Cell cycle(Molecular Biology of The Cell chapter 17 : Alberts, et al., Garland Science, 2008)Dr. Ratna Megawati Widharna, SKG, MFT

4 phases of the cell cycle(< 1 hour)(10-12 hours)

/ restriction point (late G1)(1)(2)(3)3 MaJor

ReguLATOry

TRansITIons/checkpoINTs

Cell control systemBased on connected series of biochemical switches initiates specific cell-cycle event1. binary on/off : launch events in a complete, irreversible fashion (e.g. chromosome condensation)2. Robust & reliable backup mechanism & other feature system operate effectively under a variety of conditions & even if some components fail3. highly adaptable & can be modified to suit specific cell types/ to respond to specific intracellular / extracellular signals

Cell-cycle control system depends on cyclically Activated Cyclin-Dependent Protein kinases (Cdks) Cdk activity at the G2/M checkpoint phosphorylation of protein that control chromosome condensation Nuclear envelope breakdownSpindle assemblyOther events that occur at the onset of mitosisCdk + cyclin protein kinase activityCyclin-cdk complexes triggers cell-cycle events

4 classes of cyclins(1)(2)(3)

All eucaryotic cells require 3 of these classes(1) G1/S-cyclins : activate Cdk in late G1 help trigger progression through Start commitment to cell-cycle entry their levels fall in S phase(2) S-cyclins : bind Cdks soon after progression through Start help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis contribute to control of some early mitotic events

All eucaryotic cells require 3 of these classes(3) M-cyclins : activate Cdk that stimulate entry into mitosis at the G2/M checkpointIn most cells, (4) G1-cyclins : helps govern the activities of the G1/S-cyclins which control progression through Start in late G1.

The structural basis of Cdk activation

No cyclin active site in the Cdk prot is partly obscured by a slab of prot (A)Cyclin binding slab move away from the active site partial activation of Cdk enzyme (B)Full activation of the cyclin-Cdk complex occurs when a separate kinase (CAK/Cdk-activating kinase) phosphorylates an amino acid near the entrance of the Cdk active site -> conformational change Cdk activity kinase phosphorylate its target prot effectively induce specific cell-cycle events

Phosphorylation at a pair of amino acids in the roof on the kinase active site inhibits the activity of a cyclin-Cdk complex.Phosphorylation by Wee1 inhibits Cdk activity, while dephosphorylation of these sites by a phosphatase (Cdc25) increases Cdk activityBinding of Cdk inhibitor proteins (CKIs) also regulates cyclin-Cdk complexes

Cyclin-Cdk-CKI complex reveals that CKI binding stimulates a large rearrangement in the structure of the Cdk active site, rendering it inactiveCell use CKIs primarily to help govern the activities of G1/S and S-Cdks early in the cell cycle

The cell cycle control system depends on cyclical proteolysisProgression through the metaphase-to-anaphase-transition is triggered by protein destruction, leading to the final stages of cell divisionKey regulator : anaphase-promoting complex/ Cyclosome (APC/C)APC/C catalyzes the ubiquitylation & destruction of 2 major prot:SecurinS- and M-cyclins

APC/C catalyzes the ubiquitylation & destruction of1. securin = normally protects the prot linkages that hold sister chromatid pairs together in early mitosisDestruction of securin at the metaphase-to-anaphase transition activates a protease that separates the sisters & unleashes anaphase2. The S&M cyclinsDestroying S&M cyclins INACTIVATES most Cdks in the cell many prot phosphorylated cy Cdks from S phase to early mitosis are dephosphorylated by various phosphatase that are present in the anaphase cell dephosphorylation : necessary for the completion of M phase including the final step in mitosis & the process of cytokinesis

APC/C remains active in G1 a stable period of the Cdk inactivityWhen G1/S-Cdks are activated in late G1, the APC/C is turned off allowing cyclin accumulation to start the next cell cycle

SCF (another ubiquitin ligase) Ubiquitylates certain CKI prot in late G1 helps control the activation of S-Cdks & DNA replication

