Abstract
The growth and division of eukaryotic cells are regulated by complex, multiscale networks. In this process, the mechanism of controlling cellcycle progression has to be robust against inherent noise in the system. In this paper, a hybrid stochastic model is developed to study the effects of noise on the control mechanism of the budding yeast cell cycle. The modeling approach leverages, in a single multiscale model, the advantages of two regimes: (1) the computational efficiency of a deterministic approach, and (2) the accuracy of stochastic simulations. Our results show that this hybrid stochastic model achieves high computational efficiency while generating simulation results that match very well with published experimental measurements.
Introduction
The eukaryotic cell cycle is a complex process by which a growing cell replicates its DNA and divides into two cells, each capable of repeating the process. Progression through the cycle is controlled by networks of genes, mRNAs, and proteins, with interactions that can be modeled as chemical reaction channels. To unravel the complex dynamics of multiscale reaction networks in higher organisms such as human cells, it is advisable to study singlecell organisms with molecular regulatory networks that are similar yet simpler. For instance, experimental studies and mathematical models of frog eggs^{1,2}, fission yeast^{3,4}, and budding yeast^{5,6} have shed light on mechanisms of cellcycle regulation in the cells of higher organisms. Extensive experimental studies have been conducted particularly on budding yeast (Saccharomyces cerevisiae) to explore gene regulation and signaling pathways of relevance to cell growth and division^{7,8,9}. Moreover, various modeling approaches, such as deterministic models^{10,11,12}, Boolean networks^{13,14,15,16,17,18,19}, and stochastic models^{20,21,22,23,24,25,26}, have been adopted to explore the roles of different gene and protein interactions in robust progression through the cell cycle.
Among these models, a deterministic approach is most common. In this approach, the timedependent variation of each molecular species in the biochemical reaction network is described by a nonlinear ordinary differential equation (ODE), in which the concentration of the substance is considered as a continuous quantity that evolves deterministically over time. However, the timeevolution of molecular species within the confined volume of a budding yeast cell (about 30 fL at birth) is not deterministic. Therefore, in spite of being able to reproduce certain average characteristics of cellcycle progression in yeast cell populations, a continuousdeterministic model cannot reproduce the celltocell variability observed in wetlab experiments^{27,28}. For instance, Di Talia et al.^{28} have reported that the coefficient of variation (CV = \(\frac{{\rm{standard}}\ {\rm{deviation}}}{{\rm{mean}}}\)) for G1 time of budding yeast cells (growing on glucose) is \(50 \%\).
To capture such high levels of variability, stochastic models have been built using different strategies to incorporate intrinsic and extrinsic sources of noise. In an early stochastic model of the fission yeast cell cycle proposed by Sveiczer et al.^{29}, extrinsic noise was introduced by assuming some sloppiness in the partitioning of cell volume and nucleus volume to daughter cells at division. A later model by Steuer^{30} examined the roles of intrinsic noise in cellcycle progression by adding Gaussian noise to reaction rate equations in a deterministic model. These approaches, however, do not adequately explain the root source of cellcycle variability in yeast cells, which lies in molecular fluctuations at the level of gene expression^{31,32,33}. To capture such molecularlevel noise, more accurate stochastic methods are required to explicitly model fluctuations in molecular interactions. For this purpose, the best method to implement fluctuating molecular interactions is the stochastic simulation algorithm (SSA) proposed by Gillespie^{34}. Gillespie’s algorithm is a MonteCarlo approach that numerically simulates the temporal firing of every single reaction in a chemical reaction network. An assumption of Gillespie’s method is that the propensity of every reaction in the model is described by massaction kinetics. This becomes an issue for us because most deterministic models of cellcycle regulation, such as those presented in refs ^{5,11,12,35}, incorporate complex rate laws including MichaelisMenten kinetics, Hill functions, and ultrasensitive switches. These complex phenomenological rate laws are used in deterministic models to provide sufficient nonlinearity in reaction kinetics to create bistable switches that flip on and off during progression through the cell cycle. Converting a deterministic model into a stochastic model suitable for Gillespie’s SSA by ‘unpacking’ complex rate laws into elementary reactions is a difficult problem fraught with uncertainties^{36}.
