1. Hagting et al (2002) J. Cell Biol. 157: 1125 Securin and cyclin are destroyed in metaphase

2. Securin binds to Esp1

3. The “Eureka!” experiment SU=supernatantCP=chromatin pellet Scc1 securin Uhlmann et al (1999) Nature 400: 37

4. Scc1 cleavage occurs during anaphase Non-cleavable Scc1 mutant

5. Uhlmann et al (2000) Cell 103: 375 The fabulous Scc1-TEV experiment Induce TEV expression Arrest cells in metaphase by shutting off CDC20 Measure anaphase onset

6. Scc1 cleavage Sister separation The story so far Separase

7. Stabilized Cyclin mutant Spindle disassembly in telophase Nuclear envelope assembly Chromosome decondensation Cytokinesis Anaphase chromosome movement, spindle stability Anaphase depends on cyclin destruction and Cdk substrate dephosphorylation

8. Murata-Hori et al. (2002) Mol. Biol. Cell 13:1099. GFP-Aurora B in wild-type cells GFP-Aurora B in cells with stabilized Cyclin B Aurora B is removed from kinetochores in early anaphase

9. Oliveira et al (2010) Nature Cell Biol. 12:185 Wild-type mitosis TEV-induced anaphase TEV-induced anaphase + Cdk inhibitor Wild-type mitosis The fabulous Scc1-TEV experiment, in flies

10. cohesin separase securin Cyclin-Cdk APC Cdc20 spindle disassembly, mitotic exit sister chromatids metaphaseanaphasetelophase sister segregation sister separation Cdk substrate dephosphorylation The grand summary of late mitotic control as we know it

11. cohesin separase securin Cyclin-Cdk APC Cdc20 spindle disassembly, mitotic exit sister chromatids metaphaseanaphasetelophase sister segregation sister separation Cdk substrate dephosphorylation More refinements in late mitotic control: budding yeast Cdc14 phosphatase Holt et al. (2008) Nature 454, 353.

12. cohesin separase securin Cyclin-Cdk APC Cdc20 spindle disassembly, mitotic exit sister chromatids metaphaseanaphasetelophase sister segregation sister separation Cdk substrate dephosphorylation More refinements in late mitotic control: human cells Shindo et al. (2012) Dev. Cell 23, 112.

13. What determines the timing of APC target destruction? “Spindle assembly checkpoint”

14. The spindle assembly checkpoint

15. Finding components of the spindle checkpoint …and the winners are: Mad1, Mad2, Mad3, Bub1, Bub3, et al.

16. Waters et al (1998) J. Cell Biol. 141: 1181 Mad2 at unattached kinetochores

17. Mad1-Mad2 complex at unattached kinetochore Mad2 Mad1 Unattached kinetochores catalyze the formation of Mad2-Cdc20 complexes Bub3 Mad3 Mad2 Cdc20

18. Open Mad2 (O-Mad2) Mad2 comes in two structural flavors Closed Mad2 (C-Mad2)

19. The Mad1-Mad2 complex at the unattached kinetochore Closed Mad2 (C-Mad2) Closed Mad2 (C-Mad2) Mad1

20. Open Mad2 (O-Mad2) Closed Mad2 (C-Mad2) Mad1 or Cdc20 Mad2 uses the safety belt to bind Mad1 or Cdc20 Intermediate Mad2 (I-Mad2)

21. Mapelli et al. (2007) Cell 131: 730 Mad1 C-Mad2 template Mapelli et al. (2006) EMBO J. 25: 1273 Mapelli and Musacchio (2007) Curr. Opin. Struct. Biol. 17: 716 Mad1 C-Mad2 template C-Mad2 copy Cdc20 The Musacchio Mad2 template model De Antoni et al. (2005) Curr. Biol. 15, 214 O-Mad2 Mad1 I-Mad2 C-Mad2 template

22. E2 S S NH 2 N The Anaphase-Promoting Complex: E3 extraordinaire Activator Subunit Cdc20 Substrate (e.g. securin)

23. Cdc20 Mad2 Bub3 Mad3 E2 Mad2 collaborates with Mad3-Bub3 to lock down inactive Cdc20

24. Schreiber et al. Nature 470, 227 (2011) David Barford Phil. Trans. Roy. Soc. London B 366, 3605 (2011) Apc2 Apc11 A Cdc27 Cdc23 Cdc16 Doc1 Apc2 Apc11 Cdc27 A (Cdh1) Cdc20 or Cdh1 APC under the EM

25. Schreiber et al. Nature 470, 227 (2011) David Barford Phil. Trans. Roy. Soc. London B 366, 3605 (2011) Doc1 Apc2 Apc11 S A Cdc27 Cdc23 Cdc16 Doc1 APC under the EM

26. Doc1 Cdc20 Mad2- Mad3- Bub3 Herzog et al. Science 323, 1477 (2009) Cdc20 is repositioned in the spindle checkpoint arrest

27. PP P P P P P Aurora B Low tension: Ndc80 phosphorylated, low microtubule binding affinity Spindle checkpoint on Mad1-Mad2 complex is recruited to a poorly attached (phosphorylated?) kinetochore

28. Mis-localizing Aurora B at the central or outer kinetochore causes anaphase delays (Inner kinetochore) (Central kinetochore) (Outer kinetochore) Liu et al. (2009) Science 323, 1350

29. What determines the timing of APC target destruction in flies and yeast? Spindle checkpoint

30. Maintenance of chromosome positioning through cell division H2B-CFPH2B-YFPmerge pre- bleach post- bleach image processing S phaseG2 to metaphase Gerlich et al (2003) Cell 112: 751

31. Maintenance of chromosome positioning through cell division

32. Sister pairs separate in a specific sequence CENP-A in green - Histone H2B in red Gerlich et al (2003) Cell 112: 751