Feedback Control Between S-phase and Mitosis

Feedback control ensures dependency of cell cycle events

Sensors:(i.e. Rad9)

Transducers:

Targets:

DNA Damage Checkpoints

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

:Targets

From Rieder and Salmon (1998) Trends Cell Biol. 8:310

Chromosome Movements During Mitosis

Lodish et al. Figure 13-33

What if There Were No Checkpoint?

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 Ensures the Order of Mitotic Events

Mitotic Checkpoint Inhibits Cohesion Destruction-via inhibition of APCCdc20

The Mitotic Spindle Assembly Checkpoint

Mitotic Checkpoint Also Inhibits Mitotic Exit-via inhibition of APCCdh1

aka Kinetochore CheckpointMitotic CheckpointMetaphase CheckpointSpindle Checkpoint…

From Zhou et al. (2002) J. Cell Sci. 115:3547

The Metaphase to Anaphase (M/A) Transition

Adapted From Shimoda and Solomon (2002) Cell 109:9

How do We Know There’s a Checkpoint?

Prometaphase Metaphase

Adapted From Shimoda and Solomon (2002) Cell 109:9

20 min.Variable

How do We Know There’s a Checkpoint?

Regardless of the length of Prometaphase,Metaphse lasts 20 minutes…

Prometaphase Metaphase

Cohesion is Established in S-phase

Cdc6

ORC ORC ORC

ORC

ORC

ORC

MCM Proteins

Cdc28

Cdc7

Cdc28

Replication Factors

Origin Assembly / Activation

preRC

Origin Firing Origin Inactivation

Elongating Repl. Fork

ActvieCohesin

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

Cohesion is Established in S-phase

CondensinCohesin

From Uhlman (2001) Cur. Op. Cell Biol. 13:754

Scc1

Scc3Pds5

Smc1 Smc3

Cohesion is Established in S-phase

CondensinCohesin

ChromosomeCompaction

ChromosomeCohesion

Scissile Subunit (Scc1)Cleaved at M/A

From Uhlman (2001) Cur. Op. Cell Biol. 13:754

Condensin is Required forChromosome Compaction

Cohesin is Required forChromosome Compaction

Condensin is Required forChromosome Compaction

Cohesin is Required forChromosome Compaction

Cohesin links Condensinfrom adjacent sister chromatids

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

QuickTime™ and aVideo decompressorare needed to see this picture.

From Zhou et al. (2002) J. Cell Sci. 115:3547

The Metaphase to Anaphase (M/A) Transition

20 min.

Evidence for the Existence of a Checkpoint

Needle

Micromanipulation of Chromosomes inGrasshopper Spermatocytes

Adapted From Nicklas (1997) Science 275:632

Micromanipulation of Chromosomes inGrasshopper Spermatocytes

From Nicklas (1997) Science 275:632

Adapted From Shimoda and Solomon (2002) Cell 109:9

20 min.Variable

Kinetochores Generate the Checkpoint Signal

20 min.Variable

Laser Ablate Kinetochorefrom last

Mono-oriented Chromosome

20 min

Kinetochores Generate the Checkpoint Signal

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

From Musacchio and Hardwick (2002) Nature Reviews 2:731

Unattached Kinetochores Cause a Checkpoint Delay

From Rieder and Salmon (1998) Trends Cell Biol. 8:310

Both Attachment and Tension are Monitored

3F3 EpitopePhosphorylation of unknown proteins -dims upon attachment -disappears upon tension

Both Attachment and Tension are Monitored

3F3 EpitopePresent in the

absence of tension

Mono-oriented Grasshopper Spermatocyte Chromosomes

Relaxed TensionApplied

3F3 EpitopeDisappears when

tension applied

Adapted From Nicklas (1997) Science 275:632

One More Yeast Screen: MADs and BUBs

Hoyt: Budding Uninhibited by Benomyl Bub1, Bub2, Bub3

Li: Mitotic Arrest Deficient Mad1, Mad2, Mad3

Search for Genes Responsible for Feedback Control of Mitosis

Spindle Assembly Checkpoint Components

Gene Molecular Function Checkpoint FunctionBub1 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 Generates Mad2/Cdc20 Complex

Mps1 Protein Kinase Transduction

Ipl1 Protein Kinase Corrects Mono-orientation

CENP-E Molecular Motor Trasduction, Congression

Dynein Molecular Motor Congression, Checkpoint Off

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

Potential Checkpoint Targets:

From Zhou et al. (2002) J. Cell Sci. 115:3547

APC is the Target of the Mitotic Checkpoint

APC is the Target of the Mitotic Checkpoint

Cdc2/Cyclin B

Cdc2/Cyclin B

Adapted from Peters (2002) Mol. Cell 9:931

Mad2Mad3/BubR1

Bub2

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

Spindle Assembly Checkpoint Pathways

Securin

Separase

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

Bub2 active:NO Cdc14 Release

Bub2 inactive:Cdc14 Released

Mitotic Exit is a Spindle Checkpoint Target

MitoticExit

Network(MEN)

Cdc14 Sequesteredin Nucleolus

Cdc14 Released

Cdc2/Cyclin B Cdc2/Cyclin B Cdc14

Bub2

Bub2 Prevents Cdc14 Release/Cdh1 Activation

Bub2

Cdc2/Cyclin B

Cdc2/Cyclin B

Adapted from Peters (2002) Mol. Cell 9:931

Mad2Mad3/BubR1

Bub2

APC is the Target of the Mitotic Checkpoint

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

Spindle Assembly Checkpoint Pathways

Securin

Separase

Checkpoint is Mediated by Rearrangement of Mad2 Complexes

From Millband et al. (2002) Trends Cell Biol. 12:205

Mad2 InhibitsAPCCdc20

Shah and Cleveland (2000) Cell 103:997

Mad2 Cycles Through Kinetochore and Inhibits Cdc20