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The Cell Cycle The Mechanics of the cell cycle G1 M S The cyclic process by which cells duplicate their contents and partition into two daughter cells G2 Emmy Verschuren Institute for Molecular Medicine Finland (FIMM) [email protected] February 07 2013 Chapter 17 (but not mitosis) Why understand The Mechanics of the cell cycle? The cell cycle simplified > It only takes one rogue cell and its progeny to kill a multicellular organism G1 Tumor Cells M Normal Cells Metastatic cancer invading liver G0 Start Restriction point S G2 The Cell Cycle has 2 major phases Cell cycles in yeast: a genetically tractable unicellular eukaryote G2 10-12 hours G1 < 1 hour M S G1 • Small, unicellular • Cell size and shape reflects cell cycle phase • Haploid genome makes genetics easy Figure 17-2 Molecular Biology of the Cell (© Garland Science 2008) Figure 17-5 Molecular Biology of the Cell (© Garland Science 2008) Discovery of cell division cycle - cdc - genes gain of function cdc2 mutant wild type cdc2 loss of function cdc2 mutant • Excess function triggers premature cell cycle entry > smaller (shorter) cells • Loss of function blocks cell cycle > larger (longer) cells Figure 17-6 Molecular Biology of the Cell (© Garland Science 2008) Xenopus egg biochemistry Advantages: • Large size (100.000 x more cytoplasm than average cell in the human body) • Simple S-M cell cycles without intervening gap phases Figure 17-9 Molecular Biology of the Cell (© Garland Science 2008) Xenopus egg fertilisation mimicked in a test tube Biochemical analysis of cycling sea urchin embryos The identification of MPF: Mitosis Promoting Factor (later: cyclin B) cyclins: Cyclically expressed proteins whose appearance and disappearance correlates with MPF (mitosis) activity Figure 17-10 Molecular Biology of the Cell (© Garland Science 2008) Mammalian cells proliferating in culture note Most cultured cell lines contain mutations so that they can be grown (and studied) indefinitely = immortalisation Figure 17-11 Molecular Biology of the Cell (© Garland Science 2008) Analysis of cellular DNA reveals cell cycle phase Thymidine analog bromo-deoxyuridine (BrdU) labels S phase cells > BrdU pulse Figure 17-12 Molecular Biology of the Cell (© Garland Science 2008) Cell cycle control checkpoints Two key components of the cell cycle control system Cycle control system: • Timer • Correct order • Each event only 1x per cycle • On/off switches • Robustness • Adaptability S. Pombe cdc2, Xenopus & Sea Urchin & Mammalian MPF are all the same cyclin-dependent kinase (cdk1) complex Figure 17-14 Molecular Biology of the Cell (© Garland Science 2008) Figure 17-15 Molecular Biology of the Cell (© Garland Science 2008) Cyclins are the regulatory subunits of the Cdks Core of the cell cycle control system Yeast are unlike metazoans in having only one Cdk (Cdc2/ Cdk1) which interacts with different cyclins that regulate its activity in different ways at various stages of the cell cycle •G1 cyclins promote cell cycle entry •G1/S cyclins promote passage through the late G1 restriction point and into S phase • S cyclins promote DNA replication • M cyclins promote mitosis Importantly: Cdk expression levels are constant through the cell cycle Oscillations of Cdk activities (through cyclical expression/destruction of cyclins) leads to cyclical changes in the phosphorylation of proteins that regulate DNA replication, mitosis and cytokinesis Figure 17-16 Molecular Biology of the Cell (© Garland Science 2008) Nomenclature of major cyclins and Cdks While yeasts use only 1 Cdk, vertebrates use 4 different Cdks Structural basis of Cdk activation Important The cyclin directs the Cdk activity to specific substrates > each cyclin/Cdk complex has a unique set of substrates Figure 17-17 Molecular Biology of the Cell (© Garland Science 2008) Key negative controls of the cell cycle’s mechanics Inhibitory phosphorylation of Cdks Important for checkpoint controls: 1. Inhibitory phosphorylation of Cdks by WeeI/Myt1 2. Cdk inhibition by Cdk Inhibitor (CKI) binding 3. Protein destruction catalyzed by E3 ubiquitin ligases Car engine: it is much easier to detect a little more stop than a little more start Most important negative regulator of M-Cdk activity Figure 17-18 Molecular Biology of the Cell (© Garland Science 2008) Inhibition of Cdks by Cdk Inhibitor proteins (CKIs) Control of protein proteolysis by SCF E3 ubiquitin ligase • SCF directs protein destruction in G1/S (cyclins and certain CKIs) • SCF ubiquitination requires phosphorylation of target proteins Most important negative regulator of G1/S Cdk activity Figure 17-19 Molecular Biology of the Cell (© Garland Science 2008) Control of proteolysis by APC/C E3 ubiquitin ligase Figure 17-20a Molecular Biology of the Cell (© Garland Science 2008) The core rules of the cell cycle’s mechanics • • • • • • Cyclin-dependent kinases control cell cycle transitions Cyclins are expressed in a cyclic manner G1, G1/S, S and M cyclins direct cell cycle-specific Cdk phosphorylation Cyclin binding to the Cdk provides substrate specificity Full Cdk activation