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

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.

Fig 1. Model construction and optimization workflow.

The Prior Knowledge Network (PKN) is constructed after collecting relevant information from various sources, including network databases and literature. The PKN is translated into logical functions, describing the regulatory relations among gene products. The logical model is simulated under the preferred conditions, resulting in one or more steady states, where all logical rules are satisfied. The model goes then through an optimization procedure, where the goal is to fit the resulting steady states with available experimental data by altering regulatory rules. The optimization typically includes removing outdated / low confidence links, adjusting their representation and adding new regulatory rules. The process is iterated until the simulation fits the available data. The model can then be used as a predictive tool, by performing in silico perturbations. Validation of the predictions can lead to discovery of missing regulatory links that are then added to the PKN.

Fig 2. The extended Boolean SIN model.

(A) The initial, prior knowledge network, manually re-constructed from the literature. Purple nodes represent proteins and complexes that take part in the regulation of the SIN, and pink nodes represent AND gates. Blue arrows indicate activation events and orange circles inhibition events. Other logical functions, such as AND, OR and NOT regulatory gates are also encoded in the model. SAC: spindle assembly checkpoint, APC: anaphase-promoting complex, PP: protein phosphatase. (B) The final, optimized model, which uses a 3-node representation of CDK activity. Nodes in green are used as switches, and they are turned on to represent different stages of the cell cycle: interphase, early mitosis and late mitosis. Pink nodes represent AND gates. In the case of the Cdc42p regulatory link towards Byr4p, the regulatory rule can be phrased as “NOT Cdc42p activates Byr4p”.

Fig 3. In silico steady states of the SIN, in wild type and mutated cells.

Steady states deriving from simulations performed on the final model. The boxes on the left indicate the experiments performed, which can be knock-out (KO) or over-expression (OE). When there is more than one gene in the box, it is a double perturbation. For each perturbation, 3 experiments were performed: interphase simulation (indicated as i), early mitosis (eM) and late mitosis (lM, with suffixes new and old when there are 2 resulting steady states, indicative of late mitosis asymmetry). Blue boxes correspond to active proteins, white to inactive and light blue to proteins that can be either active or inactive at the resulting steady states of the system.

Fig 4. Cdc7p over-expression in a sid4 mutant will result in septation.

(A) Steady states of in silico, double mutation experiments. The model predicts that in the absence of SIN scaffold proteins (Cdc11p or Sid4p) and over-expression of Cdc7p, the cell will septate. (B) sid4-SA1 leu1-32 was transformed with a REP1-based plasmids [80] expressing cdc7; empty vector served as a control. Cells were grown to exponential phase in EMM2 medium at 25°C containing 2mM thiamine. Expression was induced by washing with EMM2 and growth for 16h at 25°C; cells were then shifted to 36°C for 5h, fixed, and stained with DAPI and Calcofluor as described [81]. Note that the cells carrying empty vector have become elongated and multinucleated, while 75% of cells expressing cdc7 have one or more septa. The scale bar represents 10 μm. (C) The strain leu1::pADH1-cdc7 was grown to exponential phase in YE medium at 19°C. A sample was taken and cells were fixed and stained with DAPI and Calcofluor. The remainder of the culture was incubated for 5h at 36°C before fixation. Note the elevated percentage of septated cells. The scale bar represents 10 μm. (D) The indicated strains were grown to exponential phase in YE medium, counted, and diluted to 10⁶ ml^-1. 10 μl of serial 5-fold dilutions were spotted on plates, allowed to dry and then incubated at the indicated temperature until the wild-type control had formed colonies.

Fig 5. 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.