1. Cell cycle and checkpoint control

2. How do we follow the cellular processes during cell division?
Under microscopy!
Mitosis: chromosome condensation and segregation.
Time to complete one cell division cycle.
How about DNA replication?

3. Drawing of chromosomes during mitosis by Walther Flemming, circa 1880

4. When DNA is replicated?

5. How to measure DNA replication during cell division?
Pulse label H3-thymidine in non-synchronized cell population, then measure the kinetics of appearance of labeled mitotic cell!

6. 1-hour radioactive thymidine ( ) pulse (DNA synthesis).
Labeled nuclei
Labeled mitotic cell
1-hour radioactive thymidine ( ) pulse (DNA synthesis).
4% of cell nucleus are labeled. So, interphase has at least 2 phases (?). Can we see labeled mitotic cell in this experiments?

7. After pulsing, no labeled mitotic cell was observed.
When will you see the first labeled mitotic cell?
Why then the number of labeled mitotic cell increase with time?
Why the number of labeled mitotic cell decrease then?

8. G2
Tcell cycle Ts G2

9. TG1 = Tcell cycle – Ts – TG2 - Tmitosis

10. How to identify control genes of cell cycle in eukaryotes?

11. Experimental approaches to identify control genes of cell cycle in eukaryotes:
— Genetic approach
Yeast CDC (cell division control) genes
— Biochemical approach
Xenopus oocytes

13. Brief history of career development of Dr. Leland H. Hartwell
High school – sports, girls and car
Make up at a two years junior college
BS in physics from Caltech in 1961
Ph.D. in biology from MIT in 1964
Post doctor of Dr. Renato Dulbecco (Nobel Laureate in 1975 with Temin and Baltimore)
Faculty of UC Irvine : from cancer to yeast
Faculty of U of Washington, Seattle 1968-

14. Genetic approach to study the regulation of macromolecular biosynthesis (protein, RNA and DNA)
1, to pick up a good model system.
2, to isolate conditional mutant such as temperature sensitive
mutant (ts) which grows normally in low (permissive)
temperature but no longer grow at high (non permissive)
temperature.
3, to examine defect occurred in the mutant: DNA, RNA or
protein synthesis
4, to identify mutated gene which is responsible for
the phenotype of the mutant.
5, to understand how the gene works (biochemical approach).

15. Budding yeast is a good model to study cell cycle control! Because..
Why budding yeast?
Budding yeast is a good model to study cell cycle control! Because..
It’s has defined cell cycle events.
It’s easy to follow cell cycle progression by monitoring the ratio of bud size to parent cell size.

16. Yeast has both haploid and diploid life cycle
It’s easy to isolate recessive mutant in haploids and to perform complementation test in diploids.

17. How to isolated Temperature sensitive mutants of yeast
Permissive
temperature
Restrictive
Mutagenize Cells
(i.e. EMS)
Plate Yeast Cells
Grow Cells at
2 Temperatures
Make
Replica
Plates

18. Permissive
Restrictive

19. Most of ts mutant defect in macromolecular biosynthesis stop growing at non-permissive temperature with normal cell morphology
What’s wrong about this?
Too many mutants to be examined in detail.
Most mutated genes are “bore” (house keeping) genes!
Until a undergraduate student Brain Reid came…

20. The Behavior of a Temperature Sensitive cdc Mutant
cdc mutant growing
at permissive temp (23C)
cdc mutant growth arrested after
6 hrs at non-permissive temp (36C)

21. Those mutants may be cell cycle control mutants!
All mutant cell has similar staining pattern of their chromosome indicates they may be arrested at the same stage of cell cycle!
Those mutants may be cell cycle control mutants!

22. Cdc Mutants Arrest at the Same Cell Cycle Phase
Permissive (low) temperature
Restrictive (high) temperature

23. Isolation of temperature sensitive mutants of cell cycle control in budding yeast
Total 150 ts mutants with unique morphology were isolated.
Do they carry mutations on the same or different genes?
Complementation test to define how many mutated genes could be find!
Total 32 complementation groups (gene) were identified!

24. How to determine the phenotype of the cdc mutants?
To shift temperature from 23C to 36C when cells just separated after mitosis.
The initial defect is defined as the first cell cycle event that fails to occur at 36C.
Initiation of DNA synthesis (iDS)
Bud emergence (BE)
DNA synthesis (DS)
Medial nuclear division (mND)
Late nuclear migration (lND)
Cytokinesis (CK)
Cell separation (CS)

25. How are the events coordinated in the yeast cell cycle so that their sequence is fixed?

26. Two models to account for the ordering of cell cycle events
Dependent pathway: product vs substrate.
Independent pathway: signals from one single timer.

27. Two models to account for the ordering of cell cycle events
Dependent pathway: product vs substrate.
Independent pathway: signals from one single timer.

28. How to distinguish two cell cycle events are dependent or independent?
To block one gene by ts mutation and to see whether the following event will continue (independent) or stop (dependent)?

