1. Overview of the Control of the Cell Cycle
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2. The Cell Cycle
-Cell cycle control system – a network of regulatory proteins
-The cell monitors:
1. Events inside the cell
2. Conditions outside the cell

3. Phases of the Cell Cycle
G1 – Cell growth and doubling of organelles
S – Duplication of DNA
G2 – Preparation of division
M – Mitosis, Separation of chromosomes
Cytokinesis – Division into 2 daughter cells
Interphase = G1 + S + G2

4. Stages of Mitosis
Defined as structure and location of DNA

5. Control Points of the Cell Cycle
-Commitment points in the cell cycle are known as Start (in yeast) or restriction point (in mammalian cells) or checkpoints
-The cell monitors internal and external conditions
-Main Checkpoints are before S and M phases

6. Cell Cycle Control in Eucaryotes
-Time to complete the cell cycle can vary greatly between cell types within the same organism
-All eucaryotes used similar machinery and control mechanisms in the cell cycle
Model systems used to study the cell cycle
1. Yeast
2. Animal embryos
3. Cell-free systems
4. Cell culture

7. Yeast Model System Advantages include: -Reproduce rapidly
-Small genome, 1% of a mammal
-Can proliferate in a haploid state
-Nuclear envelope remains intact
-Budding yeast does not have a normal G2 pahse

8. Temperature-Sensitive Mutants
Cell-division-cell (Cdc) genes – genes important in controling the cell cycle
A mutant that cannot complete the cell cycle cannot grow and divide

9. Temperature-Sensitive Mutants
Normal Population Cdc15 Ts mutant (high temp)

10. Animal Embryo Model System
Xenopus
-Fertilized eggs are large (1mm)
-Large amounts of cell division proteins
-Rapid sequence of cell divisions after fertilization
-1st division takes 90 min
-Next 11 divisions takes 30 min each

11. Xenopus Animal Model
During early embryonic cycles the basic processes are occurring: DNA duplication and separation
Very little cell cycle control
Mid-blastula transition

12. Cell-Free Model System
-Cell-free of in vitro system
-Advantage
-Manipulate cell-cycle events
under controlled conditions

13. Cell Culture Model System
-Normal cells can be cultured for divisions before undergoing replicative cell senescence
-Immortalized cells can proliferate indefinitely
Advantage:
-Unlimited supply of genetically
homogeneous cells

14. Studying the Cell Cycle
Staining cells
-DNA-binding fluorescent dyes
-Antibodies to specific molecules
2. BrdU (bromo-deoxyuridine) incorporation
3. Tritiated thymidine incorporation
4. Flow cytometer

15. BrdU Labeling

16. Flow Cytometry
-Measures DNA content
-Thousands of cells per minute

17. Control of the Cell Cycle
-The proteins that control the cell cycle are different from the proteins that are involved in the process
-Series of checkpoints
-Each checkpoint serves as a biochemical switch

18. Control of the Cell Cycle
The control system:
1. Swithes are binary:
“On or Off” and once started the process continues to completion
2. Reliable due to back-up systems
3. Adaptable so it can be modified to different cell types or conditions

19. Key Control Components
1. Cyclin-dependent kinases (Cdk)
-Activities vary throughout the cell cycle
-The targets of phosphorylation then varies
2. Cyclins
-Control the activity of Cdk’s
-Undergo synthesis and degradation in
each cell cycle
Cycling of the cyclins creates the cycles of the cyclin/Cdk complex allowing for the varying activities resulting in progression through the cell cycle

20. Cyclin-Cdk Complexes 4 classes of cyclins
G1-cyclin
4 classes of cyclins
-All eucaryotes require 3 cyclins (G1/S, S, M)

21. Yeast – 1 Cdk
Vertebrates – 4 Cdks
Cyclin – activates the Cdk and helps direct it to the specific target

22. Cdk Activation Cdk fullly active 1. Cyclin bound
2. Phosphorylated at the active site

23. Regulation of Cdk Activity
-Cdk activity can be fine tuned through 2 phosphorylations at the top of the active site
-Important in control of M-Cdk activity

24. Inhibition of Cyclin-Cdk Complexes
-Binding of inhibitors alters the structure of the active site
-Cdk inhibitors are utilized more early in the cell cycle

25. Cyclin-Cdk Complexes APC/C Anaphase-Promoting Complex, or Cyclosome
G1-cyclin
APC/C Anaphase-Promoting Complex, or Cyclosome
-Regulated by protein destruction

26. Marking Proteins by Ubiquitin

27. Ubiquitination Process I

28. Ubiquitination Process II
There are about 300 different E2-E3 each recognizing a different degradation signal. Therefore subsets of proteins can be regulated as a group.

29. Control of Proteolysis of APC/C
-Degrading S and M-cyclins stops the Cdk activity and the Cdk’s targets become dephosphorylated and inactive
or Cdh1
or S-cyclin
-APC/C is active in G1 keeping Cdks inactive

30. Control of Proteolysis by SCF
-Degrades Cdk Inhibitors of S-Cdks and allows S phase to occur
-F-box protein is constant through the cell cycle and is used to recognize the target

31. Activation of a Ubiquitin Ligase

32. Activation of a Degradation Signal

34. Cell-Cycle Control Overview
10% of yeast genes encode mRNAs which oscillate in the cell cycle