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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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?)

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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

are needed to see this picture.

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:

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

EM sectionsshowing mothercentriole nearmidbody