1. Feedback Control Between S-phase and Mitosis
Feedback control ensures dependency of cell cycle events

2. DNA Damage Checkpoints
Sensors:
(i.e. Rad9)
Transducers:
Targets:
:Targets
From Susan Forsburg, Salk Institute

3. Chromosome Movements During Mitosis
From Rieder and Salmon (1998) Trends Cell Biol. 8:310

4. What if There Were No Checkpoint?
Lodish et al. Figure 13-33

5. The Mitotic Spindle Assembly Checkpoint
Ensures the Order of Mitotic Events
What has to Happen for Proper Chromosome Segregation?
- Sister Chromatid Cohesion established in S-phase
- Chromosome condensation in G2
- Mitotic Spindle Assembly in Mitosis
- Chromosome alignment in Mitosis
- Metaphase/Anaphase transition (M/A)
-securin degradation leads to cohesion destruction
Mitotic checkpoint has to be upstream of M/A
- Once you start destroying cohesion there’s no going back

6. The Mitotic Spindle Assembly Checkpoint
aka Kinetochore Checkpoint
Mitotic Checkpoint
Metaphase Checkpoint
Spindle Checkpoint…
Mitotic Checkpoint Inhibits Cohesion Destruction
-via inhibition of APCCdc20
Mitotic Checkpoint Also Inhibits Mitotic Exit
-via inhibition of APCCdh1

7. The Metaphase to Anaphase (M/A) Transition
From Zhou et al. (2002) J. Cell Sci. 115:3547

8. How do We Know There’s a Checkpoint?
Prometaphase
Metaphase
Adapted From Shimoda and Solomon (2002) Cell 109:9

9. Regardless of the length of Prometaphase, Metaphse lasts 20 minutes…
How do We Know There’s a Checkpoint?
20 min.
Variable
Prometaphase
Metaphase
Regardless of the length of Prometaphase,
Metaphse lasts 20 minutes…
Adapted From Shimoda and Solomon (2002) Cell 109:9

10. Cohesion is Established in S-phase
Cdc6
ORC
MCM Proteins
Cdc28
Cdc7
Replication
Factors
Origin Assembly / Activation
preRC
Origin Firing
Origin Inactivation
Elongating
Repl. Fork
Actvie
Cohesin
From Susan Forsburg, Salk Institute

11. Cohesion is Established in S-phase
Pds5
Scc3
Scc1
Smc1
Smc3
Cohesin
Condensin
From Uhlman (2001) Cur. Op. Cell Biol. 13:754

12. Scissile Subunit (Scc1)
Cohesion is Established in S-phase
Cohesin
Condensin
Chromosome
Cohesion
Chromosome
Compaction
Scissile Subunit (Scc1)
Cleaved at M/A
From Uhlman (2001) Cur. Op. Cell Biol. 13:754

13. Condensin is Required for
Chromosome Compaction
Cohesin is Required for
Chromosome Compaction

14. Condensin is Required for Chromosome Compaction
Cohesin is Required for
Chromosome Compaction
Cohesin links Condensin
from adjacent sister chromatids

15. From Susan Forsburg, Salk Institute

16. The Metaphase to Anaphase (M/A) Transition
From Zhou et al. (2002) J. Cell Sci. 115:3547

17. Evidence for the Existence of a Checkpoint
Needle
20 min.
Micromanipulation of Chromosomes in
Grasshopper Spermatocytes
Adapted From Nicklas (1997) Science 275:632

18. Micromanipulation of Chromosomes in Grasshopper Spermatocytes
From Nicklas (1997) Science 275:632

19. Kinetochores Generate the Checkpoint Signal
20 min.
Variable
Adapted From Shimoda and Solomon (2002) Cell 109:9

20. Laser Ablate Kinetochore Mono-oriented Chromosome
Kinetochores Generate the Checkpoint Signal
20 min.
Variable
20 min
Laser Ablate Kinetochore
from last
Mono-oriented Chromosome
Adapted From Shimoda and Solomon (2002) Cell 109:9

21. From Rieder et al. (1995) J. Cell Biol. 130:941

22. Laser
Anaphase
From Rieder et al. (1995) J. Cell Biol. 130:941

23. Unattached Kinetochores Cause a Checkpoint Delay
From Musacchio and Hardwick (2002) Nature Reviews 2:731

24. Both Attachment and Tension are Monitored
3F3 Epitope
Phosphorylation of
unknown proteins
-dims upon attachment
-disappears upon tension
From Rieder and Salmon (1998) Trends Cell Biol. 8:310

25. Both Attachment and Tension are Monitored
Mono-oriented Grasshopper Spermatocyte Chromosomes
Relaxed
Tension
Applied
3F3 Epitope
Present in the
absence of tension
3F3 Epitope
Disappears when
tension applied
Adapted From Nicklas (1997) Science 275:632

26. One More Yeast Screen: MADs and BUBs
Search for Genes Responsible for Feedback Control of Mitosis
Hoyt: Budding Uninhibited by Benomyl
Bub1, Bub2, Bub3
Li: Mitotic Arrest Deficient
Mad1, Mad2, Mad3

27. BubR1 (vert) Protein Kinase
Spindle Assembly Checkpoint Components
Gene
Molecular Function
Checkpoint Function
Bub1
Protein Kinase
Transduction
Bub2
Tem1 GAP
(Mitotic Exit)
Bub3
Kinetochore Scaffold
Mad1
Generates Mad2/Cdc20 Complex
Mad2
Inhibits Cdc20
Mad3/BubR1
BubR1 (vert) Protein Kinase
Mps1
Ipl1
Corrects Mono-orientation
CENP-E
Molecular Motor
Trasduction, Congression
Dynein
Congression, Checkpoint Off

28. APC is the Target of the Mitotic Checkpoint
The Metaphase to Anaphase (M/A) Transition
From Zhou et al. (2002) J. Cell Sci. 115:3547

30. Potential Checkpoint Targets:

31. APC is the Target of the Mitotic Checkpoint
From Zhou et al. (2002) J. Cell Sci. 115:3547

32. APC is the Target of the Mitotic Checkpoint
Cdc2/Cyclin B
Mad2
Mad3/BubR1
Bub2
Cdc2/Cyclin B
Adapted from Peters (2002) Mol. Cell 9:931

33. Spindle Assembly Checkpoint Pathways
Securin
Separase
Adapted From Susan Forsburg, Salk Institute

34. Mitotic Exit is a Spindle Checkpoint Target
Bub2 active:
NO Cdc14 Release
Mitotic
Exit
Network
(MEN)
Bub2 inactive:
Cdc14 Released
From Susan Forsburg, Salk Institute

35. Cdc14 Bub2 Prevents Cdc14 Release/Cdh1 Activation Bub2 Bub2
Cdc2/Cyclin B
Cdc2/Cyclin B
Cdc14
Bub2
Cdc14 Sequestered
in Nucleolus
Cdc14 Released

36. APC is the Target of the Mitotic Checkpoint
Cdc2/Cyclin B
Mad2
Mad3/BubR1
Bub2
Cdc2/Cyclin B
Adapted from Peters (2002) Mol. Cell 9:931

37. Spindle Assembly Checkpoint Pathways
Securin
Separase
From Susan Forsburg, Salk Institute

38. Checkpoint is Mediated by Rearrangement of
Mad2 Complexes
Mad2 Inhibits
APCCdc20
From Millband et al. (2002) Trends Cell Biol. 12:205

39. Mad2 Cycles Through Kinetochore and Inhibits Cdc20
Shah and Cleveland (2000) Cell 103:997