3. OVERVIEWOFTHECELLCYCLE 1055
cytokinesis
mitosis
metaphase-to-anaphasetransition
INTERPHASE
DNAreplic
chromosomes are packaged into separate nuclei at telophase.Cltokinesis then
cleaves the cell in two, so that each daughter cell inherits one of the two nuclei
(Figure l7-3). <TACT><TCAA>
Most cells require much more time to grow and double their mass of pro-
teins and organelles than they require to duplicate their chromosomes and
divide. Partly to allow more time for growth, most cell cycles have exrra gap
phases-a G1phase between M phase and S phase and a Gz phase between S
phase and mitosis. Thus, the eucaryotic cell cycle is traditionally divided into
four sequential phases:Gr, S,G2,and M. Gr, S,and G2together are called inter-
phase (Figure l7-4, and seeFigure 17-3).In a typical human cell proliferating in
culture, interphase might occupy 23 hours of a 24-hour cycle,with I hour for M
phase.Cell growth occurs throughout the cell cycle,exceptduring mitosis.
The two gap phases are more than simple time delays to allow cell growth.
They also provide time for the cell to monitor the internal and external environ-
ment to ensure that conditions are suitable and preparations are complete
before the cell commits itself to the major upheavalsof Sphaseand mitosis.The
G1 phase is especially important in this respect. Its length can vary greatly
depending on external conditions and extracellular signalsfrom other cells. If
extracellular conditions are unfavorable, for example, cells delay progress
through G1and may even enter a specializedresting stateknown as Go(Gzero),
in which they can remain for days,weeks,or even yearsbefore resuming prolif-
eration. Indeed, many cellsremain permanently in Geuntil they or the organism
dies. If extracellular conditions are favorable and signals to grow and divide are
present, cells in early G1or G0progressthrough a commitment point near the
end of G1 knorvn as Start (in yeasts) or the restriction point (in mammalian
cells).We will use the term Start for both yeast and animal cells.After passing
this point, cells are committed to DNA replication, even if the extracellular sig-
nals that stimulate cell growth and division are removed.
M PHASE
mitosis
(nuclear
division)
G2PHASE
5 PHASE
Figure17-3 Theeventsofeucaryotic
celldivisionasseenundera
microscope,Theeasilyvisibleprocesses
of nucleardivision(mitosis)andcell
division(cytokinesis),collectivelycalled
M phase,typicallyoccupyonlya small
fractionof the cellcycle.Theother,much
longer,part of the cycleisknownas
interphase,whichincludesSphaseand
the gap phases(discussedin text).The
fivestagesof mitosisareshown:an
abruptchangein the biochemicalstate
of the celloccursat the transitionfrom
metaphaseto anaphase.A cellcanpause
in metaphasebeforethistransitionpoint,
but onceit passesthispoint,the cell
carrieson to the endof mitosisand
throughcytokinesisinto interphase.
Figure17-4 Thefour phasesof the cell
cycle.In mostcells,gap phasesseparate
the majoreventsof 5 phaseand M phase'
Gristhe gapbetweenM Phaseand
5 phase,while G2isthe gaPbetween
SphaseandM phase.
cytokinesis
(cytoplasmic
(DNAreplication)
G1PHASE
4. 1056 Chapter17:TheCellCycle
Cell-CycleControllsSimilarinAllEucaryotes
Some features of the cell cycle, including the time required to complete certain
events, vary greatly from one cell type to another, even in the same organism.
The basic organization of the cycle, however, is essentially the same in all
eucaryotic cells, and all eucaryotes appear to use similar machinery and control
mechanisms to drive and regulate cell-cycle events. The proteins of the cell-
cycle control system, for example, first appeared over a billion years ago.
Remarkably, they have been so well conserved over the course of evolution that
many of them function perfectly when transferred from a human cell to a yeast
cell. we can therefore study the cell cycle and its regulation in a variety of organ-
isms and use the findings from all of them to assemblea unified picture of how
eucaryotic cells divide. In the rest of this section, we briefly review the three
eucaryotic systemsmost commonly used to study cell-cycle organization and
control: yeasts,animal embryos, and cultured mammalian cells.
Cell-CycleControlCanBeDissectedGeneticallybyAnalysisof
YeastMutants
Yeastsare tiny, single-celled fungi, with a cell-cycle control system remarkably
similar to our own. TWospeciesare generally used in studies of the cell cycle.The
fission yeast schizosaccharomycespombe is named after the African beer it is
used to produce. It is a rod-shaped cell that growsby elongation at its ends.Divi-
sion occurs when a septum, or cell plate, forms midway along the rod (Figure
r7-5A). The budding yeast Saccharomycescereuisiaeis used by both brewers
and bakers. It is an oval cell that divides by forming a bud, which first appears
during G1and grows steadily until it separatesfrom the mother cell aftei mito-
sis (Figure l7-58).
gene,becausewe avoid the complication of having a second copy of the genein
the cell.
Many important discoveriesabout cell-cycle control have come from sys-
tematic searchesfor mutations in yeaststhat inactivate genesencoding essen-
tial components of the cell-cycle control system.The genesaffectedby iome of
YEAST(Schhosaccharomycespom be)
Gr
ISTART
YEAST(5accharomycescerevisiae)
Figure17-5 A comparisonof the cell
cyclesof fissionyeastsand budding
yeasts.(A)Thefissionyeasthasa typical
eucaryoticcellcyclewith Gr,5,G2,and
M phases.Thenuclearenvelopeof the
yeastcell,unlikethatof a higher
eucaryoticcell,doesnot breakdown
duringM phase.Themicrotubulesof the
mitoticspindle(lightgreen)forminside
the nucleusandareattachedto spindle
polebodies(darkgreen)at its periphery.
Thecelldividesbyforminga partition
(knownasthecellplate)andsplittingin
two.(B)ThebuddingyeasthasnormalG1
andSphasesbut doesnot havea normal
G2phase.Instead,a microtubule-based
spindlebeginsto form latein Sphase;as
in fissionyeasts,the nuclearenvelope
remainsintactduringmitosis,andthe
spindleformswithinthe nucleus.ln
contrastwith a fissionyeastcell,the cell
dividesby budding.
G r
@
9. THECELL-CYCLECONTROLSYSTEM
lsall DNAreplicated?
lsenvironmentfavorable?
G2lMCHECKPOINT
Are all chromosomes
attachedto the spindle?
METAPHASE-TO-ANAPHASE
TRANSITION
1061
Figure'17-14Thecontrolof the cell
cycle.A cell-cyclecontrolsystemtriggers
the essentialprocessesof thecellcycle-
suchasDNAreplication,mitosis,and
cytokinesis.Thecontrolsystemis
representedhereasa centralarm-the
controller-that rotatesclockwise,
triggeringessentialprocesseswhenit
reachesspecificcheckpointson theouter
dial.Informationaboutthecompletionof
cell-cycleevents,aswellassignalsfrom
theenvironment,cancausethe control
systemto arrestthe cycleat these
checkpoints.ThemostProminent
checkDointsoccurat locationsmarked
with vellowboxes.
