MAE 545: Lecture 9 (10/15) Cell division Cell division in higher organisms in bacteria 980 PANEL 17–1: The Principle Stages of M Phase (Mitosis and Cytokinesis) in an Animal Cell PANEPANL 17–1:EL 17–1:The P 980Therinciple Principle SPANtagesE LS oftages17–1: M P haseofThe M P P(Mitosisrinciplehase (Mitosis andStages Cytokinesis) andof M Cytokinesis) Phase in an (Mitosis Animal in an and Cell Animal Cytokinesis) Cell in an Animal Cell 981 981 980 PANEL 98017–1: ThePAN PrincipleEL 17–1: Stages The Pofrinciple M Phase Stages (Mitosis of Mand Phase Cytokinesis) (Mitosis inand an Cytokinesis)Animal Cell in an Animal Cell PANEL 17–1:PANE LThe 17–1: Principle The P rincipleStages Softages M P haseof M P(Mitosishase980 (Mitosis andPAN Cytokinesis) andEL Cytokinesis)17–1: inThe an P Ainrinciplenimal an A nimalCell Stages Cell of M Phase (Mitosis and Cytokinesis) in an Animal Cell 981981 PANEL 17–1: The ThePrinciple Principle Stages Stages of Mof PMhase Phase (Mitosis (Mitosis and and Cytokinesis) Cytokinesis)Cell inin anan AAnimal Celldivision in higher organisms 981 981 PANEL 17–1:980 PANEL 17–1: The Principle Stages of M Phase (Mitosis and Cytokinesis) in an Animal Cell 1 PROPHASE 1 PROPHASE 1 PROPHASE centrosome At prophase, the replicated 1 PROPHASE centrosome At prophase, the replicated At prophase, the replicated 4 ANAPHASE Atcentrosome prophase, the replicated chromosomes, each 4 ANAPHASEdaughter chromosomes centrosome1 PROPHASEchromosomes,1 PROPHASE each chromosomes, each At anaphase, the sister centrosome At prophaseProphase, the replicatedAt prophase , the replicated daughter chromosomesAnaphase intact chromosomes, each centrosome consisting of two closely chromatids synchronouslyAt anaphase , the sister 1 PROPHASEintact consisting of two closely consisting of twoforming closely 1 PROPHASE intact Atforming prophasenuclear, the replicated1consistingPROPHASE of twoforming closely chromosomes, each chromosomes,associated sister each chromatids, 4 ANAPHASE chromatids synchronously nuclearintact 1centrosomePROPHASEcentrosome Atassociated prophase ,sister the replicated chromatids, mitotic 4 ANAPHASE separate to form two 1 PROPHASE nuclear mitotic forming At prophasemitotic, the replicatedassociatedconsistingcentrosome sister of two chromatids, closely At prophase, the replicated 4 4ANAPHASEANAPHASEdaughter chromosomes nuclear intact1 centrosomePROPHASEchromosomes,centrosomeintactenvelope eachassociated At prophase sister, the chromatids, formingreplicated spindle consistingcondense. of Outside two closely the daughter chromosomes At anaphase, the sisterseparate to form two envelope envelope spindle chromosomes,mitoticcondense. Outsidecentrosome each the spindle condense.At prophase Outside,forming the replicated the chromosomes, each daughterdaughter chromosomes chromosomesAtdaughter anaphase chromosomes,, the sister envelope nuclear chromosomes,chromosomes, each eachmitotic associated sister chromatids,associatednucleus, the sister mitotic chromatids, spindle At anaphaseAt anaphasedaughter, the sister, chromosomes,the sister consistingconsistingspindlenuclear nucleus,of two closely condense.theof two mitotic closely Outside spindle the chromosomes,mitotic each chromatidschromatidsand each synchronously is synchronously pulled slowly intact intact formingenvelopeforming consisting of twospindle closelynucleus, condense. the mitotic Outside spindle the consisting of two closely chromatids synchronously intact associatedenvelope sisterformingintact chromatids,nucleus,consisting the ofmitotic two closely spindle spindleformingcondense.assembles Outside between the the two separate to formchromatids twoand each synchronously is pulled slowly nuclear nuclear intact mitotic mitotic associatedassemblesforming sister between chromatids, the two assemblesconsisting between of two closely the two separatetoward theto form spindle two pole it nuclear intact mitoticnuclearassociatedassociated sister sisterforming chromatids, chromatids,nucleus, the mitoticmitotic spindlenucleus,associated the mitotic sister spindle chromatids, separateseparate to form totwo form two envelope envelope nuclear spindle spindlecondense.condense.centrosomes, mitoticOutsideassembles Outsidethe which the between have the twoassociated sister chromatids,centrosomes, which have daughterdaughterfaces. chromosomes,The chromosomes, kinetochoretoward the spindle pole it envelopeenvelope nuclear spindleenvelope condense. Outsidemitotic thecentrosomes, assembles betweenwhichspindle have the twocondense. Outside the daughterdaughter chromosomes, chromosomes, nucleus, nucleus,thereplicatedspindle mitotic thecentrosomes, condense.andspindlemitotic moved spindle Outside whichapart. havethe assemblesreplicated between and moved the apart.two andand microtubuleseach each is pulled is pulled get faces.slowly shorter,slowly The kinetochore envelope nucleus, the mitoticspindle spindlereplicatedcondense.centrosomes, and Outside moved which the apart.have nucleus, the mitotic spindle and each is pulled slowly assemblesassembles Forbetween simplicity, replicatedbetween nucleus,the onlytwo the thethreeand two mitotic moved spindle apart. centrosomes,For simplicity, which only threehave towardtowardand thethe the spindlespindle spindleandmicrotubules polepoleseach pole it isalso pulledit get slowly shorter, assembles between theFornucleus, tworeplicated simplicity, the and mitoticonly moved three spindle apart. assembles between the two toward the spindle pole it centrosomes,centrosomes,chromosomes whichFor assembleshave whichsimplicity, are shown.have between only In three the two replicatedchromosomes and movedare shown. apart. In faces.faces.move The The apart;kinetochore kinetochoretoward bothand the spindle polepoles it also centrosomes, which havechromosomesassemblesFor simplicity, between are only shown. the three two In faces. The kinetochore centrosomes, which have Fordiploid simplicity,centrosomes, cells, thereonly three whichwould have be microtubulesprocesses getcontributefaces. shorter,move The to apart; kinetochore both replicatedreplicateddiploid and moved cells,chromosomes and apart.theremovedreplicated would apart. areand be shown. moved diploid apart.Incentrosomes, cells, there which would have be microtubulesmicrotubules get shorter, get shorter, kinetochore kinetochorereplicated and moved apart.chromosomes are shown. Inreplicated and moved apart. microtubules get shorter, kinetochore For simplicity,Fortwo simplicity, onlycopiesdiploid three of onlyFor each cells, simplicity,three chromo- there only would three bereplicated and moved chromosomesapart.two copies of are each shown. chromo- In andand chthero the mospindleand sospindle theme poles spindlesegregation. processespoles also poles also contributealso to For simplicity, only threetwo diploid copies cells, of each there chromo- would beFor simplicity, only three shortening and the spindle poles also kinetochore chromosomeschromosomessome are present. twoshown. arecopieschromosomes In shown. Inthe of each In are chromo- shown.For In simplicity, only threediploidsome present. cells, there In the would be movemove apart; moveapart; both apart; both ch bothromo some segregation. kinetochore sometwo present. copies ofIn eachthe chromo- kinetochore shortening spindle pole condensing replicated chromosome, consisting of kinetochorecondensingchromosomesdiploid replicated cells, chromosome,are there shown. would In consisting be of twofluorescence copieschromosomes of eachmicrograph, chromo- are shown. In processesprocesses contributemove contribute apart; to bothto condensingdiploid replicated cells,diploid fluorescencechromosome, there cells,some would there consistingmicrograph, present. be would of In be the chromosomes are shown. In microtubule kinetochore processes contribute to two sister chromatidskinetochore held together along their length two sisterdiploid chromatids cells, thereheld together wouldfluorescence bealongsome theirpresent. lengthmicrograph, In the moving outwardspindle poleprocesses contribute to kinetochorekinetochore condensing replicated chromosome,two sister consisting chromatidstwo copies oftwo heldchromosomes of copies togethereachfluorescence ofchromo- eachalongtwo are chromo-copiestheirstained micrograph, length of each chromo- diploid cells, there wouldsomechromosomes be diploid present. cells, In are the therestained would be chrochmoromosochmesoromemo segregation. sosegregation.me segregation. kinetochore condensing replicated chromosome, consisting of chromosomesfluorescence are micrograph, stained shorteningshorteningshorteningmicrotubule moving outward two sister chromatids held togetherkinetochore along their lengthcondensingkinetochoretwo somereplicated copies present. of chromosome, each In thechromo- consisting of two copies of each chromo- chromosome segregation. two sistersome chromatids present.someorange held present. In thetogetherandchromosomes microtubules In the along their are arelength stained two copies of each chromo-fluorescenceorange and micrograph,microtubules are kinetochorekinetochoreshortening condensing replicated chromosome, consisting oftwo sister chromatids held togetherorange alongchromosomes theirand lengthmicrotubules are stained are kinetochore spindlespindle pole spindlepole pole condensingcondensing replicated chromosome,replicated chromosome, consisting consistingof of fluorescencegreen. some micrograph,fluorescence present. In micrograph, the chromosomessome present. are stained In the microtubulekinetochore two sister chromatids held togetherfluorescence along their lengthmicrograph,orange and microtubules aresome present. In the green. microtubulemicrotubule moving spindleoutward pole two sister chromatidstwo sister chromatids heldcondensing together held replicatedalong together their chromosome,along length their length consisting of condensingfluorescencechromosomes replicated micrograph, are chromosome, stainedgreen. orange consisting and microtubules of are movingmoving outward outward condensingchromosomes replicatedchromosomes chromosome, are green.stained are consisting stained of fluorescence micrograph,orange fluorescence and microtubules micrograph, are microtubule moving outward two sister chromatids held together along their length twochromosomes sisterorange chromatids and aremicrotubules held stained together green. are along their length two sisterorange chromatids andorange held microtubules togetherand microtubules along are their arelength chromosomes are stainedgreen. chromosomes are stained orangegreen. and microtubules are green. green. orange and microtubules areorange and microtubules are green. green. green. 2 PROMETAPHASE 2 PROMETAPHASE2 PROMETAPHASE 5 TELOPHASE Prometaphase starts Prometaphase DuringTelophase telophase, the two 2 PROMETAPHASE 2 PROMETAPHASE Prometaphase starts Prometaphase starts 5 TELOPHASE abruptly with the sets of daughterDuring chromo- telophase, the two 2 PROMETAPHASEfragments of 2 PROMETAPHASEPrometaphase starts abruptlyPrometaphase with the starts abruptly with the 5 TELOPHASEset of daughter chromosomes 2centrosomePROMETAPHASE breakdown of thefragments nuclear of fragments of 5 TELOPHASE somes arrive at the poles of 2 PROMETAPHASE nuclear envelope centrosomeabruptlyPrometaphase with the starts breakdownabruptly withof the the nuclear Prometaphasebreakdown of starts the nuclear 5 TELOPHASE at spindle pole During telophasesets of, daughterthe two chromo- at spindle 2 PROMETAPHASEcentrosome fragmentsPrometaphaseenvelope. of Chromosomes startsnuclear fragments envelope of nuclear envelope 5 TELOPHASEset of daughter chromosomesDuringthe spindle telophase and decondense., the two at spindle2 PROMETAPHASEPrometaphaseat starts spindlebreakdown abruptly of with the the nuclear breakdown of the nuclearabruptlyenvelope. with Chromosomes the contractile ringDuring telophasesets of daughter, somesthe two chromo- arrive at the poles of pole centrosome centrosomenuclear fragmentsenvelope of2 PrometaphasePROMETAPHASEnuclear starts envelope envelope.fragments Chromosomes of set of daughter chromosomesat spindle pole setsA new of daughter nuclearDuring envelope chromo- telophase , the two abruptlycan polenow with attach thebreakdown to spindle of the nuclearPrometaphase starts starting to sets of daughtersomes arrive chromo-the at spindlethe poles and of decondense. at spindle centrosome polefragmentsat spindleabruptly of nuclear withcentrosome envelopethe envelope. Chromosomes canenvelope. nownuclear attach Chromosomesenvelope to spindlebreakdowncan nowPrometaphase attach of the to nuclear spindle starts set of daughter chromosomesat spindle pole sets of daughter chromo- fragments of microtubulesabruptly via their with the set of daughter chromosomescontract contractilesomesreassembles arrive ring ataround the poles each of centrosomepole at spindle nuclearpole envelopefragmentsbreakdown atof spindle of theenvelope. nuclear Chromosomes abruptly with the envelope. Chromosomes at spindleset of pole daughter chromosomessomes arrivethe spindle at theA andpoles new decondense. ofnuclear envelope centrosome nuclear envelope breakdown of thecan nuclear now attachfragments to spindle of microtubules can now attach via their to spindle microtubules abruptly viawith their the at spindle pole contractile startingringset, co tomp letisomesng the arrive formation at the poles of at spindle centrosomepole nuclearenvelope.kinetochores envelopepole Chromosomesbreakdown canand now undergo attachof the to nuclear spindlebreakdown fragments of the nuclear of at spindle pole the spindleA new and nuclear decondense. envelope at spindle centrosomeenvelope. Chromosomesmicrotubules nuclear via envelopetheir microtubules via theircankinetochores now attach and to spindleundergo contractilestarting ringthe to contract spindle and decondense.reassembles around each pole at spindle active movement.centrosomeenvelope. Chromosomeskinetochores nuclear and undergo envelope breakdown of the nuclear contractile ring Aof new tworeassembles nuclear nucleithe andenvelope spindle around marking andeach decondense. pole at spindle can now attachmicrotubules to spindle via their envelope. Chromosomes startingcontract to Acontractile new nuclear ring envelopeset, co mpleting the formation pole can now attach toatkinetochores spindle spindle and undergoactive kinetochores movement. and undergomicrotubulesactiveenvelope. movement. via Chromosomes their starting to the end of mitosis.A new nuclearThe envelope pole microtubulescan viakinetochores now their attach andto spindle undergocan now attach to spindle contract startingreassembles toset, comp aroundleting theeach formation microtubules via theirpoleactive movement. active movement. kinetochores and undergo contract reassembles aroundof each two nuclei and marking microtubulesactive movement. via their can now attach to spindle contractset,division coofmp two ofleti the ngnucleireassembles thecytoplasm andformation marking around each kinetochores and undergo microtubules via their active movement. set, completing thethe formation end of mitosis. The kinetochores and undergokinetochores and undergo microtubules via their ofbegins twothe nuclei with end set, contractionofand mitosis. co markingmple Theti ngof the formation active movement. kinetochores and undergo of two nuclei and markingdivision of the cytoplasm active movement. active movement. kinetochores and undergo thethe end contractiledivision of mitosis.of of tworing.the The cytoplasmnuclei and marking active movement. the end of mitosis.begins The with contraction of active movement. divisionbegins of the withthe cytoplasm end contraction of mitosis. of The division of the cytoplasm interpolar centrosome beginsthe with contractile contractiondivisionthe ring.contractile of theof cytoplasm ring. begins with contraction of kinetochore chromosome in active motion microtubules nuclear envelope reassemblingthe contractilebegins ring. with contraction of centrosomecentrosome microtubule kinetochore kinetochore chromosome in active motionchromosome in active motion interpolarinterpolar around individualthe chromosomes contractile ring.the contractile ring. microtubulekinetochore microtubule chromosome in active motion kinetochorekinetochore chromosomechromosome in active in active motion motion microtubulesmicrotubules centrosomenuclear envelopenuclear reassembling envelope reassembling microtubule microtubule interpolar microtubule kinetochore chromosome in active motion interpolar centrosomearound individualaroundcentrosome chromosomes individual chromosomes kinetochore chromosome in active motion microtubules interpolar nuclear envelope reassembling kinetochore chromosome in active motionmicrotubule microtubules microtubule kinetochore kinetochore chromosome in active motionchromosome in active motion microtubules nucleararound envelope individual reassemblingnuclear chromosomes envelope reassembling microtubule microtubule around individual chromosomes microtubule kinetochore chromosome in active motion around individual chromosomes microtubule 3 METAPHASE Metaphase Cytokinesis 3 METAPHASE 3 METAPHASE 6 CYTOKINESIS 3 METAPHASE 3 METAPHASE During cytokinesis, the centrosome3 METAPHASE at 6 CYTOKINESIS 6 CYTOKINESIS cytoplasm is divided in two 3 METAPHASEspindle pole centrosome at 3 centrosomeMETAPHASE at completed nuclear envelope During cytokinesis, the centrosome at centrosome atAt metaphase, the 6 CYTOKINESIS by a contractile ring of 3 METAPHASE centrosome3 METAPHASEspindle at pole spindle pole spindle pole surroundscompleted decondensing nuclear envelope cytoplasm isDuring divided cytokinesis in two , the 3 METAPHASEspindle pole chromosomesAt are metaphase aligned , the At metaphase, the At metaphase, the 6 CYTOKINESIS6 CYTOKINESIS actin and myosin spindle pole centrosome at At metaphase, the chromosomessurrounds decondensing Duringby cytokinesis a contractilecytoplasm, the ring of is divided in two centrosome at at the equator of the chromosomes are aligned completed nuclear envelope spindle3At metaphaseMETAPHASE chromosomespole , the are aligned chromosomes are aligned chromosomes Duringcytoplasmfilaments, cytokinesisactin is andwhich dividedDuring myosin,by the pinches a cytokinesis incontractile two , thering of centrosomespindle at pole centrosome at centrosome at chromosomes are aligned completed nuclearsurrounds envelope decondensing Atspindle, metaphase chromosomesmidway, atthe the between equator are aligned of the at the equator of the Atat metaphase the equator, the of the the cellfilaments, in two whichto create pinches spindle pole spindle pole spindle pole at the equator of the completedsurrounds nuclear decondensingcompleted envelopechromosomes nuclearcytoplasm byenvelope a contractile is dividedcytoplasm ringactin in oftwo and is divided myosin in two At metaphasechromosomesthe spindle, the At poles. are metaphasespindle, aligned The midway , the betweenspindle, midway betweenchromosomes are aligned two daughters,the cell in two each to withcreate at thecentrosome equator at of the Atspindle, metaphase midway, the between spindle, midway between surroundschromosomes decondensingsurrounds decondensingby aactin contractile and myosin byring afilaments, contractileof which ring ofpinches kinetochorechromosomes microtubulesthe spindle poles.are aligned Thethe spindle poles. The two daughters, each with chromosomesat the areequator spindle,alignedspindle of the midway pole between chromosomesthe spindle poles. are aligned Theatthe the spindle equator poles. of the The chromosomes chromosomes actinfilaments,one and nucleus. myosin whichactin the pinches and cell myosin in two to create attach sister chromatidskinetochore to microtubules At metaphase, the at the equatorspindle, of midway theat spindle the between equator poles. of The the kinetochoreatkinetochore the equator microtubules microtubules of the spindle,kinetochore midway microtubules between filaments,the cellone whichin nucleus.twofilaments, pinchestotwo create daughters, which pinches each with opposite polesattach of the sister chromatids to spindle, themidway spindle betweenkinetochore poles.spindle, The midway microtubules betweenattach spindle, sister midway chromatids betweenthe attachto spindlechromosomes sister poles. chromatids The are aligned to thetwo cell daughters,in two tothe create each cell inwith two to create opposite poles of the attach sister chromatids to one nucleus. the spindlekinetochorespindle. poles. Thethe microtubules spindle poles. The oppositethe spindle poles poles. of the The kinetochoreoppositeat the poles equatormicrotubules of the of the one nucleus.two daughters, each with kinetochore attachspindle. sister chromatids to opposite poles of the two daughters, each with microtubule kinetochore kinetochoreattach microtubules sisteroppositekinetochore chromatids poles tomicrotubules of the spindle.kinetochore spindle. microtubulesattachspindle. spindle, sister chromatids midway between to one nucleus. one nucleus. kinetochore kinetochore microtubule kinetochoreattach sisteropposite chromatids polesattach of to the sister chromatids toattach sister chromatidsopposite to the polesspindle of poles.the The microtubulemicrotubule microtubulespindle. kinetochore oppositespindle. poles of theopposite poles of the opposite poles of the spindle.kinetochore microtubules contractile ring kinetochoremicrotubule re-formation of interphase kinetochorespindle. creating contractilecleavage ring re-formation of interphase microtubule kinetochore spindle. spindle. attach sister chromatids to array of microtubules nucleated kinetochore kinetochore microtubule furrow creating cleavage array of microtubules nucleated microtubule opposite poles of the by the centrosome microtubule microtubule furrowcontractile ring by the centrosome spindle. contractile ring re-formation of interphase kinetochore creating cleavage re-formation of interphasearray of microtubules(Micrographs nucleated courtesy of Julie Canman and Ted Salmon.) creating cleavagecontractilefurrow ring array of microtubules nucleated(Micrographs courtesy of Julie Canman and Ted Salmon.) Albertsmicrotubule et al., Molecular Biology of the Cellcontractile ring re-formation of interphasere-formationby the centrosome of interphase furrow creating2 cleavage by the centrosome creating cleavage array of microtubulesarray nucleated of microtubules nucleated furrow furrow by the centrosome (Micrographs courtesy of Julie Canman and Ted Salmon.) by the centrosome (Micrographs courtesy of Julie Canman and Ted Salmon.) (Micrographs courtesy(Micrographs of Julie courtesy Canman of andJulie Ted Canman Salmon.) and Ted Salmon.) Cell division

