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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
0
2
1
2
1
0
Continuous Duplication
Quantum Duplication
0
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?
1. FACS analysis Fluorescence Activated Cell Sorting
2. Incorporation of radioactive or epitope tagged nucleotides (BrdU)
3. LandmarksNuclear envelope breakdown
Condensed chromosomes Spindle elongation
Determining G0 vs G1 can be difficult.
G1Cells sense their environment - nutritional - geometrical (cell-cell contact) - physical (size am I big enough) - regulatory (growth factors)
If all is well, the cell will commit to a new cell cycle and pass START in yeast or the R-point (restriction) in mammals.
The later stages of G1 involve preparation for S phase and mitosis. These include induction of gene expression and duplication of centrosomes (MTOC).
4
S phaseCells initiate DNA synthesis.
They fire early origins early and late origins late.
Early Late
During S phase cells must have a mechanism to prevent activation of mitosis until DNA replication is completed.
In addition, chromosomes must be replicated once and only
once per S phase. Origins fire only once except in special cases.
Cell growth continues.
5
G2No major cytological events
Probably a sensing period for accumulation of information leading to the commitment to Mitosis.
Proteins needed for Mitosis are synthesized in G2.
6
MitosisProphasePrometaphaseMetaphaseAnaphase AAnaphase BTelophaseCytokinesis
Seven parts.
Each of these is carefully regulated so as to occur in the proper order.
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
Interphase Late Prophase Prometaphase
Metaphase Anaphase A Anaphase B Telophase
Early Prophase
Cell Cycle Transitions
State A State B
Cell Cycle Transitions
State A State B
Cell Cycle Transitions
State A State B
Metastable StatesMutual Incompatibility
Cell Cycle Transitions
State A State B
Inhibitory Barriers
State C
Cell Cycle Transitions
State A State B
Cell Cycle TransitionsCdc Mutants
State A State BX
Cell Cycle Transitions
State A State BXX
A Checkpoint Pathway Creates a Dependency Relationship
Cell Cycle Transitions
State A State B
Self-Reinforcement
Cell Cycle Transitions
State A State B
Self-Reinforcement
Cell Cycle Transitions
G1 S G2 Meta Ana Telo
Mitosis
Cell Cycle Transitions
G1 S G2 Meta Ana TeloCdk1SCF
Cdk1SCF
Esp1 APCAPC
Cell Cycle Transitions
G1 S G2 Meta Ana TeloCdk Cdk Esp1 APC/C
CKI Wee1 Pds1 Cdk1
Cell Cycle GeneticsYEAST The genetic system.
Advantages 1. Eukaryotic cell cycle2. Haploid---Diploid3. Transformable
4. Reverse geneticsCDC Mutants 1. Conditional lethal mutants2. Arrest the cell cycle at a unique position.
Can be used for four purposes1) Make a molecular map of the order of function of cdc protein and landmark events.
2) Make a determination of whether certain processes are dependent or not.
3) Identify genes important for a particular process.
4) Identify other genes in the process by reversion analysis.
cdc mutant
24°C
37°C
Cdk1 = cdc2 + Cdc28cdc2 was identified in S. pombe as a mutant that arrests in G2 and gives rise to long cells.
A critical experiment identified a dominant allele of cdc2 that cause the cell cycle to accelerate rather than stop, yielding shorter cells.
Why is this so important?
CDC28 was identified in S. cerevisiae-protein kinase
Two stop points G1 and G2 (the original allele had only one arrest point in G1)
How does one protein regulate two completelydifferent processes?
Cyclin A
Cyclin B
RNR
mitosis mitosis
The Relationship Between Cyclins and MPF
interphase interphase
CyclinsCyclins are periodically accumulated during the cycle, then rapidly destroyed.
MI MI MI
1 2 3
MPF activity peaked with cyclin levels. Cyclins are a regulatory component of MPF.
MPF
G1 S G2 M
G1 Cyclin/Cdk
S phase Cyclins/Cdk
MitoticExit
Mitotic Cyclins/Cdk
G1 CdkON
S CdkON
M CdkON
All CdksOFF
Cyclins are the regulatory subunit of cyclin-dependent kinases (Cdk)
Cyclins bind to Cdks and activatethe kinase and, in some circumstances, control their substrate specificity
Activation of Cdks controls certain cell cycle transitions
S. cerevisiae Cyclin/Cdk Activity
G1 G2S M
CLN1CLN2
CLB5CLB6
CLB3CLB4
CLB1CLB2CLN3
G1 Cyclins CLN Cln1, 2, 3S phase CLB Clb5, 6Mitotic CLB Clb, 1, 2, 3, 4,
Redundancy among cyclins Deletion of one Cln or Clb has little effectcln1, 2, 3, ------ G1 Arrest
clb1, 2, 3, 4 ------ G2 Arrest
Cyclins gain functions later in the cycle. Clbs can carry out the function of Clns in some circumstances
Clb1-4 can carryout the functions of Clb5,6 but Clb5,6 cannot function in place of Clb1-4.
2 Classes of Cyclins
The Start of START
Nutrients
Cell sizeSBF(Swi4/6)
MBF
Sic1
S phase
What exactly is START?
