Fluorescence Micrographs of Chromatin, Keratin, and Microtubules

1. Fluorescence Micrographs of Chromatin, Keratin, and Microtubules of mitosis of newt lung cells

2. Chapter 14 & 15Reading ListPages 560 – 569(On page 566, for controlled proteolysis, only a general understanding – no mechanism needed;Last two paragraphs on page 566 and pages 567-569 – a general understanding only- no need to memorize)Pages 642 – 646(For the extrinsic pathway on page 643 and other pathways on pages 644-646 – only a general understanding – no need to memorize)

6. Replication occurs during a defined period of the cell cycle

7. Identification of S Phase Cells by Incorporation of Radioactive Thymidine

8. Determination of Cellular DNA Content

9. Cell-fusion Experiments to Identify Cell cycle control proteins

10. Cells contain Factors that Stimulate Entry into Mitosis

11. Progesterone stimulates meiotic maturation of Xenopus oocytes in vitro

12. A diffusible factor in arrested Xenopus eggs promotes meiotic maturation in the absence of progesterone!Microinjection Assay:MPF Identified!

13. Microinjection Assay:MPF Identified!

14. MPF Activity in Xenopus oocytes, eggs,and early embryos peaks as cells enter meiosis and mitosis.MPF activity was determined by the microinjection assay and quantitated by making dilutions of cell extracts

15. The Discovery of MPF

16. Biochemical Column Chromatography characterized MPF as a heterodimeric complex composed of a cyclin subunit and a CDK subunitThe challenge was to determine how these two subunits could induce mitosis?

17. Unusual aspect of Early Xenopus embryos

18. Fluctuation of cyclin and MPF levels during the cell cycle of Xenopus

19. Experimental detection of cyclical synthesis and destruction of mitotic cyclin in sea urchin embryosA suspension of sea urchin eggs was synchronously fertilizedby the addition of sea urchin sperm, and radioactive 35 S Methionine was added. At 10 minute intervals beginning 16 minutes after fertilization, samples were taken for protein analysis by SDS-PAGE and for detection of cell cleavage by Microscopy

20. Experimental detection of cyclical synthesis and destruction of mitotic cyclin in sea urchin embryos

21. Regulation of mitotic cyclin levels in cycling Xenopus early embryonic cells

22. Budding Yeast (S.c)

23. Regulation of Cell Cycle by Budding Yeast

24. Cell Cycle studies using fission yeast S.pombe

25. S. pombe Cell Cycle Regulators

26. Recessive and dominant S.pombe cdc2 mutants have opposite phenotypes

27. Regulation of the kinase activity of S.pombe mitosis promoting factor (MPF)

28. Cdc-25 and Wee1 have opposing effects on S.pombe MPF activity

29. Targets of MPF

30. Strucural models of human CDK2 which is homologous to the S.pombe cyclin-dependent kinase (CDK)

31. Subcellular localization during the cell cycleHeLa cells injected with cyclin B1 linked to GFP G2 phase Prophase of Mitosis

32. Mammalian Cell Cycle

33. Cell Cycle Checkpoints

34. Mechanism of action of two DNA-damage checkpoints

35. P27: a Cdk inhibitor that arrests cell cycle progression p27-/- p27+/+ Thymus Gland

36. Apoptosis

37. The survival of motor neurons depends on the size of the muscle target field they innervate : Experiments with Chick Embryos In the Early 1900’s!

38. Apoptosis

39. Apoptosis: Genetic Studies in C.elegans947 non gonadal cells generated during development of the adult hermaphrodite form : 131 cells undergo cell deathcan be followed by Nomarski Interference Microscopy

40. Ced-3 gene in C.elegans is required for Cell death ced-3 mutation 131 cells survive

41. Genetic Studies in C.elegans Ced-3 mutation in C.elegans:All 131 cells survive!Ced-4 mutation in C. elegans:All 131 cells suviveCed-9mutation in C.elegans:All cells die during embryonic life – adult form never develops!Ced-9/Ced-3 Double Mutation in C.elegans:Cell Death Does Not Occur!Therefore, Ced-9 acts upstream of Ced-3 to suppress the apoptotic pathway

42. Confluence of genetic studies in Worms and Studies on Human Cancer cellsRegulator Proteins IdentifiedHuman bcl-2 gene cloned from human follicular lymphomas, since a mutated version of this gene was shown to act as a oncogene that promotes cell survival.The human Bcl2 protein was shown to be homologous to the worm CED-9 protein!A human bcl-2 transgene was able to block the extensive cell death found in ced-9 mutant worms! Thus both proteins act as regulators that suppress the apoptotic pathways.

43. Effector Proteins identifiedIn Worms, the ced-3 gene codes for the CED-3 protein which is a CaspaseEnzymeIn vertebrates, the gene ced-3 was shown to code for a caspase enzymeCaspases recognize and cleave short amino acid sequencs in many different target proteins in the cells

44. Cell Culture StudiesAdaptor Proteins identified that coordinate the action of regulatorsExpression of C.elegans CED-4 in a human kidney cell line induces rapid apoptosis!This can be blocked by coexpression of the negative regulator CED-9 (or mammalian Bcl-2) indicating that CED-9 opposes CED-4 action.CED-9 can interact directly with CED-4 Thus the pro-apoptotic function of CED-4 is directly suppressed by the anti-apoptotic function of CED-9.CED-4 also binds directly to the CED-3 caspase and promotes activation of its protease activity.Biochemical studies also indicate that CED-4 can simultaneosuly bind to both CED-9 and CED-3.Above results fit with the Genetics, which shows that the absence of CED-9 has no effect if CED-3 is also missing (ced-3/ced-9 double mutants have no cell death, like ced-3 mutants).

45. CED-9 – CED-4 Interaction

46. Overview of the evolutionarily conserved apoptotic pathway in C.elegans and vertebrates Regulator Adaptor Effector

47. For General KnowledgeProposed Intracellular Pathways leading to cell death by apoptosis or to trophic factor-mediated cell survival in mammalian cells.

48. Study Model in your Text BookChapter 15 Pages 643-646No need to memorize ModelOnly Understand it!