Mitotic Spindle Assembly Checkpoint (MSAC)

1. Mitotic Spindle Assembly Checkpoint (MSAC) System View Communication model Quantitative observations Biophysical models Molecular models

2. System View Communication model Biophysical models Molecular models

3. Basic schema of SAC • Balance between: • An inhibitory signal to prevent anaphase • Unattached kinetochores generate signals that prevent the onset of anaphase (Silenced If properly connected to microtubules ) 2.The activity of anaphase-promoting machinery - APC(anaphase-promoting complex, activated by cofactor Cdc20)

4. Focusing observations • The SAC can delay anaphase in response to a singleuncaptured chromosome, exhibiting excellent sensitivity • Quick disengagement of the spindle assembly checkpoint once all kinetochores are attached • Highly-reliable process

5. System View Communication model Quantitative observations Biophysical models Molecular models

6. Convert into Communication System • A transmitter (spindles) consistently sent out PCK (microtubules) • Consider the duplicated chromesome as receiver with multiple receiving antennas, not until all the antennas send out ACK does the PCK consider received by the transmitter.

7. Our approaches • Think without pre-knowledge of biology • Redefine “information” • How the last kinetochore can still be effective

8. System view of SAC Break SAC into three major modules • The kinetochore-localized signalling platform • The spindle attachment machinary • Cytoplasmic activities associated with APC activities.

9. System View Communication model Quantitative observations Biophysical models Molecular models

10. System View Communication model Quantitative observations Biophysical models Molecular models

11. Biophysical Models • Only one unattached kinetochore Focus on two properties of the checkpoint: 1 .The extent of cell-cycle inhibition at steady-state 2. The time it takes to increase the level c back to 90% of its maximum.

12. General Framework • c = the protein that triggers cell-cycle progression • ρ = radius of a kinetochore = 0.01μm • R = radius of a nucleus = 1 μm • Ac = the capacity of the checkpoint to inhibit the c protein (quantified by the steady-state fraction of uninhibited c, the smaller the Ac, the more efficient.) • Tb = the time it takes to increase the level c back to 90% of its maximum. • Assumptions: Tb < 3min ; Ac < 0.05

13. Direct inhibition model • Once a c molecule hits the kinetochore region, it becomes immediately inhibited (c*) • Result: Cannot support good inhibition while maintaining rapid reactivation time

14. Self-Propagating inhibition Model • An inhibited c* can bind at some rate k to an uninhibited c molecule and catalyze its inhibition. • Result: Although the inhibition is sufficiently high, the system also remains inhibited upon the binding of the last kinetochore

15. Emitted inhibition Model • Inhibition doesn’t occur solely on the kinetochore but that the kinetochore catalyze the formation of an inhibitory complex e that can diffuse away form it and inhibit the c molecule everywhere. • Activation of e occurs on the kinetochore, inactivation occurs either spontaneouly or by interaction with c

16. Pros and Cons of Biophysical Models • Provide an important function in testing hypotheses rather than revealing specific pathways • Biophysical Models are useful to understand the systems level behavior but cannot provide a clear connection to molecular mechanism

17. System View Communication model Quantitative observations Biophysical models Molecular models

18. Molecular Models • Mad2 Template model (Mad1/Mad2 sequester) -Cdc20 captured by Mad2 • MCC formation -Mad2:Cdc20 + Bub3:BubR1 => MCC • APC inhibition MCC+APC= MCC:APC (inactive) APC:cdc20 complex is the key to separate.

19. Scc1 holds the two chromatids attached • Separase protease, Esp1, degrades the cohesion. • Securin protein keep Esp1 inactive.