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

DNA Damage Checkpoints Sensors: (i.e. Rad9) Transducers: Targets: :Targets From Susan Forsburg, Salk Institute http://pingu.salk.edu/~forsburg/cclecture.html

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

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

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

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

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

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

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

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 http://pingu.salk.edu/~forsburg/cclecture.html

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

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

Condensin is Required for Chromosome Compaction Cohesin is Required for Chromosome Compaction

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

From Susan Forsburg, Salk Institute http://pingu.salk.edu/~forsburg/cclecture.html

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

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

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

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

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

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

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

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

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

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

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

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

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

Potential Checkpoint Targets:

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

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

Spindle Assembly Checkpoint Pathways Securin Separase Adapted From Susan Forsburg, Salk Institute http://pingu.salk.edu/~forsburg/cclecture.html

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 http://pingu.salk.edu/~forsburg/cclecture.html

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

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

Spindle Assembly Checkpoint Pathways Securin Separase From Susan Forsburg, Salk Institute http://pingu.salk.edu/~forsburg/cclecture.html

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

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