To address this challenge, several approaches have been tried. The simplest approach, used for example by Mura & CsikaszNagy^{37}, treats all complex rate laws directly as propensity functions of reactions and then applies the SSA. This approximation is subject to considerable errors^{36,38}. For example, Ball et al.^{21} found that the variability they observed in wetlab measurements could not be generated by this greatly simplified stochastic approach, unless some unrealistic parameter values were chosen. Later Kar et al.^{20} tried to unpack MichaelisMenten rate laws in a small (threevariable), deterministic model of the budding yeast cell cycle^{5}. Unpacking resulted in a much more complicated system with 19 species and 47 reactions. Although this simple model (with only a few key cellcycle genes) could generate noise levels that match wetlab measurements for a few key characteristics of the cell cycle, it is not feasible (in our experience) to apply this approach to more complex models with substantially more genes and proteins. Instead, we have pursued an approach in which the molecular controls of the budding yeast cell cycle are modeled directly in terms of elementary reactions (governed by the law of massaction)^{25,26}. A great advantage of this approach is that the newly designed deterministic model can be converted into its corresponding stochastic version without any approximation. A disadvantage of this approach is that we cannot repurpose our original deterministic models, which had been carefully designed and parametrized to explain a broad scope of experimental observations. Furthermore, to model the phosphorylation and dephosphorylation reactions that play such important roles in cellcycle progression introduces substantial complexity into the system. Recently we have considered a new approach that sidesteps the complications of elementary reactions and massaction rate laws and that employs a Langevintype simulation of noisy gene expression^{23}. This approach, though promising, also requires an overhaul of the original deterministic models. In order to take advantage of existing deterministic models in a framework that permits accurate stochastic simulations without ‘unpacking’, we explore a particular hybrid approach in this paper.
Gillespie’s SSA simulates every single reaction firing. In general, the time complexity of this algorithm scales proportionally with the number of reaction firings. Consequently, SSAbased models involve substantial computational complexity if a reaction network involves many fast reactions. To reduce the high computational cost of the SSA, many optimization methods^{39,40,41,42,43} and approximation methods^{44,45,46,47,48} have been proposed. Among them the hybrid stochastic approach, originally proposed by Haseltine and Rawling (HR)^{46}, performs well because it takes advantage of the multiscale features common in biochemical reaction networks. The multiscale characteristics of reaction networks have led to significant reductions in the computational cost of solving many types of stochastic systems. For example, hybrid approaches provide good approximate solutions for the chemical master equation^{49,50}.
The main idea of any hybrid approach is to divide the system into subsystems and solve each subsystem using an appropriate method. The idea of the HR hybrid approach (that we are using in this paper) is to partition the dynamical system into fast and slow reactions, based on the relative time scale of each reaction and the abundances of the reactants. Fast reactions, which fire frequently and often involve highabundance species, are partitioned into the deterministic (ODE) regime. Meanwhile, slow reactions, which are often found at the geneexpression level, fire much less frequently and are therefore simulated using the SSA. This approach was first applied by Liu et al.^{22} on the simple threevariable model of the budding yeast cell cycle, originally studied by Kar et al.^{20}. By partitioning all geneexpression reactions into the slow (SSA) regime and all proteinlevel dynamics into the fast (ODE) regime, Liu et al.^{22} were able to reproduce the noise levels that Kar et al.^{20} achieved by unpacking the original system into a much more complex one. This success motivated us to apply this approach to the very comprehensive, accurate, and complex deterministic model of yest cellcycle controls proposed by Chen et al.^{11}.
Simulation results demonstrate that, while achieving high computational efficiency, our hybrid model still matches up well with experimental measurements of the variability of cellcycle properties (cycle time, cell size, correlation coefficients), protein and mRNA abundances, and phenotypes of more than 100 mutant strains of budding yeast. Moreover, our simulations shed light on the ‘partial’ viability of mutant strains such as \(CLB2db\Delta\) \(clb5\Delta\).
Results
We develop a hybrid stochastic model of the budding yeast cell cycle, consisting of 45 proteins and 19 mRNAs. In Section Methods, we will elaborate the steps for building our hybrid stochastic model. Building on this model, we use Algorithm 1, that we introduce in Section Methods to generate sufficiently large populations of mother and daughter cells to estimate the statistical distributions of various cellcyclerelated properties of wildtype cells as well as 122 mutant strains of yeast. We evaluate our model by comparing numerical simulation results with experimental observations from the published literature.
Wildtype cell
Figure 1a, b shows a deterministic simulation of several protein and mRNA species, respectively, in our model of wildtype budding yeast cells. In early G1 phase, once the cell grows to a critical size, Cln3 and Bck2 initiate the Start event, i.e., the activation of transcription factors for Cln2 and Clb5 production. Cln2 is the cyclin responsible for bud formation. In addition, Cln2 phosphorylates Sic1 and Cdc6, a pair of cyclindependent kinase inhibitors (CKIs). Consequently, Clb5dependent kinase activity rises and initiates DNA replication in S phase. As CKIs are removed, Clb2 level rises, because Clb2 activates its own transcription factor, Mcm1, in an autocatalytic fashion. Clb2dependent kinase activity turns off the transcription factors for Cln2 and Clb5 production and is responsible for driving the cell into mitosis (M phase). Clb2 level remains high until metaphase, when the proper attachment of chromosomes to the mitotic spindle activates Cdc20. Cdc20 promotes anaphase (the separation of the two strands of replicated chromosomes to opposite poles of the mitotic spindle). At the same time, Cdc20 promotes degradation of Clb2 and Clb5 and activation of a phosphatase, Cdc14. Cdc14 plays a major role (in budding yeast) in reestablishing the dominance of CKIs in G1 phase, and in replacing Cdc20 by Cdh1 (the protein responsible for Clb2 and Clb5 degradation in G1 phase).
