The many functions of the kinetochore

1. remain attached when microtubule is growing • and shrinking growing shrinking Kinetochore The many functions of the kinetochore Microtubule - - attach sister chromatid to microtubule plus end • allow regulation of attachment to ensure • biorientation • send signals that block anaphase • when attachment is incorrect + • generate force to move chromosomes Centromere DNA

2. Kinetochores have many parts outer inner budding yeast animals

3. Spc24 Ndc80 Spc25 Nuf2 The Ndc80 complex links microtubules to inner kinetochore Tanaka and Desai Curr. Opin. Cell Biol. 2008, 20:53 microtubules microtubules + Ndc80 complex

4. The Ndc80 complex links microtubules to inner kinetochore Ciferri et al. Cell 2008, 133:427

5. The yeast Dam1/DASH complex forms rings around microtubules Westermann et al. Mol. Cell 2005, 17:277 J.J. Miranda et al. Nat. Struct. Mol. Biol. 2005, 12:138

6. The yeast Dam1/DASH complex forms rings around microtubules Ramey et al. (2011) Mol. Biol. Cell 22, 4335.

7. The yeast kinetochore at super-resolution Joglekar et al. Nature 2010, 463:446 Joglekar et al. Curr. Biol. 2009, 19:694

8. The kinetochore of higher eukaryotes Metaphase Anaphase

9. 3-D close-up of the animal kinetochore: a meshwork without rings! Dong et al. Nat. Cell Biol. 2007, 9:516 Santaguida and Musacchio. EMBO J. 2009, 28:2511

10. The animal kinetochore Dong et al. Nat. Cell Biol. 2007, 9:516 Cheeseman et al. Cell (2006) 127:983.

11. EM tomography of the animal kinetochore McIntosh et al. Cell (2008) 135:322.

12. metaphase anaphase Fibrils link kinetochore to depolymerizing MT ends McIntosh et al. Cell (2008) 135:322.

13. Using plus-end depolymerization to do the work of chromosome movement Biased diffusion Forced walk Joglekar et al. Curr. Opin. Cell Biol. 2009, 22:1

14. Only the correct bivalent attachment is stable

15. Bivalent attachment creates tension that separates the kinetochores

16. Budding yeast cells start out with syntelic attachment and then correct it

17. Mutations in the kinase Ipl1 (Aurora B) result in mono-oriented sister chromatids (40% of cells) (60% of cells) Tanaka et al. (2002) Cell108, 317

18. Models of kinetochore control by Ipl1/Aurora B Tanaka et al. (2002) Cell108, 317

19. P P P P P P Phosphorylation of Ndc80 by Aurora B reduces microtubule binding affinity

20. Aurora B lags behind when things get tense PP1 Aurora B PP1 Aurora B Andrews et al (2003) Curr. Opin. Cell Biol.15: 672 PP1 High tension Low tension

21. P P P P P P P Phosphatase PP1 Aurora B lags behind when things get tense Low tension: Ndc80 phosphorylated, low microtubule binding affinity Aurora B

22. Aurora B lags behind when things get tense High tension: Ndc80 dephosphorylated, high microtubule binding affinity Aurora B

23. TFP TFP FRET-based Aurora B activity sensor Aurora B High YFP:TFP ratio Low YFP:TFP ratio Violin et al. (2003) J. Cell Biol.161, 899 Fuller et al. (2008) Nature453, 1132 Liu et al. (2009) Science323, 1350

24. Dephosphorylation of Aurora B sensor in a tense kinetochore Phosphorylation CENP-B Mis12 Liu et al. (2009) Science323, 1350

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

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

27. Stabilized securin mutant blocks sister separation (APC activator)

28. Securin binds to Esp1

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

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

31. 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

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