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The Eukaryotic Cell Cycle: Molecules, Mechanisms and Mathematical Models John J. Tyson Biological Sciences, Virginia Tech & Virginia Bioinformatics Institute.

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Presentation on theme: "The Eukaryotic Cell Cycle: Molecules, Mechanisms and Mathematical Models John J. Tyson Biological Sciences, Virginia Tech & Virginia Bioinformatics Institute."— Presentation transcript:

1 The Eukaryotic Cell Cycle: Molecules, Mechanisms and Mathematical Models John J. Tyson Biological Sciences, Virginia Tech & Virginia Bioinformatics Institute Funding: NIH-GMS

2 S DNA synthesis G2 G1 cell division + Metaphase Anaphase Telophase Prophase The cell cycle is the sequence of events whereby a growing cell replicates all its components and divides them more-or-less evenly between two daughter cells...

3 Why study the cell cycle? All living organisms are made of cells. All cells come from previously existing cells by the process of cell growth and division.

4 Why study budding yeast?

5 S DNA synthesis G2 G1 cell division + Metaphase Anaphase Telophase Prophase Alternation of DNA synthesis and mitosis Checkpoints Balanced growth and division Robust yet noisy

6 S DNA synthesis G2 G1 cell division + Metaphase Anaphase Telophase Prophase Cdk Cln2 Clb5 Clb2 APC Cdh1 Sic1 APC Cdc20

7 Cdh1 Sic1 Clb2 Sic1 Cln2 APC-P APC Mcm1 Mcm1 A Clb5 SBF A SBF Swi5 A Swi5 Sic1 Clb5 Cdc14 Cln2 Clb2 Cdh1 Cdc20 Cdh1 Budding Yeast Chen et al. (2004) Whi5 SBF A Whi5 P Net1 Cdc14 Net1 P Cln3 Cell Size Sensor Clb2

8 Tyson & Novak, “Irreversible transitions, bistability and checkpoint controls in the eukaryotic cell cycle: a systems-level understanding,” in Handbook of Systems Biology (2012) Tyson & Novak, “Temporal Organization of the Cell Cycle,” Current Biology (2008) Kathy Chen Bela Novak Deterministic Modeling Attila Csikasz-Nagy Andrea Ciliberto

9 Cdc20 Cdh1 Sic1 Clb2 Sic1 Cln2 APC-P APC Mcm1 Mcm1 A Clb5 SBF A SBF Swi5 A Swi5 Sic1 Clb5 Cdc14 Cln2 Clb2 Cdh1 Cdc20 Cdh1 Budding Yeast Chen et al. Whi5 SBF A Whi5 P Net1 Cdc14 Net1 P Cln3 Cell Size Sensor Clb2 Deterministic Model - - differential equations X YP Y mechanism

10 differential equations Clb2-dep kinase S/A, parameter ON OFF What mechanisms flip the switch on and off? 2 steady state bifurcation diagram

11 Cdc20 Cdh1 Sic1 Clb2 Sic1 Cln2 APC-P APC Mcm1 Mcm1 A Clb5 SBF A SBF Swi5 A Swi5 Sic1 Clb5 Cdc14 Cln2 Cdh1 Cdc20 Cdh1 Budding Yeast Chen et al. Whi5 SBF A Whi5 P Net1 Cdc14 Net1 P Cln3 Clb Cdh1 Cln2 -

12 Clb2 Cln2 Entry DNA Synthesis G1 G2/M

13 Cdc20 Cdh1 Sic1 Clb2 Sic1 Cln2 APC-P APC Mcm1 Mcm1 A Clb5 SBF A SBF Swi5 A Swi5 Sic1 Clb5 Cdc14 Cln2 Clb2 Cdh1 Cdc20 Cdh1 Budding Yeast Chen et al. Whi5 SBF A Whi5 P Net1 Cdc14 Net1 P Cln3 Cell Size Sensor Clb Cdh1 Cdc14

