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Lecture 10

Lecture 10. Checkpoints. Outline: Review G1/S DNA damage/replication checkpoint spindle assembly checkpoint spindle position checkpoint. Paper: Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression.

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Lecture 10

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  1. Lecture 10 Checkpoints Outline: Review G1/S DNA damage/replication checkpoint spindle assembly checkpoint spindle position checkpoint Paper: Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression

  2. Control of G1 progression in budding yeast SCF Cdc14 (Cdh1) Mitotic exit: Cdk inactivation G1/S-Cdk activity increases - not susceptible to Sic1 S cyclin synthesis induced S-Cdk inactive until phosphorylation of Sic1 and Cdh1 by G1/S-Cdks DNA replication

  3. Possible mechanism to coordinate cell growth and cell cycle progression Cln3 synthesized in parallel with cell growth How is threshold level reached? Cells inherit fixed amount of inhibitor (DNA?)

  4. Control of G1 progression in mammalian cells G1-Cdk induced by growth factor, phosphorylates Rb E2F induces more of itself, and S phase Cyclins (E, A) S-Cdks further phosphorylate Rb More S-Cdks accumulate - DNA replication

  5. M DNA damage: S chemicals radiation normal DNA metabolism chromosome condensation during S-phase  TOAST

  6. Chromosome non-disjunction: aneuploidy  TOAST

  7. MPF

  8. caffeine treatment DNA damage Delayed entry into M-phase until damage repaired First clue about feedback control 1974 Irradiate tissue culture cells no delay: lethal chromosome damage

  9. How to identify genes involved in checkpoint function? YEAST Identify mutants that cannot recover from DNA damage 2 classes: repair deficient arrest deficient

  10. repair deficient arrest deficient

  11. Conserved elements of DNA damage and replication checkpoints I. Sensors Proteins with functional analogs in DNA replication recognize damage load onto DNA II. Transducers: = kinases ATM and ATR Chk1 and Chk2 phosphorylate substrates affecting protein activity or stability III. Effector output: cell cycle arrest activate DNA repair maintain arrest until repair complete re-initiate cell cycle progression or APOPTOSIS inhibited by caffeine

  12. Examples of pathways that block the cell cycle: ionizing radiation RAD9 RAD53 CHK1 PDS1 CDC5 (polo kinase) cohesins CLB/CDC28 anaphase entry mitosis DNA damage pathway in budding yeast  G2 or M arrest MEC1 (ATR kinase)

  13. Cdc25 Y15 ppase Another feedback pathway in fission yeast DNA damage  G2 arrest Two genes identified: chk1, rad24 Both act through cdc25

  14. Cdc25 phosphorylated by Chk1 P-Cdc25 recognized by Rad24 Rad24 transports P-Cdc25 out of nucleus Cdc25 cannot dephosphorylate nuclear Cdc2 Mechanism: Sequester Cdc25 away from Cdc2 DNA damage 14,3,3 protein with NES

  15. translocation has not been confirmed in other organisms

  16. DNA damage checkpoint in mammalian cells G1 arrest mediated by p53 p53 = tumor suppressor/transcription factor: stabilized in damaged cells  induces expression of Cdk inhibitor p21CIP  induces apoptosis

  17. Irradiation induces arrest in G1 and G2

  18. G1 checkpoint is non-functional in absence of p53

  19. Cyclin D-Cdk4,6 Cyclin E-Cdk2 ATM kinase transcription Also a faster response recently identified,  independent of p53  Cyclin D degradation and “inhibitor swap”

  20. ionizing radiation APC activated toward Cyclin D p21CIP released p21CIP inhibits Cyclin E-Cdk2

  21. cross-talk Spindle Checkpoints Kinetochore-mediated  senses when all chromosomes have been attached and properly aligned  regulates sister separation MTOC-mediated  senses proper spindle position  regulates exit from mitosis

  22. Budding yeast mutants that fail to arrest in the presence of microtubule depolymerizing drugs Hoyt lab bub: budding uninhibited by benomyl Bub1, Bub2, Bub3 Murray lab mad: mitotic arrest deficient Mad1, Mad2, Mad3 Mps1

  23. Target: Cdc20 diffusible signal Cdc20 APC Pds1, cyclins Kinetochore checkpoint Activator of APC-mediated destruction of Pds1, B Cyclins  chromosome segregation unattached kinetochore

  24. focus on Mad2 associates only with unattached kinetochores inhibits Cdc20-APC mouse knockout embryonic lethal

  25. Model  unattached kinetochores “activate” Mad2  Mad2* diffuses away and inhibits Cdc20-APC

  26. FRAP experiment: Mad2 turns over rapidly Howell et al. (2000)

  27. Questions What activates/inactivates Mad2? binding partners: Mad1, Mad3, BubR1, Bub3? What is Mad2* complex? oligomer? What generates and stops the signal??? How transduced??? somatic cells - MT attachment meiotic cells - tension dynein-dependent transport to spindle poles may turn it off

  28. Different checkpoint proteins in higher eukaryotes No Bub2 Mad3 homolog = BubR1, acquired kinase domain CENP-E ZW10/Rod (dynein interactors) All behave like Mad2: associate with unattached kinetochores Required for checkpoint signaling

  29. Motor-dependent mechanism for checkpoint signaling? CENP-E activates BubR1 kinase activity until attached to microtubules - creates “wait” signal After attachment, complexes are carried off the kinetochore by dynein Mao, Desai and Cleveland, JCB 170, 873-80 (2005)

  30. Target: Cdh1 = Hct1 Cdc14 pathway Cdh1= Hct1 APC B cyclins spindle position checkpoint Activator of APC-mediated destruction of B Cyclins  mitotic exit spindle position defect

  31. Cdc14 pathway Cfi1 sequesters Cdc14 phosphatase in the nucleolus Release of Cdc14 allows it to dephosphorylate Cdh1  targets APC to cyclins Sic1  inhibits Cdk-Cyclin activity What causes release of Cdc14?

  32. Two upstream factors identified that constitute a spindle position sensor Tem1 (small GTPase) Lte1 (GEF) Together serve as activators for Cdc14 release

  33. Tem1 (GTPase) is only found on daughter spindle pole body (SPB) tagged Tem1 Bardin, Visintin and Amon Cell 102, 21-31

  34. Lte1 (GEF) is only found in the bud

  35. telophase anaphase arrest Spindle position defect induced by dynein disruption Release of nucleolar Cdc14 and mitotic exit only occurs in cells with daughter nucleus in bud

  36. Similar spatial clues in vertebrate cells?? Piel et al. Science 291, 1550 (2001): A role for the centrosome in completion of cytokinesis: Mother centriole often moves to intercellular bridge (midbody) prior to final step in cytokinesis

  37. EM sections showing mother centriole near midbody

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