Regulating the Eukaryotic Cell Cycle

8/2/2019 Regulating the Eukaryotic Cell Cycle

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Regulating The Eukaryotic

Cell Cycle


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Cell Division-An overview

2001 Nobel Prize in Physiology orMedicine Tim Hunt, Leland Hartwelland Paul Nurse

Discoveries of key regulators of thecell cycle

fundamental molecular mechanisms

controlling the cell cycle are highlyconserved through evolution and operatein the same manner in yeasts, insects,plants, animals and humans.


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c.elegansembryos, inanaphase

CeBUB-1 aspindle

checkpointprotein

DNA in Blue


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Overview

The cell cycle comprises ordered series ofevents that lead to cell division.

Precise control of these events ensureduplication of chromosome and theirsegregation to daughter cells with highfidelity.

Regulation is crucial for normal developmentof multi-cellular organisms

Loss of control ultimately leads to cancer


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Two key events of cell cycle

chromosome replication and

segregation

are controlled by regulating thetiming of nuclear DNA replication andmitosis.


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Cell division is controlled bysurveillance mechanisms:

Prevent initiation of each step untilearlier events have been completed


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Master controllers are heterodimericprotein kinases that contain:

A regulatory subunit: cyclin (describedby T.Hunt. His work on sea urchins)

A catalytic subunit: cyclin dependentkinase (P. Nurse. Discovered inyeast)

Also CDCs (cell division cycle genes)are important during checkpointscontrolling the progression and orderthroughout the entire cycle to be


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Stages of the Cell Cycle

G1 phase: RNA and protein synthesis(9h)

S phase: DNA synthesis andchromosome replication (10h)

G2 phase: 4.5h

M phase: includes several stages (30min)

Prophase: chromosome condensation;sister chromatids are associated at


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Note: Some cellsby-pass Go and

directly enter intoG1 (i.e. bonemarrow cells).

In haploids:2n

In diploids 4n

The fate of singleparental chromosomeduring the eucaryotic

cell cycle


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Metaphase: kinetochore assembles ateach centromere. They then associatewith microtubules coming from oppositespindle pole.

Anaphase: sister chromatids separate;pulled by motor proteins along the

spindle pole. Telophase: mitotic spindle disassemble

and chromosomes decondense

Cytokinesis: physical division that

yields two daughter cells


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With the onset of mitosis, nuclearenvelope retracts into the ER and the

Golgi break down into vesicles. Nuclear envelope reforms in telophase

as chromosome decondense.

Golgi complex reforms in daughter cells Inyeastnuclear envelope doesnt break

down; spindle forms in nucleus.


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Regulation of the cellcycle

Concentration of cyclins arevariable during different phases ofcell cycle

CDKs have no kinase activity, unlessthey are associated with a cyclin.

Associated cyclin determines whichproteins are to bephosphorylatedby a CDK-cyclin complex.


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Major Checkpoints during Cellcycle

Checkpoint surveillancemechanisms operate at:

The initiation of S phase

Initiation of mitosis

Separation of daughter chromosomes atanaphase

Onset of telophase and cytokinesis


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Checkpoint mechanisms function bycontrolling cyclin-CDK activities:

Regulation of the synthesis and

degradation of cyclins P* of CDKs at specific inhibitory and

activating sites

Regulation of the synthesis of CDKinhibitors (CKI)

Regulation of APC/C ubiquitin-proteinligase


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Passage from G1-S; metaphase-

anaphase; and anaphase-telophase/cytokinesis is irreversible

Regulated by degradation of proteins (SCFand APC/C)

Cells traverse the cycle in one directiononly

In higher organisms, synthesis of G1cyclin-CDK complexes is induced by ECgrowth factors

Cells become independent of GFs in late

G1: restriction point.


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Experimental Systems

Factors regulating cell cycle arediffusible components.

Mammalian cell fusion experimentsdemonstrated that

Fusion of G1 cells with M cells ? DNAcondensation:YES

Fusion of G1 cells with S cells ? DNAreplication:YES

Fusion of G2 cells with S phase ? DNAreplication: NO


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Fusion of mitotic cell with a G1phase cell induces chromosome

condensation of the G1 phaseDNA.

