1. G2/M: Chromosome condensation SMC family of proteins structural maintenance of chromosomes large coiled coil proteins with ATPase domain interact in complexes Cohesin - maintains sister-chromatid cohesion Condensin - mitotic chromosome condensation

2. Cohesion and condensation

3. Cohesins and condensins

4. 13S Condensin SMC2 and SMC4, with xCAP-D2, xCAP-G, xCAP-H (Drosophila barren) 13S condensin induces positive writhe and supercoiling in DNA Association of 13S with chromosomes occurs exclusively in mitosis 13S in mitotic extracts: binds DNA, is phosphorylated, induces supercoil in presence of ATP and TOP1 13S in interphase extracts: binds DNA, is not phosphorylated, does not induce supercoil Immunodepletion of cdc2 decreases P of 13S and leads to loss of condensin activity; adding cdc2 back increases P of 13S in condensin activity

5. Histone H3 phosphorylation An initiating event of chromosome condensation is phosphorylation of histone H3 on ser-10 When site is mutated in Tetrahymena, there is aberrant chromosome condensation and segregation defects (but not in budding yeast!) H3 Kinase? Not MPF, may be NIMA, or aurora kinase RNAi depletion of aurora B decreases P of H3, decreases chromosome condensation, and leads to failure to recruit barren

6. Condensin, histone H3 and the cell cycle

7. Mitosis: Overview Stages Prometaphase, metaphase, anaphase, telophase Purpose: equational and equal segregation of chromatids to daughter cells Checkpoints

8. Mitosis: Overview

9. Mitosis: Prometaphase Spindle morphogenesis MTs, MT polarity and dynamic instability MT motors kinesin (+ end directed) dynein (- end directed) Centrosome and spindle assembly Centromeres and kinetochores “Dance” of the chromosomes Bipolar orientation & importance

10. The mitotic spindle

11. The centromere and kinetochore

13. MT dynamic instability

14. Mitosis: Metaphase Stable bipolar orientation achieved Role of tension Cohesin Tension-sensitive checkpoint Anaphase signal

15. How do cells get out of metaphase? Via anaphase by action of the anaphase promoting complex (APC) Injection into Xenopus oocytes of RNA encoding non-degradable cyclin B leads to sustained high MPF and metaphase arrest Ubiquitination of chromosome cohesins and cyclin targets them to the proteasome

16. Synthesis - Destruction Cycle

17. Cohesion release at M/A

18. Control of anaphase entry

19. Mitosis: Anaphase Anaphase A - separation of chromosomes Mechanisms Where is the motor? Class, 04.23.02 Anaphase B - elongation of spindle

20. Anaphase B