Difference between APC/C and SCF APC/C activity changes during the cell cycle result of changes in its association with an activating subunit-either Cdc20 during anaphase/Cdh1 from late mitosis through early G1. These subunits helps the APC/C recognize its target proteinSCF activity depends on subunits F-box prot, which help the complex recognize its target prot. Unlike APC/C activity, SCF activity is CONSTANT during the cell cycle. Ubiquitylation by SCF is controlled by changes in the phosphorylation state of its target prot, as F-box subunits recognize only specifically phosphorylated prot

Major Cell-Cycle Regulatory Proteins

Start checkpointsChromosome duplicationEarly event in mitosis

M-Cdk activationprogress G2/M checkpointEvents of early mitosisLeading to alignment of sister chromatids at the equator of the mitotic spindleFinally the APC/C + Cdc20 (activator) triggers the destruction of securin & cyclins at the metaphase-to-anaphase transition unleasing sister-chromatid segregation & completion of mitosis

S phase2 problems must be solved when initiating & completing DNA replication:1. replication must occur with extreme accuracy ( the risk of mutations in the next generation)2. every nucleotide in the genome must be copied once and only once, to prevent the damaging effects of gene amplification

To ensure that chromosome duplication occurs only once/cell cycle, the initiation of DNA replication is divided into 2 distinct stepsIn Late mitosis & early G1 when a large complex of initiator protein = PREREPLICATIVE COMPLEX (Pre-RC) assembles at origins of replication = LICENSING OF REPLICATION ORIGINS initiation of DNA synthesis is permitted only at origins containing a pre-RCONSET of S phase when components of the pre-RC nucleate the formation of a larger protein complex = PREINITIATION COMPLEX unwinds the DNA helix & loads DNA polymerases & other replication enzymes onto the DNA Strands initiating DNA synthesis pre RC is dismantled & cannot be reassembled at the origin until the following G1 origins can be activated only once/cell cycle

Assembly of the pre-RCInhibited by Cdk activityStimulated by APC/CIn late mitosis & early G1Cdk activity APC/C At the onset of S-phase activation of S-Cdk triggers the formation of a preinitiation complex initiates DNA synthesis

Pre-RC is partly dismantledS-Cdk M-Cdk APC/C Until late mitosis, new pre-RCs cannot be assembled at fired origins until the cell cycle is complete

ORC binds to replication origins throughout the cell cycleIn late mitosis and early G1, the protein Cdc6 & Cdt1 bind to the ORC at origins & help load a group of 6 related prot = Mcm prot complex pre-RC & the origin is now licensed for replicationThe 6 Mcm prot of the pre-RC form a ring around DNA serve as the major DNA helicase that unwinds the origin DNA when DNA synthesis begins & as the replication forks move out from the origin

Central purpose of pre-RC: to load the helicase that will play a central part in the subsequent DNA replication processOnce the pre-RC has assembled in G1, the replication origin is ready to fireThe activation of S-Cdk in late G1 triggers the assembly of several additional prot complexes at the origin, leading to the formation of a giant preinitiation complex that unwinds the helix & begins DNA synthesis

At the same time it initiates DNA replication, S-Cdk triggers the disassembly of some pre-RC components at the originCdks phosphorylates ORC & Cdc6 inhibition Inactivation of the APC/C in late G1 also helps turn off pre-RC assemblyIn late mitosis and early G1, the APC/C triggers the destruction of a prot GEMININ, that binds and inhibits the pre-RC component Cdt1When APC/C is turned off in late G1, GEMININ accumulates and inhibits Cdt1

At the end of mitosis, APC/C activation inactivation of Cdks and destruction of geminin pre-RC components are dephosphorylated and Cdt1 is activated allowing pre-RC assembly to prepare the cell for the S phase

Cohesins help hold sister chromatids together2 of the subunits of cohesin are members of SMC proteins (Structural Maintenance of Chromosomes)Cohesin forms giant ring-like structures

