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Hagting et al (2002) J. Cell Biol. 157 : 1125

Securin and cyclin are destroyed in metaphase. Hagting et al (2002) J. Cell Biol. 157 : 1125. Securin binds to Esp1. The “ Eureka! ” experiment. securin. Scc1. SU=supernatant. CP=chromatin pellet. Uhlmann et al (1999) Nature 400 : 37. Non-cleavable Scc1 mutant.

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Hagting et al (2002) J. Cell Biol. 157 : 1125

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

  2. Securin binds to Esp1

  3. The “Eureka!” experiment securin Scc1 SU=supernatant CP=chromatin pellet Uhlmann et al (1999) Nature400: 37

  4. Non-cleavable Scc1 mutant Scc1 cleavage occurs during anaphase Uhlmann et al (1999) Nature400: 37

  5. The fabulous Scc1-TEV experiment Arrest cells in metaphase by shutting off CDC20 Induce TEV expression Measure anaphase onset Uhlmann et al (2000) Cell103: 375

  6. Separase Separase The story so far Scc1 cleavage Sister separation

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

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

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

  10. Cdk substrate dephosphorylation The grand summary of late mitotic control as we know it APCCdc20 Cyclin-Cdk securin separase cohesin spindle disassembly, mitotic exit sister separation sister segregation sister chromatids metaphase anaphase telophase

  11. Cdk substrate dephosphorylation More refinements in late mitotic control: budding yeast APCCdc20 Cyclin-Cdk securin Cdc14 phosphatase separase cohesin spindle disassembly, mitotic exit sister separation sister segregation sister chromatids metaphase anaphase telophase Holt et al. (2008) Nature 454, 353.

  12. Cdk substrate dephosphorylation More refinements in late mitotic control: human cells APCCdc20 Cyclin-Cdk securin separase cohesin spindle disassembly, mitotic exit sister separation sister segregation sister chromatids metaphase anaphase telophase 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. Mad2 at unattached kinetochores Waters et al (1998) J. Cell Biol.141: 1181

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

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

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

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

  21. Mad1 C-Mad2 template I-Mad2 O-Mad2 Cdc20 Mad1 Cdc20 C-Mad2 copy C-Mad2 template The Musacchio Mad2 template model Mad1 C-Mad2 template De Antoni et al. (2005) Curr. Biol.15, 214 Mapelli et al. (2006) EMBO J.25: 1273 Mapelli et al. (2007) Cell131: 730 Mapelli and Musacchio (2007) Curr. Opin. Struct. Biol.17: 716

  22. E2 The Anaphase-Promoting Complex: E3 extraordinaire Substrate (e.g. securin) NH2 S S N Activator Subunit Cdc20

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

  24. APC under the EM Cdc20 or Cdh1 Cdc27 A (Cdh1) Cdc27 A Doc1 Cdc16 Apc11 Apc11 Cdc23 Doc1 Apc2 Apc2 Schreiber et al. Nature 470, 227 (2011) David Barford Phil. Trans. Roy. Soc. London B366, 3605 (2011)

  25. APC under the EM Cdc27 A S Cdc16 Doc1 Apc11 Cdc23 Doc1 Apc2 Schreiber et al. Nature 470, 227 (2011) David Barford Phil. Trans. Roy. Soc. London B366, 3605 (2011)

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

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

  28. Mis-localizing Aurora B at the central or outer kinetochore causes anaphase delays (Inner kinetochore) (Central kinetochore) (Outer kinetochore) Liu et al. (2009) Science323, 1350

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

  30. S phase G2 to metaphase Maintenance of chromosome positioning through cell division H2B-CFP H2B-YFP merge pre- bleach image processing post- bleach Gerlich et al (2003) Cell112: 751

  31. Maintenance of chromosome positioning through cell division Gerlich et al (2003) Cell112: 751

  32. Sister pairs separate in a specific sequence CENP-A in green - Histone H2B in red Gerlich et al (2003) Cell112: 751

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