STARTCHECKPOINT
lsenvironmentfavorable?
in these cells is independent of the eventsit controls, so that its timing mecha-
nisms continue to operate even if those eventsfail. In most cells,however,the
control systemdoesrespond to information receivedback from the processesit
controls. Sensors,for example, detect the completion of DNA synthesis,and if
some malfunction prevents the successfulcompletion of this process,signals
are sent to the control systemto delayprogressionto M phase.Suchdelayspro-
vide time for the machinery to be repaired and also prevent the disaster that
might result if the cycle progressedprematurely to the next stage-and segre-
gated incompletely replicated chromosomes,for example.
The cell-cyclecontrol systemis based on a connected seriesof biochemical
switches, each of which initiates a specific cell-cycle event. This system of
switchespossessesmany important engineeringfeaturesthat increasethe accu-
racy and reliability of cell-cycle progression. First, the switches are generally
binary (on/off) and launch eventsin a complete, irreversible fashion. It would
clearly be disastrous,for example, if events like chromosome condensation or
nuclear envelope breakdor.trnwere only partially initiated or started but not
completed. Second,the cell-cycle control system is remarkably robust and reli-
able,partly becausebackup mechanisms and other featuresallow the systemto
operate effectivelyunder a variety of conditions and even if some components
fail. Finally, the control system is highly adaptable and can be modified to suit
specific cell types or to respond to specific intracellular or extracellularsignals.
In most eucaryotic cells,the cell-cyclecontrol systemtriggerscell-cyclepro-
gression at three major regulatory transitions, or checkpoints (see Figure
I7-I4).The first checkpoint is Start (or the restriction point) in late Gr,where the
cell commits to cell-cycle entry and chromosome duplication, as mentioned
earlier.The second is the GzlM checkpoint, where the control system triggers
the early mitotic events that lead to chromosome alignment on the spindle in
metaphase.The third is the metaphase-to-anaphase transition, where the con-
trol systemstimulates sister-chromatid separation,leading to the completion of
mitosis and cytokinesis.The control systemblocks progressionthrough each of
these checkpoints if it detectsproblems inside or outside the cell. If the control
system sensesproblems in the completion of DNA replication, for example, it
will hold the cell at the G2iM checkpoint until those problems are solved.Simi-
larly, if extracellular conditions are not appropriate for cell proliferation, the
control system blocks progression through Start, thereby preventing cell divi-
sion until conditions become favorable.
TRIGGERANAPHASEAND
PROCEEDTO CYTOKINESIS
ENTERCELLCYCLEAND PROCEEDTO S PHASE
10. 1062 Chapter17:TheCellCycle
TheCell-CycleControlSystemDependson CyclicallyActivated
Cyclin-DependentProteinKinases(Cdks)
Central components of the cell-cyclecontrol systemare members of a family of
protein kinases knor,rmas cyclin-dependent kinases (Cdks). The activities of
these kinases rise and fall as the cell progressesthrough the cycle, leading to
cyclical changesin the phosphorylation of intracellular proteins that initiate or
regulate the major events of the cell cycle. An increase in Cdk activity at the
G2lM checkpoint, for example, increasesthe phosphorylation of proteins that
control chromosome condensation, nuclear envelope breakdown, spindle
assembly,and other eventsthat occur at the onset of mitosis.
Cyclical changes in Cdk activity are controlled by a complex array of
enzymes and other proteins that regulatethese kinases.The most important of
theseCdk regulatorsareproteins known ascyclins. Cdks,astheir name implies,
are dependent on cyclins for their activity: unless they are tightly bound to a
cyclin, they have no protein kinase activity (Figure f7-f 5). Cyclins were origi-
nally named becausethey undergo a cycle of syrthesis and degradation in each
cell cycle.The levelsof the cdk proteins, by contrast, are constant, at leastin the
simplest cell cycles.Cyclical changesin cyclin protein levelsresult in the cyclic
assembly and activation of the cyclin-cdk complexes; this activation in turn
triggerscell-cycle events.
There are four classesofcyclins, each defined by the stageofthe cell cycleat
which they bind cdks and function. All eucaryotic cells require three of these
classes(Figure l7-f 6):
l. G1/S-cyclinsactivate Cdks in late Gr and thereby help trigger progression
through Start, resulting in a commitment to cell-cycle entry. Their levels
fall in Sphase.
2. S-cyclins bind Cdks soon after progressionthrough Start and help stimu-
late chromosome duplication. S-cyclin levels remain elevateduntil mito-
sis, and these cyclins also contribute to the control of some early mitotic
events.
3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2lM
checkpoint. Mechanisms that we discusslater destroy M-cyclins in mid-
mitosis.
In most cells,a fourth classof cyclins,the Gl-cyclins, helps govern the activities
of the Gr/S cyclins,which control progressionthrough Start in late G1.
In yeastcells,a singlecdk protein binds all classesof cyclinsand triggersdif-
ferent cell-cycle events by changing cyclin partners at different stages of the
cycle.In vertebrate cells,by contrast, there are four cdks. TWointeract with Gr-
cyclins,one with G1/S-and S-cyclins,and onewith M-cyclins. In this chapter,we
simply refer to the different cyclin-Cdk complexesasG1-Cdk,Gr/S-Cdk, S-Cdk,
and M-Cdk. Thble l7-l lists the names of the individual Cdks and cyclins.
How do different cyclin-cdk complexes trigger different cell-cycle events?
The answer,at least in part, seemsto be that the cyclin protein does not simply
activateits cdk partner but also directs it to specific target proteins. As a result,
' , , G r t M
i_-metaphase-anaphase
i M G 1
APC/C
cyclin-dependent
kinase(Cdk)
Figure17-15Twokeycomponentsof
the cell-cyclecontrolsystem.When
cyclinformsa complexwith Cdk,the
proteinkinaseisactivatedto trigger
specificcell-cycleevents.Withoutcyclin,
Cdkisinactive.
Figure17-16 Cyclin-Cdkcomplexesof
the cell-cyclecontrol system.The
concentrationsof the threemajorcyclin
typesoscillateduringthecellcycle,while
the concentrationsof Cdks(not shown)
do not changeandexceedtheamounts
of cyclins.In lateG1,risingG1lS-cyclin
levelsleadto the formationof G1lS-Cdk
complexesthattriggerprogression
throughthe Startcheckpoint.S-Cdk
complexesform at the startof Sphase
andtriggerDNAreplication,aswellas
someearlymitoticevents.M-Cdk
complexesformduringG2but areheldin
an inactivestateby mechanismswe
describelater.Thesecomplexesare
activatedat theendof G2andtriggerthe
earlyeventsof mitosis.A separate
regulatoryprotein,the APC/C,whichwe
discusslater,initiatesthe metaphase-to-
anaphasetransition.
