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Lecture 10
Checkpoints
Outline:Review G1/SDNA damage/replication checkpoint spindle assembly checkpointspindle position checkpoint
Paper: Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression
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Control of G1 progression in budding yeast
Mitotic exit: Cdk inactivationG1/S-Cdk activity increases - not susceptible to Sic1S cyclin synthesis inducedS-Cdk inactive until phosphorylation of Sic1 and Cdh1
by G1/S-CdksDNA replication
(Cdh1)
Cdc14SCF
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Possible mechanism to coordinate cell growth and cell cycle progression
Cln3 synthesized in parallel with cell growthHow is threshold level reached?Cells inherit fixed amount of inhibitor (DNA?)
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Control of G1 progression in mammalian cells
G1-Cdk induced by growth factor, phosphorylates RbE2F induces more of itself, and S phase Cyclins (E, A)S-Cdks further phosphorylate RbMore S-Cdks accumulate - DNA replication
chromosome condensation during S-phase
S
M
DNA damage:
chemicalsradiationnormal DNA metabolism
TOAST
Chromosome non-disjunction:
aneuploidy
TOAST
MPF
1974
Irradiate tissue culture cells
First clue about feedback control
caffeine treatmentno delay:lethal chromosomedamage
DNA damage
Delayed entry into M-phase until damage repaired
How to identify genes involved in checkpoint function?
YEAST
Identify mutants that cannot recover from DNA damage
2 classes:repair deficientarrest deficient
repair deficient
arrest deficient
Conserved elements of DNA damage and replication checkpoints
I. Sensors Proteins with functional analogs in DNA replication
recognize damageload onto DNA
II. Transducers:= kinases
ATM and ATR
Chk1 and Chk2
phosphorylate substrates affecting protein activity or stability
cell cycle arrestactivate DNA repairmaintain arrest until repair completere-initiate cell cycle progressionor APOPTOSIS
III. Effector output:
inhibited by caffeine
DNA damage pathway in budding yeast
ionizing radiationRAD9
MEC1 (ATR kinase)
RAD53
CDC5 (polo kinase)
CLB/CDC28
mitosis
CHK1
PDS1
cohesins
anaphase entry
G2 or M arrest
Examples of pathways that block the cell cycle:
Another feedback pathway in fission yeastDNA damage G2 arrest
Two genes identified:chk1, rad24
Both act through cdc25
Cdc25Y15 ppase
DNA damage
14,3,3 proteinwith NES
Mechanism: Sequester Cdc25 away from Cdc2
Cdc25 phosphorylated by Chk1
P-Cdc25 recognized by Rad24
Rad24 transports P-Cdc25 out of nucleus
Cdc25 cannot dephosphorylate nuclear Cdc2
translocation has not been confirmed in other organisms
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
Irradiation induces arrest in G1 and G2
G1 checkpoint is non-functional in absence of p53
Also a faster response recently identified, independent of p53
Cyclin D degradation and “inhibitor swap”
ATMkinase
Cyclin D-Cdk4,6Cyclin E-Cdk2
transcription
APC activated toward Cyclin D
p21CIP released
p21CIP inhibits Cyclin E-Cdk2
ionizing radiation
Spindle Checkpoints
Kinetochore-mediated
MTOC-mediated
senses when all chromosomes have been attached and properly aligned
regulates sister separation
senses proper spindle position
regulates exit from mitosis
cross-talk
Budding yeast
mutants that fail to arrest in the presence of microtubule depolymerizing drugs
Hoyt labbub:budding uninhibited by benomyl
Murray labmad:mitotic arrest deficient
Bub1, Bub2, Bub3
Mad1, Mad2, Mad3
Mps1
Kinetochore checkpoint
Activator of APC-mediated destruction of Pds1, B Cyclins chromosome segregation
unattachedkinetochore
Pds1, cyclins
APC
Cdc20
diffusible signal
Cdc20Target:
focus on Mad2
associates only with unattached kinetochores
inhibits Cdc20-APC
mouse knockout embryonic lethal
Model
unattached kinetochores “activate” Mad2
Mad2* diffuses away and inhibits Cdc20-APC
FRAP experiment: Mad2 turns over rapidly
Howell et al. (2000)
Questions
What activates/inactivates Mad2?
binding partners: Mad1, Mad3, BubR1, Bub3?
What is Mad2*
complex? oligomer?
What generates and stops the signal???
somatic cells - MT attachmentmeiotic cells - tension
How transduced???
dynein-dependent transport to spindle polesmay turn it off
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 kinetochoresRequired for checkpoint signaling
Motor-dependent mechanism for checkpoint signaling?QuickTime™ and aTIFF (Uncompressed) decompressor
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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)
spindle position checkpoint
Activator of APC-mediated destruction of B Cyclins mitotic exit
spindle positiondefect
B cyclins
APC
Cdh1= Hct1
Cdc14 pathway
Cdh1 = Hct1Target:
Cfi1 sequesters Cdc14 phosphatase in the nucleolus
Release of Cdc14 allows it to dephosphorylate Cdh1 targets APC to cyclinsSic1 inhibits Cdk-Cyclin activity
What causes release of Cdc14?
Cdc14 pathway
Two upstream factors identified that constitute aspindle position sensor
Tem1 (small GTPase)
Lte1 (GEF)
Together serve as activators for Cdc14 release
Tem1 (GTPase) is only found on daughter spindle pole body (SPB)
taggedTem1 Bardin, Visintin and Amon
Cell 102, 21-31
Lte1 (GEF) is only found in the bud
Spindle position defect induced by dynein disruption
Release of nucleolar Cdc14 and mitotic exitonly occurs in cells with daughter nucleus in bud
anaphasearrest
telophase
Similar spatial clues in vertebrate cells??
Piel et al. Science 291, 1550 (2001):
Mother centriole often moves to intercellular bridge (midbody) prior to final step in cytokinesis
A role for the centrosome in completion of cytokinesis:
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EM sectionsshowing mothercentriole nearmidbody