Figure 1c, d shows the corresponding stochastic trajectories of our hybrid stochastic model. The stochastic trajectories in panel c correctly simulate the sequence of events predicted by the deterministic trajectories in panel a. Panel d shows considerable fluctuations in the numbers of molecules per cell of five mRNA species: three of which (\(mClb2\), \(mCdc20\), and \(mCln2\)) undergo periodic transcription during the cell cycle, and two of which (\(mCdc14\) and \(mNet1\)) are synthesized continuously throughout the cell cycle. Such high variability is expected, due to the stochastic nature of gene expression and the low abundances of mRNA molecules per cell.
We used our hybrid stochastic model to generate more than 20,000 asynchronous wildtype mother and daughter cells growing in glucose medium (massdoubling time about 100 min). These large collections of simulated cells are then used to estimate the distributions of important characteristics of the budding yeast cell cycle, including interdivision time, duration of unbudded phase (G1), duration of budded phase (SG2M), and size at birth. In addition we estimate the standard error (SE) of mean and standard error of coefficient of variation for these characteristics. Table 1 compares the computed summary statistics \(\pm\) SE for all cellcyclerelated properties with experimental data reported by Di Talia et al.^{28}. The results in Table 1 show that the model accurately reproduces the mean of these important properties of the wildtype budding yeast cell cycle. Despite the fact that the coefficients of variation reproduced by our model are generally larger than what is observed in experiment, they are in a comparable range. In accord with experimental observations, G1 phase is the noisiest phase in cell cycle, the variability in daughter cells is more than mother cells. The estimated standard errors are significantly smaller than the experimental observations. In fact, we expect such low standard errors due to the large number of simulations. We note that the standard error for volume of a cell at birth is not reported in column 4 and 6, because cell volume is not measured directly by Di Talia et al.^{28}, but rather \(V(t)\) is estimated as a function of time.
Next, we compare our simulations to the observed distributions of mRNA molecules in wildtype yeast cells. We have 11 unregulated mRNAs (\(mCdh1\), \(mTem1\), \(mCdc15\), \(mCdc14\), \(mNet1\), \(mCdc55\), \(mEsp1\), \(mSBF\), \(mMBF\), \(mMcm1\), \(mAPC\)) and eight transcriptionally regulated mRNAs (\(mClb5\), \(mCln2\), \(mClb2\), \(mSic1\), \(mCdc6\), \(mSwi5\), \(mCdc20\), \(mPds1\)) in our model. Figure 2 compares the histograms of these mRNAs with the distributions measured by Ball et al.^{27}. In the original deterministic model, MBF and SBF are described by the same algebraic equation since they were supposed to have the same dynamics^{11}. In adding \(mSBF\) and \(mMBF\) to the model, we kept the same assumption and therefore, the histograms of the two unregulated mRNAs (\(mSBF\) and \(mMBF\)) are very similar. For this reason we just include one of them (\(mSBF\)) in Fig. 2. To quantify the difference between a distribution generated by our model and the corresponding experimental observations, we use the KullbackLeibler divergence \(\left({D}_{{\rm{KL}}}=\sum _{x\in X}R(x){\log}\left(\frac{Q(x)}{R(x)}\right)\right)\) where \(R\) is the distribution from simulation and \(Q\) from experiment. The computed value of the KL divergence is reported on the topleft corner of each subplot. The smaller is \({D}_{{\rm{KL}}}\), the more similar are the two distributions.
Generally, there is a good agreement between observed and simulated mRNA distributions, except for \(mCln1+mCln2\) and \(mCdc20\). As expected, the unregulated transcripts follow Poisson distributions, which are consistent with experimental measurements. The value of \({D}_{{\rm{KL}}}\) computed for these distribution is small. The cellcycle regulated transcripts, which follow longtailed, nonPoisson distributions, are wellfit by twocomponent Poisson distributions as reported by refs ^{26,27}. (We note that in our model \(mClb2\) represents both \(mClb1\) and \(mClb2\), and \(mCln2=mCln1+mCln2\), whereas in the experiment these cyclin mRNAs are tracked independently. Therefore, we do not expect a particularly good match between the computed and observed distributions for these transcripts. As expected, the values of \({D}_{{\rm{KL}}}\) computed for these distribution are large).
Table 2 compares the average abundances of proteins as observed in ref. ^{51} and simulated by our model. We use a sufficiently large population of cells from at least 10,000 simulations to calculate the average abundance (number of molecules per cell) and the standard error of the mean for each protein. Note that, for the proteins listed in Table 2, only a single measurement has been made experimentally, so the standard error is not available for comparison. Except for the Sic1, the agreement between simulation results and experimental observations is quite reasonable.
Mutant cases
Our hybrid stochastic model is used to simulate 122 mutant strains listed in Table S5 in Supplementary Text. Prior to presenting simulation results of these mutants, we discuss the criteria for determining viability in a general stochastic model.