G,iS-cyclin
Gr/s-Cdk S-Cdk
11. THECELL.CYCLECONTROLSYSTEM
Table17-1TheMajorCyclinsandCdksofVertebratesandBuddingYeast
G1-Cdk
GrlS-Cdk
5-Cdk
M-Cdk
cyclinD* Cdk4Cdk6
cyclinE Cdkz
cyclinA Cdk2,Cdkl**
cyclinB Cdkl
Cln3
Cln1,2
clbs,6
clb1,2,3,4
cdkl"*
cdkl
cdkl
cdkl
" TherearethreeD cyclinsin mammals(cyclinsD1,D2,andD3)
**Theoriginalnameof CdklwasCdc2in bothvertebratesandflssionyeast,andCdc2Bin
buddingyeast
each cyclin-Cdk complex phosphorylates a different set of substrate proteins.
The same cyclin-Cdk complex can also induce different effects at different times
in the cycle,probably becausethe accessibilityof some Cdk substrateschanges
during the cell cycle.Certain proteins that function in mitosis, for example,may
become availablefor phosphorylation only in G2.
Studiesof the three-dimensional structuresof Cdk and cyclin proteins have
revealed that, in the absence of cyclin, the active site in the Cdk protein is partly
obscured by a slab of protein, like a stone blocking the entrance to a cave (Figure
l7-L7A). Cyclin binding causesthe slab to move away from the active site,result-
ing in partial activation of the Cdk enz)ryne(Figure l7-l7B). Full activation of the
cyclin-Cdk complex then occurs when a separate kinase, the Cdk-activating
kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active
site. This causesa small conformational change that further increasesthe activ-
ity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively
and thereby induce specific cell-cycle events (Figure I7-I7C). <TAGA>
InhibitoryPhosphorylationandCdkInhibitoryProteins(CKls)Can
SuppressCdkActivity
The rise and fall of cyclin levels is the primary determinant of Cdk activity dur-
ing the cell cycle. Severaladditional mechanisms, however, fine-tune Cdk activ-
ity at specific stagesofthe cycle.
Phosphorylation at a pair of amino acidsin the roof of the kinase active site
inhibits the activity of a cyclin-Cdk complex. Phosphorylation of thesesitesby a
protein kinase knor,vnasWeel inhibits Cdk activity, while dephosphorylation of
these sites by a phosphatase knor,rmas Cdc25 increases Cdk activity (Figure
17-18). We will seelater that this regulatory mechanism is particularly impor-
tant in the control of M-Cdk activity at the onset of mitosis.
Binding of Cdk inhibitor proteins (CKIs) also regulates cyclin-Cdk com-
plexes.The three-dimensional structure of a cyclin-Cdk-CKl complex reveals
activatingphosphate
(c)
Cdk-activatingkinase(CAK)cyclin
1063
Figure17-17Thestructuralbasisof Cdk
activation.Thesedrawingsarebasedon
three-dimensionalstructuresof human
Cdk2,asdeterminedby x-ray
crystallography.Thelocationof the bound
ATPisindicated.Theenzymeisshownin
threestates.(A)Inthe inactivestate,without
cyclinbound,the activesiteisblockedby a
regionof the proteincalledtheT-loop(red).
(B)Thebindingof cyclincausestheT-loopto
moveout of the activesite,resultingin
partialactivationof the Cdk2.(C)
Phosphorylationof Cdk2(byCAK)at a
threonineresiduein theT-loopfurther
activatesthe enzymebychangingtheshape
of theT-loop,improvingthe abilityof the
enzymeto bind itsproteinsubstrates.
activesite
(A) TNACTTVE (B) PARTLYACTIVE
12. 1064 Chapter17:TheCellCycle
that CKI binding stimulates a large rearrangement in the structure of the Cdk
active site,rendering it inactive (Figure f 7-fg). Cellsuse CKIs primarily to help
govern the activities of Gr/S- and S-Cdksearly in the cell cycle.
TheCell-CycleControlSystemDependson CyclicalProteolysis
fhereas activation of specificcyclin-Cdk complexesdrives progressionthrough
the Start and G2lM checkpoints (see Figure 17-16), progression through the
metaphase-to-anaphasetransition is triggered not by protein phosphorylation
but by protein destruction, leading to the final stagesof cell division.
The key regulator of the metaphase-to-anaphasetransition is the anaphase-
promoting complex, or cyclosome (APC/C),a member of the ubiquitin ligase
family of enzymes.As discussedin Chapter 3, many of these enzyrnesare used
in numerous cell processesto stimulate the proteolytic destruction of specific
regulatory proteins.They transfer multiple copiesof the small protein ubiquitin
to specific target proteins, resulting in their proteolytic destruction by the pro-
teasomes. Other ubiquitin ligases mark proteins for purposes other than
destruction.
The APC/C catalyzesthe ubiquitylation and destruction of two major pro-
teins.The first is securln,which normally protects the protein linkagesthat hold
sister chromatid pairs together in early mitosis. Destruction of securin at the
metaphase-to-anaphasetransition activatesa proteasethat separatesthe sisters
and unleashesanaphase.The S- and M-cyclins are the second major targetsof
the APCic. Destroying thesecyclins inactivatesmost cdks in the cell (seeFigure
17-16).As a result, the many proteins phosphorylated by Cdks from S phase to
early mitosis are dephosphorylated by various phosphatasesthat are present in
the anaphase cell. This dephosphorylation of Cdk targets is required for the
completion of M phase,including the final steps in mitosis and the processof
cytokinesis.Following its activation in mid-mitosis, the APC/c remains activein
G1,thereby providing a stableperiod of Cdk inactivity.MhenG1/S-Cdksare acti-
vated in late Gr, the APC/G is turned off, thereby allowing cyclin accumulation
to start the next cell cycle.
The cell-cycle control system also uses another ubiquitin ligase called SCF
(after the names of its three subunits). It ubiquitylates certain cKI proteins in
late G1and thereby helps control the activation of S-cdks and DNA replication.
The APC/C and SCF are both large, multisubunit complexes with some
related components, but they are regulated differently. APC/C activity changes
during the cell cycle,primarily as a result of changesin its associationwith an
activating subunit-either cdc20 during anaphase or cdhl from late mitosis
through early G1.These subunits help the APC/G recognize its target proteins
(Figure l7-2oL). SCFactivity also depends on subunits called F-box proteins,
which help the complex recognize its target proteins. unlike Apc/c activity,
however, scF activity is constant during the cell cycle. ubiquitylation by scF is
controlled instead by changesin the phosphorylation stateof its target proteins,
as F-box subunits recognize only specifically phosphorylated proteins (Figure
I7-20H.
actrve
cyclin-Cdk
complex
inactive
p27-cyclin-Cdk
comprex
Cdk activating
phosphate
INACTIVE
Figure17-18Theregulationof Cdk
activity by inhibitory phosphorylation.