Photomicrograph of the fusedcell


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Temperature sensitive mutants ofS.cerevisiae and. S. Pombe are used to

identify factors regulating cell cycle These cells are blocked at specific

stages of cell cycle

Cell division cycle mutants: cdc mutants Human cDNAs cloned into yeast

expression vectors oftencomplemented ts mutants


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4/23/12 Only cells who took up the wt gene (here CDC28) waspermitted to undergo cell division and thus colony


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Biochemical studies with eggs,ocytes and early embryos

Cell extracts from eggs, oocytes andearly embryos of amphibians and

marine invertebrates are suitable. Multiple synchronous cell cycle follow

fertilization.

Factor inducing mitosis has beendiscovered in oocyte maturationstudies in Xenopus laevis system.


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When they areActive


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When they areActive


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Oocytes can grow up to 1mmin size and stay in the frogovary up to 8 months. Whenstimulated in vitrow/Progesterone it entersinto Metaphase of Meiosis.

Half the chromosomes in 1st Polarbody

Metaphase

Highly asymmetric celldivision after homologouschromosome segregationand 1st polar bodyformation. Half thechromosome is expelled into1st PB.

Metaphase


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Fertilization releasesmetaphase arrest, andprocced into anaphase ofMeiosis II. 2nd PB isinduced via assymmetricdivision and one

chromatid of eachchromosome is expelled.

2 haploid pronuclei fuses,produces a diploid zygote.1st Mitosis is preceededby the DNA replicatn., andembryogenesis begins


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Meiotic maturation occurs under thecontrol of maturation-promotingfactor (MPF)

MPF is the key factor for the initiationof mitosis in all eukaryotic cells.

MPF activity falls as cells enterinterphase between meiosis I andmeiosis II; but rises again as cellsenter meiosis II and remains so during

metaphase II arrest.

Meiotic maturation promoted by diffusible


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Meiotic maturation promoted by diffusiblefactors

5% of Cytoplasm orisolated Mitotic Cyclin-CDK heterodimer,induces progression ofcell arrested in G2 toprogress metaphase ofMeiosis II.

This process can be repeated inthe absence of Progesterone,demonstating the MPF presencein the oocyte cytoplasm.

Microinjection of G2 arrestedoocytes: 1st MPF activityAssay.


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MPF activity peaks at the beginningof Mitosis and Meiosis

Untreated G2 arrested oocytes have low levels of MPF.Progesterone induces MPF production, and cell progressesinto Meiosis I.


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Identification of mitoticcyclin

Experiments in sea urchin showedthat new protein synthesis is required

Cyclin components of MPF was

discovered Cyclin B is synthesized continuously

during embryonic cell cycles, peaks

in mitosis and destroyed followinganaphase.

Antibody against cyclin B co-purified

it with MPF activity

Synthesis and degradation of Cyclins (Sea Urchin


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Synthesis and degradation of Cyclins (Sea Urchinegg)

NOTE: B and Clevels arecontinuouslyincreasing

Cyclin B levels and MPF


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Cyclin B levels and MPFkinase activity are

synchronous In initial 12 synchronous cell cycles,replication is very rapid and, G1 andG2 times are minimized

Oscillation of MPF activity is observedin the cytoplasm of fertilized egg,even if the nucleus is removed

All required factors for truncated cellcycles are in unfertilized egg.


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The extract of unfertilized egg inducecondensation of chromatin as well asround replication of DNA derivingfrom sperm nuclei

Cyclin B oscillation occurs as in intactcells that enter in and exit frommitosis.

MPF also was shown to P* H1; thiskinase activity then replaced oocyteinjection assay.


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MPF kinase activity was shown to riseand fall in synchrony withconcentration of cyclin B (see next

figure). Early mitotic events occurred when

MPF activity and cyclin B levelreached their highest level.

Addition of cycloheximide preventedboth activities

Cyclin B dependent cycling of MPF activity in Xenopus egg


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Cyclin B dependent cycling of MPF activity in Xenopus eggextracts


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Cyclin B functions were also assessedin RNase treatment conditions

1n sperm nuclei only replicated theirDNA, but early mitotic events did notoccur.

Addition of cyclin B mRNA restoredboth MPF kinase activity andosciilations of cyclin B..