14 Clb2 Cdc14 Exit Cell Division G1 G2/M

15 Clb2 Cln2 Cdc14 G1 S G2 M A T Clb2 Cdh1 Cln2 Cdc14

16 Clb2 Cln2 Cdc14 G1 S G2 M A T Clb2 Cdh1 Cln2 Cdc14

17 Clb2 Cln3 Cdc14 G1 S G2 M A T Clb2 Cdh1 Cln2 Cdc14 Cln3

18 Clb2 Cln3 Cdc14 G1 G2 M A T Clb2 Cdh1 Cln2 Cdc14 Cln3 S

19 Protocol to demonstrate hysteresis at Start Cross et al., Mol. Biol. Cell 13:52 (2002) Genotype: cln1  cln2  cln3  GAL-CLN3 cdc14 ts Knockout all the G1cyclins Turn on CLN3 with galactose; turn off with glucose Temperature- sensitive allele of CDC14: on at 23 o C, off at 37 o C. “Neutral” conditions: glucose at 37 o C (no Cln’s, no Cdc14) Fred Cross

20 R = raffinose G = galactose Standard for protein loading G1 cells S/G2/M cells Start with all cells in G1 Make some Cln3 Shift to neutral

21 Clb2 Cln2 Cdc14 G1 S G2 M A T Clb2 Cdh1 Cln2 Cdc14

22 Clb2 Cln2 Cdc14 G1 S G2 M A T Clb2 Cdh1 Cln2 Cdc14 Cdh1 CA

23 Clb2 Cln2 Cdc14 G1 S G2 M A T Clb2 Cdh1 Cln2 Cdc14 Cdh1 CA G1

24 Protocol to demonstrate hysteresis at Exit Lopez-Aviles et al., Nature 459:592 (2009) Genotype: MET-CDC20 GAL-CDH1 CA cdc16 ts Turn off Cdc20; block in metaphase Turn on Cdh1; degrade Clb2 and exit from mitosis Inactivate APC at 37 o C; block any further activity of Cdh1 Add galactose at 23 o C to turn on Cdh1, then raise temperature to 37 o C to turn off Cdh1 Frank Uhlmann

25 0 min 50 min 140 min min Tubulin 37 0 C MET-CDC20 GAL-CDH1 CA APC cdc16(ts) Cdh1 CA CKI CycB Gal metaphase interphase

26 S DNA synthesis G2 G1 cell division + Metaphase Anaphase Telophase Prophase Alternation of DNA synthesis and mitosis Checkpoints Balanced growth and division Robust yet noisy

27 Clb2 Cln2 Cdc14 G1 S G2 M A T DNA Damage Clb2 Cdh1 Cln2 Cdc14 Chromosome Alignment Problems

28 Clb2 Cln2 Cdc14 G1 S G2 M A T Clb2 Cdh1 Cln2 Cdc14 Growth Is this deterministic model robust in the face of the inevitable molecular noise in a tiny yeast cell (volume = 40 fL = 40 x L)

29 Table 1. Numbers of molecules (per haploid yeast cell) and half-lives for several cell cycle components. Cell cycle Gene # molecules per cellHalf-life (min) ProteinmRNAProteinmRNA CDC CLN CLB CLB SWI MCM SIC CDC Molecular Noise Budding Yeast Cells Vol = 40 fL

30 Table 1. Numbers of molecules (per haploid yeast cell) and half-lives for several cell cycle components. Cell cycle Gene # molecules per cellHalf-life (min) ProteinmRNAProteinmRNA CDC CLN CLB CLB SWI MCM SIC CDC Molecular Noise Budding Yeast Cells Vol = 40 fL

31 Table 1. Numbers of molecules (per haploid yeast cell) and half-lives for several cell cycle components. Cell cycle Gene # molecules per cellHalf-life (min) ProteinmRNAProteinmRNA CDC CLN CLB CLB SWI MCM SIC CDC Molecular Noise Budding Yeast Cells Vol = 40 fL