Theactivecyclin-Cdkcomplexisturned
off when the kinaseWeel phosphorylates
two closelyspacedsitesabovethe active
site.Removalof thesephosphatesby the
phosphataseCdc25activatesthe
cyclin-Cdkcomplex.Forsimplicity,only
oneinhibitoryphosphateisshown.CAK
addstheactivatingphosphate,asshown
in Figure17-17.
Figure17-19Theinhibitionof a
cyclin-Cdkcomplexby a CKl.This
drawingisbasedon thethree-
dimensionalstructureof the human
cyclinA-Cdk2complexboundto the
CKIp27,asdeterminedby x-ray
crystallography.Thep27bindsto both
thecyclinandCdkin the complex,
distortingthe activesiteof the Cdk.lt
alsoinsertsintotheATP-bindingsite,
furtherinhibitingtheenzymeactivity.
15. 5 PHASE
Summary
Thecell-cyclecontrol systemtriggersthe euentsof the cell cycleand ensuresthat these
euentsare properly timed and occur in the correctorder Thecontrol systemresponds
to uarious intracellular and extracellular signalsand arreststhe cyclewhen the cell
eitherfails to completean essentialcell-cycleprocessor encountersunfauorableenui-
ronmentalor intracellularconditions.
Central componentsof the cell-cyclecontrol systemare cyclin-dependentprotein
kinases(Cdks),which dependon cyclin subunitsfor their actiuity. Oscillationsin the
actiuitiesof uariouscyclin-Cdk complexescontrol uariouscell-cycleeuents.Thus,acti-
uation of S-phasecyclin-Cdk complexes(S-Cdk)initiates Sphase,while actiuation of
M-phase cyclin-Cdk complexes(M-Cdk) triggersmitosis. Themechanismsthat con-
trol the actiuities of cyclin-Cdk complexesinclude phosphorylation of the Cdk sub-
unit, binding of Cdk inhibitor proteins(CIQs),proteolysisof cyclins,and changesin the
transcription of genesencoding Cdk regulators. The cell-cyclecontrol systemalso
dependscrucially on two additional enzymecomplexes,theAPC|Cand SCFubiquitin
ligases,which catalyzethe ubiquitylation and consequentdestructionof specificregu-
latory proteins that control critical euentsin thecVcle.
SPHASE
The linear chromosomes of eucaryotic cells arevast and dyramic assembliesof
DNA and protein, and their duplication is a complex process that takes up a
major fraction of the cell cycle. Not only must the long DNA molecule of each
chromosome be duplicated accurately-a remarkablefeat in itself-but the pro-
tein packaging surrounding each region of that DNA must also be reproduced,
ensuring that the daughter cellsinherit all featuresof chromosome structure.
The central event of chromosome duplication is replication of the DNA. A
cell must solvetwo problems when initiating and completing DNA replication.
First, replication must occur with extreme accuracy to minimize the risk of
mutations in the next cell generation. Second,every nucleotide in the genome
must be copied once, and only once, to prevent the damaging effects of gene
amplification. In Chapter 5,we discussthe sophisticatedprotein machinery that
performs DNA replication with astonishing speedand accuracy.In this section,
we consider the elegantmechanisms by which the cell-cyclecontrol systemini-
tiates the replication processand, at the sametime, prevents it from happening
more than once per cycle.
S-CdkInitiatesDNAReplicationOncePerCycle
DNA replication begins at origins of replication, which are scattered at numer-
ous locations in everychromosome. During Sphase,the initiation of DNA repli-
cation occurs at these origins when specializedprotein machines (sometimes
called initiator proteins) unwind the double helix at the origin and load DNA
replication enzymes onto the two single-stranded templates. This leads to the
elongationphase of replication, when the replication machinery movesoutward
from the origin at tuvoreplicationforks (discussedin Chapter 5).
To ensure that chromosome duplication occurs only once per cell cycle,
the initiation phase of DNA replication is divided into two distinct steps that
occur at different times in the cell cycle. The first step occurs in late mitosis
and early Gr, when a large complex of initiator proteins, called the prereplica-
tive complex, or pre-RC, assemblesat origins of replication. This step is some-
times called licensingof replication origins because initiation of DNA synthe-
sis is permitted only at origins containing a pre-RC.The second step occurs at
the onset of S phase,when components of the pre-RC nucleate the formation
of a larger protein complex called the preinitiation complex. This complex
then unwinds the DNA helix and loads DNA polymerasesand other replication
enzymes onto the DNA strands,thereby initiating DNA synthesis,asdescribed
in Chapter 5. Once the replication origin has been activated in this way, the
1067
16. 1068 Chapter17:TheCellCycle
prereplicativecomplexesat replicationorigins
replication
elongation forks
M-Cdkactivation
|
.hrotoro." segregation
,.4^
I
G l
pre-Rc is dismantled and cannot be reassembledat that origin until the follow-
ing G1.As a result, origins can be activated only once per cell cycle.
The cell-cycle control system governs both assembly of the pre-RC and
assemblyof the pre-initiation complex (Figure lZ-22). Assembly of the pre-RC
is inhibited by Cdk activiry and, in most cells,is stimulated by the ApC/C. pre-
RC assemblytherefore occurs only in late mitosis and early G1,when cdk activ-
ity is low and APC/C activity is high. At the onset of S phase,activation of S-Cdk
then triggersthe formation of a preinitiation complex, which initiates DNA syn-
thesis.In addition, the pre-RC is partly dismantled. BecauseS-Cdk and M-Cdk
activities remain high (and APC/C activity remains low) until late mitosis, new
pre-RCscannot be assembledat fired origins until the cell cycleis complete.
Figure 17-23 illustrates some of the proteins involved in the initiation of
DNA replication. A key player is a large,multiprotein complex called the origin
recognition complex (oRc), which binds to replication origins throughout the
cell cycle.In late mitosis and early Gr, the proteins cdc6 and cdtl bind to the
ORCat origins and help load a group of six relatedproteins called the Mcm pro-
teins. The resulting large complex is the pre-RC,and the origin is now licensed
for replication.
The six Mcm proteins of the pre-RC form a ring around the DNA that is
thought to serve as the major DNA helicase that unwinds the origin DNA when
DNA synthesis begins and as the replication forks move out from the origin.
Thus, the central purpose of the pre-RC is to load the helicasethat will play a
central part in the subsequentDNA replication process.
Once the pre-RChas assembledin Gr, the replication origin is ready to fire.