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Is cyclin B degradation required forexit from mitosis?

When non-degradable mutant formof cyclin B was added, sperm nucleiarrested at mitosis!

Late mitotic events are blocked

Exit from Mitosis is mediated by thedegradation of Mitotic Cyclins


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4/23/12 Mitotic Cyclin B prevents cell to enter post mitotic stages

APC t l it f


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APC controls exit frommitosis

In vertebrates three proteins function likecyclin B

B type cyclins: Two closely related cyclinsB; cyclin A

In intact cells, degradation of all cyclinsstarts at late anaphase

Degradation of B-type cyclins begins withubiquitination: it is mediated by 9 aa. APC/Crecognizes destruction box located at N-

terminus ofcyclin proteins.


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Mitotic Cyclin level regulation in Xenopus


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Mitotic Cyclin level regulation in Xenopusembryonic egg

Specificity

Factor: it directspolyubiquitinylation


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G ti t di ith S


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Genetic studies with S.pombe

Identification of catalytic proteinkinase subunit of MPF was performedin genetic analyses of cell cycle in S.

pombe. In this fission yeast mitosis is tightly

regulated to coordinate cell division

with cell growth.

S. Pombe at various stages of the cell


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gcycle

Long ones:entering intomitosis

Short ones:passed throughcytokinesis


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Two types of temperature-sensitivemutants were observed in non-

permissive temperatures cdc mutants fail to progress in the cell

cycle and form long cells

wee mutants lack proteins that preventcells from dividing when they are small:yeast cells are extremely small.

Wt: cdc2 +; rm: cdc2 - ; dm: cdc2 D protein:

Cdc2


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4/23/12 Unlike mammalian cells most of its growth is during G2

phase

NOTE: nuclearenv. in notdegrading

during mitosis

MPF lik l i S


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MPF-like complex in S.pombe

Ts mutants of cdc2 produce oppositephenotypes depending on whether themutation is r (recessive) or d (dominant).

cdc2 are abnormally long; whereas cdc2D are abnormally small.

Loss of Cdc2 activity prevents entry into

mitosis; gain of Cdc2 activity stimulatesmitosis earlier than normal.

Cdc2 is a key regulator of entry into mitosis.

CDC2 as the key regulator to entry into mitosis in


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y g yS.Pombe


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Wt cdc2+ was isolated and has been shown

to encode a protein of 34 kDa with homologyto eukaryotic protein kinases.

When human homolog of cdc2 was identified itwas the 1st demonstration that key cell cycle

proteins are highlyconserved betweendistant eukaryote species.

Another characterized protein, Cdc13 showedhomology to cyclin B.

Cdc13-Cdc2 heterodimer is able to P* H1 andforms S. pombe MPF.

Cdc2: CDK1; little kinase activity unless it is bound

with cyclin B

R l ti f ki ti it f


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Regulation of kinase activity ofCDK

Ts cdc25 - mutants are delayed in enteringmitosis: long cells

Overproduction of Cdc25 causes prematureentry into mitosis by decreasing G2 length:small (wee) cells.

wee1+ mutations give rise to oppositephenotypes

Cdc25 and Wee1 stimulates and inhibitsMPF activity, respectively.

Opposing effects of cdc25 and wee1 on MPF activity ofS P b


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S.Pombe

No mitosis

Early

mitosis

More MPFactivity

Inhibited MPF

activity


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Cdc25 and Wee1 proteins regulateMPF kinase activity by P* and de-P*Cdc2 subunit of (CDK) of MPF.

Wee1 P* @ Y15 residue on CDK CAK (CDK activating kinase) P* @T161

MPF is inactive when two residues areP*

Cdc25, de-P* Y15

MPF active


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Dephosphorylates


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IMP NOTE: Y15 to Phe15 mutation that prevents P*tion by wee1,yields similar mutants. Their ability to properly regulate MPFactivity is lost.

These cells prematurely enter into mitosis..

T161 P*ted state of MPF is the highest activity state!!!

CDK-Activatedkinase

DephosphorylatesONLY @ Y15 residueonly

Cyclin binding and P* increase


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Cyclin binding and P* increaseMPF activity

Unlike yeast, vertebrates produceseveral CDKs

3D structure of human CDK2 has

been resolved and allowed us tounderstand the regulation of kinaseactivity.

