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2006-2007

Regulation of Cell

DivisionDr. Sri Widyarti

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Coordination of cell division

A multicellular organism needs tocoordinate cell division acrossdifferent tissues & organs

critical for normal growth, development &maintenance

coordinate timing of cell division

coordinate rates of cell division

not all cells can have the same cell cycle

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Frequency of cell division varies by cell type embryo

cell cycle < 20 minute

skin cells

divide frequently throughout life12-24 hours cycle

liver cellsretain ability to divide, but keep it in reservedivide once every year or two

mature nerve cells & muscle cellsdo not divide at all after maturitypermanently in G0

Frequency of cell division

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Cell Cycle

G1- Highest metabolic activity.Cytoplasmic contents increase. Proteinsynthesis, membrane synthesis

S replication of DNA. Chromosomescopied( sister chromatids form)G2- Preparation for cell division

Spindle fiber formationM Cell divisionCytokinesis division of the cytoplasm

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Checkpoint control system

Checkpoints cell cycle controlled by STOP& GO

chemical signals at critical points

signals indicate if key cellularprocesses have beencompleted correctly

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Checkpoint control system

3 major checkpoints: G1/S :can DNA synthesis begin? G2/M

has DNA synthesis been completed correctly?commitment to mitosis

spindle checkpointare all chromosomes attached to spindle?

can sister chromatids separate correctly?

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G1/S checkpoint

G1/S checkpoint is most critical primary decision point

restriction point

if cell receives GO signal, it dividesinternal signals: cell growth (size), cell nutrition

external signals: growth factors

if cell does notreceivesignal, it exits cycle &switches to G0phase

non-dividing, working state

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G0phase

M

Mitosis

G1

Gap 1

G0

Resting

G2

Gap 2

S

Synthesis

G0phase non-dividing, differentiated state

most human cells in G0phase

liver cells in G0, but can becalled back to cell

cycle by external cues

nerve & muscle cells

highly specialized

arrested in G0& can

never divide

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How do cells know when to divide? cell communication signals

chemical signals in cytoplasm give cue

signals usually mean proteins activators

inhibitors

Activation of cell division

experimental evidence: Can you explain this?

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Go-aheadsignals

Protein signals that promote cellgrowth & division internal signals

promoting factors external signals

growth factors

Primary mechanism of control phosphorylation

kinaseenzymes

either activates or inactivates cell signals

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Cell cycle signals

Cell cycle controls cyclins

regulatory proteins

levels cycle in the cell Cdks

cyclin-dependent kinasesphosphorylates cellular proteins

activates or inactivates proteins Cdk-cyclin complex

triggers passage through different stagesof cell cycle

activated Cdk

inactivated Cdk

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Cdk / G1cyclin

Cdk / G2cyclin (MPF)

G2

S

G1

CM

G2/ M checkpoint

G1/ S checkpoint

APC

ActiveInactive

ActiveInactive

InactiveActive

mitosis

cytokinesis

MPF= MitosisPromoting FactorAPC= Anaphase

Promoting Complex

Replication completedDNA integrity

Chromosomes attached

at metaphase plate

Spindle checkpoint

Growth factors

Nutritional state of cellSize of cell

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Cyclin & Cyclin-dependent kinases

CDKs & cyclin drive cellfrom one phase to nextin cell cycle

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External signals

Growth factors coordination between cells

protein signals released by bodycells that stimulate other cells

to dividedensity-dependent inhibition crowded cells stop dividing

each cell binds a bit of growthfactor

not enough activator left totrigger division in any one cell

anchorage dependence to divide cells must be attached to a

substrate

touch sensor receptors

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Example of a Growth Factor

Platelet Derived Growth Factor (PDGF) made by platelets in blood clots

binding of PDGF to cell receptors stimulatescell division in fibroblast (connective tissue)

heal wounds

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Growth Factors and Cancer

Growth factors can create cancers proto-oncogenes

normal growth factor genes that becomeoncogenes (cancer-causing) when mutated

stimulates cell growth

if switched ONcan cause cancer

example: RAS (activates cyclins)

tumor-suppressor genesinhibits cell division

if switched OFFcan cause cancer

example: p53

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Cancer & Cell Growth

Cancer is essentially a failureof cell division control unrestrained, uncontrolled cell growth