The activation of s-cdk in late G1triggersthe assemblyof severaladditional pro-
tein complexes at the origin, leading to the formation of a giant preinitiation
complex that unwinds the helix and begins DNA synthesis.
At the same time as it initiates DNA replication, S-Cdk triggers the disas-
sembly of some pre-RCcomponents at the origin. cdks phosphorylate both the
oRC and cdc6, resulting in their inhibition by various mechanisms. Further-
more, inactivation of the APC/G in late Gr also helps turn off pre-RC assembly.
In late mitosis and early G1,the APC/C triggers the destruction of a protein,
Figure17-22 Controlof chromosome
duplication.Preparationsfor DNA
replicationbeginin Grwith theassembly
of prereplicativecomplexes(pre-RCs)at
replicationorigins.S-Cdkactivationleads
to the formationof multiprotein
preinitiationcomplexesthat unwindthe
DNAat originsandbeginthe processof
DNAreplication.Two replicationforks
moveout fromeachoriginuntilthe
entirechromosomeisduplicated.
Duplicatedchromosomesarethen
segregatedin M phase.Theactivationof
replicationoriginsin Sphasealsocauses
disassemblyof the prereplicative
complex,whichdoesnot reformat the
originuntilthefollowingG1-thereby
ensuringthateachoriginisactivated
onlyoncein eachcellcycle.
17. SPHASE
u 1
COMPLETIONOF
DNA REPLICATION
prereplicativecomplex(pre-RC)
ORC(originrecognitioncomplex)
t61M
geminin, that binds and inhibits the pre-RC component Cdtl. Thus, when the
APC/C is turned off in late G1,geminin accumulatesand inhibits Cdtl.In these
various ways, S- and M-Cdk activities,combined with lowAPC/C activity,block
pre-RCformation during Sphaseand thereafter.How then, is the cell-cyclecon-
trol system reset to allow replication to occur in the next cell cycle?The answer
is simple. At the end of mitosis, APC/C activation leads to the inactivation of
Cdks and the destruction of geminin. Pre-RC components are dephosphory-
lated and Cdtl is activated,allowing pre-RCassemblyto prepare the cell for the
next Sphase.
ChromosomeDuplicationRequiresDuplicationof Chromatin
Structure
The DNA of the chromosomes is extensively packaged in a variety of protein
components, including histones and various regulatory proteins involved in
the control ofgene expression (discussedin Chapter 4). Thus, duplication ofa
1Mg
Figure17-23 Controlof the initiationof
DNAreplication.TheORCremains
associatedwith a replicationorigin
throughoutthe cellcycle.InearlyG1,
Cdc6andCdtl associatewith the ORC.
Theresultingproteincomplexthen
assemblesMcmringcomplexeson the
adjacentDNA,resultingin the formation
of the prereplicativecomplex(pre-RC).
S-Cdk(withassistancefrom another
proteinkinase,not shown)then
stimulatesthe assemblyof several
additionalproteinsat the originto form
the preinitiationcomplex.DNA
polymeraseand other replication
proteinsarerecruitedto the origin,the
Mcm proteinringsareactivatedasDNA
helicases,andDNAunwindingallows
DNAreplicationto begin.S-Cdkalso
blocksrereplicationby triggeringthe
destructionof Cdc6andthe inactivation
ofthe ORC.Cdtl isinactivatedby the
proteingeminin.GemininisanAPC/C
targetand itslevelsthereforeincreasein
5 and M phases,when APC/Cisinactive.
Thus,the componentsofthe pre-RC
(Cdc6,Cdt1,Mcm)cannotform a new
pre-RCat the originsuntilM-Cdkis
inactivatedandthe APC/Cisactivatedat
the end of mitosis(seetext).
18. 1070 Chapter17:TheCellCycle
chromosome is not simply a matter of duplicating the DNA at its core but also
requiresthe duplication of thesechromatin proteins and their proper assembly
on the DNA.
The production of chromatin proteins increasesduring S phase to provide
the raw materials needed to packagethe newly synthesizedDNA. Most impor-
tantly, S-Cdksstimulate a large increasein the synthesisof the four histone sub-
units that form the histone octamers at the core of eachnucleosome.Thesesub-
units areassembledinto nucleosomeson the DNA by nucleosome assemblyfac-
tors, which typically associatewith the replication fork and distribute nucleo-
somes on both strands of the DNA as they emerge from the DNA synthesis
machinery.
Chromatin packaginghelps to control geneexpression.In some parts of the
chromosome, the chromatin is highly condensedand is called heterochromatin,
whereasin other regionsit has a more open structure and is called euchromatin.
These differences in chromatin structure depend on a variety of mechanisms,
including modification of histone tails and the presenceof non-histone proteins
(discussedin Chapter 4). Becausethese differencesare important in generegu-
lation, it is crucial that chromatin structure, like the DNA within, is reproduced
accurately during S phase. How chromatin structure is duplicated is not well
understood, however. During DNA synthesis,histone-modifying enz).rynesand
various non-histone proteins are probably deposited onto the two new DNA
strands asthey emergefrom the replication fork, and theseproteins arethought
to help reproduce the local chromatin structure of the parent chromosome.
CohesinsHelpHold5isterChromatidsTogether
At the end of S phase,each replicated chromosome consistsof a pair of identi-
cal sister chromatids glued together along their length. This sister-chromatid
cohesion setsthe stagefor a successfulmitosis becauseit greatly facilitates the
attachment of the two sisterchromatids in a pair to opposite polesof the mitotic
spindle. Imagine how difficult it would be to achievethis bipolar attachment if
sisterchromatids were allowed to drift apart after Sphase.Indeed, defectsin sis-
ter-chromatid cohesion-in yeast mutants, for example-lead inevitably to
major errors in chromosome segregation.
Sister-chromatid cohesion depends on a large protein complex called
cohesin, which is deposited at many locations along the length of each sister
chromatid asthe DNA is replicated in s phase.TWoof the subunits of cohesin are
members of a large family of proteins calledsMC proteins (for Structural Main-
tenance of chromosomes). cohesin forms giant ring-like structures,and it has
been proposed that these might form rings that surround the two sister chro-
matids (Figure 17-24).
5mcmolecule
y'--'-} .
r
hinge
C N
ATPase
domain
Figure17-24Cohesin,Cohesinisa
proteincomplexwithfoursubunits.Two
subunits,Smcl andSmc3,arecoiled-coil
proteinswithanATPasedomainat one
end;together,theyform a large
V-shapedstructureasshown.Two
additionalsubunits,ScclandScc3,
connectthe ATPaseheaddomains
forminga ringstructurethat mayencircle
thesisterchromatidsasshown.
