The Cell Cycle Jonathon Pines Gurdon Institute [email protected] http://www.gurdon.cam.ac.uk/~pineslab/ New_Web_Site/Site/Lectures.html What does a cell need to do to proliferate? Chromosome Separation M Product - Substrate? Oscillator? S DNA replication 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 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 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’ ‘S phase’ Cyclin-CDK Cyclin-CDK S 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 cdc2 cdc13 (Cyclin B) Interphase Mitosis After Murray and Hunt, 1993. 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 Progesterone Inject cytoplasm Masui and Markert, 1971, J. Exp. Zool 177, 129. After Murray and Hunt, 1993. Cell 390 Unfertilized Fertilized A23187 NH&I UF abcdefghi jk abcdefghi jk abcdefghiik abcdefghij kkk Cyclins: Coincidence or cause? -xSea urchins: large eggs arrested in G1 of first mitotic cell cycle 116 Fertilisation causes large increase in translation One protein only translated after fertilisation and destroyed at each mitosis: Cyclin 68 Cyclin -Y 35 MPF S M S M -2 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) 21 Figure 1. The Patterns of Protein Synthesis in Eggs before and after Activation ?S-methionrne was added at a final concentration of 30 &i/ml to 15 ml of a suspension of 20,000 unfertilized eggs per milliliter. After 5 min, portrons of this suspension were fertilized, or made 10 pM in A23187 by addrtion of IO mM stock in drmethylsulfoxide. or made 10 mM in NH, Cl by addition of 1 M stock. Samples were taken for analysrs at 10 mm intervals starting at 25 min (lanes a) until 115 min (lanes j) after addition of the activator. Samples (lanes k) were taken at 127 min. The last two lanes labeled Uk and Fk are lighter exposures of the last unfedilrzed and fertilized lanes, respectively. The exposure of the autoradiograph in the unfertilrzed panel was four times longer than the other three to compensate for the low level of incorporation before fertilization. Bands X, Y, and Z are marked as examples of polypeptides whose synthesis IS reduced after fertrlization; and A, B. and C as the major nonhrstone proterns whose synthesis is activated. ing change is the appearance of the prominent new bands however, all the proteins are less strongly synthesized than A, B, and C after fertilization or activation with A23187, in activations that involve release of intracellular calcium. much as happens in Spisula (Rosenthal et al., 1980). The Synthesis of Some Proteins Stops after However, closer inspection reveals other interesting fea- Fertilization tures, of which the most unexpected is the behavior of Several proteins, like the examples labeled X, Y, and Z on protein A, which we shall call “cyclin” henceforth. It is the Figure 1, are made in the unfertilized egg but are very hard most strongly labeled protein at early times after fertiliza- to detect after fertilization, though they continue to be tion, but by 85 min after fertilization (lane g, fertilized) it made in ammonia-activated eggs. These comparisons are has almost disappeared. It gets stronger again in lanes h most easily made by examining the last three lanes of and i, only to decline again in lane k. These oscillations in Figure 1, in which light exposures of the final lanes (k) from the level of cyclin are extremely reproducible, as can be unfertilized (U) and fertilized (F) eggs are matched with the seen in Figures 2, 3, and 6, which show similar behavior final ammonia-activated lane. The decline in synthesis in in different batches of fertilized Arbacia eggs. prefertilization bands is easier to see in Figure 7, which shows a similar experiment in Lytechinus pictus. Cyclin Does Not Oscillate after Parthenogenetic Activation Cyclin Is Destroyed Periodically at a Particular Although cyclin synthesis is strongly induced by A23187 Point in the Cell Cycle or NH&I treatment of unfertilized eggs, under neither of Figure 2 correlates the oscillations in the level of cyclin with these circumstances does its level oscillate in the same the cleavage cycle. The experiment was conducted in the way as in fertilized eggs. This is probably because the same way as the previous one, except that additional eggs do not divide, a point enlarged upon below. samples from the suspension of fertilized eggs were fixed in 1% glutaraldehyde for later examination under the mi- Ammonia Activation Does Not Turn On the croscope. The dashed line in Figure 2 denotes the cleav- Synthesis of All the Proteins age index, measured as described in Experimental Pro- As mentioned above, activation of eggs with weak bases cedures. The other two curves show the relative intensities like NH4+ causes only about half the stimulation of protein of cyclin and protein B, as determined by densitometry of synthesis that A231 87 or fertilization gives. Is this because the autoradiographs. Label accumulates more or less lin- the synthesis of all the proteins is half turned on, or early in protein B, whereas cyclin (band A) falls precipi- because half the proteins are fully turned on? Figure 1 tously at the onset of cleavage, only to rise and fall again shows that protein B did not appear after ammonia acti- during the second cell cycle. Label in protein C also rose vation, though it is one of the most strongly labeled in linearly, but the data have been omitted from this figure fertilized and ionophore-activated eggs. For the most part, for clarity of presentation. Cyclin-CDK complexes define the state of the cell cycle Mitosis Initiation cdc13-cdc2 M Degrade cdc13 or cdc2 G2 G1 START 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. 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 Testing the Cdk threshold hypothesis Cyclin B (Cdc13) Analogue Sensitive Cdc2 Kinase 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 The Cell Cycle: Alternation Metaphase Initiation of Mitosis Cyclin B1-CDK1 G0 Cyclin A-CDK1/2 M G2 G1 START or R point Cyclin D-CDK4/6 Completion of DNA Synthesis S Initiation of DNA Synthesis DNA Synthesis Cyclin E-CDK2 Cyclin A-CDK2 Waves of cyclin-CDK kinase activity during the human cell cycle G1 S G2 M G1 Cyclin B-CDK1 Cyclin A-CDK2 Cyclin E-CDK2 Kinase activity Cyclin D-CDK4/6 time M A Growth Factors Cdc25 But Cdk2 is Not Essential for Mitosis Ortega et al., 2003, Nature Genetics, 35, p25-31 Cyclins E and A are partially redundant 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 Most mitotic cycles only require one Cdk Santamaria 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 Why are there multiple Cyclin-Cdks? • Partial Redundancy? S. cerevisiae paradigm • Tissue specific roles The Cell Cycle: Alternation Metaphase Initiation of Mitosis Cyclin B1-CDK1 G0 Cyclin A-CDK1/2 PoloM Aurora G2 G1 Cyclin D-CDK4/6 Completion of DNA Synthesis S Initiation of DNA Synthesis START or R point DNA Synthesis Cyclin E-CDK2 Cyclin A-CDK1 & 2 Coordination between mitotic kinases Kinase Domain PBD PBD • 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 Regulating a CDK Cyclin Proteolysis (Ubiquitin) Cyclin Binding Thr14 & Tyr15 Phosphorylation (wee1/mik1) Thr14 & Tyr15 dephosphorylation (Cdc25) T-loop Thr dephosphorylation (p24KAP) T-loop Thr phosphorylation (CAK) Inhibitor binding Inhibitor removal T14 Y15 Deactivate Activate CDK T160 Morgan 1995, Nature 374, 131.