Mitosis part 1An abrupt in M-Cdk activity at the G2/M checkpoint triggers the events of early mitosis (prophase, prometaphase, and metaphase). M-Cdk and several other mitotic protein kinase phosphorylate a variety of proteins, leading to the assembly of the mitotic spindle & its attachment to the sister chromatid pairs

Mitosis part 2The 2nd major part of mitosis begins at the metaphase-to-anaphase transition, when the APC/C triggers the destruction of securin, liberating a protease that cleaves cohesin initiates separation of the sister chromatidsThe APC/C also triggers the destruction of cyclins Cdk inactivation and the dephosphorylation of Cdk targets required for all events of late M phase , including completion of anaphase, disassembly of the mitotic spindle, & division of cell by cytokinesis

Dephosphorylation activates M-Cdk at the onset of mitosisM-Cdk activation begins with the accumulation of M-cyclinIn embryonic cell cycles, the synthesis of M-Cyclin is constant throughout the cell cycle, and M-cyclin accumulation results from the high stability of the protein in interphaseIn most cell types, M-cyclin synthesis increases during G2 and M, owing primarily to an increase in M-cyclin gene transcription

The increase in M-cyclin protein leads to a corresponding accumulation of M-Cdk (the complex of Cdk1 and M-cyclin) as the cell approaches mitosisAlthough the Cdk in these complexes is phosphorylated at an activation site by the Cdk-activating kinase (CAK), the protein kinase Wee1 holds it in inactive state by inhibitory phosphorylation at 2 neighbouring sites

Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that is primed and ready to act but is suppressed by phosphates that block the active site of kinase

What triggers the activation of the M-Cdk stockpile?Activation of protein phosphatase Cdc25, which removes the inhibitory phosphatases that restrain M-Cdk. At the same time, the inhibitory activity of the kinase Wee1 is suppressed, further ensuring that M-Cdk activity increases. The mechanism that unleash Cdc25 activity (and supress Wee1) in early mitosis are not well understood. One possibility is that the S-Cdks that are active in G2 and early prophase stimulate Cdc25

Interestingly, Cdc25 can also be activated, at least in part, by its target, M-Cdk. M-Cdk may also inhibit the inhibitory kinase Wee1. The ability of M-Cdk to activate its own activator (Cdc25) and inhibits its own inhibitor (Wee1) suggests that M-Cdk activation in mitosis involves positive feedback loopsAccording to this model, the partial activation of Cdc25 (perhaps by S-Cdk) leads to partial activation of a subpopulation of M-Cdk complexes, which then phosphorylate more Cdc25 and Wee1 molecules. This leads to more M-Cdk activation, and so on. Such mechanism would quickly promote the complete activation of all the M-Cdk complexes in the cell

Condensin helps configure duplicated chromosomes for separationRelated to that of cohesin complex (holds sister chromatid together)Contains 2 SMC subunits like those of cohesin + 3 non-SMC subunitsCondensin may form a ring-like structure that somehow uses the energy provided by ATP hydrolysis to promote the compaction & resolution of sister chromatids

CondensinAble to change the coiling of DNA molecules in a test tube important for chromosome condensation during mitosisPhosphorylation of condensin subunits by M-Cdk stimulates this coiling activity, providing 1 mechanism by which M-Cdk may promote chromosome restructuring in early mitosis

The mitotic spindle is a microtubule-based machineChromosome segregation depends on mitotic spindleThe minus end of which are focused at the 2 spindle poles, and the plus end of which radiate outward from the poles The plus end of microtubules = INTERPOLAR MICROTUBULES 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 microtubules-the kinetochore microtubules are attached to sister chromatid pairs at large protein structures called KINETOCHORES, which are located at the centromere of each sister chromatidFinally, 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 centrosomeEach centrosome consists of pericentriolar matrix that surrounds a pair of centriolesThe pericentriolar matrix nucleates a radial array of microtubules, with their fast-growing plus ends projecting outward and their minus ends assoc with the centrosome

The matrix containsA variety of proteins, includingMicrotubule-dependent motor proteinscoiled-coil proteins that link the motors to the centrosomeStructural proteinsComponents of the cell-cycle control system-tubulin ring complex responsible for nucleating microtubules