20
"t
(A)
(c)
21. ANAPHASE
s n o r l e n n g
k i n e t o c h o r e
m i c r o t ub L rl e
5 TELOPHASE
o v e r l a p
n t I c r o r L t D L t I e s
6 CYTOKINESIS
coftracill€] ilng
c r e a t l n gc l e a v a g e
frrrrow
N
-s)C-
)
-
d a L l q n t e f C n r O m O S O m e S n r" h L t h e s r s t e r
c h r o m a t i d ss v n c h r o n o u s l Y
seParateto tornl two
'
daughter chronrosomes,
a n d e a c h i s t r r u l l e ds l o w l Y
toward the sptndle Pole it
faces The krletochore
microtubules get shorter,
. a n d t h e s p i n d l ep o l e sa l s o
rr' move apart; both
processes contflbuteto
'
chromosomesellregatton
s p i n d l ep o l e
movrngoutwaro
Durin g . the two
sets of daughler chromo-
somes arrive at the poles of
t h e s p i n d l ea n d d e c o n d e n s e
A n e w n u c l e a re n v e l o p e
r e a s s e m b l e sa r o u n d e a c h
set, completi'r,Jllre [ormation
o f t w o n u c l e ra n d r n a r k i n g
t h e e n d o f n r i t o s i sT h e
d i v i s i o no f t h e c y t o p l a s m
b e g i n sw i t h ( o i l l r a c t i o no r
the contractile ring
c e n t r o s o r n e
n u c l e a re n v e l o p e r e a s s e m b l i n g
a r o u n d i n d i v i c i u a lc h r o m o s o m e s
,:..,.,
:: :1
-1..,.:
R. r } J
GiS
c o n t r a c t r l en n g
startrng to
C O N I T A C I
, : : . : t : : : . ' .
:..1
'::;, ...
'
:,;:
ta-
r.
, - t , : ' "
D u r i n g , t h e
cytoplasm is divided in two
by a contractrlerirrgof
a c t r na n d m y o s r n
f i l a m e n t s ,w h r c h p i n c h e s
the cell in two to create
two daughtcrs. each with
o n e n u c l e u s
r e - f o r m a t i o Do i n l e r p h a s e
a r r a y o f m r c r o t r ,l ) u l e st l L t c l e a t e c l
llv the centrosonre
rl.l:
r-.----:
q.-,
C
a/
s e l n ' d a U g h t e ' u l t r , , t n o s o m e s
a t s p i n d l e p o l e
-S)
- i
C2 ot
c o m p l e t e d n u c l e a re n v e l o p e
s u r r o u n d s d e c o n d e n s i n g
c h r o n t o s o m e s
(Micrographscourtesyof JulieCanman and Ted Salmon)
23. MITOStS
:'fl;;".'"';'*'#:il:?:il[::ffiffi:?ili:,T::''.",:fri:ffl:flH'alongtheirlength.Theconstrictedregionsarethecentromeres.(Courtesy
ofTerryD.Allen.)
complexes,which then phosphorylate more Cdc25 and Weel molecules. This
leads to more M-Cdk activation, and so on. Such a mechanism would quickly
promote the complete activation of all the M-Cdk complexes in the cell. As
mentioned earlier, similar molecular switches operate at various points in the
cell cycle to promote the abrupt and complete transition from one cell-cycle
state to the next.
CondensinHelpsConfigureDuplicatedChromosomesfor
Separation
At the end of S phase, the immensely long DNA molecules of the sister chro-
matids are tangled in a mass of partially catenated DNA and proteins. Any
attempt to pull the sistersapart in this statewould undoubtedly lead to breaks
in the chromosomes.To avoid this disaster,the cell devotesa greatdeal of energy
in early mitosis to gradually reorganizing the sister chromatids into relatively
short, distinct structuresthat can be pulled apart more easilyin anaphase.These
chromosomal changes involve two processes:chromosome condensation, in
which the chromatids are dramatically compacted; and sister-chromatid resolu-
tion, whereby the two sisters are resolved into distinct, separable units (Figure
17-26). Resolution results from the decatenation of the sisterDNAs, accompa-
nied by the partial removal of cohesin molecules along the chromosome arms.
As a result,when the cell reachesmetaphase,the sisterchromatids appearin the
microscope as compact, rod-like structures that arejoined tightly at their cen-
tromeric regions and only loosely along their arms.
The condensation and resolution of sister chromatids depends, at least in
part, on a five-subunit protein complex called condensin. Condensin structure
is related to that of the cohesin complex that holds sister chromatids together
(seeFigure 17-24).It contains two SMC subunits like thoseof cohesin,plus three
non-SMC subunits (Figure 17-27). Condensin may form a ring-like structure
that somehow usesthe energyprovided byATP hydrolysis to promote the com-
paction and resolution of sister chromatids. Condensin is able to change the
coiling of DNA molecules in a test tube, and this coiling activity is thought to be
important for chromosome condensation during mitosis. Interestingly, phos-
phorylation of condensin subunits by M-Cdk stimulates this coiling activity,
providing one mechanism by which M-Cdk may promote chromosome restruc-
turing in early mitosis.
TheMitoticSpindlelsa Microtubule-BasedMachine
The central event of mitosis-chromosome segregation-depends in all eucary-
otes on a complex and beautiful machine called the mitotic spindle. The spin-
dle is a bipolar array of microtubules, which pulls sister chromatids apart in
anaphase,thereby segregatingthe two setsof chromosomes to opposite ends of
the cell, where they are packaged into daughter nuclei. M-Cdk triSgers the
assembly of the spindle early in mitosis, in parallel with the chromosome
restructuring just described.Beforewe consider how the spindle assemblesand
how its microtubules attach to sisterchromatids, we briefly review the basicfea-
tures of spindle structure.
As discussedin Chapter 16,the core of the mitotic spindle is a bipolar array
of microtubules, the minus ends of which are focused at the two spindle poles,
and the plus ends of which radiate outward from the poles (Figure 17-28).
<GTCT> The plus ends of some microtubules-called the interpolar micro-
tubules-interact with the plus ends of microtubules from the other pole,
resulting in an antiparallel array in the spindle midzone. The plus ends of other
1075
1 *
DNA
Figure17-27Condensin.Condensinisa
five-subunitproteincomplexthat
resemblescohesin(seeFigure17-24).
Theheaddomainsof itstwo major
subunits,5mc2andSmc4,areheld
togetherbythreeadditionalsubunits.lt
isnot clearhowcondensincatalyzesthe
restructuringandcomPactionof
chromosomeDNA,but it mayforma ring
structurethatencirclesloopsof DNAas
shown;it canhydrolyzeATPandcoilDNA
moleculesin a testtube.