In the original deterministic model, a cell is considered viable if the following conditions are met:
 (1)
certain events, listed in ref. ^{35}, take place in a proper sequence,
 (2)
in particular, cell division occurs after budding, and
 (3)
cell mass does not exceed a predetermined threshold (\({\mathrm{mass}}\) at division \(<10\)).
The same set of rules should also apply to the stochastic model. A division is considered successful if the aforementioned viability criteria are met; otherwise, the cell is considered to be inviable. From our numerical simulations, the probability of successful division \(p\) is estimated by
Using this metric, we consider a mutant strain viable if \(p\;>\;0.75\), inviable if \(p\;<\;0.65\), and partially viable otherwise. This viability criterion is based on the following considerations.
Let \({N}_{0}\) be the initial population of cells in an experiment. After one cycle, the average number of cells that divide is \(p{N}_{0}\), while \((1p){N}_{0}\) cells exit the cycle and stop dividing. Thus, after one cycle, the total population of cells is \((1+p){N}_{0}\), of which \(2p{N}_{0}\) cells completed the previous cell cycle and \((1p){N}_{0}\) cells have ceased to divide (we call them dead cells). In our simulations, we disregard the \((1p){N}_{0}\) dead cells; hence, the number of actively dividing cells in the second cycle is \({N}_{1}=2p{N}_{0}\), and the expected number of actively dividing cells after \(k\) cycles will be \({N}_{k}={(2p)}^{k}{N}_{0}\).
In cellviability experiments, colony formation is typically assessed after 24 h growth of a series of tenfold diluted inocula. For wildtype yeast cells (\(p\approx 1\)) growing on rich glucose medium (cycle time \(\approx 12\) h), each inoculum should increase by a factor of about \({2}^{12}=4096\). The colony sizes after 24 h growth of tenfold serial dilutions would be (4000 \({N}_{0}\), 400 \({N}_{0}\), 40 \({N}_{0}\), 4 \({N}_{0}\)), of which the first would be too dense to quantify, the last would be too sparse to see, and the middle two would be used to assess viability of mutant strains. For a mutant cell with \(p\,<\,0.5\), no visible colony will grow from the initial inoculum, and the mutant will be scored ‘inviable’. For mutant strains with \(0.5<p<1\), we must consider how the colony growth assay compares to wildtype cells. For p = 0.8 the initial inoculum grows to 280 \({N}_{0}\), which is comparable to the first dilution of the wildtype cells, and we would score this mutant strain as ‘viable’. For p = 0.75 the initial inoculum grows to 130 \({N}_{0}\), which is denser than the second dilution of the wildtype cells, and we would score this mutant strain as ‘probably viable’. For p = 0.65 the initial inoculum grows to 23 \({N}_{0}\), which is less dense than the second dilution of the wildtype cells, and we would score this mutant strain as ‘hardly viable’. These calculations suggest that a mutant strain be considered viable if \(p\,\ge\, 0.7\)5 and nonviable if \(p\;\le\; 0.65\). For \(0.65\,<\, p\,<\,0.75\), the strain is identified as partially viable.
Based on these criteria, we assessed the viability of 122 mutant strains of budding yeast that were studied in the modeling paper of Chen et al.^{11}. To demonstrate the significant roles of noise in some of these mutants, we discuss two multiplemutant strains, \(cln1\Delta \ cln2\Delta \ bck2\Delta\) and \(cln3\Delta \ bck2\Delta\) multicopy \(CLN2\) (Fig. 3), in some detail. Our goal is to illustrate how we assess the viability of a mutant strain in our hybrid stochastic model. According to experimental observations, the \(cln1\Delta \ cln2\Delta \ bck2\Delta\) strain^{52} is viable. However, due to deletion of Start cyclins Cln1 and Cln2, the cell requires a longer time than normal to form a bud and hence grows to a larger size at division, in comparison with wildtype cells. Figure 3a shows that in the deterministic model the cell consistently exits mitosis and divides successfully with size larger than normal, as observed experimentally^{52}. In the hybrid stochastic simulation, however, due to the stochastic nature of the process there is a finite probability that a cell may exit the cycle and become arrested in some phase of the cell cycle. In Fig. 3b for instance the cell grows too large in G1 phase and never divides again, while in Fig. 3c, it exits mitosis and divides successfully. The probability of successful completion of the cell division cycle, in this case, is computed to be \(p\approx 0.84\). As shown in Fig. 3d, the total number of cells in our computational culture increases exponentially, with a numberdoubling time (NDT) of 140 min, which is slower than the NDT of a fully viable wildtype culture (~100 min). Therefore, we conclude that the hybrid stochastic simulation correctly confirms the viability, but the reduced growth rate, of the \(cln1\Delta \ cln2\Delta \ bck2\Delta\) strain.