?
?
?
?
CLN3uORF
Cdc28Cln1/2
Cdc28Clb5/6
Cdc28Cln3
Auto-activation Loop
Auto-activation Loop
The end of START and the start of S phase
G1 S G2 M
Cdc28Clb1-4
Cdc28Clb5/6
Cdc28Cln1/2
Nutrients
Sic1SCFSBF
MBF
Sic1 ensures that S is dependent upon G1 cyclins
Budding + Centrosome Duplication
Sic1 is made during the previous mitosis
E3
Ub
substrateUb n
E2
E1 Ub
substrateUb n
Destruction by the26S Proteasome
Ubiquitin Conjugation Cascade
S
SUb Activating Enzyme
Ub Conjugating Enzyme
Ub Ligase
Rbx1
Rbx1
Cdc34
Cdc53Skp1
Cdc34
Cdc53Skp1
E1-S-Ub
S-Ub
Cdc4
Ub
Cln/Cdc28
Signal
P
UbN
Proteasome
+
Ub N
Cdc4
PP
PP
P
Sic1 Sic1
Sic1
Sic1
Sic1 degradation through the SCFCdc4 pathway
SCFCdc4
Clb5Cdc28
Clb5Cdc28
Clb5Cdc28
Clb5Cdc28
S phase
E2
F
Cdc4
Skp1Skp1
Sic1
Function
Cdk Inhibitor
F
Grr1
Cln2 Cyclin
Substrate
F
Other F-box Proteins
Unknown Targets
The F-box Hypothesis
Cdc53
Cdc34Rbx1
F-box Proteins
Development Signaling/Transcription
Cell Cycle Control Tumorigenesis
Limb development
NFkB Activation (IB)
p27
Cdk Inhibitors (Sic1)
G1 cyclins (Cln1)
Wnt Pathway (-catenin)
Hedgehog (Ci)
Cell Fate (Notch)
Skp2 Grr1
Cdc4
Aminoacid biosynthesis (Met4)
Met30
-TRCPDactylin
Sel-10
Auxinresponse in plants
TIR1
DNA replication (Cdc6)
Bud siteselection
Plant flowering
UFO
Circadianrhythms in plants
FKF1
Fbw7
Cyclin E
Ub
Skp1
Cdc53/ Cul1
F
Apc2
substrateUb n
Cdc34
P P
E1 Ub
Rbx1 Apc11E2
RING-Finger Based Ubiquitin Ligases
SCF, VCB AnaphasePromoting Complex
substrateUb n
E1 Ub
UbE2
RING
SimpleRING-E3s
F-box Proteins BC-box Proteins 5 different Cullins
Cdc20Cdh1
MDM2CblBRCA1Parkin
HECT Family RING Superfamily
E6-AP (Angelman’s Syndrome)Smurf1 (Smad destruction)Itch (Notch destruction)Rsp5 (membrane protein endocytosis) SCFs APC Simple
RING E3s
Two Major Classes of E3s
Cell Cycle LogicMaking the cycle go forward:
SBF(Swi4/6)
Sic1
S phase
SCFCdc4
MBF
SCFGrr1
SCFCdc4
Time
Cdc28Cln1/2
Cdc28Clb5/6
Mitotic Entry
Activation of Mitotic Cyclin-Dependent Kinases
Cdk Regulation
cyclin
Cdk
CKIs
Synthesis Destruction
CAKCiv
Kinases
T14 Y15
Phosphatases
T161
Synthesis Destruction
Kinases
Wee1*Mik1Myt1
Cdc25*Pyp3
Sic1Far1Rum1
p21, p27, p57p16, p15, p18, p19
PhosphataseKap1
Cks
SCF Complexes
SCF Complexes
Cdk Inhibitors
cyclin B/Cdc2 (T160-P) ACTIVE KINASE
G2 M
Wee1Mik1
Tyrosine Kinases
Cdc25Phosphotyrosine Phosphatase
+
-
cyclin B/Cdc2
Cdc2 (Cdk1)(Cdc13) cyclin B
CAK +
Phosphorylation Regulation of Cdc2 during Mitosis
cyclin B/Cdc2 ACTIVE KINASE
cyclin B/Cdc2 Y-P (Inactive Y15-P)
Mitotic Entry in S. pombe and Mammals
cyclin B/Cdc2 (T160-P)
G2 M
Wee1Mik1
Tyrosine Kinases
Cdc25Phosphotyrosine Phosphatase
+
-
cyclin B/Cdc2
Cdc2 (Cdk1)(Cdc13) cyclin B
CAK +
cyclin B/Cdc2 (active)
cyclin B/Cdc2 Y-P (Inactive Y15-P)
-
+
Autoactivation of Cdc2 makes mitosis irreversible
Cell Cycle Logic
APC/Cyclosome
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.
3. They must divide their cell in two.
General Properties of Cell Cycle Transitions
1. Amplification mechanisms.
2. Out with the old, in with the new.
3. Overcoming inhibitory barriers- Checkpoints.
Checkpoints allow the coordination of events.
