Switches and Latches in the Control of Cell Division

Transcription

1 Switches and Latches in the Control of Cell Division Tim Hunt, Cancer Research UK Emerging Patterns, Singapore, 3 March 2015

2 All Living Things are Made of Cells (Which are very, very small)

3 Cells only ever come from other cells. An unbroken line of cell divisions, stretching back billions of years, connects us all, plants and animals alike

4 More often than not, questions beginning with Why are inane and of no service in scientific In biology, they sometimes discourse make sense.. If we ask why cells must divide, the answer can be given in terms of what happens if they do not. The answer is that they die, no matter what criterion of death we apply Dan Mazia, 1961

5 The Tree of Life

6 DNA is the Key to Life

7 Humans Comprise About 5 x Cells Each Cell Contains 2 Meters of DNA in About 50 Molecules We Each Make About 2 Million Cells per Second

8 The Central Dogma : DNA makes RNA makes Protein (makes Cells make Organisms?) 8

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11 DNA Replication Gives Two Identical Daughters Parent Daughter Daughter 11

12 And the second crucial process is accurate segregation of daughter chromosomes to daughter cells

13 Fluorescent Chromosomes Green chromosome movie Type to enter text

14 Chromosomes Arshad s spindle pole pole Centromeres Microtubules (red dots) Arshad Desai

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16 Nurse s Diagram Segregation Replication 16

17 The Completion Problem & Checkpoints DNA Damage, Replication Spindle Assembly DNA Damage Size

18 A Switch 18

19 Another On-Off Switch 19

20 A Latch: Shut the Gate, it Stays Shut 20

21 1961: Dan Mazia s Trigger Point of No Return 21 Figure 2. A generalized plan of the time flow of events in mitosis

22 The MBL, Woods Hole, 1979

23 A Female Sea Urchin Sheds Her Eggs

24 24 A Male Sea Urchin Oozes Sperm

25 Radioactivity in proteins Protein Synthesis Turns On After Fertilization Fertilized Unfertilized Time (min) 25

26 And the Eggs Begin to Divide, Synchronously 900X Speeded Up ( by Christian Sardet) 26

27 Frog Oocyte Maturation Triggered by Progesterone John Gerhart 1 mm 27

28 1971: Masui and Markert Define MPF Maturation-Promoting Factor An Enzyme that Catalyzes Cell Division!

29 MPF Levels Oscillate in Meiotic and Mitotic Cell Cycles (High in M-phase, Low in Interphase)

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32 August 1-2, 1982

33 When Cyclins go Up, do they Drive Division?When Cyclins go Away, do they Allow Progress? Concentrations Cyclin A Cyclin B Control Protein

34 Lee Hartwell s Cell Division Cycle Mutants (Genes Control Cell Cycle Transitions)

35 WT Paul Nurse s CDC Mutants cdc2 wee1 Wild-Type wee1

36 Fission Yeast cdc2 Segregation Replication CDC2 8 Budding Yeast 36

37 Focus on Cdc2/Cdc28 Controls G1-S AND G2-M Mutates to Inactive OR Hyperactive Encodes an Inactive Protein Kinase Found in Humans, Too! (Where Does Cyclin Fit In?) 37

38 A Protein Kinase Phosphate X more active!

39 The Complex Control of Cdc2 cdc2

40 The Cdk1-Wee1 Alternative Interphase Cdk1 Wee1 Mitosis The Parmenides Problem: How Can Anything Ever Change? 40

41 DNA Damage Control of Cdc2 Cdc2 41

42 SO SIMPLE WHEN YOU UNDERSTAND The Cell Makes Cyclin, MPF Turns on, and the Cell Enters Mitosis The Cell Degrades Cyclin, MPF Turns off, and the Cell Exits Mitosis

43 As far as we know, CDKs can only do something by phosphorylating target proteins How many substrates? How much phosphorylation?

