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Feedback Control Between S-phase and Mitosis
Feedback control ensures dependency of cell cycle events
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DNA Damage Checkpoints
Sensors: (i.e. Rad9) Transducers: Targets: :Targets From Susan Forsburg, Salk Institute
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Chromosome Movements During Mitosis
From Rieder and Salmon (1998) Trends Cell Biol. 8:310
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What if There Were No Checkpoint?
Lodish et al. Figure 13-33
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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
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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
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The Metaphase to Anaphase (M/A) Transition
From Zhou et al. (2002) J. Cell Sci. 115:3547
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How do We Know There’s a Checkpoint?
Prometaphase Metaphase Adapted From Shimoda and Solomon (2002) Cell 109:9
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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
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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
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Cohesion is Established in S-phase
Pds5 Scc3 Scc1 Smc1 Smc3 Cohesin Condensin From Uhlman (2001) Cur. Op. Cell Biol. 13:754
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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
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Condensin is Required for
Chromosome Compaction Cohesin is Required for Chromosome Compaction
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Condensin is Required for Chromosome Compaction
Cohesin is Required for Chromosome Compaction Cohesin links Condensin from adjacent sister chromatids
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From Susan Forsburg, Salk Institute
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The Metaphase to Anaphase (M/A) Transition
From Zhou et al. (2002) J. Cell Sci. 115:3547
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Evidence for the Existence of a Checkpoint
Needle 20 min. Micromanipulation of Chromosomes in Grasshopper Spermatocytes Adapted From Nicklas (1997) Science 275:632
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Micromanipulation of Chromosomes in Grasshopper Spermatocytes
From Nicklas (1997) Science 275:632
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Kinetochores Generate the Checkpoint Signal
20 min. Variable Adapted From Shimoda and Solomon (2002) Cell 109:9
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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
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From Rieder et al. (1995) J. Cell Biol. 130:941
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Laser Anaphase From Rieder et al. (1995) J. Cell Biol. 130:941
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Unattached Kinetochores Cause a Checkpoint Delay
From Musacchio and Hardwick (2002) Nature Reviews 2:731
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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
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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
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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
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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
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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
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Potential Checkpoint Targets:
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APC is the Target of the Mitotic Checkpoint
From Zhou et al. (2002) J. Cell Sci. 115:3547
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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
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Spindle Assembly Checkpoint Pathways
Securin Separase Adapted From Susan Forsburg, Salk Institute
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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
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Cdc14 Bub2 Prevents Cdc14 Release/Cdh1 Activation Bub2 Bub2
Cdc2/Cyclin B Cdc2/Cyclin B Cdc14 Bub2 Cdc14 Sequestered in Nucleolus Cdc14 Released
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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
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Spindle Assembly Checkpoint Pathways
Securin Separase From Susan Forsburg, Salk Institute
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Checkpoint is Mediated by Rearrangement of
Mad2 Complexes Mad2 Inhibits APCCdc20 From Millband et al. (2002) Trends Cell Biol. 12:205
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Mad2 Cycles Through Kinetochore and Inhibits Cdc20
Shah and Cleveland (2000) Cell 103:997
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