3 Growing microtubules can push centrosomes to the middle of the cell

(A) tubulin subunits growing microtubules Figure 16.51: Self-centered centrosomes. (A) Diagrams showing time sequence of a self-centering experiment. Initially, a centrosome is added to a microfabricated square well along with soluble tubulin subunits. As the centrosome nucleates growth of microtubules, individual microtubules grow and shrink in a process of dynamic instability. When a microtubule tip contacts the wall of centrosome force due to pushing on wall the chamber, it can continue to grow, (B) resulting in a pushing force on the centrosome. Eventually, as microtubules grow longer and push minutes 3 6 against the wall in all directions, the centrosome finds a stable position of the geometrical center of the well. (B) Frames from a video sequence using differential interference contrast (DIC) microscopy show this process over a period of several minutes. (Adapted from T. E. Holy et al., Proc. Natl Acad. Sci. USA 94:6228, 1997.)

10 mm R. Phillips et al., Physical this phenomenon is exploited by4 cells to set up aBiology universal of the coor- Cell dinate system whereby they are able to position their organelles at precise geographical locations in the enormous cellular volume. Specifically, if microtubules within a cell are labeled fluorescently, it can be seen that they typically emanate in a star-like array from asinglepointknownasthecentrosome.Thecentrosomeisatiny object, less than 0.5 µmacross,thatnonethelesscanpositionitself near the middle of a cell with typical sizes of tens of microns. How does the centrosome find the cell center? One possible mechanism can be demonstrated by a clever experiment illustrated in Figure 16.51 that involves isolating a centrosome and dropping it into a nanofab- ricated hole with dimensions comparable to those of a cell. The centrosome on its own diffuses around aimlessly. However, if tubu- lin is added so that microtubules can grow from the centrosome, the centrosome quickly (within minutes) zooms in to the geometric cen- ter of the hole, regardless of the hole shape. If the centrosome is grabbed with a laser trap and displaced from its central location, it will gently but insistently return to the center. The mechanism relies on the pushing forces at the tips of all of the microtubules when they run into the walls. Only when the centrosome is at the geometri- cal center of the enclosure do all of the forces cancel out. In living cells of the fission yeast Schizosaccharomyces pombe,thismechanism has been directly observed centering the nucleus halfway down this rod-shaped cell by virtue of microtubules pushing against the two opposite ends.

16.3.3 The Translocation Ratchet

Another fascinating kind of motor action introduced at the begin- ning of this chapter is associated with translocation. We argued that because of the division of cells into different compartments, there

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1 PROPHASE centrosome At prophase, the replicated chromosomes, each consisting of two closely intact forming nuclear mitotic associated sister chromatids, envelope spindle condense. Outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have replicated and moved apart. For simplicity, only three chromosomes are shown. In diploid cells, there would be kinetochore two copies of each chromo- some present. In the condensing replicated chromosome, consisting of fluorescence micrograph, two sister chromatids held together along their length chromosomes are stained orange and microtubules are green. Spindle984 Chapter is organized17: The Cell Cycle by molecular motors

2 PROMETAPHASE Prometaphase starts abruptly with the Figure 17–25 Major motor proteins of the fragments of centrosome nuclear envelope breakdown of the nuclear kinesin-14 spindle. Four major classes of microtubule- at spindle envelope. Chromosomes pole dependent motor proteins (yellow boxes) can now attach to spindle spindle microtubule microtubules via their kinesin-5 contribute to spindle assembly and function kinetochores and undergo + (see text). The colored arrows indicate the active movement. dynein dynein + direction of motor protein movement along – + – – a microtubule—blue toward the minus end – – – and red toward the plus end. – + + + centrosome kinetochore chromosome in active motion microtubule plasma sister chromatids membrane kinesin-4,10

+ 3 METAPHASE centrosome at spindle pole At metaphaseproteins,, the also called chromokinesins, are plus-end directed motors that associate chromosomes are aligned at thespindle equatorwith of the chromosome arms and push the attached chromosome away from the pole spindle, midway between the spindle poles.(or theThe pole away from the chromosome). Finally, dyneins are minus-end directed (metaphase)kinetochoremotors microtubules that, together with associated proteins, organize microtubules at various attach sister chromatids to opposite poleslocations of the in the cell. Tey linkMBoC6 the plus m17.30/17.25 ends of astral microtubules to components spindle. kinetochore microtubule of the actin cytoskeleton at the cell cortex, for example; by moving toward the minus end of the microtubules, the dynein motors pullAlberts the spindle et al., Molecular poles toward the cell cortex and away from each other. Biology of the Cell 5 Multiple Mechanisms Collaborate in the Assembly of a Bipolar Mitotic Spindle Te mitotic spindle must have two poles if it is to pull the two sets of sister chroma- tids to opposite ends of the cell in anaphase. In most animal cells, several mecha- nisms ensure the bipolarity of the spindle. One depends on centrosomes. A typi- cal animal cell enters mitosis with a pair of centrosomes, each of which nucleates a radial array of microtubules. Te two centrosomes provide prefabricated spin- dle poles that greatly facilitate bipolar spindle assembly. Te other mechanisms depend on the ability of mitotic chromosomes to nucleate and stabilize microtu- bules and on the ability of motor proteins to organize microtubules into a bipolar array. Tese “self-organization” mechanisms can produce a bipolar spindle even in cells lacking centrosomes. We now describe the steps of spindle assembly, beginning with centrosome- dependent assembly in early mitosis. We then consider the self-organization mechanisms that do not require centrosomes and become particularly important after nuclear-envelope breakdown.

Centrosome Duplication Occurs Early in the Cell Cycle Most animal cells contain a single centrosome that nucleates most of the cell’s cytoplasmic microtubules. Te centrosome duplicates when the cell enters the cell cycle, so that by the time the cell reaches mitosis there are two centrosomes. Centrosome duplication begins at about the same time as the cell enters S phase. Te G1/S-Cdk (a complex of cyclin E and Cdk2 in animal cells; see Table 17–1) that triggers cell-cycle entry also helps initiate centrosome duplication. Te two cen- trioles in the centrosome separate, and each nucleates the formation of a single new centriole, resulting in two centriole pairs within an enlarged pericentriolar matrix (Figure 17–26). Tis centrosome pair remains together on one side of the nucleus until the cell enters mitosis. Tere are interesting parallels between centrosome duplication and chromo- some duplication. Both use a semiconservative mechanism of duplication, in which the two halves separate and serve as templates for construction of a new half. Centrosomes, like chromosomes, must replicate once and only once per cell cycle, to ensure that the cell enters mitosis with only two copies: an incorrect number of centrosomes could lead to defects in spindle assembly and thus errors in chromosome segregation. MITOSIS 991

How does plus-end depolymerization drive the kinetochore toward the pole? As we discussed earlier (see Figure 17–31C), Ndc80 complexes in the kineto- chore make multiple low-afnity attachments along the side of the microtubule. Because the attachments are constantly breaking and re-forming at new sites, the kinetochore remains attached to a microtubule even as the M ITOmicrotubuleSIS depoly- 991 merizes. In principle, this could move the kinetochore toward the spindle pole. A second poleward force is provided in some cell types byHow microtubule does plus-end fdepolymerizationux, drive the kinetochore toward the pole? whereby the microtubules themselves are pulled toward Asthe we spindle discussed poles earlier and (see Figure 17–31C), Ndc80 complexes in the kineto- dismantled at their minus ends. Te mechanism underlyingchore this make poleward multiple move low-af- nity attachments along the side of the microtubule. Because the attachments are constantly breaking and re-forming at new sites, the ment is not clear, although it might depend on forces generatedkinetochore by motor remains proteins attached to a microtubule even as the microtubule depoly- and minus-end depolymerization at the spindle pole. In metaphase,merizes. In principle, the addition this could move the kinetochore toward the spindle pole. of new tubulin at the plus end of a microtubule compensates forA second the loss poleward of tubulin force is provided in some cell types by microtubule fux, at the minus end, so that microtubule length remains constantwhereby despite the microtubules the move themselves- are pulled toward the spindle poles and dismantled at their minus ends. Te mechanism underlying this poleward move- ment of microtubules toward the spindle pole (Figure 17–35ment). is Anynot clear, kinetochore although it might depend on forces generated by motor proteins that is attached to a microtubule undergoing such fux experiencesand minus-end a depolymerizationpoleward at the spindle pole. In metaphase, the addition force, which contributes to the generation of tension at theof kinetochore new tubulin at the in plusmeta end- of a microtubule compensates for the loss of tubulin at the minus end, so that microtubule length remains constant despite the move- phase. Together with the kinetochore-based forces discussedment above, of microtubules fux also towardcon- the spindle pole (Figure 17–35). Any kinetochore tributes to the poleward forces that move sister chromatidsthat after is attached they separate to a microtubule in undergoing such fux experiences a poleward anaphase. force, which contributes to the generation of tension at the kinetochore in meta- A third force acting on chromosomes is the polar ejectionphase. force Together, or polar with thewind kinetochore-based. forces discussed above, fux also con- tributes to the poleward forces that move sister chromatids after they separate in Plus-end directed kinesin-4 and 10 motors on chromosomeanaphase. arms interact with interpolar microtubules and transport the chromosomes awayA third from force the acting spindle on chromosomes is the polar ejection force, or polar wind. poles (see Figure 17–25). Tis force is particularly importantPlus-end in prometaphase directed kinesin-4 and and 10 motors on chromosome arms interact with metaphase, when it helps push chromosome arms out frominterpolar the spindle. microtubules Tis force and transport the chromosomes away from the spindle poles (see Figure 17–25). Tis force is particularly important in prometaphase and might also help align the sister-chromatid pairs at the metaphasemetaphase, platewhen it ( helpsFigure push chromosome arms out from the spindle. Tis force 17–36). might also help align the sister-chromatid pairs at the metaphase plate (Figure One of the most striking aspects of mitosis in vertebrate17–36 cells). is the continu- One of the most striking aspects of mitosis in vertebrate cells is the continu- ous oscillatory movement of the chromosomes in prometaphaseous oscillatory and metaphase. movement of the chromosomes in prometaphase and metaphase. When studied by video microscopy in newt lung cells, the movementsWhen studied by are video seen microscopy to in newt lung cells, the movements are seen to switch between two states—a poleward state, when the chromosomesswitch between aretwo states—apulled poleward state, when the chromosomes are pulled toward the pole, and an away-from-the-pole, or neutral, state,toward when the pole, the andpoleward an away-from-the-pole, or neutral, state, when the poleward forces are turned of and the polar ejection force pushes the chromosomes away forces are turned of andCurrent the polar Biology ejectionVol 15 force No 23 pushes the chromosomes away R958 Microtubules are depolymerized at spindle poles spindle pole TUBULIN spindle pole REMOVAL TUBULIN Figure 2. A hypothetical REMOVAL A below a threshold concentration of V model for force-assembly depoly Kinesin-5. Goshima et al. [2] TUBULIN Fsliding coupling at the pole. distance ADDITION acknowledge that other coupling (A) Microtubule minus ends TUBULIN Pole assumptions might be considered TUBULIN

