C ell D ivision Lecturer: Dr. Stephen Ell edge Office: T303 Phone: 798-5040 Required Reading C hapters 17 and 18in MolecularBiologyof the Cell , Third Edition Bardin AJ, Visintin R, Am on A (2000) A m echanism for co upling exit f rom mitosis to partitioning ofthe nucleus. Cell 102:21 -31.
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Cell Division
Lecturer: Dr. Stephen Elledge
Office: T303
Phone: 798-5040
Required Reading
Chapters 17 and 18 in Molecular Biology of the Cell, Third Edition
Bardin AJ, Visintin R, Amon A (2000) A mechanism for coupling exit from mitosisto partitioning of the nucleus. Cell 102:21-31.
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Cell Cycle Regulation
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2
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2
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0
Continuous Duplication
Quantum Duplication
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2
1
2
1
0
Cytoplasmic Cycle
Chromosome Cycle
Cell Growth Cytokinesis
DNA Replication Mitosis
Continuous Duplication
Quantum Duplication
The Basic Problem
S phase
MitosisG2G1
G1(G0) - S - G2 - MCell Cycle Stages
How do we determine which stage of the cycle a cell is in?
The APC is a complex ubiquitin ligase that is required for anaphase entry andmitotic exit.
Like the SCF, it has substrate specificity components called Cdc20 and Cdh1/Hct1,2 WD40 repeat proteins. The regulation of these specificity components is critical.
Anaphase Entry and Exit
APC
CohesionFactors
Mitotic Exit
ClbsCdk1
Pds1
APCCdc20 Cdh1
Clb5ChromosomesOK ?
Anaphase
Clb2*
Chromosome Cohesion
APC
Cohesin
Cdc20
Pds1
Pds1
Esp1
UbUb
Ub
Esp1
Destruction by the 26S Proteosome
Separin
Securin
Pds1 has a destruction box which allows it to be recognized by the APC
Anaphase
Separin
APC
Separin (Esp1)
Securin = Pds1Separin = Esp1Cohesin = Scc1 +
Cohesion in Mammals
Mitotic ExitAfter anaphase is complete, in order to exit mitosis and initiate cytokinesis, cells must inactivate B-type cyclin/Cdks.
High
Cyclin B/Cdk Activity
Low
Cdc14 Phosphatase
Mitosis Mitotic Exit
Low
Cyclin B/Cdk Activity
High
Cdc14 Phosphatase
Cytokinesis& G1 Entry
Mitotic ExitAfter anaphase is complete, in order to exit mitosis and initiate cytokinesis, cells must inactivate B-type cyclin/Cdks.This involves activation of the Cdh1 form of the APC.
Cdh1 Cdh1 PClbCdk1
Inactive
Cdc14(Phosphatase)
Cdh1 Active
APCCdh1
Clb/Cdk1
Cdc14Swi5 P Swi5
Active
Sic1
Cdc14Inactive
Cdc14Active
MEN
Mitotic Exit
Cytoplasmic NuclearTranscription Factor
Swi5ClbCdk1
Inactive
Cdc14 Activation for Mitotic ExitThe activation of Cdc14 is the key event in execution of mitotic exit. During S, G2 and Pre-anaphase, Cdc14 is held tethered in an inactive complex in the nucleolus.
When Anaphase is executed, Cdc14 is released and goes throughout the nucleus and cytoplasm to dephosphorylate key Cdk1 substrates.
NucleolarCdc14
Cfi1(Net1) Cdc14
Cfi1(Net1) Cdc14
ActiveInactive
MEN
The mitotic exit network (MEN) consists of several protein kinases and a G-protein Tem1. How it MEN regulated is not known.
Spindle
How Mitotic Exit is Coupled to Anaphase
Cdc15
Dbf2, Mob1
Cdc14(Nucleolus)
Cdc14(Released)
Tem1-GTP
Tem1-GDP
Sic1
Clb2
Mitotic Exit
Bfa1/Bub2 (GAP)
Lte1 (GEF)
Mitotic Exit Network
Spindle Pole Body
The Tem1-bearing SPB migrates into the daughter cell to encounter Lte1
Lte1 in Red
M
D
Tem1 in Red
Lte1 Tem1 Lte1 Tem1
Spindle in Green
Tem1
Mitotic Exit Summary
1. When anaphase occurs, Tem1 on the SPB is thrust into the daughter cell where it encounters the GEF, Lte1, Tem1 is converted to the active GTP form.
