Systems Biology of the Cell Cycle

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1 ICYSB 6th International Course in Yeast Systems Biology Göteborg, Sweden 4 June 213 Systems Biology of the Cell Cycle Assistant Professor University of Amsterdam, M.Barberis@uva.nl A Baby of UNICELLSYS (by Stefan Hohmann at the Final UNICELLSYS Meeting, Innsbruck, 4-6 March 213) 1

2 Thank to Stefan Hohmann Marija Cvijovic Life s complexity pyramid (Oltvai Z.N. & Barabási A.L., Science, 22, 298: ) 2

3 the study of the integrated and interacting network of genes, proteins and metabolites that are responsible for the normal and abnormal cellular functions as well as the emergent properties that create life aims at mechanistic understanding of cellular functions through interactions of constituent molecules in space and time Cellular Systems Biology Multidisciplinarity (from the Multidisciplinary Centre for Integrative Biology (MyCIB), The University of Nottingham, United Kingdom) 3

4 (from the Multidisciplinary Centre for Integrative Biology (MyCIB), The University of Nottingham, United Kingdom) The Cell Cycle (Morgan D.O., The Cell Cycle: Principles of Control, 27, pp. 21) 4

5 Unravel molecular mechanisms underlying Cell Cycle Control in budding yeast (Lodish H., Berk A. et al., Molecular Biology of the Cell, Sixth Edition, 28, pp. 973) 5

6 CdkActivityduringCellCyclePhases 5) As cells enter G 2, Clb1/2-Cdk1 mitotic spindle formation trigger chromosome segregation nuclear division 1) In G 1 phase, Cln3-Cdk1 senses nutritional conditions drives cell growth 2) At the G 1 /S, Cln1/2-Cdk1 activate budding 4) Later in S phase, Clb3/4-Cdk1 activate replication origins finish S initiate the formation of mitotic spindle (Lodish H., Berk A. et al., Molecular Cell Biology, Sixth Edition, 28, pp. 973) 3) At the G 1 /S, Clb5/6-Cdk1 activate pre-replication complex DNA Replication Dynamics (computational analysis and testable predictions) Cdk/Cyclin and Cki Regulation (integrating molecular biology, cell imaging and mathematical modeling) (Futcher B., Yeast, 1996, 12: ; Fitch I. et al., Mol. Biol. Cell, 1992, 3: ) ICYSB, SBI Seminar, Göteborg Dublin

7 Oscillations of Cyclins The oscillatory behavior is widely known as waves of cyclins (Morgan D.O., The Cell Cycle: Principles of Control, 27, pp. 21) (Lodish H., Berk A. et al., Molecular Cell Biology, Sixth Edition, 28, pp. 973) Oscillations of Cdk1/Cyclins and Sic1 Cdk1/Cyclins and Sic1 activities show alternate oscillations Cdk1-Clb3,4 Cdk1-Clb1,2 Substrate/Subunit Inhibitor of Cyclindependent kinase Cdk1-Clb5,6 (Zachariae W. & Nasmyth K., Genes Dev., 1999, 13: ) 7

8 UNIVERSITY Max Planck Institute OF AMSTERDAM for Molecular Genetics Humboldt University Berlin Swammerdam Institute for Life Institute Sciences for Biology Mechanisms controlling S-Cdk Activation (Morgan D.O., The Cell Cycle: Principles of Control, 27, pp. 21) Failure of Proper Timing Cancer Decrease in p27 Kip1 levels, due to its degradation, occurs in ~ 5 % of carcinomas (aggressive, high-grade tumors and poor prognosis, increased apoptosis) p27 Kip1 cytoplasmic localization correlates to enhanced cancer aggressiveness (Blain S.W. and Massagué J., Nat. Med. 22, 8: ) 8

9 Sic1 and p27 Kip1 : Two Sides of the Same Coin (Lacy E.R. et al., Nat. Struct. Mol. Biol., 24, 11: ) (Barberis M. et al., Biochem. J., 25, 387: ) Interaction Map of S. cerevisiae Cell Cycle (Kaizu K. et al., Mol. Syst. Biol. 21, 6: 415) 9

