FFK Nov 2011
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Transcript of FFK Nov 2011
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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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