Besides Y15, vertebrate CDK2 has anadditional inhibitory residue: T14

P* of both residues prevents ATP binding

3D structure of human


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Unbound to CyclinA

Block access of

substrates to-PO4 of thebound ATP

3D structure of humanCDK2

Bound ATPin ball stick

model


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unP*`te

d

T-loop is pulledaway

Availablefor P-transferrxn

-helixmovesdown


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The conformn.change uponP*tion alter thesubst-bindingsurface. This

increasesproteinsubstrateaffinity ofhuman CDKs

Summary-I


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Summary-I

1. In S.pompe, cdc2 gene encodes a CDK. This associates

with mitotic cyclin (encoded by cdc13). The resultingmitotic cyclin-CDK is equivalent toXenopus MPF.

2. Cdc2 or cdc13 mutants fail to enter mitosis. OUTCOME:elongated cells.

3. PK activity of MPF depends on the P*tion states of the 2

residues. Lowest activity: Y15 and T161 P*ted states.Mediated by Wee and CAK.

4. Greatest activity of MPF is @ T161 P*ted state. This ismediated by cdc25, since it deP*lates Y15.

5. In humans: Cyclin-CDK2 is similar toXenopus andS.pompe MPF. P*tion of activating T160 (similar toT161) induce modification @ the active site, therebyincreasing protein substrate binding affinity.

Molecular mechanisms for


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Molecular mechanisms forregulating mitotic events

The entry into mitosis is a result of P*of many substrate proteins by kinaseactivity of MPF

MPF-mediated P* regulate earlyevents of mitosis

In later stages cyclin B leveldecreases and MPF is inactivated.

Early mitotic events


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Early mitotic events

Inner lipid bilayer of nuclear membraneis supported by nuclear laminaconsisting of lamin filaments A, B, C invertebrates, (belongs to a class ofintermediate filaments of cytoskeletalproteins)

Lamin A and C derive from the samegene by alternative transcription; theyare identical except of a region 133 aa.

which is absent in lamin C

Xenopus oocyte Nuclear Lamina: it isdj i l b


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adjacent to inner nuclear membrane


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Serine residues

N-terminal-end


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Substrates of MPF

In early mitosis, P* ofSer residues inall 3 lamins causes thedepolymerization of the lamin

intermediate filaments. Lamin A and C diffuse into solution,

lamin B remains associated to themembrane by its isoprenyl anchor.

Disassembly of nuclear envelope hasbeen shown to be dependent on P* oflamin A.


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Condensation

Diffuse

Staining due to

+ve Staining

at all stages

IMP: Serto Alamutation

Other MPF substrates


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Other MPF substrates

P* ofnucleporins causes nuclear porecomplexes (NPC) to dissociate.

P* of integral inner nuclear membrane(INM) proteins decreases their affinityfor chromatin

Structural maintenance of

chromosomes (SMC) proteins isrequired for normal chromosomesegregation. These are also P*ed

MPF, P*tes Nuclear Env Proteins


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# representdifferent MPFtargets for P*,

leading to Nuc.Pore Compl(NPC)disintegration.

Inner NuclearMembrane-INM

Chromatin protn

P*tion by MPFinducescondenstn andinhibitsinteractionw/nuc env.


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Other MPF substrates (cont.)

SMC proteins are part of large multi-protein complex called condensin,which is P* as cells enter mitosis. (P* is

mediated by MPF)

If condensin is depleted by anti-SMCantibodies, egg extract can not

condense sperm chromatin.

P* of microtubules contributes tospindle formation-(also regulated by

MPF)

Other MPF substrates (cont.)


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( ) P* ofER and Golgi proteins by MPF alter

vesicle trafficking. During prophase,vesicle traffic from Golgi to ER to Cellsurface (that is seen during interphase)does not occur.

Many of the MPF substrates have beenidentified in S. Cerevisiae. By using ATPanalog that act as an inhibitor, severalsubstrates that normally P*ed by wt MPFwas found to be dephosphorylated.Proving that MPF shows kinase activity(in vivo and in vitro).

>150 proteins were identified, and their


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WT CDK- possess Phe88 (A) a

bulky group. When ATP analog isused it contains a long chain andcan not accomodated in the WTATP binding pocket (B).