distance ADDITION embedded in the pole (dark ADDITION as well, and that further evaluation Current Biology Vol 15 No 23“speckles” blue) tend to depolymerize speckles moving R958 (A) (B) (C) will be needed. Fortime example, poleward slowly when under low TUBULIN + – ADDITION sliding force (Fsliding). That recent analysis of budding yeast TUBULIN Depolymerization is done REMOVAL depolymerization occurs at and Xenopus extract spindles “speckles” Figure 2. speckles A hypothetical moving by molecular motors poleward below a threshold(D) concentration of (A) (B) B A(C) V all time may be because a Figure 17–35 Microtubuledepoly flux in the metaphasemodel spindle. for ( force-assemblyA) Tosuggest observe microtubule that tension flux,Kinesin-5. a very on small the amount Goshima of fluorescent et al. tubulin[2] is injected Fsliding into living cells so that individualdepolymerase microtubules (redcoupling form circles), with a at very the small pole. proportion of fluorescent tubulin. Such microtubules have a speckled Vdepoly kinetochore can acknowledgetrigger a bias that other coupling Fsliding appearance when viewedsuch by fluorescence as Klp10A microscopy.(A) (Kinesin- Microtubule (B) FluorescenceTUBULIN minus micrograph ends of a mitotic spindle in a living newt lung epithelial cell. The chromosomes are colored brownPole , and the tubulin speckles areREMO red. (C)VA TheL movement of individualassumptions speckles can might be followed be byconsidered time-lapse video microscopy. Images of 13),the thin acts vertical at boxed theembedded region pole (arrow) to inin (B),thetowards taken pole at sequential(dark assembly, times, show or attenuation that individual speckles move toward the poles at a rate of about 0.75 μm/min, indicating that the microtubules are moving poleward. (D) asMicrotubule well, lengthand inthat the metaphase further spindleevaluation does not (D)blue) tend to depolymerize change significantlyPole becauseincrease new tubulinthe depolymeriza- subunitsslowly are added when at theof under microtubule disassembly, low plus end atatwill thethe besame plus needed. rate ends as tubulin For subunits example, are removed from the minus end. (B and C, from T.J. Mitchison and E.D. Salmon, Nat. Cell Biol. 3:E17–21, 2001. With permission from Macmillan Publishers Ltd.) Figure 17–35 Microtubule flux in the metaphase+ spindle. (A) To observe– microtubule flux, a very small amountsliding of force fluorescent (F tubulin). That is injectedrecent analysis of budding yeast tion velocity (Vdepoly) locally. [10,11].sliding Nevertheless, the model into living cells so that individual microtubules form with a very small proportion of fluorescent(B) tubulin. If the slidingSuch depolymerizationmicrotubules forceMBoC6 ism17.41/17.35 havemakes aoccurs speckled a surprising at and prediction Xenopus extract spindles appearance when viewed by fluorescence microscopy.B (B) D.J.Fluorescence Odde, Current micrograph Biol. of a mitotic spindle in alla living may newt be lung becauseepithelial cell. a The suggest that tension on the chromosomes are colored brown, and+ the tubulin speckles are– red. (C) The movement of individualincreased, speckles then thedepolymerase can concen- be followed (red by circles),time-lapse video 15, R956-R959 (2005)Vdepoly about spindleAlberts stability etkinetochore al., Molecular that turns can trigger a bias microscopy. Images of the thin vertical boxed regionFsliding (arrow) in (B), taken at sequential times,tration show that of depolymeraseindividualsuch speckles as Klp10A move (Kinesin- toward the poles at a rate of about 0.75 μm/min, indicating that the microtubules are moving poleward. (D) Microtubule 6length13), in the acts metaphase at theout spindle to pole be true.does toBiology nottowards of the Cell assembly, or attenuation Current Biology increases locally so that the change significantly because new tubulin subunits are added at the microtubule plus end at the samePole rate asincrease tubulin subunitsthe depolymeriza- are removed fromof the disassembly, at the plus ends minus end. (B and C, from T.J. Mitchison and E.D. Salmon, Nat. Cell Biol. 3:E17–21, 2001. With permission from Macmillan PublishersSpindle Ltd.)length regulation has depolymerizationtion velocity rate (Vdepoly) locally. [10,11]. Nevertheless, the model (B) If the slidingbeen force studied is mostmakes extensively a surprising in prediction increases. In their report,MBoC6 Goshima m17.41/17.35 et al. [2] are justifiably nonspecific about the detailed mechanism of coupling,+ focusing instead on its– mathematical form and generalincreased, impor- then buddingthe concen- yeast [12].about As inspindle the S2 stability that turns tration of depolymerase out to be true. tance in order to have a steady-state length at all. The cartoonCurrent here Biology is meantincreases for illus- locallycells, so that the the spindle length in budding tration purposes only, depicting just one way in which the mathematicaldepolymerization model of rate Spindle length regulation has yeast is fairly constantbeen studied in most extensively in Goshima et al.increases.[2] for force-assembly In their report, coupling Goshima could et al operate. [2] are mechanistically. justifiably nonspecific about the detailed mechanism of coupling, focusing instead on its mathematical form and generalmetaphase impor- [13]. buddingBut there yeastis no [12]. As in the S2 tance in order to have a steady-state length at all. The cartoon here is meant for illus- cells, the spindle length in budding tration purposes only, depicting just one way in which the mathematicalevidence model forof minus-endyeast is fairly constant in extended periodsGoshima (minutes), et al. [2] for force-assemblysignificant coupling increase could in operate spindle mechanistically.disassembly, simplifyingmetaphase analysis [13]. But there is no whereas the viscoelastic relaxation length. Interestingly, perturbing the of the system [14].evidence Also, only for four minus-end extended periods (minutes), significant increase in spindle disassembly, simplifying analysis time is estimatedwhereas to bethe on viscoelastic the relaxationconcentrationlength. of Klp61F Interestingly, (Kinesin- perturbingmotors enterthe theof nucleus, the system Cin8p [14]. Also, only four order of milliseconds,time is estimated suggesting to be on the5), which is thoughtconcentration to be the ofmain Klp61F (Kinesin-5),(Kinesin- Kip1pmotors (Kinesin-5), enter the nucleus, Cin8p that the elasticorder forces of milliseconds, dominate the suggestingforce generator5), in which the spindle, is thought had to beKar3p the main (Kinesin-14),(Kinesin-5), and Kip3p Kip1p (Kinesin-5), that the elastic forces dominate the force generator in the spindle, had Kar3p (Kinesin-14), and Kip3p force balanceforce [6]. balance In general, [6]. In general,very little effectvery on littlespindle effect length on spindle(Kinesin-8), length and from(Kinesin-8), deletion and from deletion viscous dragviscous forces drag are probably forces are probablyover a broad rangeover a of broad range of mutant analysis, mutantit was deduced analysis, it was deduced negligible, negligible,as a 1 µm radius as a 1sphere µm radiusconcentrations. sphere concentrations. Below a critical Below a thatcritical Cin8p and Kip1pthat Cin8p act as andsliding Kip1p act as sliding (modelling a chromosome) moving concentration of Klp61F, however, motors to elongate the spindle, (modelling ata chromosome) 1 µm per minute moving experiencesconcentration a the of Klp61F, spindle however, completely collapsedmotors to elongatewhile the Kar3p spindle, acts antagonistically at 1 µm perdrag minute force experiences of ~1 fN, aabout 1000-the spindle completelyinto a monopolar collapsed array ofwhile Kar3p actsto antagonistically shorten spindles [15]. The drag force foldof ~1 less fN, aboutthan the 1000- stall forceinto of a a monopolarmicrotubules. array of to shorten spindlesbudding [15]. The yeast system also permits single molecular motor [7]. To explain this effect, Goshima rough estimation of the magnitude fold less thanWhat the stall Goshima force of et a al. [2] havemicrotubules.et al. [2] used a previouslybudding yeast systemof the alsoinward permits tensional force on single molecularnow done motor is [7]. perform a perturbationTo explain thispublished effect, Goshimamathematical roughmodel estimation for chromatin, of the magnitude which from What Goshimaanalysis et whereal. [2] have the concentrationset al. [2] usedspindle a previously length during anaphase,of the inward tensionalchromosome force on marking experiments of putative spindle length extending it to metaphase by is estimated in metaphase to be now done isregulators perform a are perturbation systematicallypublished mathematicalincluding spindle model for elasticity,chromatin, whichsufficient from to force nucleosome analysis wheredecreased, the concentrations through RNA spindle lengthkinetochore during anaphase, microtubules,chromosome and markingrelease experiments [16,17]. From in vitro of putative interferencespindle length (RNAi), or increased,extending it tosister metaphase chromatid by cohesionis estimated[8,9]. in metaphasestudies with to belaser tweezers, this through overexpression, and the Even with these effects included, suggests an average tensional regulators areeffect systematically on spindle length measuredincluding spindlehowever, elasticity, the model did sufficientnot settle to forceforce nucleosome exceeding 15 pN per decreased,quantitatively through RNA using an automatedkinetochore microtubules,to a steady-state and length,release but [16,17]. Fromchromosome in vitro [18]. Given that there interferencemicroscope (RNAi), or increased, and image analysissister chromatidrather cohesion grew to[8,9]. infinity. Tostudies with laserare tweezers, 16 chromosomes, this then the system. Starting with an initial data establish a steady-state, the total inward tensional force is through overexpression,set of about one and millionthe cells,Even their with theseauthors effects then included, consideredsuggests a an averageestimated tensional to be >240 pN, implying effect on spindlemachine length identified measured a ‘small’ however,subset the ‘couplingmodel did assumption,’not settle whereforce exceeding the an15 outwardpN per extensional force of quantitativelythat using was anmitotic, automated so that thousandsto a steady-stateKinesin-5 length, sliding but activitychromosome promotes [18].>240 Given pN, that neglecting there net tension or of mitotic cells were analyzed. microtubule depolymerization. For compression exerted from outside microscopeThey and foundimage that,analysis in S2 cells,rather it was grew toconcreteness infinity. To and illustrationare 16 chromosomes,the spindle. then the system. Startingthe regulators with an initial of microtubule data establish a steady-state,purposes only, the one potentialtotal inward tensionalIn the force Xenopus is extract system, set of aboutassembly one million dynamics cells, their that hadauthors the then consideredscenario is asketched in Figureestimated 2. to be >240the addition pN, implying of monastrol, a drug strongest effect on spindle length. This coupling, where the that interferes with Eg5 (Kinesin-5) machine identifiedThese regulators a ‘small’ subset included the‘coupling assumption,’depolymerization where the rate dependsan outward extensionalfunction, force was of recently reported to that was mitotic,assembly-promoting so that thousands moleculesKinesin-5 slidingexponentially activity promotes on the sliding>240 force, pN, neglectingcause net a tension ‘switch-like or transition’ EB1, Msps (Dis1/XMAP215/TOG), saturating to a maximum rate at from bipolar to monopolar as a of mitotic cellsand wereMast/Orbit analyzed. (CLASP), andmicrotubule the depolymerization.large forces, enabled For a steady-compression exertedfunction from of outside increasing monastrol They founddisassembly-promoting that, in S2 cells, it was moleculesconcretenessstate and illustration spindle length to bethe spindle. concentration, with little effect on the regulatorsKlp10A of microtubule (Kinesin-13) and Klp67Apurposes only,achieved. one potential In the Xenopusspindleextract length system, noted at sub- (Kinesin-8). Eliminating sister Interestingly, the model also threshold concentrations [19]. assembly dynamicschromatid that cohesion had the also ledscenario to a is sketchedpredicts in the Figure collapse 2. of thethe spindleaddition of monastrol,Interestingly, a drug even though spindle strongest effect on spindle length. This coupling, where the that interferes with Eg5 (Kinesin-5) These regulators included the depolymerization rate depends function, was recently reported to assembly-promoting molecules exponentially on the sliding force, cause a ‘switch-like transition’ EB1, Msps (Dis1/XMAP215/TOG), saturating to a maximum rate at from bipolar to monopolar as a and Mast/Orbit (CLASP), and the large forces, enabled a steady- function of increasing monastrol disassembly-promoting molecules state spindle length to be concentration, with little effect on Klp10A (Kinesin-13) and Klp67A achieved. spindle length noted at sub- (Kinesin-8). Eliminating sister Interestingly, the model also threshold concentrations [19]. chromatid cohesion also led to a predicts the collapse of the spindle Interestingly, even though spindle 988 Chapter 17: The Cell Cycle

replicated chromosome kinetochore anaphase chromatid direction of chromatid movement

centromere region MITOSIS of chromosome kinetochore 989

microtubules embedded in + kinetochore (A) (B) (C) kinetochore(D) (E) microtubules + + + + nuclear + + envelope (C) 1 µm

(A) + (B) chromatid spindle + pole + + plus ends coming from two directions. However, the kinetochores do not instantly Figure 17–30 The kinetochore. achieve the correct ‘end-on’ microtubule attachment to both spindle poles. (A) A fluorescence micrograph of a metaphase chromosome stained with a early prometaphase: lateral kinetochore mid prometaphase: metaphase: late prophase Instead, detailed studies with light and electron microscopy show that most initial DNA-binding fluorescent dye and with attachments, chromosome arms pushed outwards end-on attachment bi-orientation attachments are unstable lateral attachments, in which a kinetochore attaches to human autoantibodies that react with the side of a passing microtubule, with assistance from kinesin motor proteins in specific kinetochore proteins. The two Figure 17-32 Chromosomethe outer attachmentkinetochore. to Soon,the mitotic however, spindle the in dynamic animal cells. microtubule (A) In late plusprophase ends of cap most- animalkinetochores, cells, the one associated with each mitotic spindle poles have moved to opposite sides of the nuclear envelope, with an array of overlapping microtubulessister chromatid, between are stained red. ture the kinetochores in the correct end-on orientation (Figure 17–32). (B) A drawing of a metaphase chromosome them. (B) Following nuclearAnother envelope attachment breakdown, mechanism the sister-chromatid also plays pairsa part, are particularly exposed to thein the large absence number of dynamic plus MBoC6 m17.36/17.30 showing its two sister chromatids ends of microtubulesof radiating centrosomes. from the Careful spindle microscopic poles. In most analysis cases, the suggests kinetochores that shortare first microtubules attached to the sidesattached of these to the plus ends of kinetochore microtubules, whilein at thethe vicinitysame time of thethe arms chromosomes of the chromosomes become areembedded pushed outward in the plus-end-bindingfrom the spindle interior, microtubules. preventing Each kinetochore forms a the arms from blocking microtubule access to the kinetochores. (C) Eventually, the laterally-attached sister chromatids are sites of the kinetochore. Polymerization at these plus ends then results in growth plaque on the surface of the centromere. arranged in a ring around the outside of the spindle. Most of the microtubules are concentrated in this ring, so that(C) the Electron spindle micrograph of an anaphase is relatively hollow inside.of the (microtubulesD) Dynamic micr awayotubule from plus the ends kinetochore. eventually encounter Te minus the ends kinetochores of these inkineto an end-on- chromatidorientation with and microtubules attached to are captured and stabilized.chore microtubules (E) Stable end-on are eventually attachment cross-linked to both poles to results other in minus bi-orientation ends and. Additional focused micr otubulesits kinetochore. are While most kinetochores have a trilaminar structure, the one shown attached to the kinetochore,by motor resulting proteins in at a thekinetochore spindle fiberpole containing (see Figure 10–40 17–28). microtubules. Microtubules attach to here (from a green alga) has an unusually complex structure with additional layers. Bi-orientation Is Achieved by Trial and Error (A, courtesy of B.R. Brinkley; C, from 988 Chapter 17:they The separateCell Cycle in anaphase. How is this mode of attachment, called bi-orienta- J.D. Pickett-Heaps and L.C. Fowke, chromosomesTe success of mitosis demands via that sister kinetochore chromatids in a pair attach to opposite Aust. J. Biol. Sci. 23:71–92, 1970. With MBoC6 n17.301/17.30 tion, achieved? What prevents thepoles attachment of the mitotic of bothspindle, kinetochores so that they moveto the to same opposite ends of the cell when permission from CSIRO.) spindle polereplicated or the attachment of one kinetochore to both spindle poles? Part of the answerchromosome is that sister kinetochores are constructed in a back-to-back orienta- kinetochore tion that reduces the likelihood that both kinetochores can face the same spindle kinetochore anaphase chromatid direction of pole. Nevertheless, incorrect attachments do occur, and elegant regulatorychromatid mech- kinetochore movement anisms have evolved to correct them. plus end of microtubule centromere region of chromosomeIncorrect attachmentskinetochore are corrected by a system of trial and error that is based