Next we consider the inviable mutant strain \(cln3\Delta \ bck2\Delta\) multicopy \(CLN2\)^{53}. Figure 3e shows that in the deterministic model the mutant cell is arrested in G1 phase and grows without dividing until it dies. In the hybrid stochastic simulation, although many of the cells become arrested in G1 (see Fig. 3f), some cells manage to exit G1 phase, complete the cell cycle, commence a new cycle and divide a few times (see Fig. 3g). Nonetheless, according to Fig. 3h the total number of cells in our computational culture declines with time, because the probability of cell division is only \(p\approx 0.40\). Therefore, we conclude that the hybrid stochastic simulation correctly confirms the inviability of \(cln3\Delta \ bck2\Delta\) multicopy \(CLN2\) strain.
Based on our hybrid stochastic simulations of all 122 mutant strains in Chen’s data base, we find that the model successfully reproduces the phenotypes of 103 of these strains. Our results for all mutant strains are reported in Table S5 in Supplementary Text.
\(CLB2db\Delta \ clb5\Delta\) is a mutant with an interesting stochastic phenotype: it is inviable when grown on glucose medium but ‘partially viable’ when grown on raffinose (a sugar that supports a slower growth rate than glucose)^{54}. Due to deletion of the destruction box of \(CLB2\), Clb2 protein is in excess at telophase and the cell is unable to exit mitosis and divide, even in the absence of Clb5 protein (due to deletion of the \(CLB5\) gene). When growing on raffinose, however, many of these mutant cells (approximately \(60 \%\)–\(75 \%\)) are able to exit mitosis and commence a new cycle, whereas the remaining cells (\(25 \%\)–\(40 \%\)) are arrested in telophase and never reenter the cell cycle^{21}. In fact, the NDT of the doublemutant cells (250–300 min) is observed to be much longer than the NDT of wildtype cells (160 min) growing in raffinose medium^{21}.
Simulation results of Chen’s deterministic model predict that \(CLB2db\Delta \ clb5\Delta\) cells are inviable on glucose and viable on galactose and raffinose media. Clearly, we cannot expect a deterministic model to capture the stochastic properties of such a ‘partially viable’ mutant strain. Our hybrid stochastic model, however, describes the phenotype in exquisite detail. The probability of division for \(CLB2db\Delta \ clb5\Delta\) strain is \(p\approx 0.68\) confirming the partial viability of the mutant according to our viability criterion. Figure 4a shows the cumulative probability, \(P(T)\), of cycle times for wildtype and \(CLB2db\Delta \ clb5\Delta\) mutant cells growing in raffinose. (\(P(T)\) is the probability that the cycle time of a randomly chosen cell is longer than a specified time, \(T\).) As shown in Fig. 4a, \(P(T)\) for the mutant cells levels off at approximately \(35 \%\) as \(T\) increases, whereas, for wildtype cells, \(P(T)\) drops steadily (below \(5 \%\)) as \(T\) increases beyond 250 min. Cumulative distributions of cycle times computed by our hybrid stochastic model (black lines) are in excellent agreement with the experimental distributions (redbluegreen lines) for both wildtype and the doublemutant cells.
Another interesting mutant strain is \(CLB1clb2\Delta \ cdh1\Delta\), for which the \(CLB1\) gene is intact and \(CLB2\) and \(CDH1\) genes are deleted. Due to the mutual antagonism between Clb2 and Cdh1, deletion of both genes, \(CDH1\) and \(CLB2\), might be consistent with viability of the doublemutant strain, provided Clb1 is still functional. Indeed, experimental observations show that \(CLB1clb2\Delta \ cdh1\Delta\) cells are poorly viable in glucose medium and viable when growing on galactose^{54}. Chen’s deterministic model does not capture this phenotype; the model predicts the mutant cells to be viable in both media. However, simulation results of our hybrid stochastic model (Fig. 4b) can reproduce the observed phenotype. The probability of division computed for a population of cells growing in glucose is \(\approx\!0.62\), which suggests poor viability. In galactose the corresponding probability of division is \(\approx\!0.79\), which indicates that the mutant grows well in this medium. Figure 4b confirms a faster increase in cell number in the slower growth medium (galactose), which is in agreement with experimental observations.
Discussion
In this paper we present a hybrid stochastic model of the molecular mechanism controlling progression through the budding yeast cell cycle. Our model provides a good match with experimental observations of many important characteristics of the budding yeast cell cycle, including interdivision time, cell size, and the phenotypes of more than 100 mutant strains. Compared with other approaches to stochastic modeling, our hybrid stochastic approach has several advantages. In a multiscale regulatory network such as cellcycle controls, the major source of intrinsic noise can be attributed to low copy numbers of mRNA species in the geneprotein regulatory network. In fact, in budding yeast cells there are only 5–10 copies of each mRNA species encoding the production of corresponding proteins at levels of 500–5000 molecules per cell. In such circumstances, small fluctuations in the population of mRNAs will result in substantial fluctuations in the corresponding protein levels. With this in mind, the key idea of the hybrid scheme is to partition the dynamics of mRNA species into the stochastic regime, in order to capture the major effects of random fluctuations in mRNA numbers, and to keep the protein dynamics in the deterministic framework, to achieve greater simulation efficiency. In addition, in this scheme it is not necessary to reformulate the complex rate laws governing protein interactions as elementary massaction rate laws, which is a great advantage from a modeling standpoint.
