 25%-75% range for the population, the line indicates the median. The bars indicate 10% and 90% range for the population, and dots indicate more extreme individual values. The y-axis shows fluorescence intensity on an arbitrary scale. (C) Cells bearing the leu1::cdc16-HA allele were induced to express fin1 (ON) by growing them in defined minimal medium [81] in the presence (OFF) or absence (ON) of 2mM thiamine. Protein extracts were prepared 27h after induction and analyzed by western blotting using monoclonal antibody 12CA5. The anti-α-tubulin monoclonal antibody TAT-1 [90] was used as a control.
Mentions: In the PKN of the model there were no negative regulators targeting GAP components during early mitosis, which resulted in suboptimal outcomes during the simulations of early mitosis; i.e. the simulation would produce a steady state where the GAP remained active in early mitosis. Since removal of the SIN GAP from the SPB is an early step in the activation of the SIN after entry into mitosis [12,74,89], we modeled whether GAP components could be regulated by fin1. Since Cdc16p contains several sites matching the established consensus for mammalian Nek2 (one of the orthologues of nimA), the effect of increased expression of fin1 on Cdc16p localization was investigated by in vivo experiments. Expression of fin1 from the medium strength nmt-41 promoter [80] resulted in partial displacement of Cdc16p-GFP from SPBs in interphase cells (Fig 5A). Quantification of the SPB-associated signal of Cdc16p-GFP in interphase cells revealed that it was significantly decreased upon expression of fin1 (Fig 5B). This was not due to a significant alteration of the steady state level of Cdc16p (Fig 5C).

Bottom Line: In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach.The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set.Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.

View Article: PubMed Central - PubMed

Affiliation: Vital-IT Group, Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland.

ABSTRACT
Cytokinesis in fission yeast is controlled by the Septation Initiation Network (SIN), a protein kinase signaling network using the spindle pole body as scaffold. In order to describe the qualitative behavior of the system and predict unknown mutant behaviors we decided to adopt a Boolean modeling approach. In this paper, we report the construction of an extended, Boolean model of the SIN, comprising most SIN components and regulators as individual, experimentally testable nodes. The model uses CDK activity levels as control nodes for the simulation of SIN related events in different stages of the cell cycle. The model was optimized using single knock-out experiments of known phenotypic effect as a training set, and was able to correctly predict a double knock-out test set. Moreover, the model has made in silico predictions that have been validated in vivo, providing new insights into the regulation and hierarchical organization of the SIN.

No MeSH data available.


Related in: MedlinePlus