Mutant CDK has Gly instead of

Phe, and N6 benzyl ATP canexhibit high kinase activity.

IMP. NOTE: An ATP inhibitor: possesses abulky substitution @ the N6 position of

Adenine. It prevent kinase activity of the


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Initiation of anaphase

Degradation of cyclin B and thedecrease in MPF activity are requiredfor chromosome decondensation, but

not for chromosome segregation. Is ubiquitin-dependent degradation of

another protein required for

chromosome segregation?

Onset of Anaphase depends on p(ubiquitination) of proteins otherthan Cyc-B


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y

Xenopus Egg and Sperm extracts are used along w/othercomponents: Rho-labelled Tubulin, to visualize microtubules. T=0:condensed Chr & spindle visible post mixing of extracts. @T=40`: NoTubulin is visible, since Cyclin B is degraded. DNA is diffused by

40min.

DiffuseDNAstaining

Nostaining


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Here, please note that mut.mRNA of Cyclin Bis added to the extract,and even after 80` Tubulin is still visible (i.e. No depolymerization)and DNA is not decondensed. This is due to the presence of nondegraded Cyclin B. Its presence prevented Chr. decondensation butDID NOT prevented the Chr. segragation. The spindle microtubule

depolymerization is required during Telophase !!!


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Addition of peptide, dose dependently delayed, and completelyinhibited chromosome segregation following spindle formation isinitiated (15` or 35` post-spindle formation, top vs bottom,

respectively). WHY?

Because, peptide is competitively inhibit APC/C mediatedp(ubiquitination) of Cyclin B and other target proteins whose


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p(ubiquitination) of Cyclin B and other target proteins whosedegradation is required for chromosome segragation

At metaphase sister chromatids donot separate because:

Opposite forces pull the kinetochores

toward the opposite poles and pushspindle poles apart (state oftension)

Sister chromatids are held togetherby cohesins (SMC antibody assay!)

Cohesin linkage of sister chromosomes: A workingmodel


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Association occurs @ lateG1

Cohesin complex is circular (similar to other SMC complexes!). # ofcohesins around sister chromatids is not known!!!. Passage of replicationfork through cohesin rings link together sister chromatids. In vertebrates:

Cohesins are released from Chr. arms, during prophase and early

@ late metaphase: nearcentromere


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In yeast and vertebrates, cohesinsremain associated only atcentromeric regions

MPF-mediated P* of cohesin results intheir dissociation from chromosomalarms

A specific protein phosphatase PP2Amediates this association.

Localization of PP2A @ the centromere


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(human metaphase chromosome is used forthe staining)

PP2Asubtype

B56a (green)

Centromore specific markerprotein (labeled W/ a reddye)

C h i Cl


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Cohesin Cleavage

How is sister chromatid separationregulated to initiate anaphase?

Genetic studies in S. cerevisiae

proposed a model for the regulationof sister chromatid separation.

Cdc20, another specificity factorfor

APC, is activated before Cdh1 anddirects APC to securin, the inhibitorofseparase.

REGULATION OF COHESIN CLEAVAGE


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REGULATION OF COHESIN CLEAVAGE

Separase (a protease), can cleave kleisin su. of cohesin complex.But this is inhibited before anaphase by the SECURIN. When thespindle is poorly assembled and all kinetochores are attached tomicrotubule spindles, CDC security factor associates w/ APC andp(ubiquitinylate) securin. This freeds Separase, and cleaves Kleisin,and breaks cohesin complex. Sister chromatids then can be pulled

towards opposite poles...

SUMMARY


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SUMMARYLamin filament depolymerization take place during earlymitosis.

Lamin A, B, & C P*tion is catalyzed by MPF. Dissociationof nuclear pores led to nuclear env. Disassembly and itsretraction into ER

P*tion of condensin complexes by MPF (or a kinaseregulated by MPF) promotes chromosome condensationearly in mitosis

Sister chromatids formed by DNA replication (in S-

phase), linked to centromere via cohesin complexes thatcontain DNAbinding SMC proteins.