Microtubule-dependent motor proteins govern spindle assembly and functionKinesin-related prot : move toward the plus end of microtubulesDynein : move toward the minus end of microtubulesKinesin-5 protein: contains 2 motor domains that interact with plus ends of antiparallel microtubules in the spindle midzone slide the 2 antiparallel microtubules past each other toward the spindle poles, forcing the poles apart

Kinesin-14 protein: minus end directed motors with a single motor domain & other domains that can interact with a different microtubule. They can cross-link antiparallel microtubules at the spindle midzone and tend to pull the poles togetherKinesin 4 & kinesin 10 : chromokinesin plus end directed motors associated with chromosome arms and push the attached chromosome away from the pole (/ the pole away from chromosome)

Dynein : minus end directed-motors together with associated protein: organize microtubules at various cellular locationsThey link the plus ends of astral microtubules to components of the actin cytoskeleton at the cell cortex, e.g. by moving toward the minus end of the microtubules, the dynein motors pull the spindle poles toward the cell cortex and away from each other

2 mechanisms collaborate in the assembly of a bipolar mitotic spindle1. Depends on the ability of mitotic chromosomes to nucleate and stabilize microtubules and on the ability of the various motor proteins to organize microtubules into a bipolar array, with minus ends focused at 2 spindle poles and plus ends interacting with each other in the spindle midzone

2 mechanisms collaborate in the assembly of a bipolar mitotic spindle2. depends on the ability of centrosomes to help form the spindle polesEach of the pair of centrosomes nucleates a radial array of microtubulesThe 2 centromeres facilitate bipolar spindle assembly by providing a pair of fabricated spindle poles

Centrosomes duplication occurs early in the cell cycleCentrosome duplication begins at about the same time as the cell enters S phaseThe G1/S-Cdk (complex of cyclin E and Cdk2 in animal cells) that triggers cell cycle entry also initiates centrosome duplicationThe 2 centrioles in the centrosome separate, and each nucleates the formation of a single new centriole 2 centriole pairs within an enlarged pericentrilar matrix. This centrosome pair remains together on 1 side of the nucleus until the cell enters mitosis

Centrosome ~ chromosome duplicationUse a semi-conservative mechanism of duplication 2 halves separate and serve as templates for construction of a new halfMust replicate once and only once per cell cycle to ensure that the cell enters mitosis with only 2 copies

M-Cdk initiates spindle assembly in prophase M-Cdk activity initiates spindle assembly2 centrosomes move apart along the nuclear envelope & the plus ends of the microtubules between them interdigitate to form the interpolar microtubules of the developing spindleThe amount of tubulin ring complexes in each centrosomes greatly, the ability of centrosomes to nucleate new microtubules = CENTROSOMES MATURATION

M-Cdk initiates spindle assembly in prophase (2)Minus end directed dynein motor protein at the plus ends of astral microtubules provide the major force these motors are anchored at the cell cortex/on the nuclear envelope and their movement toward the microtubule minus end pulls the centrosome apartInteraction between the centrosomal microtubules & the cell cortex allow actin-myosin bundles in the cortex to pull the centrosomes further apart

Finally, kinesin 5 motors cross-link the overlapping, antiparralel ends of interpolar microtubules and push the poles apartDynein & kinesin-5 motors : centrosome separation and spindle lengthKinesin 14 protein : minus end directed motors and interact with a microtubule from 1 pole while traveling toward the minus end of an antiparallel microtubule from the other pole they tend to pull the poles together

M-Cdk and Aurora A phosphorylate kinesin-5 motors and stimulate them to drive centrosome separationAurora-A and Plk also phosphorylate components of the centrosome promote its maturation

The completion of spindle assembly in animal cells requires nuclear envelope breakdownBegin when M-Cdk phosphorylates several subunits the giant nuclear pore complexes in the nuclear envelopeInitiates the disassembly of nuclear pore complexes and their dissociation from the envelopeM-Cdk also phosphorylates components of the nuclear lamina, the structural framework that lies beneath the envelope leads to disassembly of the nuclear lamina & the breakdown of the envelope membranes into small vesicles