32 Table 1. Numbers of molecules (per haploid yeast cell) and half-lives for several cell cycle components. Cell cycle Gene # molecules per cellHalf-life (min) ProteinmRNAProteinmRNA CDC CLN CLB CLB SWI MCM SIC CDC Molecular Noise Budding Yeast Cells Vol = 40 fL

33 Table 1. Numbers of molecules (per haploid yeast cell) and half-lives for several cell cycle components. Cell cycle Gene # molecules per cellHalf-life (min) ProteinmRNAProteinmRNA CDC CLN CLB CLB SWI MCM SIC CDC Molecular Noise Budding Yeast Cells Vol = 40 fL

34 Birth-Death Process

35 Transcription-Translation Coupling Swain, Paulsson, etc.

36 How variable is the yeast cell cycle? Di Talia et al., Nature (2007) G1 Duration Mean = 16 min CV = 48% S/G2/M Duration Mean = 74 min CV = 19% Cycle Time Mother Daughter 87 min ± 14% 112 min ± 22% Div 68 fL ± 19%

37 Budding: Myo1-GFP Cell size: ACT1pr-DsRed Di Talia et al., Nature (2007) Whi5 exit: Whi5-GFP Cell size: ACT1pr-DsRed Daughter Cells

38 Debashis Barik & Sandip Kar Jean PeccoudYang Cao Mark Paul Bill Baumann Stochastic Modeling

39 Multisite Phosphorylation Model (Barik, et al.) Clb2 Cdh1 bistable switch

40 Multisite Phosphorylation Model (Barik, et al.) Cell size control Clb2 Cdh1 Cln2

41 Multisite Phosphorylation Model (Barik, et al.) Clb2 Cdh1 Cln2Cdc14

42 Deterministic calculations  The model consists of 58 species, 176 reactions and 68 parameters  Mass-action kinetics for all reactions  At division daughter cells get 40% of total volume and mothers get 60%

43 Stochastic calculations  The model consists of 58 species, 176 reactions and 68 parameters  Mass-action kinetics for all reactions  Protein populations: ~1000’s of molecules per gene product  mRNA populations: ~10 molecules per gene transcript  mRNA half-lives: ~ 2 min  Reactions are simulated using Gillespie’s SSA

44 Experimental data from: Di Talia et al., Nature (2007) MotherDaughter Cycle Time (min)Expt87 ± 14%112 ± 22% Model89 ± 20%114 ± 22% G1 duration (min)Expt16 ± 50%37 ± 50% Model21 ± 48%41 ± 48% (fL)Expt40 ± 18%28 ± 20% Model41 ± 23%28 ± 23%

45 Daughter cells ModelDi Talia et al. Mother cells Model Di Talia et al.

46 Daughter cells

47 Expt.: Di Talia et al, Nature (2007)

48 Summary Cell cycle control in eukaryotes can be framed as a dynamical system that gives a coherent and accurate account of the basic physiological properties of proliferating cells. The control system seems to be operating at the very limits permitted by molecular fluctuations in yeast-sized cells. A realistic stochastic model is perfectly consistent with detailed quantitative measurements of cell cycle variability.

49 Computation Theory Experiment Current Biology 18:R759 (2008) Proc Natl Acad Sci 106:6471 (2009) Mol Syst Biol 6:405 (2010) Handbk of Syst Biol (to appear)

50 Budding: Myo1-GFP Cell size: ACT1pr-DsRed Whi5 exit: Whi5-GFP Cell size: ACT1pr-DsRed Di Talia et al., Nature (2007) Whi5 Cyclin Whi5P Start Exit BE DNA synth

51 Daughter cell Di Talia et al., Nature (2007) T 2 = T G1 – T 1 = constant

52 Mother cells

53

54 Expt.: Di Talia et al, Nature (2007) T G1 T1T1 Daughter cells T2T2 T 1 = Time when Whi5 exits from nucleus


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