requires phosphorylation by CAK Cdk inhibition can be achieved by WeeI inhibitory phosphorylation, CKI binding or cyclin proteolysis • The SCF ubiquitin ligase mediates destruction in G1/S and requires phosphorylation of substrates • The APC/C ubiquitin ligase mediates destruction in M and requires binding of activator proteins • Cyclic transcription and gene expression also controls the cell cycle • APC/C directs protein destruction in M phase Figure 17-20b Molecular Biology of the Cell (© Garland Science 2008) The Mechanics of the cell cycle How do cyclin-Cdk switches initiate DNA replication in S phase and chromosome segregation and cell division in M phase? S- cdk low The initiation of DNA replication only once per cell cycle s-cdk high What ensures that this only happens once per cell cycle? Figure 17-23 Molecular Biology of the Cell (© Garland Science 2008) DNA replication alternates with chromosome segregation DNA REplication Inhibitory circuits Mitosis REplication origin licencing Figure 17-22 Molecular Biology of the Cell (© Garland Science 2008) These mechanisms ensure that S phase •! Starts in the right cell •! Starts on the right DNA site •! Starts at the right time •! Occurs only once per cycle MCM proteins as cancer biomarkers Squamous intraepithelial lesions The Mechanics of the cell cycle How do cyclin-Cdk switches initiate chromosome segregation and cell division in M phase? What ensures that this only happens once per cell cycle? Laskey and Coleman, Nature Reviews Cancer, 2005 G2/M cell cycle progression in less than a snapshot the stages of mitosis Activation of cyclin B/cdk1 and other mitotic kinases drives mitosis through phosphorylation of key mitotic regulators This is important for: I. Nuclear envelope breakdown and setting up a bipolar mitotic spindle > Mitosis entry II. Protein destruction feedback > Mitosis exit Anti-Mitotics: TAXOL 32 However: in vivo cell proliferation rates are low PREDECT public-private partnership to improve cancer models Partner & EFPIA clinical samples MBoC Jan 2012 (17 years since discovery of Taxol) 2D/3D on Plastic 3D in Matrix 3D Co-culture 3D Bioreactor Tissue Slices Mouse models Towards Systems Pathology Tissues Responses in the tumour microenvironment in vivo are complex, and need to be understood to understand and model therapy responses Cells www.predect.eu somatic cell proliferation To start the cycle: the mechanics of mitogenesis Proliferation is not normally determined by nutrient availability Strategic control of proliferation is through social signals (mitogens) Cells make their major decision about proliferation in late G1 Mitogens and nutrients required M M Mitogens G1 G1 G0 What do mitogens do biologically ? SS G2 M M G2 Mitogens engage (and maintain) expression of multiple genes that promote or enable cell proliferation G1-CDK E2F transcription factors Cell cycle entry Restriction Point (R) Three choices: 1. Proceed through cycle 2. Stay put for the time being 3. Enter long term quiescent state (e.g. G0 or terminal differentiation) Mitogens stimulate G1-Cdk through transcriptional responses Mitogens are found in blood serum, e.g. PDGF, EGF Inhibition of E2F G1/S transcription by Rb family pocket proteins Rb/p107/p130 ‘pocket’ Mitogens force cells to (re)-enter the cell cycle!! cyclin D Figure 17-62 Molecular Biology of the Cell (© Garland Science 2008) e2f peptide > Progressive phosphorylation of pRb proteins by G1-, G1/S-, S- and M-Cdk inactivates pRb and liberates E2Fs to activate transcription E2F TARGET GENES Positive feedback loops drive S phase entry CELL CYCLE Rb ‘pocket’ cyclin D e2f peptide E2Fs G1 S/G2 cyclin D1 Cycin D3 Jun Myc N-Myc Aurora B kinase cyclin A1 Cdk1 Cdc20 Cks1 Cks2 Hec Ki-67 KIF4A KNSL4 Polo kinase Prc1 Smc2L1 Smc4L1 Stk12 G1/S Cyclin E1 Cyclin E2 Cdc25A Cdk2 E2F1 E2F2 E2F3 NPAT Myb Mybl2 TFDP1 DNA SYNTHESIS CHECKPOINTS DNA REPAIR DEVELOPMENT Ask Cdc14B Cdc45L Cdc6 Cdc7L1 Cdt1 Dck Dhfr Dut Lig1 Mcm2 Mcm3 Mcm4 Mcm5 Mcm6 Mcm7 Orc1L PCNA Pola Pold1 Prim2A RFC1 BRCA1 BRCA2 Bub1 Bub1B Bub3 CENPE Chk1 Mad2L1 p53 TTK Bard1 Cstf1 Fen1 Mgmt Mlh1 Msh2 Msh6 Pms2 Prkdc Rad51 Rad54L Ung1 Ung2 Bapx1 Eed En2 Ezh2 Fos Hey1 HoxA4 HoxA5 HoxA7 HoxA9 HoxA10 HoxA11 HoxB9 HoxD8 Pitx1 Six1 Suz12 RFC2 RFC3 RFC4 RPA1 RPA2 RPA3 Rrm1 Rrm2 TK1 Top2A Tyms ANTI-PROLIFERATIVE APOPTOSIS CDKN1 CDKN2 E2F7 Rb RbL1 Apaf1 Bad Bak1 Bcl-2 Bid Bok Caspase 3 Caspase 7 Caspase 8 MAP3K14 MAP3K5 p73 DIFFERENTIATION Bmp2 Fts TgfA PparGC1 JunB Tead4 Figure 17-62 Molecular Biology of the Cell (© Garland Science 2008) DNA damage activates the p53 transcription factor p53 activation halts the cell cycle so that there is time to repair the damage Figure 17-63 Molecular Biology of the Cell (© Garland Science 2008) Active p53 arrests the cell cycle through the p21 CKI > The cell cycle arrest allows for the DNA damage to be repaired > If damage is severe or not repairable, cell death or senescence ensues Figure 17-63 Molecular Biology of the Cell (© Garland Science 2008) The cancer conundrum: “cell-intrinsic tumour suppression” Out of control mitogen stimulation does NOT necessarily stimulate cell division