In this paper, we have applied our hybrid stochastic method to a detailed molecular mechanism of cellcycle controls in budding yeast^{11}. To apply our scheme to Chen’s model, which is a deterministic model of protein interactions, we first had to extend the model to include mRNA species that are transcribed from cellcycle genes and translated into proteins. Then we carried out comprehensive simulations of wildtype yeast cells and more than 100 mutant strains, using both the deterministic and hybrid ODE/SSA models. Our stochastic model predicts the statistical properties of many different cellcycle variables, including interdivision times, size at birth, and the abundances of specific mRNAs and proteins, and our stochastic simulations are in accord with most experimental observations, including detailed phenotypic characteristics of 103 out of 122 mutant cases. (Although Chen’s deterministic model may seem to ‘score’ better on the ‘viability’ of mutant strains, it is not so highly constrained as our stochastic model by consideration of the statistical properties of these mutant cells, especially the characteristics of ‘partially viable’ mutants.) In addition, our results prove that our hybrid approach to stochastic/deterministic simulations can achieve a good tradeoff between accuracy and efficiency of numerical simulations. FORTRAN code takes about 15 min to simulate 10,000 cell cycles on an Intel i73770 processor with 16G memory running a Linux environment. A similar system using a fully stochastic model may take more than one day (for example, when the FORTRAN code of Barik et al.^{26} is run using the same work station, it takes more than 30 h to generate a population of 10,000 yeast cells).
Method
Deterministic model
A comprehensive continuousdeterministic model of the budding yeast cell cycle was developed by Chen et al.^{11} in 2004. By integrating the findings of decades of experimental studies, Chen’s model provides an accurate mathematical description of the cell division cycle of budding yeast. The protein regulatory network of Chen’s model focuses primarily on the mutual antagonism between mitotic Btype cyclins (Clb16) and G1 phase stabilizers (Cdh1, Sic1, and Cdc6). During the growth and division of yeast cells, this antagonism leads to transitions between two coexisting steady states called Start (G1\(\to\) S) and Exit (M\(\to\) G1). A detailed description of the budding yeast cellcycle model is given in Supplementary Information.
Chen’s mathematical model reproduces the average cellcycle properties (including cycle time, G1 duration, and cell size at division) of wildtype budding yeast cells and the variant cellcycle phenotypes of more than 100 mutant strains. Our goal is to develop a hybrid (stochasticdeterministic) version of this large regulatory network, in order to quantify the variabilities observed in cellcycle characteristics and mutant phenotypes within a computationally efficient framework.
Since Chen’s model is formulated in terms of normalized (dimensionless) concentrations of proteins, the first step to this goal is to convert the state variables of Chen’s model into integer numbers of molecules per cell. This conversion facilitates comparison of our numerical simulation results with observed data from singlecell experiments. Furthermore, it is necessary because, in Gillespie’s SSA, state variables are discrete (species populations) rather than continuous (species concentrations). Since a hybrid model involves both SSA and ODEs, it is important that we assure consistency between units of state variables in both the stochastic and deterministic regimes. Therefore, we calculate \({S}_{i}\), the number of molecules of species \(i\) in a cell, from the corresponding normalized concentration, \({[{{\rm{S}}}_{i}]}_{{\rm{n}}}\), by (1):
where \([{{\rm{S}}}_{i}]\) is the actual concentration of species \(i\) (in nanomoles/liter = \(1{0}^{9}\) mol/L), \({C}_{i}\) is the ‘characteristic’ concentration of species \(i\) (used to convert between actual concentration and ‘normalized’ concentration), \(V(t)\) is the volume of the cell (in femtoliters = \(1{0}^{15}\) L), and \({N}_{{\rm{A}}}=0.6\) is Avogadro’s number (when concentration is expressed in nM and volume in fL). One simplifying assumption made in published models^{21,23} is to use a constant volume for the size of cell. However, this unrealistic assumption introduces errors into the model because cell size (\(V\)) increases exponentially during a cycle (\(V(t)=V(0){{\rm{e}}}^{{k}_{{\rm{g}}}t}\), where \({k}_{{\rm{g}}}\) is the specific growth rate of yeast cells).
