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The Cell Cycle

The Cell Cycle. Jonathon Pines Gurdon Institute j.pines@gurdon.cam.ac.uk http://www.gurdon.cam.ac.uk/~pineslab/New_Web_Site/Site/Lectures.html. Outline. How do we know there is a cell cycle? How is the cell cycle controlled? What goes wrong in cancer?.

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The Cell Cycle

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  1. The Cell Cycle Jonathon Pines Gurdon Institute j.pines@gurdon.cam.ac.uk http://www.gurdon.cam.ac.uk/~pineslab/New_Web_Site/Site/Lectures.html

  2. Outline How do we know there is a cell cycle? How is the cell cycle controlled? What goes wrong in cancer?

  3. What does a cell need to do to proliferate? Chromosome Separation M Product - Substrate? Oscillator? S DNA replication

  4. How do we know there is a cell cycle? G1 or S or G2 M M = + G1 S S + = S G2 G2 delay = + • Rao & Johnson, 1970, Nature 225, 159-164; Johnson & Rao, 1970, Nature 226, 717-722 After Murray and Hunt, 1993 M phase and S phase are different cellular states

  5. Problems to be solved:Alternation and Completion Chromosome Separation M Murray and Hunt, 1993 The Cell Cycle: an introduction. Morgan, D.O., 2007 The Cell Cycle: Principles of Control S DNA replication

  6. Cyclin-CDK complexes drive the cell cycle • Three converging lines of evidence: Yeast genetics Xenopus meiosis Translational control in sea urchin eggs M ‘M phase’ Cyclin-CDK ‘S phase’ Cyclin-CDK S

  7. How can you isolate cell cycle regulators? Schizosaccharomyces pombe (fission yeast): Position in cell cycle related to length of cell Screen for genes that accelerate or slow down the cell cycle cdc2 is a conserved protein kinase required at 2 points in cell cycle and with a wee allele Wee1, mik1 cdc25 cdc13 (Cyclin B) cdc2 Interphase Mitosis After Murray and Hunt, 1993.

  8. Progesterone Inject cytoplasm MPF: a potent trigger for mitosis Xenopus laevis: arrested in G2 of meiosis I, egg arrested in metaphase of meiosis II Factor in egg cytoplasm forces oocyte to enter M phase - M phase promoting factor (MPF) Self amplifies and does not require protein synthesis: pre-MPF in oocyte Universal property of M phase cells Masui and Markert, 1971, J. Exp. Zool 177, 129. After Murray and Hunt, 1993.

  9. Cyclin MPF S M S M Cyclins: Coincidence or cause? Sea urchins: large eggs arrested in G1 of first mitotic cell cycle Fertilisation causes large increase in translation One protein only translated after fertilisation and destroyed at each mitosis: Cyclin Evans et al. 1993. Cell 33, 389 MPF eventually shown to consist of cyclin and Cdc2 (Dorée and Hunt, 2002, J Cell Sci, 115, 2461-4)

  10. Cyclin-CDK complexes define the state of the cell cycle Mitosis Initiation cdc13-cdc2 M Degrade cdc13 or cdc2 START G2 G1 S Re-initiate DNA synthesis DNA Synthesis Initiation DNA Synthesis cig2-cdc2 Hayles et al., 1994, Cell 78 , 813 and Broek et al., 1991, Nature 349 , 388.

  11. The cell cycle as alternation of CDK activity Low CDK activity: Assemble and fire origins of replication High CDK activity: Repress origins of replication Build mitotic apparatus • S. pombe paradigm - the Threshold hypothesis • Stern and Nurse, 1996, Trends Genet. 12, p345-350 Norton and Diffley, 2000, Molecular Cell, 5, 85-95

  12. Cyclin B (Cdc13) Analogue Sensitive Cdc2 Kinase Testing the Cdk threshold hypothesis Cyclin-cdc2 fusion drives cell cycle in absence other cyclins Control amount of kinase activity through analogue sensitive Cdc2 Low concentrations of inhibitor block mitosis High concentrations block DNA replication Add different inhibitor concentrations to drive cell cycle with non-degradable Cdc13 Block G2 cells and release into low concentrations of inhibitor - Cells re-replicate Coudreuse & Nurse, 2010 Nature 468, 1074-1079

  13. The Cell Cycle: Alternation Metaphase Initiation of Mitosis Cyclin B1-CDK1 G 0 M Cyclin A-CDK1/2 START or R point G 2 G 1 Cyclin D-CDK4/6 S Completion of DNA Synthesis Initiation of DNA Synthesis DNA Synthesis Cyclin E-CDK2 Cyclin A-CDK2

  14. Cyclin E-CDK2 G 1 Cyclin A-CDK2 S G 2 M G 1 Cyclin B-CDK1 Cdc25 GrowthFactors Waves of cyclin-CDK kinase activity during the human cell cycle Kinase activity time M A