on a simple principle: incorrect attachments are highly unstable andmicrotubules do not last, whereas correct attachments become locked in place. How does theembedded kinetochore in kinetochore sense a correct attachment?kinetochore Te answer appears to be tension (Figure 17–33). When a sister-chromatidmicrotubules pair is properly bi-oriented on the spindle, the two kine- tochores are pulled in opposite directions by strong poleward forces. Sister-chro- Ndc80 matid cohesion resists these poleward forces, creating high levels of tension within complex the kinetochores. When chromosomes(C) are incorrectly attached—when both sis- (A) (B)1 µm depolymerization(C) of kinetochore ter chromatids are attached to the same spindle100 pole, nm for example—tension is low (A) (B) chromatid Figure 17–31 Microtubule attachmentmicrotubules sites in the kinetochore. (A) Inat this spindleelectron micrograph of a mammalian kinetochore, the chromosome is on the right, and the plus ends of multiple microtubules are embedded in the outer kinetochore on the left. (B) Electron tomography (discussed plus ends coming from two directions. However, the kinetochoresin Chapter do not 9) was instantly used to constructFigure 17–30 a low-resolutionpoles The kinetochore. three-dimensionalproduces image tension of the outer kinetochore in (A). Several microtubules (in multiple achieve the correct ‘end-on’ microtubule attachment to colors both) are spindle embedded poles. in fibrous(A) Amaterial fluorescence of the micrograph kinetochore, of a which is thoughtFigure to be 17–33 composed Alternative of the N dc80forms complex of and other proteins. (C) Each microtubule is attached to themetaphase kinetochore chromosome by interactions stained with with multiple a copies of the Ndc80 complex (blue). This complex binds to the sides of the Instead, detailed studies with light and electron microscopy show that most initial kinetochore attachment to the spindle microtubule near its plus end,DNA allowing-binding polymerization fluorescent dye and and depolymerization with poles. to occur (A) whileInitially the, a microtubule single microtubule remains attached to the kinetochore. attachments are unstable lateral attachments, in which a kinetochore(A and B, from attaches Y. Dong to et al.,human Nature autoantibodies Cell Biol. 9:516–522, that react 2007.with With permission from Macmillan Publishers Ltd.) the side of a passing microtubule, with assistance from kinesin motor proteins in specific kinetochore proteins. The two from a spindle pole binds to one the outer kinetochore. Soon, however, the dynamic microtubule plus ends cap- kinetochores, one associated with each kinetochore in a sister-chromatid pair. sister chromatid, are stained red. ture the kinetochores in the correct end-on orientation (Figure 17–32). Additional microtubules can then bind to (B) A drawing of a metaphase chromosome Tension sensed by sister Another attachment mechanism also plays a part, particularly in the absence the chromosome in various ways. MBoC6 m17.36/17.30 (A) UNSTABLE showing its two sister chromatids of centrosomes. Careful microscopic analysis suggests that short microtubules attached to the plus ends of kinetochore (B) A microtubule from the same spindle in the vicinity of the chromosomes become embedded in the plus-end-binding microtubules. Each kinetochore forms a pole kinetochorescan attach to the other sister increases sites of the kinetochore. Polymerization at these plus ends then results in growth plaque on the surface of the centromere. kinetochore, or (C) microtubules from (C) Electron micrograph of an anaphase both spindlebinding poles can attach affinity to the same for of the microtubules away from the kinetochore. Te minus ends of these kineto- chromatid with microtubules attached to chore microtubules are eventually cross-linked to other minus ends and focused its kinetochore. While most kinetochores kinetochore. These incorrect attachments by motor proteins at the spindle pole (see Figure 17–28). have a trilaminar structure, the one shown microtubulesare unstable, however, so that and one of thelocks them here (from a green alga) has an unusually two microtubules tends to dissociate. complex structure with additional layers. (D)in When the a micr otubulecorrect from the oppositeattachment Bi-orientation Is Achieved by Trial and Error (A, courtesy of B.R. Brinkley; C, from J.D. Pickett-Heaps and L.C. Fowke, pole binds to the second kinetochore, Te success of mitosis demands that sister chromatids in a pair attach to opposite Aust. J. Biol. Sci. 23:71–92, 1970. With the sister kinetochores are thought to poles of the mitotic spindle, so that they move to opposite ends of the cell when permission from CSIRO.)MBoC6 m17.37/17.31 sense tension across their microtubule- binding sites. This triggers an increase in microtubule binding affinity, thereby locking (B) UNSTABLE (C) UNSTABLE (D) STABLE the correct attachmentAlberts in place. et al., Molecular

plus end of microtubule 7 Biology of the Cell

MBOC6 m17.39/17.33

Ndc80 complex

(A) (B) (C) kinetochore 100 nm Figure 17–31 Microtubule attachment sites in the kinetochore. (A) In this electron micrograph of a mammalian kinetochore, the chromosome is on the right, and the plus ends of multiple microtubules are embedded in the outer kinetochore on the left. (B) Electron tomography (discussed in Chapter 9) was used to construct a low-resolution three-dimensional image of the outer kinetochore in (A). Several microtubules (in multiple colors) are embedded in fibrous material of the kinetochore, which is thought to be composed of the Ndc80 complex and other proteins. (C) Each microtubule is attached to the kinetochore by interactions with multiple copies of the Ndc80 complex (blue). This complex binds to the sides of the microtubule near its plus end, allowing polymerization and depolymerization to occur while the microtubule remains attached to the kinetochore. (A and B, from Y. Dong et al., Nature Cell Biol. 9:516–522, 2007. With permission from Macmillan Publishers Ltd.)

MBoC6 m17.37/17.31 996 Chapter 17: The Cell Cycle

the proteins that hold the sister chromatids together. APC/C also promotes cyclin destruction and thus the inactivation of M-Cdk. Te resulting dephosphorylation of Cdk targets is required for the events that complete mitosis, including the disassem- bly of the spindle and the re-formation of the nuclear envelope. CYTOKINESIS Te fnal step in the cell cycle is cytokinesis, the division of the cytoplasm in two. In most cells, cytokinesis follows every mitosis, although some cells, such as early Drosophila embryos and some mammalian hepatocytes and heart muscle cells, undergo mitosis without cytokinesis and thereby acquire multiple nuclei. In most animal cells, cytokinesis begins in anaphase and ends shortly after the comple- tion of mitosis in telophase. Te frst visible change of cytokinesis in an animal cell is the sudden appear- ance of a pucker, or cleavage furrow, on the cell surface. Te furrow rapidly deep- ens and spreads around the cell until it completely divides the cell in two. Te structure underlying this process is the contractile ring—a dynamic assembly composed of actin flaments, myosin II flaments, and many structural and regula- tory proteins. During anaphase, the ring assembles just beneath the plasma mem- brane (Figure 17–41; see also Panel 17–1). Te ring gradually contracts, and, at the same time, fusion of intracellular vesicles with the plasma membrane inserts new membrane adjacent to the ring. Tis addition of membrane compensates for the increase in surface area that accompanies cytoplasmic division. When ring contraction is completed, membrane insertion and fusion seal the gap between the daughter cells.

Actin and Myosin II in the Contractile Ring Generate the Force for Cytokinesis MITOSIS Cell division 995

Once all chromosomesIn interphase are cells, correctly actin attachedand myosin to microtubules,II flaments form they a corticalbreak network underly- into pair ofing chromatids, the plasma which membrane.ANAPHASE are then A In somepulled cells, towards they alsocentrosomes form large cytoplasmicANAPHASE bundles B called stress fbers (discussed in Chapter 16). As cells enter mitosis, these arrays of actin and myosin disassemble; much of the actin reorganizes, and myosin II flaments are released. As the sister chromatids separate in anaphase, actin and myosin II begin to accumulate in the rapidly assembling contractile ring (Figure 17–42), which also contains numerous other proteins that provide structural sup- port or assist in ring assembly. Assembly of the contractile ring results in part from the local formation of new actin flaments, which depends on formin proteins that Contractile ring involving actin and myosin motors divides the cell in two

Alberts et al., Molecular Biology of the Cell Segregatedactin and Chromosomes myosin8 filaments ofAre the Packaged in Daughter Nuclei at Figure 17–40 The two processes of contractile ring anaphase in mammalian cells. Separated (A)Telophase sister chromatids move toward the poles in anaphase A. In anaphase B, the two By the end of anaphase, the daughter chromosomes have segregated into two spindle poles move apart. equal groups at opposite ends of the cell. In telophase, the fnal stage of mitosis, the two sets of chromosomes are packaged into a pair of daughter nuclei. Te frst major event of telophase is the disassembly of the mitotic spindle, followed by the re-formation of the nuclear envelope. Initially,(B) nuclear membrane fragments (C) associate with the surface of individual chromosomes. Tese membrane frag- MBoC6 m17.46/17.40 200 µm 25 µm ments fuse to partly enclose clusters of chromosomes and then coalesce to re- form the complete nuclear envelope. Nuclear pore complexes are incorporated Figureinto 17–41 the Cytokinesis. envelope, the (A nuclear) The actin–myosin lamina re-forms, bundles and of the the contractile envelope oncering are again oriented as shown, so that their contraction pulls the membrane inward.becomes (B) In this continuous low-magnification with the scanning endoplasmic electron reticulum. micrograph Once of thea cleaving nuclear frog enve egg,- the cleavage furrow is especially prominent, as the cell is unusuallylope has large. re-formed, The furrowing the pore of the complexes cell membrane pump inis causednuclear byproteins, the activity the nucleusof the contractile ring underneath it. (C) The surface of a furrow at higher expands,magnification. and the(B and mitotic C, from chromosomes H.W. Beams are and reorganized R.G. Kessel, into Am. their Sci. interphase 64:279–290, 1976. With permission from Sigma Xi.) 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 two. MBoC6 m17.49/17.41 We saw earlier that phosphorylation of various proteins by M-Cdk promotes spindle assembly, chromosome condensation, and nuclear-envelope breakdown in early mitosis. It is thus not surprising that the dephosphorylation of these same proteins is required for spindle disassembly and the re-formation of daughter nuclei in telophase. In principle, these dephosphorylations and the completion of mitosis could be triggered by the inactivation of Cdks, the activation of phos- phatases, or both. Although Cdk inactivation—resulting primarily from cyclin destruction—is mainly responsible in most cells, some cells also rely on activa- tion of phosphatases. In budding yeast, for example, the completion of mitosis depends on the activation of a phosphatase called Cdc14, which dephosphory- lates a subset of Cdk substrates involved in anaphase and telophase.

Summary M-Cdk triggers the events of early mitosis, including chromosome condensation, assembly of the mitotic spindle, and bipolar attachment of the sister-chromatid pairs to microtubules of the spindle. Spindle formation in animal cells depends largely on the ability of mitotic chromosomes to stimulate local microtubule nucle- ation and stability, as well as on the ability of motor proteins to organize micro- tubules into a bipolar array. Many cells also use centrosomes to facilitate spindle assembly. Anaphase is triggered by the APC/C, which stimulates the destruction of Current Biology 1980

Current Biology 1980 Spindle length is similar across the cells

Spindles in Drosophila cells

10µm cell nuclei microtubules Figure 1. High-Throughput, Semiautomated Measurement of Metaphase Spindle Length of Drosophila S2 Cells spindle poles (A) A raw image taken by the automated ImageXpress microscope with a 403 objective. Control GFP-tubulin cells were stained with g-tubulin (red) and chromosomes (blue). GFP is shown in green. (B) Enhanced signals of g-tubulin and chromosomes (see Supplemental Experimental Procedures). Two metaphase cells and one anaphase cell G. Goshimawere et detectedal., Current by the existence of two g-tubulin signals and chromosome masses in between. Most of the interphase cells were not stained by Biol. 15, 1979-1988g-tubulin antibody. (2005) (C) A gallery of metaphase cells. After9 automated detection of g-tubulin-stained cells, all the nonmetaphase cells were eliminated by manual selection. Cell shape was estimated by the boundary of the diffuse GFP signals in the cytoplasm (white circle), and GFP expression level was quantified as the signal intensity within the area. The bar represents 10 mm. (D)DistributionofmetaphasespindlelengthofcontrolGFP-tubulincells.Datafrom703mitoticcellswerecombined.Averagelength(11.5 6 2.0 mm) was well reproduced in each experiment (see Table S1). (E) Spindle length was plotted against GFP intensity. No statistically significant correlation was found between GFP level and spindle length, in- dicating that GFP-tubulin expression itself did not perturb the spindle. Figure 1. High-Throughput, Semiautomated Measurement of Metaphase Spindle Length of Drosophila S2 Cells (A) A raw image taken by the automated ImageXpress microscope with a 403 objective. Control GFP-tubulin cells were stained with g-tubulin (red) and chromosomes (blue). GFP is shown in green. exerted primarily at kinetochores [17, 21–24]. Klp10A for control GFP-tubulin-expressing cells [3]. Cells were (B) Enhanced signals of g-tubulin and chromosomes (see Supplemental Experimentaland EB1, Procedures on the). other Two metaphase hand, are cells concentrated and one anaphase at the cell cen- plated onto concanavalin-A-coated, 96-well glass-bot- were detected by the existence of two g-tubulin signals and chromosome masses in between. Most of the interphase cells were not stained by g-tubulin antibody. trosome, although both are likely to modulate MT assem- tom dishes and then fixed and stained for g-tubulin (as (C) A gallery of metaphase cells. After automated detection of g-tubulin-stainedbly throughout cells, all the nonmetaphase the mitotic cells cytoplasm were eliminated as well by manual[3, 4, 16]. a marker of centrosome locations) and chromosomes selection. Cell shape was estimated by the boundary of the diffuse GFP signalsTo in interfere the cytoplasm with (white sister-chromosome circle), and GFP expression tension, level was we de- (Figure 1A). For the majority of our experiments, images quantified as the signal intensity within the area. The bar represents 10 mm. pleted the critical sister-chromatid-cohesion protein, were collected with a high-throughput automated mi- (D)DistributionofmetaphasespindlelengthofcontrolGFP-tubulincells.Datafrom703mitoticcellswerecombined.Averagelength(11.5 6 2.0 mm) was well reproduced in each experiment (see Table S1). Rad21 (cohesin) [15]. Our results show that modulators croscope, and a semiautomated image-analysis proce- (E) Spindle length was plotted against GFP intensity. No statistically significantof correlation MT dynamics was found and between chromatid GFP level cohesion and spindle are length, the in- major dure was used to identify the rare mitotic cells in the im- dicating that GFP-tubulin expression itself did not perturb the spindle. governors of spindle length. In contrast, bipolar spindle ages. This algorithm, which took advantage of the fact length is robust to changes in MT sliding forces pro- that most interphase S2 cells do not exhibit punctuate exerted primarily at kinetochores [17, 21–24]. Klp10A forduced control by GFP-tubulin-expressing the Kinesin-5. We produce cells [3]. a Cells mathematical were signals of g-tubulin [3], recognizes g-tubulin spots and and EB1, on the other hand, are concentrated at the cen- platedmodel onto that concanavalin-A-coated, provides a framework 96-well for glass-bot- understanding identifies a cell as mitotic if it contains a pair of g-tubulin trosome, although both are likely to modulate MT assem- tomthese dishes observations. and then fixed and stained for g-tubulin (as spots with a DAPI-staining chromosomal mass in be- bly throughout the mitotic cytoplasm as well [3, 4, 16]. a marker of centrosome locations) and chromosomes tween (Figure 1B). During this process, g-tubulin spot– To interfere with sister-chromosome tension, we de- (FigureResults 1A). and For Discussion the majority of our experiments, images spot distances were measured automatically. Potential pleted the critical sister-chromatid-cohesion protein, were collected with a high-throughput automated mi- metaphase cells were then displayed into visual galler- Rad21 (cohesin) [15]. Our results show that modulators croscope, and a semiautomated image-analysis proce- Mitotic cells are relatively rare in the S2-cell population ies, and a manual selection was performed to eliminate of MT dynamics and chromatid cohesion are the major dure was used to identify the rare mitotic cells in the im- (1%–3%). Therefore, we required a method to obtain prophase, anaphase, and aberrant interphase cells as governors of spindle length. In contrast, bipolar spindle ages. This algorithm, which took advantage of the fact length is robust to changes in MT sliding forces pro- thatimages most of interphase sufficient S2 numbers cells do not of exhibit mitotic punctuate spindles for well as metaphase cells with multipolar spindles, which duced by the Kinesin-5. We produce a mathematical signalsquantitation of g-tubulin and statistical[3], recognizes comparisong-tubulin among spots and different had been also selected by the automated selection pro- model that provides a framework for understanding identifiesRNAi treatments. a cell as mitotic In most if it contains cases, we a pair employed of g-tubulin a high- cess (see Figure S1 for an example of manual selection). these observations. spotsthroughput with a DAPI-stainingprocedure of imagechromosomal collection mass and in analysis, be- The average distribution of the spindle length (herein de- tweenas summarized (Figure 1B). below During and this in process,Figureg 1-tubulin(and Figure spot– S1 in finedascentrosome-to-centrosomedistance)in703con- Results and Discussion spotthe Supplemental distances were Data measuredavailable automatically. with this articlePotential online) trol cells from several experiments was 11.5 6 2.0 mm metaphase cells were then displayed into visual galler- Mitotic cells are relatively rare in the S2-cell population ies, and a manual selection was performed to eliminate (1%–3%). Therefore, we required a method to obtain prophase, anaphase, and aberrant interphase cells as images of sufficient numbers of mitotic spindles for well as metaphase cells with multipolar spindles, which quantitation and statistical comparison among different had been also selected by the automated selection pro- RNAi treatments. In most cases, we employed a high- cess (see Figure S1 for an example of manual selection). throughput procedure of image collection and analysis, The average distribution of the spindle length (herein de- as summarized below and in Figure 1 (and Figure S1 in finedascentrosome-to-centrosomedistance)in703con- the Supplemental Data available with this article online) trol cells from several experiments was 11.5 6 2.0 mm Length Control of the Metaphase Spindle How various1981 factors affect the spindle length?