10 Overview of the Cell Cycle Control System NUCLEUS 19 Far Far1 5 Kinetic Model Cdk1-Cln3-Far1 37 Cdk1-Cln3-Far1-p 31 Cdk1-Cln Cln3 21 Cdk Cln3 16 Cdk1 6 7 CYTOPLASM Far1-p Degradation 23 SBF/MBF- Whi5-p 39 Whi5-p SBF/MBF-Whi Whi5 SBF/MBF Cdk1-Cln1,2 CLN1,2 mrna CLB5,6 mrna Cdk1-Clb5,6-Sic1-6p 38 Cdk1-Clb5,6-Sic1 41 Sic1-6p Cdk1-Clb5,6 Degradation DNA SYNTHESIS Whi5 1 CLN1,2 mrna Cln1, CLB5,6 4 Clb5,6 mrna Whi5-p Degradation Cdk1-Cln1,2 47 Cdk1-Clb5,6-Sic1 32 Cdk1-Clb5, Sic1 9 BUDDING (Barberis M. et al., PLoS Comput. Biol., 27, 3: e64) 1

11 Model Improvement Compartmentalization Volume Changes cytoplasm r dmi nijv j dt nucleus j 1 dm r cyt Volnuc nijv j ktr mnuc dt Volcyt j 1 Cell size growth during the G 1 phase 2 cytoplasm dmcyt dvolcyt mcyt 1 vi nucleus dt dt Volcyt i P P S r dmnuc Volcyt nijv j ktr mcyt dt Volnuc j 1 Model Description by Mass Action Kinetics Systems equations v 1 v 2 p S 1 1 p 2 S 4 v p 3 3 S 2 v 4 p 4 S p v N n ij Network properties r dsi nijv j dt j 1 r number of reactions S i metabolite concentrations v j reaction rates n ij stoichiometric coefficients Individual reaction properties v v S p, p d[s1]/ dt = v[1] v[2] = p 1 p 2 S1 d[s2]/ dt = v[3] v[4] = p 3 S1S4 p 4 S2 d[s3]/ dt = v[5] = p 5 S2 d[s4]/ dt = v[3] + v[4] = d[s2]/ dt 1 S1 S[t].5 S2 S3 S Time 11

12 Modeling Pipeline Time courses for: Cln3, Far1, Clb5, Sic1 Localization of: Clb5 and Sic1 (Rossi, R.L. et al., Cell Cycle, 25, 4: ) (Alberghina, L. et al., J Cell Biol, 24, 167: ) Protein levels 4 3,5 3 2,5 2 1,5 1,5 Sic1 Clb5 Clb5/Sic Time (minutes) 4 3,5 GLUCOSE 2% 3 2,5 Sic1 2 Clb5 1,5 Clb5/Sic1 1, ETHANOL 2% Time (minutes) Protein levels Modeling Pipeline Kinetic constants (BIAcore analysis) for: binding of Sic1 to the Cdk-cyclin complex N-term Sic1 ( ) C-term Cyclin A Cdk2 (Barberis, M. et al., Biochem. J., 25, 387: ) 12

13 How the Information is generated? Monitoring of changes in mass concentration on the chip surface Simulated Time Courses for Protein and Protein Complex Concentrations Cln3 activity Study properties of small modules, e.g. Cln3 activity, transcription factors regulation, Cln1-2 and Clb5-6 activities, Sic1 localization, Cln3 activity Concentration ( M).6 Cdk1Cln3 nuc.4.2 Cdk1Cln3-Far1 nuc Time (minutes) 13

14 Simulated Time Courses for Protein and Protein Complex Concentrations Transcription factors regulation Study properties of small modules, e.g. Cln3 activity, transcription factors regulation, Cln1-2 and Clb5-6 activities, Sic1 localization, Concentration ( M) SBF/MBF nuc SBF/MBF-Whi5 nuc Time (minutes) Transcription factors regulation Reproducing Experimental Data Glucose Ethanol Concentration ( M) Sic1 tot Clb5tot Concentration ( M) Sic1 tot Clb5 tot Time (minutes) Time (minutes) Experimental dynamics of Sic1 Experimental dynamics of Clb5 Simulated dynamics of Sic1 Simulated dynamics of Clb5 (Barberis M. et al., PLoS Comput. Biol., 27, 3: e64) 14