ATPasedomain
24. spindlepole replicated
1076 Chapter17:TheCellCycle
chromosome kinetochore
(sisterchromatids) ,/
motor
+ protern
astralmicrotubules kinetochoremicrotubules interoolarmicrotubules
microtubules-the kinetochore microtubules-are attached to sister chro-
matid pairs at large protein structures called kinetochores,which are located at
the centromereof each sister chromatid. Finally, many spindles also contain
astral microtubules that radiate outward from the poles and contact the cell
cortex, helping to position the spindle in the cell.
In most somatic animal cells, each spindle pole is focused at a protein
organelle called the centrosome (discussedin chapter l6). Each centrosome
consistsof a cloud of amorphous material (called the pericentriolar matrix) that
surrounds a pair of centrioles(Figure lz-29). The pericentriolar matrix nucle-
ates a radial array of microtubules, with their fast-growing plus ends projecting
outward and their minus ends associatedwith the centrosome.The matrix con-
tains a variety of proteins, including microtubule-dependent motor proteins,
l p m
Figure| 7-29Thecentrosome.(A)Electronmicrographof an S-phasemammaliancellin culture,
showinga duplicatedcentrosome.Eachcentrosomecontainsa pairof centrioles;althoughthe
centrioleshaveduplicated,theyremaintogetherin a singlecomplex,asshownin the drawingof the
micrographin (B).Onecentrioleof eachcentriolepairhasbeencut in crosssection,whilethe other
iscut in longitudinalsection,indicatingthatthetwo membersof eachpairarealignedat right
anglesto eachother.Thetwo halvesof the replicatedcentrosome,eachconsistingof a centriolepair
surroundedby pericentriolarmatrix,willsplitandmigrateapartto initiatetheformationof thetwo
polesof the mitoticspindlewhenthe cellentersM phase.(C)Electronmicrographof a centriolepair
that hasbeenisolatedfroma cell.Thetwo centrioleshavepartlyseparatedduringthe isolation
procedurebut remaintetheredtogetherbyfinefibers,whichkeepthe centriolepairtogetheruntilit
istimeforthemto separate.Bothcentriolesarecut longitudinally,andit cannow beseenthatthe
two havedifferentstructures:the mothercentrioleislargerandmorecomplexthanthe daughter
centriole,andonlythe mothercentrioleisassociatedwith pericentriolarmatrixthat nucleates
microtubules.Eachdaughtercentriolewill matureduringthe nextcellcycle,whenit will replicateto
giveriseto itsown daughtercentriole.(A,fromM.McGill,D.p.Highfield,T.M.Monahanand
B.R.Brinkley,J.ultrastruct.Res.57:43-53,1976.with permissionfrom Academicpress;C,from
M.Paintrandet al.,J.Struct.Biol.108107-128,1992.with permissionfrom Elsevier.)
centro50me
Figure17-28Thethreeclassesof
microtubulesof the mitoticspindlein
an animalcell.Theplusendsof the
microtubulesprojectawayfrom the
centrosomes,whilethe minusendsare
anchoredat the spindlepoles,whichin
thisexampleareorganizedby
centrosomes.Kinetochoremicrotubules
connectthe spindlepoleswith the
kinetochoresof sisterchromatids,while
interpolarmicrotubulesfromthe two
polesinterdigitateat the spindleequator.
Astralmicrotubulesradiateout fromthe
polesintothe cytoplasmandusually
interactwith thecellcortex,helpingto
positionthe spindlein the cell.
200nm
microtubule pericentriolarmatrix pairof centrioles
25. MlroSlS 1477
coiled-coilproteinsthat link the motorsto the centrosome,structuralproteins,
and componentsof the cell-cyclecontrolsystem.Most important,it contains
the y-tubulin ring complex,which is the componentmainly responsiblefor
nucleatingmicrotubules(discussedin Chapter16).
Somecells-notably the cellsof higherplantsandthe ooc)'tesof manyver-
tebrates-do not havecentrosomes,and microtubule-dependentmotor pro-
teinsandotherproteinsassociatedwith microtubuleminusendsorganizeand
focusthespindlepoles.
Microtubule-DependentMotorProteinsGovernSpindle
AssemblyandFunction
The assemblyand function of the mitotic spindle depend on numerous micro-
tubule-dependent motor proteins. As discussedin Chapter 16, these proteins
belong to two families-the kinesin-related proteins, which usually move
toward the plus end of microtubules, and dyneins, which move toward the
minus end. In the mitotic spindle, these motor proteins generallyoperate at or
near the ends of the microtubules. Four major types of motor proteins-kinesin-
5, kinesin-14,kinesins-4and 10, and dynein-are particularly important in spin-
dle assemblyand function (Figure f 7-30).
Kinesin-S proteins contain two motor domains that interact with the plus
ends of antiparallel microtubules in the spindle midzone. Because the two
motor domains move toward the plus ends of the microtubules, they slide the
two antiparallel microtubules past each other toward the spindle poles, forcing
the poles apart. Kinesin-l4 proteins, by contrast,areminus-end directed motors
with a singlemotor domain and other domains that can interact with a different
microtubule. They can cross-link antiparallel interpolar microtubules at the
spindle midzone and tend to pull the poles together. Kinesin-4 and kinesin-10
proteins, also called chromokinesins,areplus-end directed motors that associ-
ate with chromosome arms and push the attached chromosome away from the
pole (or the pole away from the chromosome). Finally, dyneins are minus-end
directed motors that, together with associatedproteins, organize microtubules
at various cellular locations. They link the plus ends of astral microtubules to
components of the actin cytoskeletonat the cell cortex,for example;by moving
toward the minus end of the microtubules, the dgrein motors pull the spindle
poles toward the cell cortex and away from each other.
TwoMechanismsCollaborateintheAssemblyof a BipolarMitotic
Spindle
The mitotic spindle must have two poles if it is to pull the two setsof sisterchro-
matids to opposite ends of the cell in anaphase.In animal cells, the primary
focus of this chapter,two mechanisms collaborateto ensurethe bipolarity of the
spindle. One depends on the ability of mitotic chromosomes to nucleate and
stabilize microtubules and on the ability of the various motor proteins just
described to organize microtubules into a bipolar array, with minus ends
spindlemicrotubule
kinesin-14
kinesin-5
Figure17-30Majormotor proteinsof
the spindle.Fourmajorclassesof
microtubule-dependentmotorproteins
(yel/owboxes)contributeto spindle
assemblyandfunction(seetext).The
coloredarrowsindicatethedirectionof
motormovementalonga microtubule-
b/uetowardthe minusend,andred
towardthe plusend.
kinesin-4,10 /
sisterchromatids
centrosome
27. MITOSIS 1079
(B) (c)
2 p m
Figure17-32Theinfluenceofopposingmotorproteinsonspindlelengthinbuddingyeast.(A)Adifferential-
interference-contrastmicrographofa mitoticyeastcell.Thespindleishighlightedingreen,andthepositionofthespindle
polesisindicatedbyredarrows.Thenuclearenvelopedoesnotbreakdownduringmitosisinyeasts,andthespindleforms
insidethenucleus.In(B-D),themitoticspindleshavebeenstainedwithfluorescentanti-tubulinantibodies.(B)Normal
yeasrcells.(C)Overexpressionoftheminus-end-directedmotorproteinKar3(akinesin-14protein)leadsto abnormally
shortspindles.(D)Overexpressionoftheplus-end-directedmotorproteinCinS(akinesin-5protein)leadsto abnormally
longspindles.Thus,itseemsthatabalancebetweenopposingmotorproteinsdeterminesspindlelengthinthesecells.