Microtubules instability increases greatly in mitosis (interphase)Dinamyc instability:Growth shrinkage : CatastropheShrinkage growth : RescueNew microtubules are continually being created to balance the loss of those that disappear completely by depolymerizationLong microtubules a >>> # of shorter & more dynamic microtubules surrounding each centromere

Prometaphase T1/2 microtubules Metaphase dramatically In microtubule instability Ability of centrosomes to nucleate microtubuleM-Cdk initiates these changes by phosphorylating 2 classes of protein that control microtubule dynamics these contains microtubule-dependent motor protein and microtubule-associated protein (MAPs)Dense and dynamic array of spindle microtubules that are ideally suited for capturing sister chromatids

2 classes of protein govern microtubule dynamics in mitosisCATASTROPHE Factors : destabilize microtubule arrays by frequency of catastrophes. One of these protein : kinesin-related protein that does not function as a motorMAP : stabilizing microtubules : frequency of rescues shrinkage to growth, growth rate, shrinkage rate of microtubules

Bi-orientation is achieved by trial and errorSister kinetochores are constructed in a back-to back orientation that reduces the likelihood that both kinetochores can face the same spindle poleIncorrect attachment are highly unstable while correct attachments are locked in placeTension when a sister chromatid pair is properly bi-oriented on the spindle, the 2 kinetochores are pulled in opposite directions by strong poleward forces

Sister-chromatid cohesion resists these poleward forces high level of tension within kinetochoresHigh tension at the kinetochore shuts off the inhibitory signal, strengthening microtubule attachment formation a thick kinetochore fiber composed of multiple microtubules

Aurora BGenerate the inhibitory signal that reduces the strength of microtubule attachment in the absence of tensionIt phosphorylates several components of the microtubule attachment site, sites affinity for a microtubule plus endInactivated when bi-orientation occurs kinetochore phosphorylation and affinity of the attachment site

The APC/C triggers sister-chromatid separation and the completion of mitosisAfter M-Cdk has triggered the complex rearrangements that occur in early mitosis, the cell cycle reaches its climax with the separation of the sister chromatids at the metaphase-to-anaphase transitionAlthough M-Cdk activity sets the stage for this event, the anaphase-promoting complex (APC/C) throws the switch that initiates sister-chromatid separation by ubiquitylating several mitotic regulatory proteins and thereby trigerring their destruction

The APC/C triggers sister-chromatid separation and the completion of mitosisDuring metaphase, cohesins holding the sister chromatids together resist the poleward forces that pull the sister chromatid apart. Anaphase begins with a sudden disruption of sister-chromatid cohesion, which allows the sisters to separate and mote to opposite poles of the spindleThe APC/C initiates the process by targeting the inhibitory protein securin for destruction.

Before anaphase, securin binds to and inhibits the activity of a protease, called separaseThe destruction of securin at the end of metaphase releases separase which is then free to cleave one of the subunits of cohesinThe cohesin fall away and the sister chromatids abruptly and synchronously separate

In addition to securin, the APC/C also targets the S and M-cyclins for destruction, leading to the loss of most Cdk activity in anaphaseCdk inactivation allows phosphatases to dephosphorylates the many Cdk target substrates in the cell, as rewuired for the completion of mitosis and cytokinesis

The APC/C triggers sister-chromatid separation and the completion of mitosisIf the APC/C triggers anaphase, what activates the APC/C?APC/C activation requires the protein Cdc20, which binds and activates the APC/C in mitosisAt least 2 processes regulate Cdc20 and its association with the APC/C

1. Cdc20 synthesis increases as the cell approaches mitosis, owing to an increase in the transcription of its gene2. Phosphorylation of the APC/C helps Cdc20 kinases that phosphorylates and thus activate the APC/C is M-Cdk. M-Cdk is not only triggers the early mitotic events leading up to metaphase, but it also sets the stage for progression into anaphase

