1. Announcements Monday, April 16: the cell cycle, pp. 586-598. Wednesday, April 18: protein synthesis, pp. 684-695. Friday, April 20: protein targeting, pp. 695-711. –Homework problems 19-10, 14 due.

2. Day 39 Outline/Objectives Regulation of the cell cycle A.MPF B.Cyclin C.Cell cycle checkpoints 1.G2 2.Spindle assembly 3.G1 After reading the text, attending lecture, and reviewing lecture notes, you should be able to: Discuss the evidence for and components of mitosis-promoting factor. Discuss the three main cell cycle checkpoints (G1, spindle assembly, and G2) and how passage is controlled through the these checkpoints.

3. Regulation of the Cell Cycle: Cell Cycle Checkpoints E.g. Oocytes Differentiating cells

4. Evidence that Cytoplasmic Signals Control the Cell Cycle

5. Evidence for a Maturation (or Mitosis) Promoting Factor (MPF) – Masui et al. 1971

6. The Discovery of Cyclin Tim Hunt (Nobel Prize co-winner, 2001) and MBL Physiology class, Woods Hole, MA, in 1982 Fertilized sea urchin eggs in presence of 35 S-methionine Ran SDS-PAGE gel and autoradiography: Saw the following results: 0 15 30 45 60 75 90 105 120 135 All other proteins “Cyclin” Minutes post-fertilization Mitosis 1 Mitosis 2

7. Synthesis and Degradation of Cyclin correlates with MPF Activity

8. Cell Division Cycle (cdc) Mutants in Yeast (late 1980’s) Hartwell (Nobel Prize, 2001) et al., working with budding yeast S. cerevisiae –Found temperature-sensitive mutants stuck in some point of cell cycle Nurse (Nobel Prize, 2001) et al., working with fission yeast S. pombe –Found gene they called cdc2, essential for passing G2 checkpoint –cdc2 turned out to be a new protein kinase - cyclin dependant kinase (Cdk) - with counterparts in all eukaryotic cells.

9. G2 Checkpoint Control by MPF Active MPF = Mitotic Cdk + mitotic cyclin Cdk is cyclin-dependant kinase MPF controls G2  M by phosphorylating and activating proteins involving in: –Chromosome condensation –Nuclear envelope breakdown –Spindle assembly –It’s own self-destruction

10. G2 checkpoint

11. Spindle Assembly Checkpoint Controls Metaphase  Anaphase MPF (+) anaphase promoting complex, which destroys: 1.Securin, which allows separin protease to cleave cohesin. 2.Mitotic cyclin, which causes loss of MPF activity, leading to chromosome decondensation and envelope reformation. Mad2 signal stops coming from kinetochore MT’s once attached, also (+) APC. (+)

12. G1 checkpoint Controlled by G1 Cdk-cyclin G1 cyclin levels also vary with the cell cycle Many additional levels of phosphorylation, dephosphor- ylation regulate.

13. Growth Factor Signaling Through the Ras Pathway  crossing of G1 checkpoint Ras*, Raf* MAPK cascade Activation of nuclear TFs Activation of G1 Cdk cyclin genes: G1  S

14. Summary: Cell Cycle Control A “clock” is running within the cell - of synthesis and degradation of cyclins - which activate cyclin-dependant kinases (Cdk’s), which activate other proteins to cause checkpoint transitions. The “clock” is adjusted (cyclins and Cdk’s are regulated) by other proteins, based on: –External signals (growth factors) –Internal signals (correct completion of previous step)