(A,courtesyofKerryBloom;B-D,fromW.Saunders,V.LengyelandM.A.Hoyt,Mol.Biol.CellS:1025-1033,1997.With
permissionfromAmericanSocietyforCellBiology.)
envelope,and the plus ends of the microtubules between them interdigitate to
form the interpolar microtubules of the developing spindle. At the same time,
the amount of y-tubulin ring complexes in each centrosome increasesgreatly,
increasing the ability of the centrosomesto nucleate new microtubules, a pro-
cesscalled centrosomematuration.
Multiple motor proteins drive the separation of centrosomesin early mito-
sis.In prophase, minus-end directed dlnein motor proteins at the plus ends of
astral microtubules provide the major force.These motors are anchored at the
cell cortex or on the nuclear envelope,and their movement toward the micro-
tubule minus end pulls the centrosomes apart (see Figure 17-30). Following
nuclear envelope breakdown at the end of prophase, interactions between the
centrosomal microtubules and the cell cortex allow actin-myosin bundles in the
cortex to pull the centrosomesfurther apart. Finally,kinesin-5 motors cross-link
the overlapping,antiparallel ends of interpolar microtubules and push the poles
apart (seeFigure 17-30).
The balance of opposing forces generatedby different types of motor pro-
teins determines the final length of the spindle. Dynein and kinesin-s motors
generallypromote centrosome separation and increasespindle length. Kinesin-
14 proteins do the opposite: they are minus-end directed motors and interact
with a microtubule from one pole while traveling toward the minus end of an
antiparallel microtubule from the other pole; as a result, they tend to pull the
poles together. It is not clear how the cell regulates the balance of opposing
forcesto generatethe appropriate spindle length (Figure 17-32).
M-Cdk and other mitotic protein kinasesare required for centrosome sepa-
ration and maturation. M-Cdk and aurora-A phosphorylate kinesin-S motors
and stimulate them to drive centrosomeseparation.Aurora-A and PIkalsophos-
phorylate components of the centrosome and thereby promote its maturation.
TheCompletionof SpindleAssemblyin AnimalCellsRequires
NuclearEnvelopeBreakdown
The centrosomesand microtubules of animal cellsarelocated in the cytoplasm,
separated from the chromosomes by the double membrane barrier of the
nuclear envelope (discussedin Chapter 12). Clearly, the attachment of sister
chromatids to the spindle requiresthe removal of this barrier. In addition, many
of the motor proteins and microtubule regulatorsthat promote spindle assem-
bly are associatedwith the chromosomes inside the nucleus. Nuclear envelope
breakdor.rmallows theseproteins to carry out their important functions in spin-
dle assemblv.
overexpressionof
CinSp
(D)
overexpressionof
Ka13p
normalspindles
28. 1080 Chapter17:TheCellCycle
Nuclear envelope breakdown is a complex, multi-step process that is
thought to begin when M-cdk phosphorylates several subunits of the giant
nuclear pore complexes in the nuclear envelope.This initiates the disassembly
of nuclear pore complexes and their dissociation from the envelope. M-Cdk
also phosphorylates components of the nuclear lamina, the structural frame-
work that lies beneath the envelope.The phosphorylation of theselamina com-
ponents and of severalinner nuclear envelope proteins leadsto disassemblyof
the nuclear lamina and the breakdown of the envelope membranes into small
vesicles.
MicrotubuleInstabilitylncreasesGreatlyin Mitosis
Most animal cells in interphase contain a cytoplasmic array of microtubules
radiating out from the singlecentrosome.As discussedin chapter 16,the micro-
tubules of this interphase array are in a state of dynamic instability, in which
individual microtubules are either growing or shrinking and stochastically
switch between the two states.The switch from growth to shrinkage is called a
catastrophe, and the switch from shrinkage to growth is called a rescue(seeFig-
ure l6-16). New microtubules are continually being created to balance the loss
of those that disappear completely by depolymerization.
Entry into mitosis signalsan abrupt change in the cell'smicrotubules. The
interphase array of few, long microtubules is converted to a larger number of
shorter and more dynamic microtubules surrounding each centrosome.During
prophase,and particularly in prometaphaseand metaphase(seepanel l7-l), the
half-life of microtubules decreases dramatically. This increase in microtubule
instability, coupled with the increasedability of centrosomesto nucleate micro-
tubules as mentioned earlier, results in remarkably dense and dlmamic arrays of
spindle microtubules that are ideally suited for capturing sisterchromatids.
M-Cdk initiates these changesin microtubule behavior, at least in part, by
phosphorylating two classesof proteins that control microtubule dynamics (dis-
cussed in chapter 16).These include microtubule-dependent motor proteins
and microtubule-associated proteins (MAPs). Experiments using cell-free
Xenopuseggextracts,which reproduce many of the changesthat occur in intact
cellsduring the cell cycle,have revealedthe roles of theseregulatorsin control-
ling microtubule dyrramics.If centrosomesand fluorescenttubulin are added to
theseextracts,fluorescentmicrotubules nucleatefrom the centrosomes,and we
can observethe behavior of individual microtubules by time-lapse fluorescence
video microscopy. The microtubules in mitotic extracts differ from those in
interphase extractsprimarily by the increasedrate of catastrophes,in which the
microtubules switch abruptly from slow growth to rapid shortening.
TWo classes of proteins govern microtubule dlrramics in mitosis. proteins
called catastrophe factors destabilize microtubule arrays by increasing the fre-
quency of catastrophes(seeFigure 16-16). one of these proteins is a kinesin-
related protein that does not function as a motor. MAps, by contrast, have the
opposite effect, stabilizing microtubules in various ways:they can increasethe fre-
quency of rescues,in which microtubules switch from shrinkage to growth, or they
can either increasethe growth rate or decreasethe shrinkage rate of microtubules.
Thus, in principle, changes in catastrophe factors and MAps can make micro-
tubules much more dFramic in M phase by increasing total microtubule depoly-
merization rates,decreasingtotal microtubule polymerization rates,or both.
constant throughout the cell cycle,the balance between the two opposing activ-
ities of the MAP and catastrophe factor would shift, increasing the dynamic
instability of the microtubules.
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