What control is lost?

lose checkpoint stops gene p53plays a key role in G1/S restriction point

p53 protein halts cell division if it detects damaged DNA options:

stimulates repair enzymes to fix DNA

forces cell into G0resting stagekeeps cell in G1arrestcauses apoptosis of damaged cell

ALLcancers have to shut down p53 activity

p53 discovered at Stony Brook by Dr. Arnold Levine

p53 is theCell Cycle

Enforcer

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DNA damage is causedby heat, radiation, orchemicals.

p53 allows cells

with repairedDNA to divide.

Step 1

DNA damage iscaused by heat,radiation, orchemicals.

Step 1 Step 2

Damaged cells continue to divide.

If other damage accumulates, thecell can turn cancerous.

Step 3p53 triggers the destructionof cells damaged beyondrepair.

ABNORMALp53

NORMALp53

abnormalp53 protein

cancercell

Step 3The p53 protein fails to stopcell division and repair DNA.Cell divides without repair to

damaged DNA.

Cell division stops, andp53 triggers enzymes torepair damaged region.

Step 2

DNA repair enzymep53protein

p53protein

p53 master regulatorgene

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Chromosome overview

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Chromsome structure

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Metaphase Structure

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Go

Many times a cell will leave the cell cycle, temporarilyor permanently. It exits the cycle at G1 and enters astage designated G0 (G zero). A G0 cell is oftencalled "quiescent", but that is probably more a

reflection of the interests of the scientists studyingthe cell cycle than the cell itself. Many G0 cells areanything but quiescent. They are busy carrying outtheir functions in the organism. e.g., secretion,attacking pathogens.

Often G0 cells are terminally differentiated: theywill never reenter the cell cycle but instead will carryout their function in the organism until they die.

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Cyclins and cell cycle regulation

The passage of a cellthrough the cellcycle is controlled

by proteins in thecytoplasm.

Among the mainplayers in animal

cells are:

Cyclins

G1 cyclin(cyclin D)

S-phase cyclins

(cyclins E and A)mitotic cyclins(cyclins B and A)

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Cyclin dependent kinases

Their levels dependupon the stage ofthe cell cycle

They add phosphatesto cyclins during thephases of the cell

cycle

Cyclin-dependentkinases(Cdks)

G1 Cdk(Cdk4)

S-phase Cdk(Cdk2)M-phase Cdk(Cdk1)

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Cyclins - events

G1-cyclins+ Cdks signal the cell to preparethe chromosomes for replication.S-phase promoting factor(SPF) includes

cyclin A + Cdk2

enters the nucleus andpreparesthe cell to duplicate its DNA (andits centrosomes).As DNA replication continues, cyclin E is

destroyed, and the level of mitotic cyclinsbegins to rise (in G2).

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Oncogenes

All the checkpoints examined requirethe services of a complex of proteins.Mutations in the genes encoding some of

these have been associated with cancer;that is, they are oncogenes. This shouldnot be surprising since checkpoint

failures allow the cell to continuedividing despite damage to its integrity.

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Cyclins and cell cycle regulations

Spindle fibers have three

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Spindle fibers have threedestinations:

Some attach to one kinetochoreof a dyadwith those growing from the oppositecentrosome binding to the other kinetochore

of that dyad.Some bind to the arms of the chromosomes.

Still others continue growing from the twocentrosomes until they extend between eachother in a region of overlap.

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How Spindle Fibers Work

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Microtubules

Grow at each end bythe polymerizationof tubulin dimers

(powered by thehydrolysis of GTP),and

Shrink at each end

by the release oftubulin dimers(depolymerization)

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All types of spindle fibersparticipate in

the assembly of the chromosomes at the metaphaseplate at metaphase. Proposed mechanism (the diagramshows only 1 and 2):Microtubules attached to opposite sides of the dyad

shrink or grow until they are of equal length.Microtubules motors attached to the kinetochoresmove them toward the minusend of shrinkingmicrotubules (a dynein); toward the plusend of lengtheningmicrotubules (a kinesin).

The chromosome armsuse a different kinesinto moveto the metaphase plate.