At the onset of Anaphase, the APC/C is directed by cdk20to polyubiquitinylate securin (subsequently degraded byproteasomes. This activates separase, which cleaveskleisin, thereby unlinking sister chromatids

Reassembly of nuclear


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Reassembly of nuclearenvelope and cytokinesis

After the inactivation of MPF in lateanaphase by phosphatases, proteinsreturn back to their state in interphasecells. (Cdc14)

De-P* inner nuclear membrane proteinsreassociate with decondensed

chromosomes Protrusion of ER regions toward

chromatin fuse with each other and

form a double membrane around each Reassembly of Nuclear Envelope duringTelophase


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p

ER extensionsreach andassociate w/ the

decondensingChromosome

Then, fuse w/one another.


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Fusion of ER extensions occurs in

similar manner to that of vesicles andtarget membranes in secretorypathway

Ran.GTP is required for nuclear porecomplex formation and and fusion of ERprojections

Membrane fusion is stimulated at thesurfaces of decondensing chromosomesbecause Ran.GEF is associated withchromatin.


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ER extensions first surround eachdecondensed chromosomes forming mininuclei called karyomeres

Fusion of karyomeres at each spindle poleforms two daugther cell nuclei eachcontaining a full set of chromosomes

De-P* lamins A and C are transportedthrough nuclear pores and reassemble withlamin B in the membrane deriving from theER (isoprenyl anchor)


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MPF activity P* myosin light chain andinhibits the ability of myosin toassociate with actin filaments.

In late anaphase, after MPF isinactivated, contractile machinery isactivated.

This allows cleavage furrow to formand cytokinesis to proceed.

A mechanism that assures that

cytokinesis does not occur until

-Ubiquitin Protein


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Ubiquitin-Protein

Ligase Control of Sphase. In this section our focus is on the G1

to S transition.

This phase is START, similar to mitosis,it is also controlled by cyclin-CDKs.

The roles of G1 Cyclin-CDKs and S-Phase cyclin-CDKs to initiate DNAreplication once/cell cycle and how itresets after mitosis will be discussed.

Genetic studies with S


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cerevisiae

Entering S phase and replication ofDNA is the key decision for cells tocomplete the cell cycle.

S cerevisiae cells replicate by buddingand have to reach a proper sizebefore dividing.

They remain in G1 phase

In late G1 phase the point where cellsare committed to replicate their DNA

is START


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4/23/12 SEM of S.cerevisiae @ different stages of the cellc cle.

About todivide

Newlybud

The larger the bud @ the end of G1 phase, thefurther along in the cycle the cell is!!


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further along in the cycle the cell is!!

Size!


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G1 and S cyclin-CDKs:

Initiate DNA synthesis

Ensure that DNA replication occurs onlyonce per cell cycle

How the cell cycle resets after mitosis,

in preparation for the next celldivision.

A CDK is critical for S-phase entry in S.

cerevisiae

CDK for S phase entry


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CDK for S phase entry

Particular cdc mutants arrest with thesame size at non-permissivetemperature.

No bud: cdc28 (arrest just before theemergence of the new bud from the mothercell).

Intermediate or larger buds: cdc7 (arrest

just before the mother cell and budseparates).

In S. Cerevisiae

Wt: CDC28

Size dependence!


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Size dependence!

At non-permissive temperatures: Large cdc28 mutants continue through

the cell cycle and undergo mitosis

Passes START at the time of shift Small cdc28 mutants do not enter S

phase

Blocked in G1; continue to grow, but cannot bud

wt CDC28 complemented these

mutants, and have been shown to

CDC28 is a CDK in


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S.cerevisiae CDC28 was found to be homologous

to S. pombecdc2+ gene.

These are also functionally analogous

and can substitute each other. Why are mutant phenotypes ofcdc2

- in S. pombe and cdc28 mutants in

S. cerevisiae different?

S Phase Promoting Factors


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S Phase-Promoting Factors

By analogy to MPF activity, it is thoughtthat S cerevisiae contains an SPF thatP* and regulates DNA replication.

It is likely that SPF is a heterodimer,which consists of a CDK and anassociated cyclin

Where is SPF supposed to act?


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Ts studies; complementation with wildtype genes led to the discovery of

CLN1; CLN2 and CLN3

These are related proteins, eachcontaining 100 aa. region homologous toB-type cyclins (cyclin domain) of seaurchin, xenopus, human, & S.pombe.