Second, we extend the protein regulatory network in Chen’s model to include the dynamics of 11 regulated and eight unregulated mRNAs. This extension is necessary because the major source of intrinsic noise in yeast cells is the small number of mRNA molecules per cell per gene^{27}. Experimental observations^{28} in yeast cells with increased dosage of genes suggest that the dominant source of variability with respect to cellcycle time and cell size at division is the low copy number of mRNA and protein molecules in a cell, specifically in G1 phase. However, Chen’s model did not incorporate the turnover of mRNA molecules, and thus it cannot account for fluctuations stemming from transcriptional noise. For these reasons, Chen’s original model must be supplemented with appropriate synthesis and degradation rates for each mRNA, as well as realistic rates of translation from mRNA to protein. In our model, based on experimental observations in^{55}, we assigned halflife times for mRNAs in the range of 5–10 min, except for \(mCln2\) and \(mClb2\), which were assigned shorter halflives (3 and 2 min, respectively). The synthesis rate of each mRNA was then estimated to match the mRNA averageabundance measurements in Ball et al.^{27}.
Third, we modified Chen’s model by introducing ODEs for the concentrations of Cln3 and Bck2 proteins. In Chen’s original model, the normalized concentration of Cln3 and Bck2 were assumed to be given by steadystate algebraic equations (2) and (3),
where \({C}_{0}\) determines the maximum concentration of Cln3, \({D}_{{\rm{n}}3}\) is the dosage of the \(CLN3\) gene, \({J}_{{\rm{n}}3}\) and \({B}_{0}\) are constants, and mass is the ‘size’ of a cell. We replaced the algebraic equations (2) and (3) by ODEs in (4) and (5).
The synthesis (\({k}_{{\rm{s,n}}3}\), \({k}_{{\rm{s,k2}}}\)) and degradation (\({k}_{{\rm{d,n}}3}\), \({k}_{{\rm{d,k2}}}\)) rate constants were estimated so that the halflives and average abundances of these proteins match with experimental data^{51}. The reason for this change is to model the unbalanced partitioning of Cln3 molecules between daughter and mother cells at cell division. According to experimental observations, the concentration of Cln3 in a newborn daughter cell is about three times less than its concentration in the mother cell^{56,57}, indicating that mother cells get more than their ‘fair share’ of Cln3 molecules at cell separation. As a consequence of this unequal partitioning of Cln3 between mother and daughter cells at division, the G1 time of mother cells is much shorter and the G1 time of daughter cells is much longer (on average) than would otherwise be expected. By including Cln3 and Bck2 as state variables in the model, we can apply an asymmetric partitioning rule with ratio of 20:80 to daughter and mother cells at cell division. We note that this ratio is set to 40:60 for all other proteins and mRNAs, according to observations in^{28}.
Finally, we comment that the quadratic dependence of Cln3 and Bck2 synthesis rates on cell size is introduced to account for the major influence that these two proteins have on cell size at the G1/S transition^{58,59}. Because the rate of synthesis of these two proteins increases quadratically with cell volume, there is a strong size control on the G1/S transition in our model.
In summary, the variables, equations, parameter values and reaction propensities in our model are provided in Tables S1–S4 in Supplementary Text.
Hybrid stochastic model
As we mentioned in Section 1, the regulatory network of the budding yeast cellcycle is a multiscale system: both the numbers of molecules of mRNAs and proteins and the propensities of individual reactions vary by orders of magnitude. For instance, in budding yeast cells, there are 500–5000 copies of each protein encoded by only 5–10 copies of the corresponding mRNA. Furthermore, the synthesis and degradation of mRNA species occur much less frequently than the phosphorylation and dephosphorylation of proteins in the cell. The Haseltine and Rawling (HR) hybrid method leverages these large scale differences to improve the efficiency of stochastic simulations without sacrificing accuracy of the computations. The HR method divides the system into subsystems, each including species and reactions with similar scales, and applies an appropriate simulation method to each subsystem. This partitioning is done by using predefined thresholds for propensities of reactions and abundances of reactants. In this way the system is divided into four disjoint regions: (I) slow reactions with lowabundance reactants, (II) slow reactions with highabundance reactants, (III) fast reactions with lowabundance reactants, and (IV) fast reactions with highabundance reactants. Then an appropriate simulation method is chosen for each region^{22,40,46}. We follow the strategy proposed Liu et al.^{22} where the dynamics of all mRNAs (region I) is simulated by SSA, and the other three regions (II, III, and IV) are modeled with ODEs. We shall refer to this partitioning as the ‘Liu strategy’. We notice that the partitioning thresholds in Liu strategy are predefined and static. That is, while it is not guaranteed, the fast and slow sets are assumed to remain the same during the simulation.
In order to demonstrate the scale difference in our partitioning strategy, we approximate the propensity function of every reaction by its corresponding rate law function (obtaining a stochastic model with 145 reactions) and track the firing frequency of each of these reactions in a test run of Gillespie’s SSA. Of 18 million reactions fired in one cell cycle, only about 34,000 \((0.2\%)\) involve mRNA turnover, and \(99.8\%\) represent fast reactions of protein posttranslational modifications. Based on this test run, we estimate that our HR hybrid scheme will run at least 100 times faster than a bruteforce Gillespie simulation of a fully stochastic model. In section 3 we show that our hybrid stochastic model, using the Liu strategy, still generates accurate results that agree well with experimental observations.