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Microtubule Motors

There are two major groups ofmicrotubule motors:

kinesins(most of these move towardthe plus end of the microtubules) and

dyneins(which move toward the minusend).

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Kinesins and dyneins

The sister kinetochores separate and,carrying their attached chromatid,move along the microtubules powered byminus-end motors, dyneins, while the

microtubules themselves shorten (probably atboth ends).The overlapping spindle fibers move past eachother (pushing the poles farther apart)

powered by plus-end motors, the "bipolar"kinesins.In this way the sister chromatids end up atopposite poles.

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Anaphase promoting complex

The anaphase-promoting complex(APC). (The APC is also called thecyclosome, and the complex is often

designated as the APC/C.) The APC/CTriggers the events leadingtodestruction of the cohesinsthusallowing the sister chromatids toseparate;Degrades the mitotic cyclin B.

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Proteosome Core Particle

The core particle ismade of 2 copies ofeach of 14 differentproteins.These are assembledin groups of 7forming a ring.

The 4 rings arestacked on eachother (like 4 donuts)

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Proteosome Regulatory Particle

There are two identicalRPs, one at each end ofthe core particle.Each is made of 14

different proteins (noneof them the same asthose in the CP).6 of these are ATPases.Some of the subunitshave sites thatrecognize the smallprotein ubiquitin.

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Ubiquitin

A small protein (76 amino acids)

Conserved throughout all the kingdomsof life; that is, virtually identical insequence whether in bacteria, yeast, ormammals.

Used by all these creatures to targetproteins for destruction.

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Proteosome and proteins

Are conjugated to a molecule of ubiquitinwhichbinds to the terminal amino group of alysineresidue.Additional molecules of ubiquitin bind to the

first forming a chain.The complex binds to ubiquitin-recognizingsite(s) on the regulatory particle.The protein is unfolded by the ATPases using

the energy of ATPThe unfolded protein is translocated into thecentral cavity of the core particle.

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Proteosomes and Particles(2)

Several active sites on the inner surface of the twomiddle "donuts" break various specific peptide bondsof the chain.This produces a set of peptides averaging about 8amino acids long.

These leave the core particle by an unknown routewherethey may be further broken down into individualamino acids by peptidases in the cytosol orin mammals, they may be incorporated in a class Ihistocompatibility molecule to be presented to theimmune system as a potential antigen[see below].The regulatory particle releases the ubiquitins forreuse.

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Spindle Checkpoints

Spindle checkpoints. Some of thesethat have been discovered

Detect any failure of spindle fibers toattach to kinetochores and arrest thecell in metaphase (M checkpoint example);

Detect improper alignment of thespindle itself and block cytokinesis;

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Mitosis - Prophase

The two centrosomesof the cell, each withits pair of centrioles, move to opposite "poles"of the cell.The mitotic spindleforms. This is an array ofspindle fibers, each containing ~20microtubules. Microtubules are synthesizedfrom tubulin monomers in the cytoplasm andgrow out from each centrosome.The chromosomes become shorter and morecompact.

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Mitosis - Prometaphase

The nuclear envelopedisintegrates because of thedissolution of the laminsthat stabilize its innermembrane.A protein structure, the kinetochore, appears at the

centromereof each chromatid.With the breakdown of the nuclear envelope, spindlefibers attach to the kinetochores as well as to thearms of the chromosomes.For each dyad, one of the kinetochores is attached toone pole, the second (or sister) chromatid to theopposite pole. Failure of a kinetochore to becomeattached to a spindle fiber interrupts the process.

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Metaphase

At metaphase all thedyads have reachedan equilibriumposition midway

between the polescalled themetaphase plate.The chromosomes

are at their mostcompact at thistime.

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Anaphase

The sisterkinetochoressuddenly separate

and each moves toits respective poledragging itsattached chromatid

(chromosome)behind it.

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Telophase

The chromosomesreach the poles

A nuclear envelope

reforms around eachcluster of

These return totheir more extendedform.

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Cytokineses

In animal cells,a belt ofactin filamentsformsaround the perimeter of thecell, midway between thepoles. The interaction of

actin and a myosin(not theone found in skeletalmuscle) tightens the belt,and the cell is pinched intotwo daughter cells.

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