IMP.NOTE: CLN3 is Mid G1 cyclin, anddepend on the nutrient level. Identified w/a different approach.


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WT cells produce normal CDK & associates w/G1-Cylins,forming normal SPF resulting colonies at both Temp.


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CD28ts cells produce mut. CDK, and have low affinity toG1-Cylins. At 25C they produce enough SPF supportingcolony formation, but not @ T=36C


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3 CDKs formed:WT, CLN1 andCLN2.

High-copy G1-cylins, offset thelow affinity CDKand form enoughcyc-CDKs (SPF)leading to S-phaseentry. Coloniesformed at bothTemp.

Gene knock-out experiments:


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Gene knock-out experiments:

S cerevisiae that contain any of 3 G1

cyclin genes can grow in richmedium.

Over production of G1 cyclins

decreases the fractions of cells inG1.

Overproduction of G1-CDK drive cellsprematurely through START.

In the absence of all three G1-cyclins,cells become arrested @ G1.Documenting that G1-Cyclin-CDK

heterodimer (or SPF) is re uired for


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Yeast expression vector: under the control of a strongGAL1 promoter, and is turned off under GLU+.

To determine the proportions of cells in G1 and G2,cells were stained by a fluorescent dye that binds toDNA and sorted by FACS (Propidium Iodide:PI)

Since the content s of DNA in G2 cells is 2x more thanG1 cells than these 2 populations can be identified bythe Fluorescence intensities of these phases.

PI Staining of cells and analyses byFACS


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Addition ofGLU: noeffect oncelldistribution

Over expression ofG1-cyclin, reduced

the time of G1phase and most ofthe cells were in S-or G2 phase.

When shut-off: cellphases turned

normal

Mutant cells w/ the G1 cyclinprovided by the vector enter

mostly into S and G2 phases (i.esimilar to case B).

When Glu is provided and thevectror is shut down: all cellscompleted cycle and arrested @G1.

Indicating that G1cyclin is

In WT yeast cells Gln3 is a midG1 C li


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G1 Cyclin

CLN3 translation is inhibited at anupstream open reading frame (ORF) inthe absence of nutrients. It is a highlyunstable protein.

This causes instability and fluctuationsin Cln3 levels.

Thus: Cln3-CDK is tightly regulated by

nutrients. Mid G1-cyclin CDK complex, P* and

activates two related TF (SPF and MBF)

and induce transcription of the late G1


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Regulating CLN3 level by nutrients,modulates length of G1 phase in

S.cerevisiae. SBF and MBF also induce transcription

other genes required for DNA

replication including, DNA polymerase subunits; RPA subunits

(eukaryotic ssDNA binding protein); DNAligase


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Cdh1 is a substrate of late G1 cyclins(Cln1-CDK; Cln2-CDK).

MBF also stimulates transcription of

CLB5 and CLB6, which encode B-typecyclins.

These are required for the initiation

of DNA synthesis. Clb5 and Clb6 are S-phase cyclins.


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APC is inactivated early in the G1. This allows S-phase cyclins-CDK.

complexes to accumulate in late G1.

Cdh1 is kept P* by late G1 cyclins, aswell as by S-phase cyclins.

Thus: Cdh1 remains inhibited untilthe late anaphase.

S-phase inhibitor


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S phase inhibitor

As S-phase cyclin-CDK complexesaccumulate in late G1, they areimmediately inactivated by an inhibitor,Sic1.

Sic1 has no effect on G1 cyclins; it is S-phase inhibitor.

In S. cerevisae, entry into S phasedepends on the degradation of Sic1.

SCF ubiquitin ligase polyubiquitinates

and tar ets Sic1 to roteosomal

Cln1and 2CDK ) ACTIVE


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Inhibitor:

preventDNAreplicationi i i i

@ late G1: assembly iscompleted.

It also P* Cdh1. Dissociatesfrom APC/C complex andprevent further degradation ofB-type Cyclins during late G1.

Clb5 andclb6 CDKs

CDKs)

SCFubiquitinlagase

ACTIVE

Initiation of DNAsynthesis by P*pre-initiation

complexes

Regulating the Eukaryotic Cell Cycle

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