Algorithm 1
Proposed in Liu et al.^{22} describes the hybrid ODE/SSA algorithm adopted in this paper, which is a variant of the original HR hybrid method^{46}. Our hybrid stochastic simulation code that implements Algorithm 1 (a FORTRAN file) is available in Supplementary Code.
Algorithm 1 Hybrid Stochastic Simulation Algorithm
\(HYBRID({{\mathcal{R}}}_{{{{\mathrm{fast}}}}},{{\mathcal{R}}}_{{{{\mathrm{slow}}}}})\)

\(t\leftarrow 0\)

While t < T do

Calculate the propensity function, \({a}_{i}\), for all reactions in slow subset \(i=1,...,k\).

Calculate total propensity function: \({a}_{0}({\boldsymbol{s}},t)={\sum }_{j = 1}^{k}{a}_{j}({\boldsymbol{s}},t)\).

Generate two uniform random variables \({r}_{1}\) and \({r}_{2}\) in \({\rm{U}}(0,1)\).

Integrate the ODE system until an event occurs at time \(t+\tau\) such that
$${\int }_{t}^{t+\tau }{a}_{0}({\boldsymbol{s}},x){\rm{d}}x+{\rm{ln}}({r}_{1})=0.$$(6) 
Select the smallest \(\mu\) such that: \({\sum }_{i = 1}^{\mu }{a}_{i}({\boldsymbol{s}},t)\;>\;\;{r}_{2}{a}_{0}({\boldsymbol{s}},t).\)

Update the state variables according to \({\mu }_{th}\) reaction in \({{\mathcal{R}}}_{{\rm{slow}}}.\)

end While
Hybrid stochastic simulation algorithm
Consider a wellstirred system with \(N\) species in a set \({\mathcal{S}}\) that interact with each other through \(M\) reaction channels in a set \({\mathcal{R}}\). The reactions in \({\mathcal{R}}\) are partitioned into two disjoint subsets of fast and slow reactions denoted by \({{\mathcal{R}}}_{{\rm{fast}}}\) and \({{\mathcal{R}}}_{{\rm{slow}}}\), respectively. The subset \({{\mathcal{R}}}_{{\rm{slow}}}\) includes \(k\) reactions, which are simulated using SSA, while the remaining \(M\)\(k\) fast reactions in \({{\mathcal{R}}}_{{\rm{fast}}}\) are governed by ODEs. Let \({a}_{j}({\boldsymbol{s}},t)\) be the propensity function of the \(j\)th reaction in \({{\mathcal{R}}}_{{\rm{slow}}}\), where \({\boldsymbol{s}}=({S}_{1}(t),\ldots ,{S}_{N}(t))\) is the state vector with each element \({S}_{i}(t)\) representing the number of molecules of species \(i\) at time \(t\). In addition, let \({{\boldsymbol{v}}}_{j}=({v}_{j1},\ldots ,{v}_{jN})\) be the statechange vector of the \(j\)th reaction, where \({v}_{ji}\) denotes the change in the population of species \(i\) when reaction \(j\) fires. Let \(\tau\) be the jump interval to the next slow reaction and \(\mu\) be the index of the reaction that fires. The algorithm only needs to simulate the firings of slow reactions, while integrating the fast subset of ODEs simultaneously in Eq. (6). When a slow reaction fires, the corresponding state variables are updated. In this way the hybrid algorithm generates trajectories of state variables as the system proceeds in time. More details on implementation can be found in refs ^{60,61,62}.
Reporting summary
Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.
Data availability
Supplementary information includes two files: one for the Supplementary Text and one for the Supplementary Code. In the Supplementary Text, we present more details for the cellcycle model used in this paper. Tables S1–S4 in Supplementary Text list the timedependent variables, differential equations, reactions and propensity functions, and parameter values. In Table S5 in Supplementary Text we compare simulation results of 122 mutant strains with the observed phenotypes in experiment. The Supplementary Code file includes our hybrid model code in FORTRAN and statistical analysis code in MATLAB. The experimental datasets used in Fig. 2 and Fig. 4a are available from the corresponding author upon request.
Code availability
The datasets generated and analyzed during the current study are reproducible using the FORTRAN and MATLAB codes provided in the Supplementary Code.
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Acknowledgements
This work was partially supported by the National Science Foundation under awards CCF0953590, CCF1526666, and MCB1613741. Dr. Peccoud and Dr. Tyson’s work was also partially supported by NIH under award GM078989.
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M.A. and Y.C. conceived of the presented idea. M.A. developed the simulation code and performed the computations. J.P. provided the experimental data. Y.C. and J.J.T. verified the analytical methods and supervised the findings of this work. All authors discussed the results and contributed to the final manuscript.
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Correspondence to John J. Tyson or Yang Cao.
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Ahmadian, M., Tyson, J.J., Peccoud, J. et al. A hybrid stochastic model of the budding yeast cell cycle. npj Syst Biol Appl 6, 7 (2020). https://doi.org/10.1038/s415400200126z
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