Certain factors are removed with RNA interference

Figure 2. Metaphase Spindle Length after RNAi (A) Average length of metaphase spindle after single or double RNAi-knockdown of eight proteins. RNAi treatment of each gene was repeated at least once, and in each experi- ment, relative average length to the accom- panying control was obtained. Combined da- taset of all the treatments is described in this graph. Error bars represent the 95% con- fident interval. See Table S1 for individual datasets. (B) Representative spindle images after RNAi of indicated genes. These images were taken manually with a 633 oil immersion objective. The bar represents 10 mm.

(Figure 1D). This length distribution was broader than separation from the kMT for EB1 RNAi is small, only ac- G. Goshimathose et al. observed, Current in yeast or fly embryos [6, 10]. However, counting for a 0.2 mm increase to the measurement of using a sufficient sample, we obtained consistent aver- spindle length [26]. In the case of Klp67A, the centro- Biol. 15, 1979-1988age metaphase (2005) spindle length among experiments some is detached, but is not always localized along 10 (10.95–11.84 mm; Table S1). By plotting spindle length the axis. Indeed, by comparing measurements of cen- versus GFP intensity for individual cells, we also estab- trosome-to-centrosome distance to the distances of lished that the spindle length was independent of GFP- the minus ends of the kMT in 32 bipolar spindles, we find tubulin expression levels (Figure 1E). that the centrosome-to-centrosome distance in Klp67A Next, we applied the same procedure to examine RNAi cells underestimates spindle length by 0.3 mm metaphase spindle length in cells depleted of various compared with control cells. Nevertheless, these effects proteins by RNAi (Figure 2; see also Table S1 for statis- are small and do not affect our conclusions that EB1 and tical summary). One general caveat of RNAi approach is Klp67A RNAi treatments shorten and lengthen spindle that the targeted proteins are not completely depleted length, respectively. and that the observed phenotypes (especially ‘‘no phe- If spindle length is controlled by a balance of MT poly- notype’’) could be still ‘‘hypomorphic’’ as a result of merization and depolymerization, we reasoned that a the residual proteins. Although this is also the case of phenotype generated by depletion of the MT-depolymer- our study, we confirmed significant protein-level reduc- izing protein Klp67A might be rescued by depletion of tion for most of the genes (Figure S2A) and could ob- MT-polymerizing proteins (e.g., Msps or Mast). In accor- serve specific spindle/chromosome phenotypes (Figure dance with this idea, the simultaneous knockdown of 2B). First, we found that reduction of Rad21, a protein Klp67A and Msps or Mast produced an average length essential for sister-chromatid cohesion, led to longer that was intermediate between single Klp67A and single spindles, as documented in yeast [10]. After Rad21 Msps or Mast knockdowns (Figure 2A). RNAi, anti-Cid staining (an inner-kinetochore marker) re- The important role played by MT-polymerization dy- vealed no paired sister-kinetochore dots, and thin chro- namics in determining spindle length was also discov- mosomal masses were scattered, strongly suggesting ered recently in Xenopus egg extracts by Mitchison and the precocious separation of sister chromatids (Figure colleagues, who also argued that an unidentified non- S4A) [15]. Statistically significant effects also were ob- MT tensile element, possibly a ‘‘spindle matrix’’ [27], served for regulators of MT dynamics. In agreement with may constrain spindle length [28]. Although we cannot previous qualitative descriptions [3, 4, 16, 17, 25], RNAi exclude the existence of such an element, our results of the MT stabilizers EB1, Msps, and Mast caused short- have not uncovered evidence for its existence. For ex- ening of metaphase spindle (61%–83%; all p values < ample, shortening of MT length by EB1 or Msps RNAi 0.0007), whereas knockdowns of MT depolymerases produced short metaphase spindles without signifi- (Klp10A and Klp67A) caused expansion (125%–147%, cantly perturbing its shape (Figure 2B). all p values < 0.004). Notably, the outer-kinetochore- Previous studies have shown that the inhibition of a enriched regulators of MT plus ends (Klp67A, Mast) minus-end-directed kinesin (Kinesin-14) causes spindle affected the length more severely than the global or elongation in yeast [5, 8]. Dynein inhibition causes dras- centrosome-localized MT regulators EB1, Msps, and tic spindle elongation or shortening depending upon the Klp10A. Klp67A [3] and EB1 [16] RNAi sometimes causes cell type [6, 9]. However, RNAi of Ncd (Kinesin 14) or centrosome detachment from the kinetochore microtu- Dhc64C (cytoplasmic DHC) only slightly changed the bules (kMTs), and this detachment could skew our mea- spindle size in S2 cells (3%–17% increase; Figure 2A sured centrosome-to-centrosome distance from the ac- and Table S1). The slight increase after dynein knock- tual spindle length. However, the degree of centrosome down can be explained by the effect of detachment of Current Biology 1982

centrosomes from spindle poles [29, 30] because we a measurable parameter. Collectively, these quantitative recently found in another study that detachment of the comparisons of spindle length following RNAi-mediated centrosomes from the minus end of kMT alone accounts protein depletion indicate that MT polymer dynamics for an average 14% increase in the centrosome-to- and sister-chromatid cohesion more dramatically affect centrosomedistance[26].Thespindle-lengthincreaseaf- metaphase spindle length than depletion of minus-end- ter dynein RNAi is less than that observed by Morales- directed motor proteins. Mulia and Scholey [31], for reasons that are not clear. We next examined whether increasing the levels of MT However, these authors also noted a partial misrecruit- sliding motors (Klp61F, Ncd) or of MT depolymerases ment of the Klp10A depolymerase to the poles, a misre- (Klp10A, Klp67A) influences spindle length (Figure 3). cruitment that may have contributed to this effect. The To correlate expression level and spindle length on a lack of dramatic effect upon Ncd RNAi is unlikely to be cell-to-cell basis, we expressed GFP-tagged kinesins due to residual proteins because we noted splaying of from an inducible promoter (Figure 3B) and measured kinetochore fibers (a characteristic of Ncd RNAi knock- GFP levels and spindle lengths for individual cells. In down [3, 26]) in the majority of the mitotic spindles exam- support of our assumption that the exogenous fusion ined (80%), found significant depletion (>90%) of Ncd at proteins are physiologically functional, we observed the population level by immunoblot analysis, and found that moderate expression of these kinesin-GFP fusions that 33 of 36 mitotic spindles scored had no detectable rescued the loss-of-function spindle phenotypes pro- immunofluorescence signal for Ncd after Ncd RNAi. duced by depletion of the corresponding endogenous However, the slight increase of the spindle length might kinesin by RNAi, and this rescue suggests that GFP tag- be explained by the reduced spindle elasticity upon un- ging did not destroy the kinesin’s function [22]. More- focusing of kinetochore fibers (discussed in Box 1). De- over, overexpressed Klp67A-GFP localized primarily to pletion of Klp61F (Kinesin-5) by RNAi produced monop- kinetochores and kinetochore MTs (kMTs), and over- olar spindles [3], so the length of bipolar spindles was not expressed Ncd-GFP and Klp61F-GFP localized primarily

Box 1. Mathematical Force-Balance Model of Metaphase Spindle-Length Control

Here, we outline a simple dynamical force-balance 1. Fsliding (green): The Kinesin-5-dependent force model of the determinants of S2-cell metaphase that slides apart antiparallel MTs (e.g., be- spindle length, a model that is a modification of an tween antiparallel interpolar (ip) MTs or be- earlier quantitative model that explains spindle-pole tween ipMT and kMT [B2]) and acts to separate dynamics during anaphase B [B1]. As described be- centrosomes and also on chromosomes low, an important aspect of this model is that Kinesin- through kMTs. 5-driven MT sliding activity is coupled to MT depoly- 2. Ftension (purple): Spindle elasticity, an assumed merization, and this coupling guarantees stability of restoring force that behaves as a Hookean the steady-state solution and allows robustness of spring and opposes spindle extension (possi- spindle stability to changes in MT dynamics. bly MT elasticity, MT crosslinking, or a ‘‘spindle We first define three state variables that are impor- matrix’’). tant for tracking spindle dynamics: spindle lengths 3. Fkt (yellow): A force (in addition to Fsliding) that (S), length of overlapping region of antiparallel MTs pulls the kinetochore toward the pole and pulls (L), and MT sliding velocity (Vsliding)(Figure A, left). centrosomes inward (Figure B) and that could In Figure A (right), we describe the total force acting be due to Hill sleeve mechanism [B3], minus- directly on the centrosome. Outward force, Fsliding, is end-directed motors at the kinetochore, opposed by Ftension and Fkt. or MT depolymerization at the pole. In addition, we define the following forces (1–3 be- 4. Vpoly: The rate of MT polymerization at the plus low) and parametersModel for of microtubule spindle polymerization length control ends of antiparallel ipMTs as determined by (4–5) relevant to the metaphase spindle (Figure A): dynamic instability of MTs and as a function

Figure A. Schematic Representation of the Forces Acting on the Meta- phase Spindle

Continued on following page

G. Goshima et al., Current Biol. 15, 1979-1988 (2005) 11 Current Biology 1982

centrosomes from spindle poles [29, 30] because we a measurable parameter. Collectively, these quantitative recently found in another study that detachment of the comparisons of spindle length following RNAi-mediated centrosomes from the minus end of kMT alone accounts protein depletion indicate that MT polymer dynamics for an average 14% increase in the centrosome-to- and sister-chromatid cohesion more dramatically affect centrosomedistance[26].Thespindle-lengthincreaseaf- metaphase spindle length than depletion of minus-end- ter dynein RNAi is less than that observed by Morales- directed motor proteins. Mulia and Scholey [31], for reasons that are not clear. We next examined whether increasing the levels of MT However, these authors also noted a partial misrecruit- sliding motors (Klp61F, Ncd) or of MT depolymerases ment of the Klp10A depolymerase to the poles, a misre- (Klp10A, Klp67A) influences spindle length (Figure 3). cruitment that may have contributed to this effect. The To correlate expression level and spindle length on a lack of dramatic effect upon Ncd RNAi is unlikely to be cell-to-cell basis, we expressed GFP-tagged kinesins due to residual proteins because we noted splaying of from an inducible promoter (Figure 3B) and measured kinetochore fibers (a characteristic of Ncd RNAi knock- GFP levels and spindle lengths for individual cells. In down [3, 26]) in the majority of the mitotic spindles exam- support of our assumption that the exogenous fusion ined (80%), found significant depletion (>90%) of Ncd at proteins are physiologically functional, we observed the population level by immunoblot analysis, and found that moderate expression of these kinesin-GFP fusions that 33 of 36 mitotic spindles scored had no detectable rescued the loss-of-function spindle phenotypes pro- immunofluorescence signal for Ncd after Ncd RNAi. duced by depletion of the corresponding endogenous However, the slight increase of the spindle length might kinesin by RNAi, and this rescue suggests that GFP tag- be explained by the reduced spindle elasticity upon un- ging did not destroy the kinesin’s function [22]. More- focusing of kinetochore fibers (discussed in Box 1). De- over, overexpressed Klp67A-GFP localized primarily to pletion of Klp61F (Kinesin-5) by RNAi produced monop- kinetochores and kinetochore MTs (kMTs), and over- olar spindles [3], so the length of bipolar spindles was not expressed Ncd-GFP and Klp61F-GFP localized primarily

Box 1. Mathematical Force-Balance Model of Metaphase Spindle-Length Control

Here, we outline a simple dynamical force-balance 1. Fsliding (green): The Kinesin-5-dependent force model of the determinants of S2-cell metaphase that slides apart antiparallel MTs (e.g., be- spindle length, a model that is a modification of an tween antiparallel interpolar (ip) MTs or be- earlier quantitative model that explains spindle-pole tween ipMT and kMT [B2]) and acts to separate dynamics during anaphase B [B1]. As described be- centrosomes and also on chromosomes low, an important aspect of this model is that Kinesin- through kMTs. 5-driven MT sliding activity is coupled to MT depoly- 2. Ftension (purple): Spindle elasticity, an assumed merization, and this coupling guarantees stability of restoring force that behaves as a Hookean the steady-state solution and allows robustness of spring and opposes spindle extension (possi- spindle stability to changes in MT dynamics. bly MT elasticity, MT crosslinking, or a ‘‘spindle We first define three state variables that are impor- matrix’’). tant for tracking spindle dynamics: spindle lengths 3. Fkt (yellow): A force (in addition to Fsliding) that (S), length of overlapping region of antiparallel MTs pulls the kinetochore toward the pole and pulls (L), and MT sliding velocity (Vsliding)(Figure A, left). centrosomes inward (Figure B) and that could In Figure A (right), we describe the total force acting be due to Hill sleeve mechanism [B3], minus- directly on the centrosome. Outward force, Fsliding, is end-directed motors at the kinetochore, opposed by Ftension and Fkt. or MT depolymerization at the pole. In addition, we define the following forces (1–3 be- 4. Vpoly: The rate of MT polymerization at the plus low) and parameters of microtubule polymerization ends of antiparallel ipMTs as determined by (4–5) relevant to the metaphase spindle (Figure A): dynamic instability of MTs and as a function Sliding force pushes centrosomes apart

Figure A. Schematic Representation G. Goshima et al., Current of the Forces Acting on the Meta- Biol. 15, 1979-1988 (2005) phase Spindle

letters to nature

v a halt movementContinued almost entirely. on following These results are page consistent with a sliding 60 role for load in lowering the effective rate of ATP binding. At Fsliding = ↵L 1 2 mM ATP (max) 800 limiting ATP, kb becomes much lower than kcat, and therefore the v ! 50 5 µM ATP stall force becomes independent of the latter. However, at 2 mM sliding M ATP ATP, the stall force probably depends on both k and k , as K rises µ 40 600 b cat m beyond ,300 mM at loads of 7 pN or more. Although the stall force ), 5 ), 2 mM ATP

–1 30 –1 may depend on additional factors at all ATP levels, stall forces are 400 expected to rise continuously from ,5.5 pN to ,7.0 pN as the ATP 20 concentration is raised. 200 To further test this supposition, we measured the ATP depen- ↵ proportional to density 10 dence of kinesin stall force by two additional methods, using either 17 Velocity (nm s Velocity (nm s Velocity an optical position clamp or a ®xed trap. The position clamp , 0 0 of sliding motors 01234567 based on an optical trapping interferometer, is well suited to Load (pN) isometric measurements of stall force16, and was used previously b to study force generation by individual RNA polymerase molecules18. Position clamp measurements on single kinesin 8 K. Visscher et al., Nature 400, 184-189 (1999) motors gave stall forces that rose with ATP concentration, as 12 anticipated (Fig. 3b). An inherent problem encountered in these 7 experiments was the dramatic decline in kinesin processivity at loads approaching stall12. Rapid detachment of beads from the microtubule made the collection of a large data set prohibitive, 6 and accounted for much of the experimental scatter (Fig. 3b). To