29. Six events comprise a dependent pathway of cell cycle progression

30. Six events comprise a dependent pathway of cell cycle progression
CS CK lND mND DS IDS

31. A common step (cdc28) controls both pathways

32. Properties of S. cerevisiae cdc28 (START) Mutants
\Figures_Hi-res\ch14\cell3e14130.jpg

33. Very short period for mitosis
What Start is doing?
Very short period for mitosis
\Figures_Hi-res\ch14\cell3e14051.jpg

34. Regulation of Animal Cell Cycles by Growth Factors
\Figures_Hi-res\ch14\cell3e14060.jpg

35. Mating factor
This model system allows scientist to work out biochemical function of each gene product involved in cell cycle regulation for the following 30 years!

36. LH Hartwell, J Culotti, JR Pringle, BJ Reid Science 183: 46-51; 1974
Genetic Control of the Cell Division Cycle in Yeast A model to account for the order of cell cycle events is deduced from the phenotypes of yeast mutants
LH Hartwell, J Culotti, JR Pringle, BJ Reid
Science 183: 46-51; 1974

38. S. pombe (the fission yeast) has a well defined process for mitosis.
Schizosaccharomyces pombe
(Fission Yeast)
S. pombe (the fission yeast) has a well defined process for mitosis.

39. Cell Cycle of Fission Yeast
\Figures_Hi-res\ch14\cell3e14071.jpg

40. Two kinds of yeast

41. A dominant wee mutant turned out
to be an allele of CDC2
cdc2ts
Cdc2wee2
From Susan Forsburg, Salk Institute
LOF mutations in CDC2 fail to enter mitosis and become large
GOF mutations are wee-initiate mitosis prematurely, get small

42. What is function of cdc2 and CDC28 in cell cycle control?G2 S G1
Genetic model in fission yeast
CDC28
Genetic model in budding yeast
What is function of cdc2 and CDC28 in cell cycle control?

44. Evans et al (1983) Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell 33:
System: the sea urchin Arbacia punctulata. After fertilization, it undergoes 8 very rapid cell divisions, which require continual protein synthesis.
Very simple experiment: add 35-S-methionine to eggs. Then extract proteins and examine the synthesis of "new" (radiolabeled) protein.

45. “Protein A” rises and falls in advance of each cycle of cell division.
Cleavage index
Hunt names the protein “cyclin.”

46. Evidence for cytoplasmic signals in cell cycle regulation

47. South African clawed frog (Xenopus laevis) and its big eggs

48. “Maturation Promoting Factor” (MPF) in the cytosol of mature egg!
Evidence for the existence of soluble factors in the Xenopus oocyte to trigger meiosis
“Maturation Promoting Factor” (MPF) in the cytosol of mature egg!

49. The oscillation of MPF activity during the cell cycle
mitosis
interphase
MPF activity
Seen in all eukaryotic cells

50. Further purification work showed that MPF consisted of a kinase subunit and a regulatory subunit – cyclin.
Cdk: Cyclin-dependent protein kinase

51. Targets of MPF
\Figures_Hi-res\ch14\cell3e14250.jpg

52. A simplified view of the core of the cell cycle control system: dynamic level of cyclin proteins determine whether the Cdk is active or not!
Synthesis and degradation of specific cyclins become another key control point!

54. How to make sure the cell cycle progression is in a right process?
What happen if some key events of cell cycle is going wrong?

55. DNA damage will delay mitosis: why and how?
Two possible models
Some control genes to check!

57. Hartwell proposed “checkpoint” for these control mechanisms
A few mutants of yeast have been identified to relief dependence of ordered events of cell cycle
Hartwell proposed “checkpoint” for these control mechanisms

58. Dependence of mitosis on DNA synthesis
cdc9: DNA ligase
Cell cycle arrest at G2/M at non-permissive temperature
RAD9 was isolated as mutant that permit cell division of cells with DNA damaged induced by x-irradiation
Should be viable at high temperature.
Has higher ( x21) rate of chromosome lost than the wild type cells
Rad9 mutant can relief cdc9 induced mitotic arrest

59. RAD9 is a checkpoint of cell cycle progression What happen when RAD59 mutation relief dependence of mitosis on DNA synthesis?
In the absence of RAD9, DNA ligase deficient cells die much more rapidly at the non-permissive temperature.
Mitotic catastrophe!

60. Is checkpoint essential?
For somatic cells
No, but it is very important for normal cells!
No checkpoint makes low fidelity in DNA replication and separation during cell division.
Accumulation of somatic mutation and aneuploidy causes cancer formation!

61. Is checkpoint essential?
For embryonic cell cycle
Absolute no! because….

62. Why embryonic cell cycle don’t need checkpoing?
What’s wrong to have checkpoint?
Synchrony and speed of cell division is the priority!
How to handle “damaged product-defective cells”?
When cell divisions slow down (mid-blastula transition in Xenopus and division 14 of Drosophla), abnormal nuclei may fall into the interior of the egg and do not contribute to the larval cells.
The Drosophila embryo has capacity to replace lost nuclei

63. Why always negative regulation?
Cell Cycle Checkpoints
Why always negative regulation?

64. A Mechanism Involving Negative Regulation is More Robust
Very different
situations
Anaphase:
Not very
different
situations
Anaphase:
GO x 5
GO x 6

65. How do Cell Cycle Checkpoints Work?
Zhou and Elledge Nature 408, (2000)