The ability of M-Cdk to promote Cdc20/APC/C activity creates a negative feedback loop: M-Cdk sets in motion a regulatory process that leads to cyclin destruction and thus its own activation

During metaphase, cohesins holding the sister chromatids together resist the poleward forces that pull the sister chromatids apart.Anaphase begins with a sudden disruption of sister-chromatid cohesion, which allows the sisters to separate and move to opposite poles of the spindleThe APC/C initiates the process by targeting the inhibitory protein securin for destruction

Before anaphase, securin binds to and inhibits the activity of a protease called separaseThe destruction of securin at the end of metaphase releases separase, which is then free to cleave one of the subunits of cohesin. The cohesin falls away and the sister chromatids abruptly and synchronously separate

Unattached chromosomes block sister-chromatid separation : The spindle assembly checkpointSpindle assembly checkpoint mechanism that is activated by drug treatment and blocks progression through the metaphase-to-anaphase transitionThe checkpoint mechanism ensures that cells do not enter anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle

Any kinetochore that is not properly attached to the spindle sends out a negative signal that blockes Cdc20-APC/C activation and thus blocks the metaphase-to-anaphase transition. Only when the last kinetochore is properly attached is this block removed, allowing sister-chromatid separation to occurInappropriately attached kinetochores somehow generate a diffusible signal that inhibits Cdc-20-APC/C activity throughout the cell

Several proteins, including Mad2 are recruited to unattached kinetochores and are required for spindle assembly checkpoint to functionUnattached kinetochore acts like an enzyme that catalyzes the change in the conformation of Mad2 so that Mad2 can bind and inhibit Cdc20-APC/CThe destruction of securin in mamalian somatic cells begins moments after the last sister chromatid pair becomes bi-oriented on the spindle and anaphase begins 20 minutes later

Chromosomes segregate in anaphase A and BSudden loss of sister-chromatid cohesion at the onset of anaphase sister-chroatied separation allows forces of the mitotic spindle to pull the sisters to opposite poles of the cell = CHROMOSOME SEGREGATIONChromosomes move by 2 independent and overlapping processes:Anaphase AAnaphase B

Anaphase AInitial poleward movement of the chromosomesAccompanied by shortening of kinetochore microtubulesChromosome movement depends on the combination of 2 major poleward force1. Force generated by microtubule depolymerization at the kinetochore results in loss of tubulin subunits at the plus end as the kinetochore moves toward the pole2. Provided by microtubule flux, which is the poleward movement of the microtubules toward the spindle pole, where minus end depolymerization occurs

Anaphase BSeparation of the spindle poles themselves, which begins after the sister chromatids have separated and the daughter chromosomes have moved some distance apartSpindle pole separation depends on motor-driven mechanismPlus end directed kinesin 5 motor proteins, which cross-linke the overlapping plus ends of the interpolar microtubules, push the poles apartIn addition, dynein motors that anchor astral microtubule plus ends to the cell cortex pull the poles apart

Completion of a normal anaphase depends on the dephosphorylation of Cdk substrates, which in most cells results from the APC/C dependent destruction of cyclinsIf M-cyclin destruction is prevented-by the production of a mutatnt form that is not recognized by APC/C, e.g. sister-chromatid separation generally occurs but the chormosome movements and microtubule behaviour of anaphase are abNormal

Segregated chromosomes are packaged in daughter nuclei at telophase2 sets of chromosome are packaged into a pair of daughter nuclei1st major event : disassembly of the mitotic spindle, followed by re-formation of the nuclear envelopeInitially, nuclear membrane fragments associate with the surface of individual chromosomes

These membrane fragments fuse to partly enclose clusters of chromosomes and then coalesce to re-form the complete nuclear envelopeNuclear pore complexes are incorporated into the envelopeThe nuclear lamina re-formsAnd the envelope once again become continuous with the endoplasmic reticulum

Once the nuclear envelope has re-formed, the pore complexes pump in nuclear proteins, the nucleus expands and the condensed mitotic chromosomes are reorganized into their interphase state, allowing gene transcription to resume. A new nucleus has been created, and mitosis is complete. All that remains is for the cell to complete its division into 2.