Stall force (pN) Stall force con®rm the position clamp measurements, we also measured stall Position clamp forces using a ®xed optical trap. Kinesin-coated beads moving 5 Fixed trap outwards in a stationary trap experience increasing loads until they arrive at a position where forward progress ceases: the ®nal 10 100 1,000 displacement, multiplied by the trap stiffness, supplies a measure of ATP concentration (µM) the stall force12. Kinesin stall forces determined by this method exhibit a similar increase with ATP concentration (Fig. 3b). Figure 3 Load dependence of motility. a, Average bead velocity, v How is the kinesin ATPase pathway coupled to mechanical (mean 6 s:e:m:), versus applied load for ®xed ATP concentrations (red triangles, movement under load? One possibility is that kinesin coupling is left axis, 5 mM ATP, N ˆ 19±57; blue circles, right axis, 2 mM ATP, N ˆ 37±87). The tight, involving no futile hydrolysis events that fail to generate velocity point at (5.6 pN, 5 mM ATP) is likely to represent an overestimate because movement, even under load (that is, e ˆ 1). In this case, any changes beads which stalled completely (v ˆ 0) were indistinguishable from beads lack- in the rates of load-dependent transitions must account entirely for ing active motors, and so were not included in the analysis. b, Stall force the observed decreases in velocity under load. However, more (mean 6 s:e:m:) versus ATP concentration, measured either with the position complicated scenarios may be entertained. Although the rise in 17,18 clamp (red triangles) or with a ®xed optical trap (blue circles). Stalls had to last a Km with load eliminates the simpler versions of loose coupling, minimum of 2 s to be included in the analysis. Data points represent an average of mechanochemical coupling in kinesin might display an admixture either 12±29 (position clamp) or 6±70 (®xed trap) stalls. of both tight and loose elements, with load-dependent transitions and unproductive hydrolyses conspiring jointly to lower the velo- city. Another candidate mechanism for loose coupling involves an traps10±12 or microneedles13, all of which reported nearly linear increase in the frequency of 8-nm backwards steps10±12 with load. decreases in velocity with increased load, for both limiting and However, the latter mechanism may be discounted because the saturating ATP concentrations. In fact, such linear force±velocity fraction of backwards steps found in this study (data not shown) relations formed the basis of an argument for a load-independent and by others remains only ,5±10% at both negligible8,11 and 10 11,12 Km (refs 10, 19) which supported a simple, loosely coupled picture . substantial loads. However, the earlier studies lacked the improved resolution To study the coupling mechanism under load, we performed a afforded by the force clamp, and subtle differences in shape were ¯uctuation analysis23,24 of kinesin movement subjected to the force not distinguished within experimental error. Data from all previous clamp. Fluctuation analysis, which relies on the processive property studies consistently showed a lower stall force at limiting ATP than exhibited by some molecular motors, has been used previously to at saturating ATP,although the difference in force was insuf®cient in study ATP coupling in kinesin near zero load8, as well as the duty any given experiment to have been considered signi®cant. More- ratio of the bacterial rotary motor under torque25. Fluctuations in over, a greater discrepancy (,1 pN) between the stall force at displacement are quanti®ed by a randomness parameter, r, which limiting and saturating ATP was observed when the limiting measures the temporal irregularity of mechanical advances. For an concentration of ATP was ,35-fold lower than the unloaded Km ensemble of displacement records, x(t), r is de®ned by (ref. 13), as compared to when limiting ATP values were only ,6- x2 t† 2 x t† 2 fold lower (,0.5 pN, refs 10±12). We therefore measured velocity as r ˆ lim h i h i t ` d x t† a function of load in the force clamp at both 2 mM and 5 mM ATP, ! h i choosing the lower ATP level to be .17-fold below the unloaded Km where d is the motor step size, and the angle brackets denote an to highlight any potential differences. ensemble average23,24. A perfectly clocklike motor displays no step- Force±velocity curves obtained with the force clamp displayed ping irregularity, and has r ˆ 0, whereas a `Poisson' motor with one different shapes at limiting and saturating ATP levels (Fig. 3a), and biochemical transition moves at exponentially distributed intervals, they also showed different stall forces. The 5-mM curve is nearly and has r ˆ 1. In general, r-1 provides a continuous measure of the linear, with a stall force of ,5.5 pN. In contrast, the 2-mM curve is number of rate-limiting transitions in the overall mechanochemical concave down, and supports a greater stall force of ,7.0 pN. cycle. Determinations of randomness are insensitive to thermal or Strikingly, a 5-pN load leads to a ,40% decline in speed for other stationary noise and can therefore be performed even when 2 mM ATP, whereas at 5 mM ATP an identical load is suf®cient to individual steps are not resolved. When steps can be discerned amid

© 1999 Macmillan Magazines Ltd 186 NATURE | VOL 400 | 8 JULY 1999 | www.nature.com Current Biology 1982

centrosomes from spindle poles [29, 30] because we a measurable parameter. Collectively, these quantitative recently found in another study that detachment of the comparisons of spindle length following RNAi-mediated centrosomes from the minus end of kMT alone accounts protein depletion indicate that MT polymer dynamics for an average 14% increase in the centrosome-to- and sister-chromatid cohesion more dramatically affect centrosomedistance[26].Thespindle-lengthincreaseaf- metaphase spindle length than depletion of minus-end- ter dynein RNAi is less than that observed by Morales- directed motor proteins. Mulia and Scholey [31], for reasons that are not clear. We next examined whether increasing the levels of MT However, these authors also noted a partial misrecruit- sliding motors (Klp61F, Ncd) or of MT depolymerases ment of the Klp10A depolymerase to the poles, a misre- (Klp10A, Klp67A) influences spindle length (Figure 3). cruitment that may have contributed to this effect. The To correlate expression level and spindle length on a lack of dramatic effect upon Ncd RNAi is unlikely to be cell-to-cell basis, we expressed GFP-tagged kinesins due to residual proteins because we noted splaying of from an inducible promoter (Figure 3B) and measured kinetochore fibers (a characteristic of Ncd RNAi knock- GFP levels and spindle lengths for individual cells. In down [3, 26]) in the majority of the mitotic spindles exam- support of our assumption that the exogenous fusion ined (80%), found significant depletion (>90%) of Ncd at proteins are physiologically functional, we observed the population level by immunoblot analysis, and found that moderate expression of these kinesin-GFP fusions that 33 of 36 mitotic spindles scored had no detectable rescued the loss-of-function spindle phenotypes pro- immunofluorescence signal for Ncd after Ncd RNAi. duced by depletion of the corresponding endogenous However, the slight increase of the spindle length might kinesin by RNAi, and this rescue suggests that GFP tag- be explained by the reduced spindle elasticity upon un- ging did not destroy the kinesin’s function [22]. More- focusing of kinetochore fibers (discussed in Box 1). De- over, overexpressed Klp67A-GFP localized primarily to pletion of Klp61F (Kinesin-5) by RNAi produced monop- kinetochores and kinetochore MTs (kMTs), and over- olar spindles [3], so the length of bipolar spindles was not expressed Ncd-GFP and Klp61F-GFP localized primarily

Box 1. Mathematical Force-Balance Model of Metaphase Spindle-Length Control

Here, we outline a simple dynamical force-balance 1. Fsliding (green): The Kinesin-5-dependent force model of the determinants of S2-cell metaphase that slides apart antiparallel MTs (e.g., be- spindle length, a model that is a modification of an tween antiparallel interpolar (ip) MTs or be- earlier quantitative model that explains spindle-pole tween ipMT and kMT [B2]) and acts to separate dynamics during anaphase B [B1]. As described be- centrosomes and also on chromosomes low, an important aspect of this model is that Kinesin- through kMTs. 5-driven MT sliding activity is coupled to MT depoly- 2. Ftension (purple): Spindle elasticity, an assumed merization, and this coupling guarantees stability of restoring force that behaves as a Hookean the steady-state solution and allows robustness of spring and opposes spindle extension (possi- spindle stability to changes in MT dynamics. bly MT elasticity, MT crosslinking, or a ‘‘spindle We first define three state variables that are impor- matrix’’). tant for tracking spindle dynamics: spindle lengths 3. Fkt (yellow): A force (in addition to Fsliding) that (S), length of overlapping region of antiparallel MTs pulls the kinetochore toward the pole and pulls (L), and MT sliding velocity (Vsliding)(Figure A, left). centrosomes inward (Figure B) and that could In Figure A (right), we describe the total force acting be due to Hill sleeve mechanism [B3], minus- directly on the centrosome. Outward force, Fsliding, is end-directed motors at the kinetochore, opposed by Ftension and Fkt. or MT depolymerization at the pole. In addition, we define the following forces (1–3 be- 4. Vpoly: The rate of MT polymerization at the plus low) and parameters of microtubule polymerization ends of antiparallel ipMTs as determined by (4–5) relevant to the metaphase spindle (Figure A): dynamic instability of MTs and as a function Kinetochore pulls centrosome inwards

Figure A. Schematic Representation of the Forces Acting on the Meta- phase Spindle

constant tension force Continued on following page Fkt = Fkt,0 between chromosomes and centrosomes

G. Goshima et al., Current Biol. 15, 1979-1988 (2005) 13 Current Biology 1982

centrosomes from spindle poles [29, 30] because we a measurable parameter. Collectively, these quantitative recently found in another study that detachment of the comparisons of spindle length following RNAi-mediated centrosomes from the minus end of kMT alone accounts protein depletion indicate that MT polymer dynamics for an average 14% increase in the centrosome-to- and sister-chromatid cohesion more dramatically affect centrosomedistance[26].Thespindle-lengthincreaseaf- metaphase spindle length than depletion of minus-end- ter dynein RNAi is less than that observed by Morales- directed motor proteins. Mulia and Scholey [31], for reasons that are not clear. We next examined whether increasing the levels of MT However, these authors also noted a partial misrecruit- sliding motors (Klp61F, Ncd) or of MT depolymerases ment of the Klp10A depolymerase to the poles, a misre- (Klp10A, Klp67A) influences spindle length (Figure 3). cruitment that may have contributed to this effect. The To correlate expression level and spindle length on a lack of dramatic effect upon Ncd RNAi is unlikely to be cell-to-cell basis, we expressed GFP-tagged kinesins due to residual proteins because we noted splaying of from an inducible promoter (Figure 3B) and measured kinetochore fibers (a characteristic of Ncd RNAi knock- GFP levels and spindle lengths for individual cells. In down [3, 26]) in the majority of the mitotic spindles exam- support of our assumption that the exogenous fusion ined (80%), found significant depletion (>90%) of Ncd at proteins are physiologically functional, we observed the population level by immunoblot analysis, and found that moderate expression of these kinesin-GFP fusions that 33 of 36 mitotic spindles scored had no detectable rescued the loss-of-function spindle phenotypes pro- immunofluorescence signal for Ncd after Ncd RNAi. duced by depletion of the corresponding endogenous However, the slight increase of the spindle length might kinesin by RNAi, and this rescue suggests that GFP tag- be explained by the reduced spindle elasticity upon un- ging did not destroy the kinesin’s function [22]. More- focusing of kinetochore fibers (discussed in Box 1). De- over, overexpressed Klp67A-GFP localized primarily to pletion of Klp61F (Kinesin-5) by RNAi produced monop- kinetochores and kinetochore MTs (kMTs), and over- olar spindles [3], so the length of bipolar spindles was not expressed Ncd-GFP and Klp61F-GFP localized primarily

Box 1. Mathematical Force-Balance Model of Metaphase Spindle-Length Control

Here, we outline a simple dynamical force-balance 1. Fsliding (green): The Kinesin-5-dependent force model of the determinants of S2-cell metaphase that slides apart antiparallel MTs (e.g., be- spindle length, a model that is a modification of an tween antiparallel interpolar (ip) MTs or be- earlier quantitative model that explains spindle-pole tween ipMT and kMT [B2]) and acts to separate dynamics during anaphase B [B1]. As described be- centrosomes and also on chromosomes low, an important aspect of this model is that Kinesin- through kMTs. 5-driven MT sliding activity is coupled to MT depoly- 2. Ftension (purple): Spindle elasticity, an assumed merization, and this coupling guarantees stability of restoring force that behaves as a Hookean the steady-state solution and allows robustness of spring and opposes spindle extension (possi- spindle stability to changes in MT dynamics. bly MT elasticity, MT crosslinking, or a ‘‘spindle We first define three state variables that are impor- matrix’’). tant for tracking spindle dynamics: spindle lengths 3. Fkt (yellow): A force (in addition to Fsliding) that (S), length of overlapping region of antiparallel MTs pulls the kinetochore toward the pole and pulls (L), and MT sliding velocity (Vsliding)(Figure A, left). centrosomes inward (Figure B) and that could In Figure A (right), we describe the total force acting be due to Hill sleeve mechanism [B3], minus- directly on the centrosome. Outward force, Fsliding, is end-directed motors at the kinetochore, opposed by Ftension and Fkt. or MT depolymerization at the pole. In addition, we define the following forces (1–3 be- 4. Vpoly: The rate of MT polymerization at the plus low) and parameters of microtubule polymerization ends of antiparallel ipMTs as determined by (4–5)Restoring relevant to the metaphasespring spindle forces (Figure try A): to keepdynamic instability of MTs and as a function spindle at rest length S0

Figure A. Schematic Representation of the Forces Acting on the Meta- phase Spindle

S0 Continued on following page

F = (S S ) tension 0

G. Goshima et al., Current Biol. 15, 1979-1988 (2005) 14 Current Biology 1982

centrosomes from spindle poles [29, 30] because we a measurable parameter. Collectively, these quantitative recently found in another study that detachment of the comparisons of spindle length following RNAi-mediated centrosomes from the minus end of kMT alone accounts protein depletion indicate that MT polymer dynamics for an average 14% increase in the centrosome-to- and sister-chromatid cohesion more dramatically affect centrosomedistance[26].Thespindle-lengthincreaseaf- metaphase spindle length than depletion of minus-end- ter dynein RNAi is less than that observed by Morales- directed motor proteins. Mulia and Scholey [31], for reasons that are not clear. We next examined whether increasing the levels of MT However, these authors also noted a partial misrecruit- sliding motors (Klp61F, Ncd) or of MT depolymerases ment of the Klp10A depolymerase to the poles, a misre- (Klp10A, Klp67A) influences spindle length (Figure 3). cruitment that may have contributed to this effect. The To correlate expression level and spindle length on a lack of dramatic effect upon Ncd RNAi is unlikely to be cell-to-cell basis, we expressed GFP-tagged kinesins due to residual proteins because we noted splaying of from an inducible promoter (Figure 3B) and measured kinetochore fibers (a characteristic of Ncd RNAi knock- GFP levels and spindle lengths for individual cells. In down [3, 26]) in the majority of the mitotic spindles exam- support of our assumption that the exogenous fusion ined (80%), found significant depletion (>90%) of Ncd at proteins are physiologically functional, we observed the population level by immunoblot analysis, and found that moderate expression of these kinesin-GFP fusions that 33 of 36 mitotic spindles scored had no detectable rescued the loss-of-function spindle phenotypes pro- immunofluorescence signal for Ncd after Ncd RNAi. duced by depletion of the corresponding endogenous However, the slight increase of the spindle length might kinesin by RNAi, and this rescue suggests that GFP tag- be explained by the reduced spindle elasticity upon un- ging did not destroy the kinesin’s function [22]. More- focusing of kinetochore fibers (discussed in Box 1). De- over, overexpressed Klp67A-GFP localized primarily to pletion of Klp61F (Kinesin-5) by RNAi produced monop- kinetochores and kinetochore MTs (kMTs), and over- olar spindles [3], so the length of bipolar spindles was not expressed Ncd-GFP and Klp61F-GFP localized primarily

Box 1. Mathematical Force-Balance Model of Metaphase Spindle-Length Control

Here, we outline a simple dynamical force-balance 1. Fsliding (green): The Kinesin-5-dependent force model of the determinants of S2-cell metaphase that slides apart antiparallel MTs (e.g., be- spindle length, a model that is a modification of an tween antiparallel interpolar (ip) MTs or be- earlier quantitative model that explains spindle-pole tween ipMT and kMT [B2]) and acts to separate dynamics during anaphase B [B1]. As described be- centrosomes and also on chromosomes low, an important aspect of this model is that Kinesin- through kMTs. 5-driven MT sliding activity is coupled to MT depoly- 2. Ftension (purple): Spindle elasticity, an assumed merization, and this coupling guarantees stability of restoring force that behaves as a Hookean the steady-state solution and allows robustness of spring and opposes spindle extension (possi- spindle stability to changes in MT dynamics. bly MT elasticity, MT crosslinking, or a ‘‘spindle We first define three state variables that are impor- matrix’’). tant for tracking spindle dynamics: spindle lengths 3. Fkt (yellow): A force (in addition to Fsliding) that (S), length of overlapping region of antiparallel MTs pulls the kinetochore toward the pole and pulls (L), and MT sliding velocity (Vsliding)(Figure A, left). centrosomes inward (Figure B) and that could In Figure A (right), we describe the total force acting be due to Hill sleeve mechanism [B3], minus- directly on the centrosome. Outward force, Fsliding, is end-directed motors at the kinetochore, opposed by Ftension and Fkt. or MT depolymerization at the pole. In addition, we define the following forces (1–3 be- 4. Vpoly: The rate of MT polymerization at the plus low) and parameters of microtubule polymerization ends of antiparallel ipMTs as determined by (4–5) relevant to the metaphase spindle (Figure A): dynamic instability of MTs and as a function Model for spindle length control Figure A. Schematic Representation of the Forces Acting on the Meta- phase Spindle

v dL F = ↵L 1 sliding =2(vpoly vsliding) Continued on following page sliding (max) dt v ! sliding dS =2(v v ) Fkt = Fkt,0 dt sliding depoly F = (S S ) tension 0 dL dS dS 2(F F F ) + =2(v v ) = sliding kt tension dt dt poly depoly dt µ assuming viscous drag No steady state if rates of G. Goshima et al., Current microtubule polymerization and Biol. 15, 1979-1988 (2005) depolymerization are different! 15 Current Biology Vol 15 No 23 DepolymerizationR958 rate depends on the sliding force Figure 2. A hypothetical A below a threshold concentration of Vdepoly model for force-assembly Kinesin-5. Goshima et al. [2] Fsliding coupling at the pole. acknowledge that other coupling (A) Microtubule minus ends Pole assumptions might be considered embedded in the pole (dark blue) tend to depolymerize as well, and that further evaluation slowly when under low will be needed. For example, + – sliding force (Fsliding). That recent analysis of budding yeast depolymerization occurs at and Xenopus extract spindles B all may be because a suggest that tension on the depolymerase (red circles), Vdepoly kinetochore can trigger a bias Fsliding such as Klp10A (Kinesin- 13), acts at the pole to towards assembly, or attenuation