15 Model Validation Model Validation 15

16 Model Validation Concentration ( M).6 OE-CLN WT.2.1 cln Time (minutes) Model Validation by altering the Gene Dosage Concentration ( M) A Cdk1-Cln1,2 cyt OE-CLN3 WT cln Concentration ( M) B Cdk1-Clb5,6 nuc.6 OE-CLN WT.2.1 cln Time (minutes) Time (minutes) C Cdk1-Cln1,2 cyt D Cdk1-Clb5,6 nuc Concentration ( M) far1 WT.2.1 OE-FAR Concentration ( M) far1 WT OE-FAR Time (minutes) Time (minutes) 16

17 SIMPLER??? modeling is a very concise way of thinking about a system and generating hypothesis Model of Sic1 Expression and Degradation Clb5 nuc Sic1 Clb5 cyt SIC1 mrna (Barberis M.*, Beck C.* et al., Mol. BioSyst., 211, 7: ) Stochastic Model 17

18 Transcriptional Noise at the G1/S Transition Mean Sic1 Q CV, Q = noise SD Sic1 CV Sic1 = CV Sic1 SD Sic1 mean Sic1 Q = CV Sic1 CV SIC1 mrna Initial SIC1 mrna SIC1 mrna transcription SIC1 mrna degradation (Barberis M.*, Beck C.* et al., Mol. BioSyst., 211, 7: ) Low SIC1 mrna ensures Robust G1/S Timing Initial SIC1 mrna = 2 Initial SIC1 mrna = 6 Initial SIC1 mrna = 1 Mean Sic1 Q SD Sic1 CV Sic1 Initial SIC1 mrna Max mean Sic1 32 molecules 35 molecules 38 molecules Max noise Q Max Timing noise Q 39 min 45 min 5 min (Barberis M.*, Beck C.* et al., Mol. BioSyst., 211, 7: ) 18

19 How many SIC1 mrna Molecules? MS2 + GFP (x3) SIC1 ORF x12 loops 3 UTR Majority of cells show or 1 SIC1 mrnas Observed range between and 1 mrnas (Barberis M. et al., Mol. BioSyst., 211, 7: ) Oscillations of Cdk1/Cyclins and Sic1 Cdk1/Cyclins and Sic1 activities show alternate oscillations Cdk1-Clb3,4 Cdk1-Clb1,2 Cdk1-Clb5,6 (Zachariae W. & Nasmyth K., Genes Dev., 1999, 13: ) 19

20 UNIVERSITY Max Planck Institute OF AMSTERDAM for Molecular Genetics Humboldt University Berlin Swammerdam Institute for Life Institute Sciences for Biology Which Mechanisms control Cdk1/Clb Waves? (Morgan D.O., The Cell Cycle: Principles of Control, 27, pp. 21) ODE Model of Cdk1/Clb Activity Cyclins Clb5, Clb6 Clb3, Clb4 Clb1, Clb2 Active Complexes Cyclin-Dependent Kinase (CDK) Cyclin-Dependent Kinase Inhibitor (CKI) Cdk1 (Cdc28) Sic1 Inactive Complexes 2

21 Clb5 Clb6 MBF CLB5,6 C A TF Clb3 Clb4 CLB3,4 B Fkh2 D Clb1 Clb2 CLB1,2 Potential Transcriptional Regulation of Clb Cyclins 26 Ensemble Modeling of Sic1 Involvement Sic1 C 2 1 C 2 C 3 1 C 1 Cdk1-Clb5,6 3 Cdk1-Clb5,6-Sic1 5 Sic1 A 6 Clb5, C 1 C 2 C 3 7 C 2 Cdk1-Clb3, Cdk1-Clb3,4-Sic1 Clb3, C 1 C 2 C 3 Sic1 C B 9 C 3 Cdk1-Clb1,2 D 1 12 Cdk1-Clb1,2-Sic1 Clb1, Sic1 21