Earlier : phosphorylation of various proteins by M-Cdk promotes spindle assembly, chromosome condensation and nuclear envelope break-down in early mitosisDephosphorylation of these same proteins is required for spindle disassembly and the re-formation of daughter nuclei in telophaseThese dephosphorylation and the completion of mitosis could be triggered by inactivation of Cdks, the activation of phosphatases, or both

Although Cdk inactivation-resulting primarily from cyclin destruction-si mainly responsible in most cells, some cells also rely on activation of phosphatases

meiosisLoss of arm cohesion in meiosis I depends on APC/C activation leads to securin destruction , separase activation and cohesin cleavage along the armsIn contrast to mitosis, cohesin complexes near the centromeres remain uncleaved in meiosis I because cohesin in that region is protected from separase

Sister-chromatid pairs therefore remained linked at their centromeres throughtout meiosis I, allowing their correct bi-orientation on the spindle in meiosis IIThe mechanisms that block cohesin cleavage at the centromere in meiosis I are removed in meiosis IIAt the onset of anaphase II, APC/C activation therefore triggers centromeric cohesin cleavage and sister-chromatid separation

CytokinesisThe final step in the cell cycle = division of cytoplasm1st visible change : sudden appearance of a pucker/cleavage furrow, on the cell surfaceThe furrow rapidly deepens and spreads the arround the cell until it completely divides the cell in 2The process underlying this process: contractile ring-a dynamic assembly composed of actin filaments, myosin II filaments, and many structural and regulatory proteinsDuring anaphase, the ring assembles just beneath the plasma membrane

The ring gradually contracts, and, at the same time, fusion of intracellular vesicles with the plasma membrane inserts new membrane adjacent to the ringThis addition of membrane compensates for the increase in surface area that accompanies cytoplasmic divisionWhen ring contraction is completed, membrane insertion and fusion seal the gap between the daughter cellsThus, cytokinesis can be considered to occur in 4 stages : initiation, contraction, membrane insertion and completion

Local activation of RhoA triggers assembly and contraction of the contractile ring RhoA, a small GTPase of the Ras superfamily controls the assembly and function of the contractile ring at the site of cleavageRhoA is activated at cell cortex at the future division site, where it promotes actin filament formation, myosin II assembly, and ring contraction. It promotes actin filament formation by activating formins, and it promotes myosin II assembly and contractions by activating multiple protein kinase, including the Rho-activated kinase Rock

These kinases phosphorylate the regulatory myosin light chain (RMLC), which is one of the subunits of myosin IIPhosphorylation of the RMLC stimulates bipolay myosin II filament formation and motor activity, thereby promoting the assembly and contraction of the actin-myosin ring

Like other GTPases, RhoA is inactive when bound to GDP and active when bound to GTPThe local activation of RhoA at the cleavage furrow is though to depend on a Rho guanine nucleotide exchange factor (RhoGEF), which is found at the cell cortex at the future division site and stimulates the release of GDP and binding of GTP to RhoA

The microtubules of the mitotic spindle determine the plane of animal cell divisionCytokinesis must occur only after the 2 sets of chromosomes are fully segregated from @ other, and the site of division must be placed between the 2 sets of daughter chromosomes ensuring that @ daughter cell receives a complete setCorrect timing & positioning of cytokinesis depend on mitotic spindle

Cytokinesis occurs at the correct timeDuring anaphase, the spindle generates signals that initiate furrow formation at a position midway between the spindle poles ensuring that division occurs between the 2 sets of separated chromosomes contributes to correct timing of cytokinesis in late mitosisDephosphorylation of Cdk substrates, which depends on cyclin destruction in metaphase and anaphase initiates cytokinesis