Pole increase the depolymeriza- of disassembly, at the plus ends tion velocity (Vdepoly) locally. [10,11]. Nevertheless, the model (B) If the sliding force is makes a surprising prediction + – increased, then the concen- about spindle stability that turns tration of depolymerase out to be true. Current Biology increases locally so that the depolymerization rate Spindle length regulation has (max) increases. In their0 report, Goshima et al. [2] areF slidingjustifiably/ nonspecific about the detailed been studied most extensively in vdepoly = vdep + vdep 1 e mechanism of coupling, focusing instead on its mathematical form and general impor- budding yeast [12]. As in the S2 tance in order to have a steady-state⇣ length at all. The cartoon⌘ here is meant for illus- cells, the spindle length in budding tration purposes only, depicting just one way in which the mathematical model of yeast is fairly constant in Goshima et al. [2] for force-assembly coupling could operate mechanistically. D.J. Odde, Current Biol. G. Goshima et al., Current metaphase [13]. But there is no 15, R956-R959 (2005) Biol. 15, 1979-1988 (2005) evidence for minus-end extended periods (minutes),16 significant increase in spindle disassembly, simplifying analysis whereas the viscoelastic relaxation length. Interestingly, perturbing the of the system [14]. Also, only four time is estimated to be on the concentration of Klp61F (Kinesin- motors enter the nucleus, Cin8p order of milliseconds, suggesting 5), which is thought to be the main (Kinesin-5), Kip1p (Kinesin-5), that the elastic forces dominate the force generator in the spindle, had Kar3p (Kinesin-14), and Kip3p force balance [6]. In general, very little effect on spindle length (Kinesin-8), and from deletion viscous drag forces are probably over a broad range of mutant analysis, it was deduced negligible, as a 1 µm radius sphere concentrations. Below a critical that Cin8p and Kip1p act as sliding (modelling a chromosome) moving concentration of Klp61F, however, motors to elongate the spindle, at 1 µm per minute experiences a the spindle completely collapsed while Kar3p acts antagonistically drag force of ~1 fN, about 1000- into a monopolar array of to shorten spindles [15]. The fold less than the stall force of a microtubules. budding yeast system also permits single molecular motor [7]. To explain this effect, Goshima rough estimation of the magnitude What Goshima et al. [2] have et al. [2] used a previously of the inward tensional force on now done is perform a perturbation published mathematical model for chromatin, which from analysis where the concentrations spindle length during anaphase, chromosome marking experiments of putative spindle length extending it to metaphase by is estimated in metaphase to be regulators are systematically including spindle elasticity, sufficient to force nucleosome decreased, through RNA kinetochore microtubules, and release [16,17]. From in vitro interference (RNAi), or increased, sister chromatid cohesion [8,9]. studies with laser tweezers, this through overexpression, and the Even with these effects included, suggests an average tensional effect on spindle length measured however, the model did not settle force exceeding 15 pN per quantitatively using an automated to a steady-state length, but chromosome [18]. Given that there microscope and image analysis rather grew to infinity. To are 16 chromosomes, then the system. Starting with an initial data establish a steady-state, the total inward tensional force is set of about one million cells, their authors then considered a estimated to be >240 pN, implying machine identified a ‘small’ subset ‘coupling assumption,’ where the an outward extensional force of that was mitotic, so that thousands Kinesin-5 sliding activity promotes >240 pN, neglecting net tension or of mitotic cells were analyzed. microtubule depolymerization. For compression exerted from outside They found that, in S2 cells, it was concreteness and illustration the spindle. the regulators of microtubule purposes only, one potential In the Xenopus extract system, assembly dynamics that had the scenario is sketched in Figure 2. the addition of monastrol, a drug strongest effect on spindle length. This coupling, where the that interferes with Eg5 (Kinesin-5) These regulators included the depolymerization rate depends function, was recently reported to assembly-promoting molecules exponentially on the sliding force, cause a ‘switch-like transition’ EB1, Msps (Dis1/XMAP215/TOG), saturating to a maximum rate at from bipolar to monopolar as a and Mast/Orbit (CLASP), and the large forces, enabled a steady- function of increasing monastrol disassembly-promoting molecules state spindle length to be concentration, with little effect on Klp10A (Kinesin-13) and Klp67A achieved. spindle length noted at sub- (Kinesin-8). Eliminating sister Interestingly, the model also threshold concentrations [19]. chromatid cohesion also led to a predicts the collapse of the spindle Interestingly, even though spindle Length Control of the Metaphase Spindle 1983

Current Biologyof ‘‘general’’ MT regulators (e.g., EB1 or mini- velocity of Kinesin-5. b is the Hookean-spring con- 1982 spindles). stant of spindle elasticity, and S0 is the spring’s rest 5. Vdepol: The rate of depolymerization of all MTs length. For simplicity, we assume a constant force at their minus ends, this may be mainly regu- on the kinetochore (Fkt,0). The assumption of linear centrosomeslated from spindle by Kinesin-13 poles [29, 30] (Klp10A)because we that actsa measurable as a MT parameter. Collectively,force-extension these quantitative forces for the spindle spring ele- recently found in another study that detachment of the comparisons of spindle length following RNAi-mediated centrosomesdepolymerase. from the minus end of kMT alone accounts protein depletion indicatements that MT polymerand of dynamics linear force-velocity curves for Kine- for anForces average 14% exerted increase by in astral the centrosome-to- MTs [B4–B6]andor sister-chromatid chromo- cohesionsin-5 more activity dramatically serve affect to simplify the system and ensure centrosomedistance[26].Thespindle-lengthincreaseaf- metaphase spindle length than depletion of minus-end- ter dyneinkinesins RNAi is[B7] less thanmay that also observed play by a Morales- role in spindle-lengthdirected motor proteins. the existence of analytical solutions. Muliadetermination, and Scholey [31], for but reasons they that were are not not clear. consideredWe next in examined this whether increasingFrom these the levels equations, of MT it is clear that in order to However, these authors also noted a partial misrecruit- sliding motors (Klp61F, Ncd) or of MT depolymerases mentstudy of the Klp10A for simplicity. depolymerase We to the do poles, not a misre- take into(Klp10A, account Klp67A) an influencesreach spindle a length stable (Figure steady 3). state in which spindle length cruitmenteventual that may limitation have contributed of the to size this effect. of the The spindleTo correlate (i.e., lim- expression leveland and overlap spindle length length on remain a constant with time (ds/dt = lackited of dramatic amount effect of upon tubulin Ncd RNAi or is other unlikely spindle to be componentscell-to-cell basis, we expressed0, dL/ GFP-taggeddt = 0), net kinesins polymerization and depolymeriza- due to residual proteins because we noted splaying of from an inducible promoter (Figure 3B) and measured kinetochoreor size fibers of the(a characteristic cell), although of Ncd RNAi these knock- factorsGFP may levels also and spindle lengthstion must for individual be equal cells. (InVpoly = Vdepol). The set of assump- downcome[3, 26]) ininto the majority play in of the living mitotic cells. spindles exam- support of our assumptiontions that the presented exogenous fusion above state that polymerization and ined (80%), found significant depletion (>90%) of Ncd at proteins are physiologically functional, we observed the populationThe microtubule level by immunoblot polymerization/depolymerization analysis, and found that moderate expressiondepolymerization of these kinesin-GFP fusions are constant parameters that natu- thatrates 33 of 36 can mitotic be spindles combined scored had with no detectable the rate ofrescued MT sliding, the loss-of-functionrally spindle produce phenotypes an unstable pro- steady state in which every immunofluorescence signal for Ncd after Ncd RNAi. duced by depletion of the corresponding endogenous However,Vsliding the, slight to form increase two of the simple spindle kinematic length might equations:kinesin by RNAi, and this rescueperturbation suggests that to GFP MT tag- polymerization or depolymeriza- be explained by the reduced spindle elasticity upon un- ging did not destroy the kinesin’stion will function result[22]. in More- deviations from the steady state. focusing of kinetochore fibersdS (discussed in Box 1). De- over, overexpressed Klp67A-GFP localized primarily to =2 Vsliding 2 Vdepol (1) However, our experiments showed that we can per- pletion of Klp61F (Kinesin-5)dt by RNAi produced monop- kinetochores and kinetochore MTs (kMTs), and over- olar spindles [3], so the length of bipolar spindles was not expressed Ncd-GFP and Klp61F-GFPturb only localized polymerization primarily ordepolymerizationby RNAi, ÿ Á yet the spindle still reaches a new steady state with dL =2 Vpoly 2 Vsliding (2) a different constant length (e.g., EB1 RNAi decreases Box 1. Mathematical Force-Balancedt Model of Metaphase Spindle-Length Control Vpoly but probably not Vdepol). ÿ Á Here,This we set outline of kinematic a simple dynamical equations force-balance should be coupled1. Fsliding (green): to The Kinesin-5-dependentTo generate force a stable steady state, we propose an model of the determinants of S2-cell metaphase that slides apart antiparalleladditional MTs assumption (e.g., be- that the depolymerization rate spindlea third length, force-balance a model that is equation, a modification which of an is basedtween on the antiparallel interpolar (ip) MTs or be- earlierlow Reynold’s quantitative model number that explains approximation spindle-pole that appliestween ipMT at and kMT([B2]Vdepol) and) acts is dependent to separate on the extent of the sliding force dynamics during anaphase B [B1]. As described be- centrosomes and also on chromosomes (Fsliding). Here, we proposed one plausible mechanism low,subcellular an important scalesaspect of this (force model for is that movement, Kinesin- F =through velocity kMTs. 5-drivenof movement, MT sliding activity dS/dt is coupled3 the to drag MT depoly- coefficient2. ofFtension sepa-(purple): Spindleto elasticity, explain an this assumed dependency on the basis of the as- merization, and this coupling guarantees stability of restoring force thatsumption behaves as aof Hookean a higher concentration of Klp10A at the therating steady-state the poles, solutionm and). allows robustness of spring and opposes spindle extension (possi- spindle stability to changes in MT dynamics. bly MT elasticity, MTcentrosomes crosslinking, or a ‘‘spindle than elsewhere, combined with a poly- We first definedS three state2 1 variables that are impor- matrix’’). mer ratchet theory [B8]. If a force of magnitude = m 2 Fsliding 2 Fkt 2 Ftension (3) tant for trackingdt spindle dynamics: spindle lengths 3. Fkt (yellow): A force (in addition to Fsliding) that (S), length of overlapping region of antiparallel MTs  pulls the kinetochoreF towardsliding thepushes pole and pulls MT toward the centrosome, then, as (L), and MT sliding velocity (Vsliding)(Figure A, left). centrosomes inwarda (Figure result B) and of thatBrownian could motion, the probability density In FigureThe A above(right), we forces describe can the total be force defined acting as follows:be due to Hill sleevefor mechanism the minus[B3], minus- end to be at distance x away from the directly on the centrosome. Outward force, Fsliding, is end-directed motors at the kinetochore, F x opposed by F and F . or MT depolymerization at the pole. sliding tension kt V 2 Nk T sliding centrosome is e b , where kbT is the thermal en- In addition, we defineFsliding the following= aL 1 forces2 (1–3 be- 4. Vpoly: The(4) rate of MT polymerization at the plus low) and parameters of microtubule polymerizationV ends of antiparallel ipMTs as determined by  sliding, max ergy [B9] and N is the number of antiparallel overlap- (4–5) relevant to theModel metaphase spindle for (Figure spindle A): dynamic length instability control ofping MTs MTs.and as a The function implicit assumption here is that the Ftension = b S 2 S0 (5) sliding force per single MT is the arithmetic mean, ð Þ F Figure A.sliding Schematic, and Representation we are not taking into account differences of the ForcesN Acting on the Meta-v(max) phasebetween Spindle differentdep MT populations. Integrating this Fkt = Fkt,0 (6) ln (max) 0 v +v vpoly Lprobability= ✓ dep densitydep from 0◆ to d, we get the probability that↵ this MT endv ispoly not farther than distance d from the The sliding force parameter a represents Kinesin-5 1 (max) v F d sliding 2 sliding force per unit length of overlap (L)(pN/mm), and depolymerizing✓ motor:◆ P x

Fslidingd 2 Nk T LS ? 

Here we define Vd, 0 as sliding-force-independent basal depolymerization at the pole. Our FSM/kymo- global Figureproportional B. Parameter Sensitivity Analysis intension Steady State between graph analysis of GFP-tubulin suggests that this is to density of spindle centrosomes and G. Goshima et al., Current sliding motors spring kinetochores constant Biol. 15, 1979-1988 (2005) 17 Continued on following page Current Biology 1986

Figure 4. Bistability of the Spindle Morphol- ogy but Robustness of Metaphase Spindle Length to Alteration of Kinesin-5 Concentra- tion (A) RNAi knockdown of endogenous Klp61F with dsRNA targeting 50 UTR region was Spindle bistability combined with ectopic expression of Klp61F- GFP. Immunoblot was performed by anti- Klp61F antibody. (B) Spindle ‘‘bistability.’’ At top, representa- tive images of monopolar, monastral bipolar, and bipolar spindles are shown. Red shows g-tubulin, and green shows Klp61F-GFP. The bar represents 10 mm. At bottom, frequency of monopolar spindles over total monopolar and no normalnormal bipolar spindles is shown by blue line; frequency was calculated on the basis of the spindle distributionspindle of 238 spindles into 15 bins. Length distributions of the normal bipolar spindle (green dots) and monastral bipolar spindle (green open circles; g-tubulin staining at only one pole of the bipolar spindle) after various levels of Klp61F-GFP expression are also plotted. Abrupt decrease in monopolar spindle frequency is observed at a critical expression level of Klp61F. The length of monastral bipolar spindles was measured by doubling the distance between g-tubulin and center of the chromosome mass.

concentration of metaphase is 25% longer (1.0 6 0.13 mm [n = 23], p < ward fluxG. inGoshima S2 cells because et al., RNAiCurrent of Klp61F (Kinesin- 0.0001) than in colchicine-treatedsliding cells, motors which lack 5) causes a bipolar-to-monopolar spindle collapse and kMT tension (0.8 6 0.13 mm [n = 47]) (Figure S4A). a concomitantBiol. 15, significant 1979-1988 reduction (2005) in the rate of pole- Time-lapse imaging of Klp67A-GFP (as an18 outer-kineto- ward flux (Figure S5, Movies S2 and S3). Given these as- chore marker) also showed that metaphase sister-kinet- sumptions, the quantitative model explains how chang- ochore distance rapidly decreased upon colchicine ing the rate of MT polymerization/depolymerization can treatment (n = 6, Figure S4B, Movie S1). These data sup- produce changes in the metaphase spindle length with- port the idea that sister kinetochores are under tension out changing its stability while the length can remain in- during metaphase in S2 cells. sensitive to a wide range of Kinesin-5 concentration. Combining these forces via a simple force-balance A novel outcome of our model is that it predicts ‘‘bi- model [13], we show that these assumptions by them- stability’’ in spindle architecture. Specifically, at low selves are not sufficient to allow a stable metaphase concentrations of Klp61F (Kinesin-5), monopolar spin- steady-state (Box 1). We further tested what other as- dles are created, but as the concentration of this motor sumptions allow the steady state to be achieved. We increases, an abrupt transition to bipolar spindle forma- found that the addition of an assumption that the rate tion takes place [Box 1, equation (11) and Figure B]. To of MT depolymerization at spindle poles (which contrib- test this prediction of spindle bistability, we examined utes to poleward flux [1, 4, 35]) is proportional to the the relationship between Klp61F expression and spindle motor-generated sliding forces is sufficient to allow sta- length over a wide range of motor concentrations by bility of the metaphase steady state [Box 1, equation (7)]. RNAi-depleting endogenous Klp61F by using dsRNA di- This feature also enables the spindle length to remain rected against its 50 UTR and then expressing varying constant despite the overexpression of the Kinesin-5 levels of Klp61F-GFP from a plasmid with an inducible motor. Although this assumption remains speculative in promoter (Figure 4A and [22]). We assume that the ex- Drosophila cells, it is supported by recent experiments pressed fusion protein is active, an assumption that is showing that the rate of poleward flux in meiotic spin- supported by its ability to rescue bipolar spindle assem- dles assembled in Xenopus extracts correlates with bly in RNAi-treated cells at appropriate concentrations Kinesin-5 activity in a dose-dependent manner [18]. (Figure 4B). Significantly, we found that there was an Kinesin-5 activity also appears to be an important factor abrupt increase in the percentage of cells that exhibited in maintaining spindle bipolarity and generating pole- bipolar spindles at a critical level of Klp61F-GFP protein Cell division in bacteria