22 Ensemble Modeling to study Sic1 Involvement Sic1 potentially regulates the Appearance of Clb Cyclins Concentration (a.u.) Clb5,6 tot Clb3,4 tot Clb1,2 tot Global Sensitivity Analysis: is the time delay independent on the parameter choice? The timing of Clb cyclins appearance is Sic1-dependent Time (minutes) (Barberis M. et al., Biotechnol. Adv., 212, 3: 18-13) 22

23 (t 1,2 -t 5,6 ) (t 3,4 -t 5,6 ) (t 1,2 -t 3,4 ) t 5,6 t 3,4 t 1,2 Cyclin levels Clb5,6 Clb3,4 Clb1,2 G 1 S G 2 M Cell cycle progression The Time Delay is Sic1-dependent t 1,2 t 5,6 (min) [Version 1] t Clb1,2 t Clb5,6 r = t 1,2 t 3,4 (min) [Version 1] t Clb1,2 t Clb3,4 r = t 1,2 t 5,6 (min) [Version 3] t 1,2 t 3,4 (min) [Version 3] The timing of appearance of Clb cyclins is due to the binding of Sic1 to all Cdk1/Clb complexes (Barberis M. et al., Biotechnol. Adv., 212, 3: 18-13) 23

24 Structural Modeling of Sic1-Cdk1/Clb Interaction Sic1-KID Leu 224 Clb5 Val 225 Cdk1 Interfaces have steric features that allow the formation of a stable ternary complex (Barberis M. et al., Biochem. J., 25, 387: ; Barberis M. et al., Biochem. Biophys. Res. Commun., 25, 336: ) (Barberis M., Adv. Exp. Med. Biol., 212, 736: ; Barberis M., FEBS J., 212, 279: ) Sic1 interacts with all Clb Cyclins (Barberis M. et al., Biotechnol. Adv., 212, 3: 18-13) 24

25 Förster Resonance Energy Transfer (FRET) (courtesy of Andreas Herrmann, Humboldt University Berlin) Fluorescence Lifetime Imaging Microscopy (FLIM) (courtesy of Andreas Herrmann, Humboldt University Berlin, Germany) 25

26 mcer myfp m, A26K mutation preventing any potential dimerization of fluorescent proteins Transient Sic1-Clb Interactions in Living Cells (Schreiber G.*, Barberis M.* et al., FASEB J., 212, 26: ) 26

27 Efficiency of Energy Transfer E (%) E 1 DA D τ DA, average lifetime of the donor measured in presence of the acceptor τ D, average lifetime of the donor expressed alone error bar = SEM (Standard Error of the Measurement) (Schreiber G.*, Barberis M.* et al., FASEB J., 212, 26: ) Does Sic1 regulate Timing and Oscillatory Behavior of Clb Cyclins? SIC1 sic1 Concentration (a.u.) Clb5,6 tot Clb3,4 tot Clb1,2 tot Concentration (a.u.) Clb1,2 tot Clb3,4 tot Clb5,6 tot Time (minutes) Time (minutes) (Barberis M. et al., Biotechnol. Adv., 212, 3: 18-13) 27

28 Sic1 may be Responsible for Clb Timing during Cell Cycle Progression Time (min) 1C/Bud 1C/Bud SIC1 sic1δ G1 S G2 M Clb2 Clb3 Clb5 Sic1 Clb2 Clb3 Clb5 Wild type strain shows Clb periodicity, which is delayed but still present in the SIC1-P strain /46 46/53 14/81 34/67 65/29 74/9 97/4 99/3 1/ 42/74 49/73 5/58 58/47 63/38 67/25 7/14 76/14 8/13 In the sic1δ strain, timing of Clb appearance is lost, although cells proceed into S phase SIC1 sic1δ (Barberis M. et al., Biotechnol. Adv., 212, 3: 18-13) Hcm1 is a Transcriptional Activator of S Phase (Pramila T. et al., Genes Dev., 26, 2: ) 28