  15. But Cdk2 is Not Essential for Mitosis Cyclins E and A are partially redundant Ortega et al., 2003, Nature Genetics, 35, p25-31 Cyclin E is only essential for endo-replication Geng et al., 2003 Cell 114, 431-443 Cyclin A is only essential in early embryos & stem cells Kalaszczynska et al., 2009 Cell 138, 352-365

  16. Most mitotic cycles only require one CdkSantamaria et al., 2007, Nature 448, 811-815 • Looks like dividing animal cells are just like fission yeast • But for cells to differentiate and form particular tissues they need specialised Cdks • Cdk2 - spermatogenesis, oogenesis • Cdk4 - pancreatic beta cells, pituitary, mammary epithelium • Cdk4 or Cdk6 - haematopoesis, cell size

  17. Why are there multiple Cyclin-Cdks? • Partial Redundancy? S. cerevisiae paradigm • Tissue specific roles

  18. The Cell Cycle: Alternation Metaphase Initiation of Mitosis Cyclin B1-CDK1 G 0 Polo Aurora M Cyclin A-CDK1/2 G 2 G 1 Cyclin D-CDK4/6 S Completion of DNA Synthesis Initiation of DNA Synthesis START or R point DNA Synthesis Cyclin E-CDK2 Cyclin A-CDK1 & 2

  19. Kinase Domain PBD PBD Coordination between mitotic kinases • Polo box binds to S-pT or S-pS - Elia et al., 2003, Science 299, p1228 • Often generated by Cdk (S-T-P) • Thus Cdk phosphorylation generates Plk substrate Eg: Cdk1 phosphorylates INCENP to recruit Plk1 to kinetochores - Goto et al., 2006, Nature Cell Biology 8, p180

  20. Cyclin Proteolysis (Ubiquitin) Thr14 & Tyr15 Phosphorylation (wee1/mik1) Deactivate Inhibitor binding KAP Activate Inhibitor removal Thr14 & Tyr15 dephosphorylation (Cdc25) Cyclin Binding T-loop Thr dephosphorylation (p24 ) T-loop Thr phosphorylation (CAK) Regulating a CDK T14 Y15 CDK T160 Morgan 1995, Nature 374, 131.

  21. O F F P P Cyclin-CDK inactivation: the paradigm N-term ATP Cyclin Box P KINASE Cyclin Fold 2 C-term Cks ON

  22. Sites not well conserved Not inhibitory SP SP SP SP SP Conserved inhibitory sites Wee1 - Generating a Robust Switch • Fission yeast wee1 and mik1 prevent premature mitosis • mik1 stabilised by unreplicated DNA • Wee1 in animal cells blocks mitosis in interphase Ultrasensitive response to inhibition by Cdk1 - bistable state SP SP SP SP SP Kinase Domain Kinase Domain Kim & Ferrel, 2007, Cell 128, 1133-1145

  23. O Cki F F Cyclin-CDK inactivation: the Paradigm 2 N-term ATP Cyclin Box P KINASE Cyclin Fold 2 C-term Cks ON

  24. CKIs inhibit both the CDK and the cyclin Russo et al, 1996 Nature 382, 325.

  25. Yeast CKI Paradigm: Cell Cycle Co-ordination - Sic1 Cln2-Cdc28 Clb5-Cdc28 Cdc4 Cdc34 - SCF Sic1 Sic1 p40 p40 Degradation Cdc28-Clb2 Cdc28-Clb5/6 M G1 S Phase

  26. Sic1 Inactivation has Parallels with Wee1 Inactivation Sic1 is processively phosphorylated by Cln2 and Clb5 Clb5 phosphorylation generates a positive feedback loop Kõivomägi et al., 2011, Nature 480, 128–131

  27. Generating a Robust Switch also requires Regulating Antagonistic Phosphatases How to turn off a specific complex?

  28. Generating a Robust Switch - Target a Specific Phosphatase Complex with an Inhibitor Mochida et al., 2010 Science 330, p1670

  29. States of the Cell Cycle are generated by Proteolysis Different complement of proteins present in different cell cycle states

  30. The Cell Cycle is Co-ordinated by Ubiquitin-dependent Proteolysis Effectively an interplay between the SCF and the APC/C SCF = Skp1 + Cullin + F-box protein APC/C = Anaphase Promoting Complex/Cyclosome

  31. D E S T R U C T I O N Ubq 2 6 S P r o t e o s o m e Ubq Ubq Ubiquitination UBC Ubiquitin Ligase E1 E 2 E 3 A T P A D P + P i E1 E 3 E2 Ubq Ubq S u b s t r a t e

  32. F-Box/ BTB/ UBQ DOC1 E2 SOCS Box Schematic of RING E3 ligases COP9/Signalosome Substrate RING Nedd8 Cullin Mammalian cells: 69 F box proteins - bind to Cul 1 >200 BTB proteins - bind to Cul 3 >50 SOCS box proteins - bind to Cul2/5 >15 DDB1 proteins - bind to Cul 4 F-box proteins Fbw = WD40 domain: Fbw1 = bTrcp1, Fbw2 = bTrcp2, Fbw7 = Cdc4 Fbl = leucine rich domain: Fbl1 = Skp2 Fbx = other domains

