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

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Presentation on theme: "Feedback Control Between S-phase and Mitosis Feedback control ensures dependency of cell cycle events."— Presentation transcript:

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

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

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

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

5 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 theres no going back The Mitotic Spindle Assembly Checkpoint Ensures the Order of Mitotic Events

6 Mitotic Checkpoint Inhibits Cohesion Destruction -via inhibition of APC Cdc20 The Mitotic Spindle Assembly Checkpoint Mitotic Checkpoint Also Inhibits Mitotic Exit -via inhibition of APC Cdh1 akaKinetochore Checkpoint Mitotic Checkpoint Metaphase Checkpoint Spindle Checkpoint…

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

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

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

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

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

12 Cohesion is Established in S-phase Condensin Cohesin Chromosome Compaction Chromosome Cohesion 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 From Zhou et al. (2002) J. Cell Sci. 115:3547 The Metaphase to Anaphase (M/A) Transition

17 20 min. Evidence for the Existence of a Checkpoint Needle 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 Adapted From Shimoda and Solomon (2002) Cell 109:9 20 min.Variable Kinetochores Generate the Checkpoint Signal

20 20 min.Variable Laser Ablate Kinetochore from last Mono-oriented Chromosome 20 min Kinetochores Generate the Checkpoint Signal 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 From Musacchio and Hardwick (2002) Nature Reviews 2:731 Unattached Kinetochores Cause a Checkpoint Delay

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

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

26 One More Yeast Screen: MADs and BUBs Hoyt: B udding U ninhibited by B enomyl Bub1, Bub2, Bub3 Li: M itotic A rrest D eficient Mad1, Mad2, Mad3 Search for Genes Responsible for Feedback Control of Mitosis

27 Spindle Assembly Checkpoint Components Gene Molecular FunctionCheckpoint Function Bub1 Protein KinaseTransduction Bub2 Tem1 GAP(Mitotic Exit) Bub3 Kinetochore Scaffold Mad1 Generates Mad2/Cdc20 Complex Mad2 Inhibits Cdc20 Mad3/BubR1 BubR1 (vert) Protein KinaseGenerates Mad2/Cdc20 Complex Mps1 Protein KinaseTransduction Ipl1 Protein KinaseCorrects Mono-orientation CENP-E Molecular MotorTrasduction, Congression Dynein Molecular MotorCongression, Checkpoint Off

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

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30 Potential Checkpoint Targets:

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

32 Cdc2/Cyclin B Adapted from Peters (2002) Mol. Cell 9:931 Mad2 Mad3/BubR1 Bub2

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

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

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

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

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

38 Checkpoint is Mediated by Rearrangement of Mad2 Complexes From Millband et al. (2002) Trends Cell Biol. 12:205 Mad2 Inhibits APC Cdc20

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

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