29 The Sir2 Histone Deacetylase Sir2 is a NAD-dependent histone deacetylase involved in silencing telomeres and mating locus, maintaining genome integrity and blocking inappropriate gene expression sir2δ shortens life span, whereas SIR2 extra copy extend lifespan Calorie excess Calorie restriction (Guarente L., Genes Dev., 2, 14: ) Sir2 interacts and influences Hcm1 Localization Hcm1-HA Is Sir2 involved in the regulation of Fkh1/2 for the timing of mitotic events? (Rodriguez-Colman M.J. et al., J. Biol. Chem., 21, 285: ) 29

30 Sir2 interacts with Fkh1 and Fkh2 (Linke C.,, Barberis M.* and Krobitsch S.*, under revision) (Rodriguez-Colman M.J. et al., J. Biol. Chem., 21, 285: ) Sir2 interacts with Fkh Transcription Factors ICYSB, SBI Seminar, Göteborg Dublin

31 Sir2 inhibits Cell Growth in a Different Extent SDII SDIV pbtm + pact pbtm-fkh1 + pact pbtm-fkh1 + pact-sir2 pbtm-fkh2 + pact pbtm-fkh2 + pact-sir2 pbtm-hcm1 + pact pbtm-hcm1 + pact-sir2 pbtm + pact-sir2 Sir2 inhibition: Fkh1 > Fkh2 > Hcm1 (Linke C.,, Barberis M.* and Krobitsch S.*, under revision) Sir2 binds to CLB2 Promoter Fold change Exponential phase G1 phase S phase G2/M phase ACT1 CLB2 Sir2 binds to CLB2 promoter in G1 and M phases (Linke C.,, Barberis M.* and Krobitsch S.*, under revision) 31

32 Ndd1-VC+ VN-Fkh2 Sir2-VC+ VN-Fkh1 (Linke C.,, Barberis M.* and Krobitsch S.*, under revision) Sir2_VC UNIVERSITY + OF AMSTERDAM 1 Swammerdam 2 3 Institute for Life 4 VN_Fkh1 Sciences 5 Transmitted light Venus FACS Transmitted light Venus FACS 32

33 Sir2 binds to CLB2 Promoter via Fkh1/Fkh2 Fold change Exponential phase Stationary phase 2 mm H 2 O 2 Fold change ACT1 wild type fkh1 fkh2 CLB2 (Linke C.,, Barberis M.* and Krobitsch S.*, under revision) ACT1 CLB2 Sir2 Nuclear Localization requires Fkh1/Fkh2 (Linke C.,, Barberis M.* and Krobitsch S.*, under revision) 33

34 Activation and Repression in Clb Regulation Fkh1 Fkh1 Sir2 Hcm1 Ndd1 Clb2 Sir2 Physical Interaction Transcriptional activation Transcriptional repression Fkh2 Fkh2 (Linke C.,, Barberis M.* and Krobitsch S.*, under revision) Conclusion Timing of Cdk1/Clb activation is achieved by coupling multiple mechanisms: (i) Sic1-dependent Clb inhibition (ii) Sir2-dependent inhibition via Fkh1/Fkh2 34

35 SYSTEMS Microscopy Computational PPIs Modeling DNAPIs Structural Modeling Biochemical Assays BIOLOGY (Barberis M., FEBS J., 212, 279: ) ICYSB, SBI Seminar, Göteborg Dublin

36 From Ph.D. Student to Assistant Professor Prof. Lilia Alberghina Prof. Edda Klipp Prof. Hans Westerhoff Christian Linke (Ph.D., Post-doc) Gabriele Schreiber (Post-doc) Aouefa Amoussouvi (Ph.D.) Miquel Á. Adrover (Ph.D.) Alberto González-Novo (Post-doc) Silvia Scolari (Ph.D.) Christine Klaus (Technician) Christian Diener (Ph.D.) Thomas Spiesser (Master, Ph.D.) Adriana Supady (Master) Claudia Beck (Bachelor) 36

37 Thank You! Questions welcome. 37