44 Interphase Mitosis MPF There are actually hundreds of substrates, many with multiple phosphorylation sites P 44 P P P

45 How Do You Get Out Of Mitosis? Mitosis Interphase Who Does This? Geneticists: PP1 Biochemists: PP2A P P P P P P P P

46 The Futile Cycle Problem... Kinase Either/Or NOT Both At Once Phosphatase 46

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48 Kinase Phosphatase 48

49 Satoru Mochida in Okinawa Satoru Mochida 49

50 Frog Egg Extract: Pure Cytoplasm Spin Lohka & Maller Murray Ohsumi 50

51 Cell-Free Cell Cycles! 51

52 Regulated Kinase or Regulated Phosphatase? Or Both? Apc3 Cyclin B2 H1 kinase Kinase Either Phosphatase Or Phosphatase Kinase

53 A Mitotic Phosphatase Assay Mitotic Phosphatase Substrate S50 of Cdc20 Maltose-Binding Protein sslntsantstlspmkasnrshsss + Cyclin A-Cdk2 and -[ 32 PO4]ATP MBP

54 Two Phosphatase Activities: Calcineurin and Phosphatase X Cyclosporin A Okadaic Acid 54

55 Phosphatase X Turns Off in Mitosis Phosphatase activity 55

56 What is Phosphatase X? How is it Regulated? 56

57 Depletion of PP2A-B55δ by Antibodies Removes Phosphatase X Activity

58 How Does Control Work? Two Possible Explanations... Model 1 PP2A-B55 is directly inhibited by phosphorylation 58

59 Model 2: PP2A-B55δ is inhibited by a pseudo-substrate (A Phosphorylatable Phosphatase Inhibitor)

60 Clues from Reading and Genetics Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila Jiangtao Yu,1 Shawna L. Fleming,2 Byron Williams,1 Erika V. Williams,1 ZeXiao Li,1 Patrizia Somma,4 Conly L. Rieder,2,3 and Michael L. Goldberg

61 Greatwall Kinase is a Phosphatase Inhibitor 2009 (But Greatwall does NOT Phosphorylate PP2A 61

62 ARPP19 and Endosulfine Greatwall Site 62

63 Clues from Flies The authors were puzzled, because Cdk1 activity was high, yet the cells were unable to enter meiosis... 63

64 Ensa Gets Phosphorylated in Mitosis in the Cycling Extract Apc3 Cyclin B Cdc25 Wee1 Ensa Greatwall But everything happens at the same time!

65 Ensa Depletion Prevents Mitosis Control Δ-Ensa 65

66 Depleting PP2A-B55-δ Accelerates Entry into Mitosis, and Keeps the Extract There Mock B min Apc3 CycB2 B CDK targets (S P -P-X-K/R) Cdc2Y15 P H1 kinase Is PP2A-B55 the Main Mitotic Phosphatase?

67 Bela Novak 67

68 A Tricky Ambiguity: Phosphatases Work Both Downstream And Upstream of CDK1 And There Are Two Positive Feedback Loops 68

69 New Facts 1. Greatwall is an essential gene in mice. 2. But somatic cells show no problems entering mitosis. 3. Mouse oocytes lacking Greatwall enter meiosis I absolutely normally and complete MI, but after that they can t keep Cdc2 kinase ON and fail to perform meiosis 4. Yeast II. have the same system, but it controls survival in the face of starvation, not cell division. 5. THIS DOES NOT MAKE SENSE! 69

70 A Latch with a Delay Timer? ESSENTIAL for Entering Mitosis, AND for Staying in Mitosis

71 There is only one way to find things out... You know, the proper method for inquiring after the properties of things is to deduce them from experiments Isaac Newton, 8th July 1672

72 Pierre Gilles de Gennes Science is clearly a form of art, with the same invention and the same doubts. There are major differences, however: one is the difficulty of communication. An Indian playing his flute in the streets of Bogota invents a new tune: with ten seconds, with and ten passer-by seconds, may and be passer-by struck it may possibly be struck for their by it whole possibly f life. But in our trade, a beautiful discovery can be transmitted only to people who have been through a long specialised education. We must do our best to keep in contact with our fellow citizens, but we often fail.

73 Pierre Gilles de Gennes (2) The analogy between chemists and sculptors, or physicists and printers, is rather close. The great scientists of the quantum period produced heroic pictures of the whole universe. After this, there is a natural trend towards artists of small details. Then, the competition of black-and-white with ten seconds, and passer-by may be struck by it possibly f photography with painting catalyses a deep change. Exact reproductions of nature become standard operation. What is discovered at this stage, however, is the difficulty of extracting a single vision from a scene, an impression. We have similar problems in soft-matter physics. Simulations and other numerical exercises are the analogue of photography. What we need is a simple impressionist vision of complex phenomena, ignoring many details.

74 Pierre Gilles de Gennes (3) Incidentally, the artistic professions suffer from many parasites: among others, the art critics, or commentators. Fortunately, we do not have the counterparts of art critic in with ten seconds, and passer-by may be struck by it possibly f our sciences (although some referees tend to mimic this style ). From Soft Interfaces, CUP.

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