1. You may not believe it but by the end of the semester This will make sense! Hanahan and Weinberg, Cell 100:57-70 (2000)

2. Cell cycle and its control

3. Cells must be able to proliferate - during development - wound healing - stem cells in blood, small intestine, immune system For cells to copy themselves they need to: - Grow; make more stuff; e.g. proteins, lipids - Copy their genetic material - Segregate contents to daughter cells, especially… - Segregate replicated chromosomes to daughter cells

4. Many of the images in the cell cycle part of the course are taken from The Cell Cycle, by David O Morgan (New Science Press) Interphase cells duplicate chromosomes Mitosis cells segregate duplicated chromosomes into two daughter cells The Cell Cycle

5. Interphase has 3 periods: G1, S, G2 G1: cells decide whether to divide or not: - Have I grown big enough to enter the cell cycle? - Am I OK? Restriction Point / START

6. S: chromosomes are duplicated G2: cell prepare to enter mitosis by asking: - Have I completed DNA synthesis properly? - Am I OK? Execution of these decisions commits a cell to complete a full division cycle

7. The main jobs of the cell cycle: 1.To accurately transmit the genetic information! 2.To maintain normal ploidy; i.e. diploidy! Regulatory mechanisms: - Accuracy in the “assembly line” (e.g. DNA polymerase) - Extrinsic regulatory mechanisms (all processes follow a correct order)

8. Let’s remind ourselves some basic stuff Starting with the S phase

9. Early G1 Pre-replicative complex (origin licensing) Early S Activation of helicase; Assembly of pre-initiation complex Helicase

10. DNA does not come naked It is packed into chromatin Mainly, histone proteins Thus, duplicating chromosome = duplicating DNA and duplicating histones In addition, we need to repack the duplicated DNA

11. Histone synthesis increases sharply during the S phase Increase in transcription, in processing, and in stability

13. Chromatin Inheritance -Telomeres Cis-elements: sequences recruiting proteins that modify histones -Centromere Epigenetic mechanisms, not clearly understood Reproducing chromatin organization during the S phase

15. Mitosis During the S phase, the duplicated DNA is rearranged through cohesion to form two sister-chromatids attached to each other by cohesins Gradually, the cohesins will be removed to allow sister- chromatid separation

16. - Sister-chromatids condense - Centrosomes move to opposite poles of the cell, nucleating microtubules (MTs) - Nuclear envelope breakdown Prophase

17. Prometaphase - Nuclear envelope breakdown is completed - The centrosomes nucleate MTs towards each other, forming the spindle MTs - The growing (+) ends of the MTs capture the chromosomes at the site of the centromere through a protein complex called the kinteochore

18. Kerry Bloom Kinetochore Microtubule Kinetochore Centromere Ted Salmon

19. Prometaphase - Nuclear envelope breakdown is completed - The centrosomes nucleate MTs towards each other, forming the spindle MTs - The growing (+) ends of the MTs capture the chromosomes at the site of the centromere through a protein complex called the kinteochore

20. At the end of the day: Metaphase

21. Now, we are ready for Anaphase

22. Anaphase (A+ B) Salmon lab

24. Silverman-Gavrila lab Mitosis Prophase Chromatid condensation Prometaphase Kinetochore-MTs binding Spindle assembly Metaphase Chromosomes align at the midline Telophase and Cytokinesis Birth of two daughter cells Anaphase Segregation of sister-chromatids

25. Cell cycle is controlled Cells can be fused

26. - Fuse S phase cell with G1 cell: The G1 nucleus enters S phase Rao and Johnson (1970) Cell fusion experiments - Fuse M phase cell with interphase cell: Interphase nucleus enters M

27. Cell cycle has a clock, regulated by promoting factors and checkpoints

28. For example, anaphase- metaphase transition will take place only if ALL the kinetochores are attached to MTs If the checkpoint regulators are compromised, unattached chromosome might be lagging behind, resulting in aneuploidy

29. G1

30. Cyclin Dependent Kinases Regulate the Cell Cycle

31. Experimental Systems Important for Cell Cycle Studies Arbacia punctulata Xenopus laevisSchizosaccharomyces pombe Saccharomyces cerevisiae

32. Budding Yeast: Saccharomyces cerevisiae

33. Lee Hartwell Hartwell was interested in the protein synthesis machinery Budding Yeast: a genetic eukaryotic model organism Let’s look for mutants that cannot synthesize proteins

34. Isolating temperature sensitive mutants in haploid yeast

35. Lee Hartwell Budding Yeast: Saccharomyces cerevisiae Serendipity, our old friend Brian Reid, an undergrad, needs to look at a microscope to follow a mutant. They realize that bud size stores information about the cell cycle Brian Reid

36. Permissive (low) temperature (mixed population of cells in different stages of the cell cycle) Restrictive (high) temperature An assay for isolating cdc mutants cdc: cell division cycle mutants

37. cdc mutant growing at permissive temp cdc mutant growth arrested after 6 hrs at restrictive temp Temperature sensitive cdc mutant

38. Genetic and descriptive analysis discover the interactions between the mutants

39. DNA How to clone cdc genes in yeast? Let’s say you have a candidate sequence cdc28 (-) If the candidate sequence complements (rescues) the mutated phenotype: that’s your gene! WT

40. How to Clone cdc Genes in Yeast Gene Z

41. Many of the cdc genes encode proteins needed for DNA replication

42. cdc28 gene encodes a kinase

43. Sir Paul Nurse Fission yeast: Schizosaccharomyces pombe

44. cdc genes encode proteins needed for the G2-M transition: studies in s. pombe cdc2 D = gain of function mutant

45. Cloning cdc2 The same approach used in budding yeasts: complementation by a library Only using a budding yeast library

46. START/Restriction Point Cdc2 (fission) Cdc28 (budding)

47. This is all great Yeast are really cute and interesting Can we really learn something from that about humans? Schizosaccharomyces pombe

48. Sir Paul Nurse Crazy idea Let’s try to complement (rescue) the cdc2 (-) mutant of pombe with a human cDNA library It worked for us with budding yeast genes. Why not try human genes?

49. Human cdc2 rescues cdc2 mutants Elongated cdc2 mutants, failing to undergo mitosis cdc2 mutants, complemented by a human cdc2 gene Melanie Lee

50. Summary - A genetic approach in fission and budding yeasts reveal genes that are essential in promoting the cells through the cell cycle -These genes encode kinases proteins and are called CDKs for Cyclin-Dependent Kinases Cdk1 = the protein encoded by cdc2/CDC28

51. Tim Hunt Woods Hole Marine Biological Laboratory

53. can be stimulated to lay lots of eggs Sea urchins The summer project: to follow protein synthesis upon fertilization by following incorporation of S 35 - Met and getting samples every 10’

54. Proteins X,Y,Z are synthesized only in unfertilized eggs Proteins A,B,C are synthesized upon fertilization

55. Protein A disappears 10’ before completion of mitosis mitosis In clams two proteins, A and B, express this cyclic behavior

56. Cyclins are synthesized and degraded in a cyclic manner and with correlation to the cell cycle Protein Level Time cyclin A cyclin B MMM Something needs to go away in order for the cell cycle to proceed

57. Yeast genetics Needed for promoting cells through the cell cycle CDK Biochemistry in sea urchin Appear in correlation with the cell cycle Cyclin Time to bring them together