2. Active Tem1 activates Cdc15 and MEN, which causes the release of the Cdc14 phosphatase from the nucleolus where it is inhibited.
3. Cdc14 dephosphorylates Cdh1 to activate the APC to destroy Clbs, it also activates the synthesis of Sic1, a Cdk inhibitor.
4. Together, the APC and SIC1 turn off Cdk activity to initiate mitotic exit.
Cell move from high CDK, low CDC14 state to a Low CDK, high Cdc14 state.
To re-enter the next cell cycle they need to turn off Cdc14 to re-establish the null state, CDK off, Cdc14 off, APC off, making cells permissive for Clb activation of S phase.
How does the cycle move forward?- Positive amplification loops- Feedback inhibition
1) Clns activate their own transcription
2) Once Clns provide sufficient activity to pass START, they activate a ubiquitin proteolysis pathway that destroys an inhibitor of Clb kinase activity, Sic1.
3) Clb/Cdc28 kinase activate Clb transcription and repress Cln transcription.
4) Clb/kinase activate S phase.
5) Once S phase is complete, Clb kinases activate mitosis.
6) Once chromosomes are properly aligned at the metaphase plate, a ubiquitin proteolysis pathway is activated that destroys Clbs but not Clns and resets the cycle.
7)Clb destruction allows PRC complexes to form.
8) Cln kinase activity is required to shut off the Clb proteolysis pathway to allow S entryin the next cell cycle. This allows Pds1 to be synthesized again which recruits Esp1 into the nucleus.
In MammalsCyclin B/Cdc2 can help activate itself by turning on an activating phosphatase and turning off an inhibitory kinase
The Rao and Johnson Cell Fusion Experiments Cell Cycle Regulation
M cells + G1, S, or G2 cells
S cells + G1
S cells + G2
M
G1 cells enter S
G2 cells do not enter S, but do not enter mitosis until the S-phase nucleus has entered G2.- Block to re-replication- Inhibitor of mitosis produced by S phase cells
G1 cells + G2 Like S above.- G1 cells also block mitosis
- Mitotic state is dominant.
Cell Cycle Checkpoints
Definition: "A checkpoint is a biochemical pathway that ensures dependence of one process on another process that is otherwise biochemically unrelated."
B C
D EA
Damage ExtrinsicMechanism
IntrinsicMechanism
Why are checkpoints important?
Checkpoints control the order and timing of events. In some cases the natural timing of events can allow the proper order of events in the absence of a checkpoint. However, the fidelity is often compromised.
The accumulation of errors, whether due to entering DNA replication in the presence of damage, or mis-segregating a chromosome is deleterious to the reproductive fitness of unicellular organisms, and in multicellular organisms may lead to uncontrolled cell proliferation and cancer.
Checkpoints in S. c.DNA Damage CheckpointsSpindle Assembly CheckpointsS phase CheckpointsSize CheckpointsG1/M CheckpointMorphology CheckpointMeiotic Checkpoints
Checkpoints are defined by loss of function mutations that relieve the dependency of two events.
cdc13 ts mutants
cdc13 rad9 mutants
The Spindle Assembly CheckpointThe proper assembly of a spindle is sensed by a group of proteins called Mad or Bub located on the kinetochore. These proteins send a signal to inhibit the APC.
Metaphase Anaphase A/B
Pds1APC
Esp1
Scc1
Misaligned Chromosomes
Mps1 Mad1,2,3 Bub1,2,3
Cdc20
Mutant Hunt - benomyl sensitive mutants thatcontinue to cycle in the presence of benomyl.
WT
mad or bub mutants
ben
ben
The Spindle Assembly Checkpoint
What is being sensed?
Kinetochore - Microtubule Attachment
Tension and bipolar attachment
When tension is not present at sister chromatids, a Mad/Bub-dependent phosphorylation occurs on the kinetochore. This is thought to be part of the signal used to turn off the APC.
