Chapter 17

1. Chapter 17 • The Cell Cycle and Programmed Cell Death • Overview • Molecular Parts list • Intracellular Control Mechanisms • Apoptosis • Extracellular Control

2. Overview of the Cell cycle

3. Metaphase Details - Classical Stages from Microscopy

4. Interphase:3 generic sub-phases G1 - gap1 or growth S - synthesis G2 - gap 2

5. Alternative cell cycles and cytokinesis strategies in 2 yeast species

6. Genetic analysis of cell cycle control with temperature sensitive cdc mutants in yeast Ts cell-division-cycle (cdc) mutants are conditional, growth inhibited at high temp only Cdc mutants distinguished from others by microscopy. How? Cdc mutants can be organized into complementation groups. How?

7. S. cerevisiae cdc15 cell cycle arrest A - wild type -non-synchronized cells B - cdc15 cells grown at the restrictive temperature

8. Huge Xenopus oocytes for cell cycle biochemistry Fertilized eggs undergo synchronous cell division without transcription Oocytes or eggs can be injected with proteins, RNA, inhibitors etc.

9. Cell-free oocyte cytoplasm can “cycle “ in a test tube Fractionation of an active extract can lead to identification of cell cycle components

10. Identifying cell-cycle phases in cell populations Recent DNA synthesis can be detected by pulse labeling Brief exposure to 3H thymidine or Br-dUTP can identify cells active in DNA replication Flow cytometry can characterize (and separate) cells based on DNA content

11. The Laundromat model of the cell cycle Desirable control features Unidirectionality Feedback to make sure that necessary prior processes are complete Error recognition - in case things don’t work normally External controls to modify the cycle for different cell types/situations

12. Generic cell cycle checkpoints

13. Cyclins and cyclin dependent kinases (Cdk’s) control the cell cycle Cyclins - regulatory subunits Synthesized and degraded cyclically Cdks - catalytic subunits Kinase (phosphotransferase) activity is dependent on cyclins Cdk levels remain relatively constant

14. Fictitious “Core” cyclin Cdk control cycle S cyclin synthesized during G1 Activates Cdk for phosphorylation of S-phase targets (DNA replication) S-cyclin degradation system also activated M cyclin synthesized during G2 Activates Cdk for phosphorylation of M-phase targets (segregation) M-cyclin degradation system also activated

15. G1/S cyclins commit the cell to a new round of replication S cyclins trigger initiation of replication M cyclins promote mitosis G1 cyclins (optional) initiate a new cell Multiple cyclins and Cdks are widely conserved

16. Structural basis of Cdk Activation

17. Other modulators of Cyclin/Cdk Activity Some kinases inhibit Cyclin/Cdk activity Wee1 (small cell mutant) inhibits by phosphorylation Cdc25 phosphatase reverses the effect Cdk inhibitory protens (CKIs) inhibit by direct binding

18. Regulated Proteolysis Anaphase promoting complex (APC) targets M-cyclin SCF targets CKI

19. Ubiquitinylation can target proteins for destruction

20. Intracellular Control Mechanisms S phase Cyclin/Cdk complexes issue DNA replication licenses M phase Cyclin/Cdk complexes trigger entry into mitosis DNA replication checkpoint Chromosome separation potentiated by M-cdk Triggered by proteolysis (APC) Spindle-attachement checkpoint M-Cdk inactivation required for exit from mitosis Cdks are inactived by CKIs in G1

21. Cell fusion experiments can detect positive and negative regulatory components

22. S-Phase Cyclin–Cdk Complexes (S-Cdks) Initiate DNA Replication Once Per Cycle Unphosphorylated ORC binds to origins Cdc6 binds to ORC in G1 Mcm (mini chromosome maintenance) proteins assemble to complete the Pre replication complex (PreRC) S-Cdk phosphorylates Cdc6 and subsequently ORC and Mcm ORC remains phosphorylated until the end of the cell cycle After replication Mcm is exported from the nucleus

23. Origin recognition complexes are phosphorylated by S-Cdks

24. The Activation of M-Phase Cyclin–Cdk Complexes (M-Cdks) Triggers Entry into Mitosis M-cyclin levels rise in G2 M-Cdk is held in check by phosphorylation until Cdc25 phosphatase produces a small amount of active M-Cdk M-Cdk activates Cdc25, inactivatesWee1 and thus promotes a burst of Active M-Cdk

25. Activation of M-Cdk to begin mitosis

26. The DNA replication checkpoint Hydroxyurea blocks replication Caffeine prevents proper checkpoint signaling

27. Proteolysis APC destroys securin releasing separase Separase destroys cohesin and allows chromosome separation that begins anaphase Degradation of M-Cyclin is required for completion of anaphase

28. APC

29. Exit from Mitosis Requires the Inactivation of M-Cdk

30. Sphase initiation

31. Nutritional control

32. DNA damage sensed by P53

33. Cell cycle overview

34. Apoptosis Programmed cell death is a normal cellular function used in Development to remove excess cells Adulthood to maintain the proper cell numbers Disease to remove damaged cells Caspases are proteases that cleave each other and cellular components to disassemble cells Caspase activity can be triggered by internal or external signals

35. Sculpting in Development

36. Necrosis vs Apoptosis Injured Cells (A) can undergo Necrosis spilling their contents and triggering inflammatory responses Appoptosis (B & C) results in the orderly destruction of the cell and packaging into membrane bound vesicles

37. Caspases trigger apoptosis by a cascade of proteolyis

38. Extracellular Activation of Apoptosis

39. Intracellular Activation of Apoptosis

40. Mitochondrial permeability is controlled by Bcl-2 family proteins Bcl-2 family proteins assemble into homo and hetero oligomers Bcl-2 and Bcl-Xl block cytochrome C release from mitochondria and inhibit apoptosis Bad, Bax and Bak promote apoptosis IAP (inhibitors of apoptosis) inhibit caspases, synthesized by viruses to delay apoptosis until after viral replication is complete

41. Extracellular control of cell size, cell division and cell death Regulated by extracellular signals Mitogens - stimulate cell division Growth factors - stimulate cell growth Survival Factors - inhibit apoptosis Signals are transduced through Cell surface receptors

42. Platelet Derived Growth Factor PDGF is released by platelets in at the site of a wound (triggered by a protease cascade of clotting factors) Blood Serum (cleared by clotting) but not plasma (cleared by centrifugation before clotting) supports growth of epidermal cells

43. Mitogen Signal Transduction Pathway

44. Overactivity of cell cycle stimulators normally triggers arrest and apoptosis

45. Normal somatic cells have limited growth potential Progressive increases in Cdk inhibitors (CKIs) Progressive shortening of telomeres in cells that do not express telomerase Telomeres shortened with each replication cycle Non-telomere sequences exposed at chromosome ends are recognized asDNA damage

46. Growth Factors A signal transduction through phosphatidyl inositol pathway Kinase cascade leads to increased translation Some factors stimulate both growth and cell cycle progression

47. Nerve growth factors can influence both rate and direction of growth

48. Neural pathfinding depends on cell growth and apoptosis Cell growth initially responds to gradients of growth factors. Not all nerve cells reach their target Neurons that do not make it (or make it late) die

49. Death by neglect: continuous stimulation required to prevent Apoptosis

50. Normal cells need both mitogens and “anchorage”- to enter a new cell cycle