  33. Nedd8 closes the gap between E2 and substrate Nedd8 Duda et al., 2008 Cell 134, 995-1006

  34. Interplay between the SCF and the APC/C • SCFSkp2 is degraded by the APC/C • Allows p27 (Cdk inhibitor) to accumulate in G1 phase Bashir et al., 2004, Nature 428, 190-193

  35. E 3 APC7 S u b s t r a t e Ubq APC3 APC10 Ubq Apc3 APC6 APC11 APC8 Ubq Apc6 Apc8 APC4 APC2 APC5 E 3 APC1 Ubiquitination: Mitosis Destruction box proteins UBC (UBC10/UBC5 + Ube2S) E 2 Cyclin B Cdc20 Securins E2 P APC/C Ubq Pines, J. 2011. Nature Reviews Molecular and Cell Biology 12, 427-438

  36. Cytokinesis Chromosome Attachment Chromatid Separation Spindle Disassembly NEBD Spindle checkpoint Cyclin A Securin Plk1 Cdc20 UbcH10 Aurora A Cyclin B1 Co-ordinating Mitosis by Proteolysis Antephase Pro-metaphase Metaphase Anaphase Telophase Pines, J. 2006, Trends Cell Biol., 16, 55-63

  37. APC10 and Cdc20 form the Destruction Box receptor EM reconstructions Da Fonseca et al., 2011, Nature 470, 274-278

  38. APC/C specificity: two co-activators Cdc20 Cdh1 Required for correct G1 phase & to degrade Aurora kinases Essential for Mitosis Regulated by spindle checkpoint Regulated by phosphorylation (by CDKs) • proteolysis by Cdh1 • Rca1/Emi1 Proliferating cells Somatic & Differentiated cells (brain & trophectoderm) Yu, H. 2007, Mol Cell., 27, 3-16; Garci-Higuera et al., 2008, Nature Cell Biology, 10, 802-811 Floyd et al., 2009 Curr Biol., 18, 1649-1658

  39. Completion: Timing and Checkpoints Chromosome Separation M S DNA replication

  40. Completion: Timing Budding yeast mitosis (at least under laboratory conditions) Embryonic cell cycles Xenopus (all you need is cyclin B) Drosophila Problem: High error rate Inflexible

  41. Completion: Checkpoints First defined by Weinert and Hartwell, 1989, Science 246, 629. “The events of the cell cycle .. are ordered into dependent pathways in which the initiation of late events depends on the completion of early events. Some dependencies can be relieved by mutation ….suggesting that the dependency is due to a control mechanism and not an intrinsic feature of the events themselves. Control mechanisms enforcing dependency in the cell cycle are here called checkpoints.”

  42. M S Checkpoints:S phase checkpointG2 Checkpoints Ds breaks UVMorphogenesis CheckpointProphase CheckpointSpindle Checkpoint

  43. Checkpoints Think about the biology • DNA damage: budding yeast arrest in mitosis fission yeast and animal cells in G2 What should be the phosphorylation state of Cdk1? What should be the targets of the checkpoint?

  44. Checkpoints • Budding yeast do use Cdk1 Y18 phosphorylation • Part of the morphogenesis checkpoint to prevent budding in inappropriate conditions • Major regulation is on the stability of Swe1p • Degradation of Swe1 accompanied by relocalisation to bud neck via binding to Hsl7p McMillan et al., 2002, Mol. Biol. Cell., 13, p3560-75

  45. Summary The cell cycle is driven by alternation of high and low Cdk activity Robust switches are driven by ultrasensitivity and phosphatase feedback loops Alternation in Cyclin-Cdk activity is underpinned by proteolysis In somatic cells checkpoints ensure that the switch is not thrown until previous stage is complete (embryos often rely on timing)

  46. The Cell Cycle & Development • Cells must proliferate only in response to the correct cues • Proliferation, growth and differentiation must be coordinated to generate tissues and organs of the correct size and structure

  47. To Cycle or Not? • The decision to proliferate or differentiate or become quiescent is made between mitosis and DNA replication • The Restriction point: the time when cells are committed to replicate their DNA and divide

  48. The Restriction Point

  49. Committment • D-type Cyclins are important signal transducers • D-type Cyclins are particularly important in specialised cell types • E.g.: Retina, Cyclin D1 binds to notch promoter - Bienvenu et al., 2010 Nature 463, 374 • D-type Cyclins are basically there to turn on E-types • Rescue Cyclin D1 KO with a knock-in of Cyclin E - Geng et al., 1999 Cell 97. 767-777

  50. Committment Turning on genes required for S phase and mitosis requires de-repressing the E2F-family

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