Topic 2: The Cell Cycle

1. Topic 2:The Cell Cycle

2. Overview • Cell cycle phases • Review of DNA Replication • Mitosis • Cytokinesis • Cell Cycle Regulation

3. Phases of the Cell Cycle • Key concepts • Undergoes cell division only when asked to • Makes sure cell division steps proceed with quality control • Makes 2 daughter cells with exact same chromosomes and chromosome number

4. Four Phases of Cell Cycle • G1—interphase • S phase—synthesis phase • G2---continuum of interphase • M phase---mitosis

5. M phase • Mitosis • Division of DNA and cytoplasm into 2 daughter cells • 6 phases • Prophase • Prometaphase • Metaphase • Anaphase • Telophase • Cytokinesis

6. G1 phase • Cells growing but not dividing • DNA not replicated

7. S phase • DNA replication occurring • Centrosome is replicated

8. G2 phase • Cells are growing but not dividing • DNA is duplicated • Cell preparing for mitosis

9. Cell cycle length • Tissue culture • Generation time • S phase • Exposure to radioactive thymidine • G1—cells arrested in G1 are said to be in G0

10. S phase

11. Replication Licensing • Ensures that any given DNA replicates once and only once in a given S phase • Binding of minichromosome maintenance proteins (MCM) to replication origin in presence of origin recogntion complex (ORC) and helicase loaders • Once replication begins, MCM displaces and Cdkphosphorylates ORC and helicase loaders • Geminin blocks binding of MCM to DNA

12. Replication Licensing

14. Telomeres

15. Telomerase • present in germ cells, few other proliferating cells • In other cells, telomeres shorten with each cell division • Too short for capping proteins • apoptosis

16. Mitosis Review

17. Parts of the Chromosome

18. Phases of Mitosis • Prophase • Prometaphase • Metaphase • Anaphase • Telophase • Cytokinesis

19. Prophase • Interphase MTs are replaced by astral MTs • Condensation of the chromosomes • Centrosomes to opposite sides of nucleus

20. Prometaphase • Fragmentation of nuclear envelope • Each chromatid has a kinetochore and attached to opposite spindle poles • Polymerization/depolymerization of MTs move chromosomes to metaphase plate

21. Metaphase • Chromosomes aligned at metaphase plate • All chromatids are attached to spindle MTs

22. Anaphase • Separation of sister chromatids • Chromatids pulled to opposite poles • Signal is activation of the anaphase promoting complex • Chromosomes are moved by MT shortening

24. Telophase • Chromosomes decondense • Reassembly of nuclear envelope • Contractile ring is assembled

25. Microtubules of Mitosis • Mitotic spindle • 3 classes of microtubules • Kinetochore MTs • Polar MTs • Astral MTs

26. Kinetochore MTs • Finds chromosomes and attaches to the kinetochore at the centromere • Moves chromosomes around the cell • Attached to kinesin-13 • Kinesin-13 attached at kinetochore • Encourages MT depolymerization

27. Polar MTs • Extend from MTOC to opposite pole • Overlaps in an anti-parallel manner • Causes cell to elongate • Kinesins drive apart MTs from opposite poles

28. Astral MTs • Extends from spindle to cell cortex • Anchors centrosome to cell

29. Motor Proteins

30. Cytokinesis • Cell cortex assembles into cytokinetic furrow • Ring of actin and myosin II that bisects the mitotic spindle • Myosin II walks along the actin filaments • Contractile ring constricted thru action of myosin and actin • Assembly/disassembly of actin-myosin complex regulated by Rho

33. Cell Division can be Asymmetrical • Usually symmetrical • Exception (examples) • Yeast • Frog embryos • Development of egg cells

34. Cell Cycle Regulation • Generation time variable in multi-cellular organisms • Rapidly dividing • Slowly dividing • Not dividing • Dividing when induced

36. TOR • Target of rapamycin • Central role in signalling network that controls cell size • Coordinates cell cycle progression • Activated in presence of nutrients and growth factors • Facilitates entry into S phase

37. Experimental Evidence for Cell Cycle Regulation • Heterokaryon Experiments • Xenopus Experiments • Yeast mutants

38. Heterokaryon Expts

39. Maturation Promoting Factor

40. S. pombe studies • Mutant yeast • If small cells—too many divisions • Wee-1 • Encodes inhibitory kinase • Big yeast cells—not enough cell divisions • Cdc-25 • Activating phosphatase

41. Further Refinements • Isolation and purification of protein components • Maturation promoter factor • Consists of cyclin and cyclin-dependent kinase (CdK)

42. CDK and cyclins • CDK activated by cyclins • CDK levels remain constant throughout the cell cycle • Cyclins usually cycle • Different cyclins required at different phases of the cell cycle • Cyclins A and B contain destruction box; Cyclins C and D a PEST sequenceubiquitin-mediated cyclin proteolysis at the end of a cell cycle phase

43. Cyclin/Cdks • Cyclin—no enzyme activity • Bind to kinase to activate them • Cdk—catalytic subunit of the complex

44. Activation of Cdks • Cyclin necessary but not sufficient • Must get a specific set of phosphorylations

46. Cell Cycle Checkpoints • Transition from one phase to another in an orderly fashion • Key regulators are cyclin-dependent kinases (CDK) • Family of serine/threonine protein kinases

48. Checkpoint ControlRestriction Point • Passing thru restriction point commits cell to division • Senses availability of • Nutrients • Presence of growth factors • Cell size

49. DNA Damage Checkpoints • Check for DNA damage and provides time for repair • Occur before cell enters S phase (G1-S checkpoint) • After DNA replication (G2-M checkpoint)

50. G1-S checkpoint • Cell arrest is p53-dependent • DNA damage results in rapid increase in p53 levels