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Coupling two numerical modules of the cell cycle

Study the activation of G1-S transition and regulation of G2-M transition, focusing on CycE/CycD oscillations and molecular interactions. Anticipate possible coupling problems and modifications needed for desired behavior. Utilize computer assistance for model verification and refinement.

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Coupling two numerical modules of the cell cycle

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  1. Coupling two numerical modules of the cell cycle Work in progress … INRIA Rocquencourt

  2. Module 1: G1-S transition based on Aguda and Tang, 1999, Cell Prolif.

  3. Module 1: G1-S transition CycE p16 p27 Rb E2F CycD

  4. Module 2: G2-M transition Wee1P Wee1 .. .. CycB CycB* APC APC Cdc25P .. .. .. .. Cdc25 .. .. based on Qu, 2001, Biophysical J.

  5. Module 2: G2-M transition APC CycB Wee1

  6. G1-S G2-M

  7. What do the modules do? • Module 1: 1. Study of the activation of the G1-S transition (through that of CycE) in different conditions: - if CycD present then CycE activates else CycE stays inactive - effect of the amount of inhibitors (p27 and p16) 2. No emphasis on the oscillations of the cyclins … • Module 2: 1. Emphasis on G2-M transition and regulation of CycB (oscillatory behavior) 2. Regulation of CycB activity through phosphorylation by Wee1 and dephosphorylation by Cdc25

  8. Coupling the two modules:What to expect … From each module Define what they will be used for: - Do we need to look inside the module (details of biochemical reactions)? Are we only interested in the output of the module (black box)? - What behaviors do we want to keep (activations…) or change (oscillations…)? From the model of the two modules Define what you will want to see: - All molecules (concentration) of module 1 should oscillate period(CycE,24), oscil(CycE) - CycD peaks before CycE and CycE peaks before CycB peak_order(CycD,CycE,CycB) - Length of G1 phase should be around 1/3 of the cell cycle phase_shift(CycD,CycE,8) …

  9. Coupling the two modules:Checklist Define things to check and anticipate possible coupling problems:

  10. Phosphorylation of Rb and release of E2F from the complex Rb-E2F km1*[E2F]*[Rb] for E2F+Rb=>RbE2F. km1*[E2F]*[Rb] for E2F+Rb=>RbE2F. k1p*[CycD]*[RbE2F] for RbE2F=[CycD]=>E2F+RbP. k1pp*[CycDp27]*[RbE2F] for RbE2F=[CycDp27]=>E2F+RbP. k1*[aCycE]*[RbE2F] for RbE2F=[aCycE]=>E2F+RbP. RbE2F CycDp27 CycD CycE CycB k1ppp*[CycB]*[RbE2F] for RbE2F=[CycB]=>E2F+RbP. Rb E2F RbP Coupling the two modules:Find the link… In module 1, Rb gets phosphorylated and inactivated by all the cdk/cyclin complexes (referred to as CycD, CycE) => CycB probably participates in the phosphorylation of components of module 1 in which other cyclins are involved LINK: Module 2 => Module 1 Unidirectional link: most properties of Module 2 remain unchanged

  11. How is CycE activated? Modify kinetics of CycE Reduce amplitude of CycE activation Simulations CycE CycB - Synthesis decreased (1.42 => 0.5) - Degradation increased (0.1 => 0.1) - Activation /inactivation from iCycE to aCycE modified (1 => 0.1 and 0.1 => 1) => 4 parameters modified

  12. How is CycE controlled (upstream)? Try to modify what regulates CycE synthesis: Rb/E2F interaction CycE does not oscillate yet Simulations CycB CycE - Synthesis increased of E2F, Rb - Degradation increased of E2F, Rb - Association between Rb and E2F stronger =>7 parameters modified

  13. p16 and p27 do not oscillate yet Why do p16 and p27 remain constant? Modify kinetics of p16 and p27 Simulations CycB CycE - Synthesis increased for p16 and decreased for p27 - Degradation increased for p16 and decreased for p27 => 5 parameters modified

  14. Simulations CycB CycE CycE CycD [CycD] is close to zero and CycE activates even when [CycD]~0 Why is CycD never activating? Modify kinetics of CycD to be continued …

  15. Problems met during the early coupling • Questions to ask before the coupling: • Are there common elements in the modules we want to couple? • Are the two modules on the same scale of time and concentration? • What do individual modules do? Are they compatible? • Is the coupling unidirectional? Can one of the module be considered as a "black box" where only the output matters? …

  16. Problems met during the early coupling • Things to do once these questions are answered: • Define precisely what is expected in the coupled model. • Define the link(s) between the modules. • In the coupled model, verify, modify and compare the activity of all molecules to their expected properties. • Understand the subparts of the modules (e.g. what regulates the activity of the cyclins, how to lower the activity of CycE, what makes CycE or CycD oscillate, …) to get an idea of what can be modified to obtain the saught behavior. • Make sure that, after each change, the molecules continue to behave as desired, and that the global properties of the model are conserved (mutant phenotypes …).

  17. Effort of formalization should assist in the coupling: Help from the computer in the verification process (model checking) and modification process (model revision)

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