How does the mitotic spindle specify the site of division?1. Astral stimulation model : postulates that the astral microtubules carry furrow-inducing signals to the cell cortex, where they are somehow focused into a ring halfway between the spindle poles experiment in large embryonic cells cleavage furrow forms midway between 2 aster, even when the 2 centrosomes nucleating the asters are not connected to each other by a mitotic spindle (Fig 17-54)

How does the mitotic spindle specify the site of division?2. Central spindle stimulation model : spindle midzone/central spindle generates a furrow-inducing signal that specifies the site of furrow formation at the cell cortexThe overlapping interpolar microtubules of the central spindle associate with numerous signaling proteins, including proteins that may stimulate RhoA

How does the mitotic spindle specify the site of division?3. astral microtubules promote the local relaxation of actin-myosin bundles at the cell cortexCortical relazation is minimal at the spindle equator promoting cortical contraction at that site

Membrane-enclosed organelles must be distributed to daughter cell during cytokinesisHow do the various membrane-enclosed organelles segregate when a cell divides?Organelles e.g mitochondria and chloroplasts are usu present in large enough number sto be safely inheritedThe ER in interphase cell is continuous with the nuclear membrane and is organized by the microtubule cytoskeleton

Upon entry into M phase, the reorganization of the microtubules and breakdown of the nuclear envelope releases the ER.ER remains largely intact and is cut in 2 during cytokinesisThe Golgi apparatus is reorganized and fragmented during mitosis. Golgi fragments associate with the spindle poles & r thereby distributed to opposite ends of the spindle, ensuring that @ daughter cell inherits the materials needed to reconstruct the Golgi in telophase

Some cells reposition their spindle to divide asymmetricallyMost animal cells divide symmetrically : the contractile ring forms around the equator of the parent cell, producing 2 daughter cells of equal size and with the same componentsThis symmetry results from the placement of the mitotic spindle, which in most cases tends to center itself in the cytoplasm

Mitosis can occur without cytokinesis

The G1 phase is a stable state of Cdk inactivityKey regulatory event in late M phase : inactivation of Cdks, which is driven primarily by APC//C dependent cyclin destructionInactivation of Cdks in late M phase has many functions :It triggers the events of late mitosisPromotes cytokinesis enables the synthesis of prereplicative complexes at DNA replication originsProvides a mechanism for resetting the cell-cycle control system to a state of Cdk inactivity as the cell prepares to enter a new cell cycle

In most cells, this state of Cdk inactivity generates a G1 gap phase, during which the cell grows and monitors its environment before committing a new divisionThe destruction of M-cyclin in late mitosis soon leads to inactivation of all APC/C activity in an embryonic cellThis APC/C inactivation immediately after mitosis is especially useful in rapid embryonic cell cycles, as it allows the cell to quickly begin accumulating new M-cyclin for the next cycle (Fig 17-60A)

Rapid cyclin accumulation immediately after mitosis is not useful, for cells with cell cycles containing a G1 phaseMechanism 1 : use another APC/C activating protein, called Cdh1, a close relative of Cdc20

Cell CycleZack CookStart the Tour

InterphaseProphaseCytokinesisTelophaseAnaphaseMetaphaseEnd Show

InterphaseCells MatureChromosomes copy themselvesBack

ProphaseNucleur Membrane DisappearsSpindle Fibers Form at the cells polesBack

MetaphaseChromosomes line up in the middle of cells2 spindle fibers attach to each of the 23 chromosome pairsBack

AnaphaseChromosome pairs splitSpindle Fibers pull chromosomes to each poleBack

Telophase2 nuclei form at polesMitosis completeMembrane pinches in, dividing the cellBack

Cytokinesis2 new daughter cells are formedBack

Mitotic chromosomes promote bipolar spindle assembly

Kinetochores attach sister chromatids to the spindle

Start checkpoint : cell commits to cell-cycle entry & chromosome duplicationG2/M checkpoint : control system triggers the early events that lead to chromosome alignment on the spindle in metaphaseMetaphase to anaphase transition : stimulates sister-chromatid separation completion of mitosis and cytokinesis*Central component of the cell-cycle control system : CdkCdk regulator : cyclin*