Signal Transduction
Signal
Sensor
Transducer
Effector
SENSORS
TRANSDUCERS
SIGNALS
EFFECTORS
TranscriptionCell Cycle ArrestApoptosis DNA Repair
STOPSTOP
PCNA- & RFC-like Proteins
Mediators (BRCT proteins, Mrc1/Claspin)
Kinases:PIK ATM + ATR
PK CHK1 and CHK2
Conserved Families
DNA Damage Response Pathways
ATR
Chk2
PP
G1 MG2S
p53
DNA replicationproteins?
The DNA Damage Response in Humans
Chk1
Claspin
P P
PP
ATRIP
PP
p21Cdc25
Cdc2/Cyclin B
BRCA1BLMNBS1Repair Proteins
Rad9
Rad1
Hus1
P P
RFC Rad17
PC
DNA Damage Checkpoints - Sensing Damage
RFC Rad17
RFC Rad17
Rad9
Rad1
Hus1
ATP
Rad9
Rad1
Hus1
ATR
ATRIP
PP
ATR
ATRIP
PP
RFC Rad17
Rad9
Rad1
Hus1
Rad9
Rad1
Hus1
ATR
ATRIP
PP
P P P P
P P
ATR and RC-PC Engagement Activates Checkpoint
Chk1 Chk2 BRCA1 Nbs1
Checkpoint Responses
Mediators
G1 Arrest in Mammals
Cdk activity is rate limiting for S phase entry and is the target for checkpoint control.
DNA Damage
p53 p53*
Apoptosis
orp21 G1 Cyclin
Cdks
p53 levels increase in response to DNA damage and activate transcription of p21
ATM/ATR
Chk2
Mdm2
?Cdc25A
Chk1,2
How is p53 activated? - Relief of repression.
MDM2 binds p53 and targets it for ubiquitin-mediated proteolysis.
p53 transcriptionally regulates MDM2 to make a feedback loop.
p53 Mdm2 transcription
Mdm2
In response to DNA damage, both p53 and Mdm2 are phosphorylated, causing a disruption in Mdm2 binding, thereby allowing p53 to both increase in abundance and become transcriptionally active.
During activation, p53 increases the amount of Mdm2 protein to return to low p53 levels when the signal is eventually turned off.
This also explains why p53 levels are so high in tumors in which p53 is mutant, no Mdm2 is made.
RING Finger Ubiquitin Ligase
Active
Cdc25C
Cdc25 is regulated by Chk1 phosphorylation
Cdc25C
Cytoplasmic
PSer216
14-3-3
S/G2
Inactive
Nuclear
Mitosis
Chk1Ser216
DNA Damage
ON OFF
ATR
G2 Arrest in Mammals
G2 Arrest in S. pombe + MammalsCdk activity is rate limiting for entry into Mitosis and is the target for checkpoint control.
DNA Damage
Chk1
Cdc25
Chk1*
Mitosis
cyclin BCdc2 Y-P OFF
OFF ON
cyclin BCdc2 ON
ATM or ATR
Chk2 Chk2*
Cdc25 P
14-3-3 Cytoplasm OFF
Nucleus ON
p53* p21
Wee1
MEC1 DDC2
RAD53
Anaphase Entry Mitotic Exit
ESP1
Mechanism of pre-anaphase arrest in response to DNA damage
RAD9
PDS1
CHK1
CDK
rad3 rad26
Mitotic Entry
crb2
chk1
cdc2
cdc25
S. pombeMammals
S. cerevisiae
*
Chk1 phosphorylation of Pds1 protects it from degradation by the APCCdc20
*
ATR
Mediator
Chk Kinases
Effectors
G1 Cdk
Mitosis
G1 S G2 Meta AnaA AnaB Tele
S Cdk
Sic1
APCCdc20
Overall Organization of the Cell Cycle
Pds1 B/Cdk1
Replication Checkpoint
M Cdk
APCCdh1
SCF
SCFAPCON
APCOFF
?
B/Cdk1OFF
B/Cdk1ON
APCONB/Cdk1OFF
SpindleCheckpoint
M Cdk
Cdc14
APCON ON
Cdc20APC
Cdh1
General Points
Cells need to do only a few things absolutely right
1. They must duplicate their chromosomes precisely,i.e. completely but only once per cycle.
2. They must segregate their chromosomes precisely.