19 Genetic information in bacteria

One large A few small circular DNA circular plasmids

Plasmids carry additional genes that have recently evolved and may benefit survival (e.g. antibiotic resistance)

20 PERSP ECTIVES DNA replication and segregation

Spontaneous demixing due to that bacterial cytoskeletal proteins such as absteric excluded volume interactions plasmid partition protein M (ParM) and Genome-sized ParA are the machinery for active transport dsDNA of low-copy-number plasmids and ori loci, respectively. The system consisting of ParA, Blob Stretching and twisting Blobs of newly synthesized DNA ParB and parS (the binding site for ParB) is widely conserved in many bacteria and con- tributes to ori segregation in C. crescentus Supercoiled omosome and oriII segregation in V. cholerae. plectonemes Stabilization by nucleoid- and ation However, although ParA plays an impor- associated proteins eplic tant part in chromosome segregation, its role Nucleoid- is likely to be limited, for the following rea- associated protein sons. In C. crescentus, ParA is not required Topologically once ori loci are separated at the beginning independent Close packing of the structural unit string of blobs Simultaneous r demixing of the chr of the cell cycle27. The absence of ParA alone has little effect on chromosome segregation in B. subtilis4, and there is no ParA homo- logue in E. coli. Also, as we discuss below, eukaryotic sister chromatids demix and move ~0.5 μm apart before their separation Molecular crowding and nucleoid compaction by spindles, without any active pushing or create a concentric shell in the nucleoid periphery 28 S. Jun and A. Wright, Nat. Rev. pulling of the replicated DNA . A potential Wikipedia Figure 2 | Physical model of a bacterial chromosome and its segregation.Microbiology a | A8, reductionist 600-607 (2010)model problem with active transport of chromo- Nature Reviews | Microbiology of the Escherichia coli chromosome. First, we stretch21 a bacterial-genome-sized naked double-stranded somal ori loci for organisms that undergo DNA (dsDNA). This breaks the DNA into a series of blobs, the total volume of which gradually decreases multifork replication is that simple transport as pulling continues. In parallel, we also twist the DNA to match the supercoil density of a bacterial of multiple copies of ori may be harmful to chromosome. As a result, the DNA blobs will consist of supercoiled plectonemes (a shape of the DNA the cell unless it also solves the ‘hierarchy in which the two strands are intertwined). We stop the simultaneous pulling and twisting processes dilemma’ of positioning an ori depending on when the total volume of the blobs equals the target volume of the nucleoid inside the cell. Next, we its identity. An obvious experimental test of ‘sprinkle’ the chromosome with nucleoid-associated proteins. These stabilize the supercoiled DNA this idea would be to insert an active plasmid blobs as topologically independent structural units of the chromosome. Finally, we connect the two segregation system, such as ParM or the ends of the chromosome to make it circular, and then pack it tightly in the cell. For the simpler case of ParAB–parS system, into the E. coli chromo- chains without supercoiling, the phase diagram in FIG. 1 provides a model for the close-packed organi- zation and segregatability of the chains inside the cell. In general, supercoiling will only increase the some. We predict that chromosome segrega- tendency for chromosome demixing because of the branched structure that it induces. b | The con- tion during multifork replication in such centric shell model predicts extrusion of the newly synthesized DNA (blue and red) to the periphery an organism will be defective. We propose of the nucleoid15. The newly replicated DNA is extruded to the periphery of the unreplicated nucleoid that organisms encoding ParA have adopted (grey) and forms a string of DNA blobs in the order of replication, promoted by SMC (structural main- this protein from plasmids for a more effi- tenance of chromosomes) proteins and other nucleoid-associated proteins. In our model, the cient segregation of the ori domain, which is two strings of blobs repel each other and drift apart owing to the excluded-volume interaction and comparable in size to plasmids, rather than conformational entropy. for segregation of the bulk chromosomes. C. crescentus is smaller than E. coli and has a smaller cytoplasmic space surround- Several proteins are involved in active move DNA in the cell. The finding of a ing its nucleoid, so the outer concentric shell transport of DNA inside a bacterium. fixed replication factory in B. subtilis led of this species may not be large enough for Examples include SpoIIIE, which moves to the proposal of an ‘extrusion–capture’ fast diffusion of newly replicated DNA. In DNA into the forespore during sporulation model30, which assumed that the energy this case, ParA might be needed to ensure of B. subtilis, and FtsK, which resolves dim- released during replication could contrib- rapid segregation of the duplicated ori loci ers and carries out other ‘rescue’ tasks in ute to chromosome partitioning. Recent in the early stage of the replication cycle, E. coli 29. However, it is important to realize work, however, has shown that replication when newly synthesized DNA would oth- that these proteins translocate DNA at the forks and their associated replisomes are erwise be kinetically trapped in the cell (see last step of segregation by taking advantage both independent and highly dynamic Supplementary information S4 (box)). The of the directionality that is provided by in the cell, making their role in segrega- advantage of having an outer shell leads us to the septum, which is an entirely different tion unlikely31–33. It was suggested that predict that, even in C. crescentus, duplicated process from segregation of replicating the negative effects of streptolydigin on ori loci will move in the periphery of the chromosomes. chromosome segregation implicate RNA nucleoid. Finally, as the cell grows and rep- polymerases in chromosome segregation34. lication continues, reducing the volume of Segregation models based on replication, In addition, it has been proposed that tran- the dense, unreplicated-DNA core, an outer transcription, translation and tethering. sertion (the insertion of polypeptides into shell is not needed for entropy-driven segre- In addition to the active DNA transport membranes during translation), instead of gation15. The C. crescentus cell cycle is longer models, both the force of DNA ejection polymerases themselves, could segregate than those of E. coli and B. subtilis, which by DNA polymerase and the tethering of the replicating chromosomes35. Definitive will further help segregation by entropy. DNA polymerase have been proposed to experimental support for these proposals is

604 | AUGUST 2010 | VOLUME 8 www.nature.com/reviews/micro © 2010 Macmillan Publishers Limited. All rights reserved PERSP ECTIVES

Box 2 | Chain molecules in strong confinement or at high concentrations

The effect of a cylindrical confinement on a chain is equivalent to applying a a Stretched chain = string of entropic springs tension, which pulls and stretches the long molecule (see the figure, part a). In confined in a flexible tube of fixed width polymer physics, the stretched chain is described as a series of entropic springs (or ‘blobs’), the size of which is determined by the width of the tube. There is a Force linear relationship between the total contour length of the chain and the total Force number of blobs, and the free energy stored in the chain is directly proportional to the number of blobs. In other words, a blob is also a basic unit of free energy D stored in a polymer and has a thermal energy of kBT (where T is the room temperature (~300 Kelvin) and kB is the Boltzmann constant (2 cal per mol K) 52,53 regardless of its physical size (for comparison, ATP hydrolysis releases ~12 kBT b of energy, and a hydrogen bond is several kBT). In other words, blobs can have different sizes but the same free energy, as long as the self-avoidance condition is met. When the concentration of the chain is very high, transverse motions of a chain segment embedded in the meshwork of polymers is hindered. In this case, the polymer motion is best understood as sliding in a conceptual tube embedded in a polymer meshwork, also known as ‘reptation’ (see the figure, part b). Reptation is slow; it takes much longer to travel the same absolute distance by reptation in c DirTopoisomeraseectionality from global polymer r1epulsion and can 2 release tension along the a meshwork than by diffusion in the absence of the meshwork, and the difference compensDNAate for mistak andes by typespeed II topoisomer upase the separation process between the rates increases in proportion to the length of the chain itself. Chain connectivity and excluded-volume interactions provide directionality for the segregation of intermingled chains54 (see the figure, part c). Type II topoisomerases do not need to provide or know the directionality. By occasional random strand-passing, these enzymes can accelerate the kinetics of chromosome segregation because reptation is a much slower process (see the figure, part c). However, too high a concentration of topoisomerase means that the two chains Separation by reptation is slow will not know their directionality, because the excluded-volume interaction between the chains and the enzyme effectively vanishes (by virtue of the high concentration of the enzyme) and the forward and reverse topoisomerase reactions will cancel each other out. We thus predict that there will be an optimal range of type II topoisomerase concentrations at which chromosome segregation is Separation by type II topoisomerase can be fastest, because the entropy-driven directionality and topoisomerase-dependent fast despite occasional reverse strand-passing ‘free pass’ are in balance. physical model of the E. coli nucleoid: the isolated nucleoid nor the size of the not confined andNa thetur entropice Reviews |repulsion Microbiolog y the size of the structural unit (also called the structural unit have been measured for betweenS. Jun andthem A. isWright, weak. Nat. In Rev.the absence of correlation length; ξ); the Flory radius these organisms, although we expect anyMicrobiology specific interactions 8, 600-607 (2010) with the chromo- of gyration (RF) of the isolated nucleoid B. subtilis and C. crescentus to belong to some (such as ‘hitchhiking’), or without22 (namely, the diameter of the fully expanded regime III, considering that both organisms dedicated segregation machinery, plas- nucleoid released from the lysed cell); have similar cell dimensions and genome mids will distribute randomly inside the and the length and width (L and D) of the sizes to E. coli. Future studies are needed to cell because of their small sizes. Indeed, nucleoid inside the cell. The most impor- measure ξ for the nucleoids in these organ- recent experimental results from studies tant parameter is ξ, which can be measured isms, perhaps using fluorescence correlation of the high mobility and random distribu- as described in Supplementary information spectroscopy techniques similar to those tion of the RK2 plasmid, which lacks a S3 (box). used in REF. 11 (explained in Supplementary partitioning system, are fully consistent Experimentally, E. coli str. B/r H266 is information S3 (box)) in order to fully test with our prediction12. the only organism for which these param- our predictions. eters have been measured10,11 (see BOX 2 and The next question concerns plas- Segregation in round cells. Perfect symme- Supplementary information S3 (box)). They mid segregation in bacteria. Note that try of cell shape means that the confined are: D = 0.24 μm, L = 1.39 μm, RF = 3.3 μm; plasmids make little contribution to the chains do not have any preferred confor- and ξ = 87 nm. Thus, E. coli str. B/r H266 amount of DNA in the cell because of mations between mixing and segregation, belongs to regime III, in which the strongly their small sizes. Indeed, RF for plasmids although their global reorganization is 13 compressed individual chromosomes gain is typically one-tenth of the RF value readily achieved . Few data are available maximum conformational entropy when for the chromosome. This separates the about chromosome organization in spheri- they remain segregated and linearly ordered plasmids from the chromosomes by one cal bacteria, so we can only speculate about (FIG. 1c; see Supplementary information S4 order of magnitude along the diagonal possible contributing factors to segrega- (box)). Similarly, our phase diagram can be line between the two axes in the phase tion. We think that supercoiling is the most used to make a prediction for other bacteria diagram (FIG. 1c; see Supplementary infor- important factor, as it gives rise to the such as Vibrio cholerae (which has two chro- mation S4 (box)). As a result, plasmids branched structure of the bacterial chro- mosomes), Bacillus subtilis and Caulobacter belong to the lower part of regime V in mosome14. We did not take into account crescentus. At present, neither the size of the phase diagram, in which polymers are the topological complexity of the polymer

602 | AUGUST 2010 | VOLUME 8 www.nature.com/reviews/micro © 2010 Macmillan Publishers Limited. All rights reserved Bacteria divide faster than DNA replicates

Under normal conditions E. coli divides every 15-20 min In E. coli it takes ~40 min to replicate DNA

How can bacteria divide faster than DNA replicates?

Multiple replication forks!

Bacteria starts replicating DNA for their daughters, grand daughters, etc.

23 FUNCTION AND ORIGIN OF THE CYTOSKELETON 897

(A) (B) 1 µm 1 µm 1 µm 1 µm

Figure 16–8 Actin homologs in bacteria determine cell shape. (A) The MreB protein forms abundant patches made up of many short, interwoven linear or helical filaments that are seen to move circumferentially along the length of the bacterium and are associated with sites of cell wall synthesis. (B) The common soil bacterium Bacillus subtilis normally forms cells with a regular rodlike shape when viewed by scanning electron microscopy (left). In contrast, B. subtilis cells lacking the actin homolog MreB or Mbl grow in distorted or twisted shapes and eventually die (center and right). (A, from P. Vats and L. Rothfield, Proc. Natl Acad. Sci. USA 104:17795–17800, 2007. With permission from National Academy of Sciences; B, from A. Chastanet and R. Carballido-Lopez, Front. Biosci. 4S:1582–1606, 2012. With permission Frontiers in Bioscience.) plant cells (see Figure 19–65). As with FtsZ, MreB and Mbl flaments are highly dynamic, with half-lives of a few minutes, and nucleotide hydrolysis accompanies the polymerization process. Mutations disrupting MreB or Mbl expression cause extreme abnormalities in cell shape and defects in chromosome segregation (Fig- ure 16–8B). Relatives of MreB and Mbl have more specialized roles. A particularly intriguing bacterial actin homolog is ParM, which is encoded by a gene on certain bacterial plasmids that also carry genes responsible for antibioticMBoC6 resistancem16.26/16.08 and cause the spread of multidrug resistance in epidemics. Bacterial plasmids typically encode all the gene products that are necessary for their own segregation, presumably as a strategy to ensure their inheritance and propagation in bacterial hosts fol- lowing plasmid replication. ParM assembles into flaments that associate at each end with a copy of the plasmid, and growth of the ParM flament pushes the rep- licated plasmid copies apart (Figure 16–9). Tis spindle-like structure apparently arises from the selective stabilization of flaments that bind to specialized proteins recruited to the origins of replication on the plasmids. A distant relative of both tubulin and FtsZ, called TubZ, has a similar function in other bacterial species. Tus, self-association of nucleotide-binding proteins into dynamic flaments is used in all cells, and the actin and tubulin families are very ancient, predating Figure 16–9 Role of the actin homolog the split between the eukaryotic and bacterial kingdoms. ParM in plasmid segregation in bacteria. At least one bacterial species, Caulobacter crescentus, appears to harbor a pro- (A) Some bacterial drug-resistance tein with signifcantPlasmid structural similarity segregation to the third major class of cytoskeletal plasmids (orange) encode an actin flaments found in animal cells, the intermediate flaments. A protein called cres- homolog, ParM, that will spontaneously centin forms a flamentous structure that infuences the unusual crescent shape nucleate to form small, dynamic filaments Plasmids are too small to spontaneously (green) throughout the bacterial cytoplasm. of this species; when the gene encoding crescentin is deleted, the Caulobacter A second plasmid-encoded protein cells grow as straightsegregate rods (Figure on different16–10). sides of bacteria called ParR (blue) binds to specific DNA sequences in the plasmid and also stabilizes the dynamic ends of the ParM filaments. When the plasmid duplicates, both ends of the ParM filaments become stabilized, and the growing ParM filaments push the duplicated plasmids to opposite ends of the cell. (B) In these bacterial plasmid cells harboring a drug-resistance plasmid, the plasmids are labeled in red and the ParM protein in green. Left, a short ParM ParM filament bundle connects the two daughter plasmids shortly after their duplication. Right, the fully assembled ParM filament has pushed the duplicated plasmids to the cell poles. (A, adapted from E.C. Garner, ParM plasmid origin of ParM ParR C.S. Campbell and R.D. Mullins, Science monomers replication filaments proteins 306:1021–1025, 2004; B, from J. Møller- (A) (B) Jensen et al., Mol. Cell 12:1477–1487, 2 µm 2003. With permission from Elsevier.) ParM is analogous to actin (assembly by ATP hydrolysis)

Alberts et al., Molecular Biology of the Cell 24 MBoC6 m16.27/16.09 896 Chapter 16: The Cytoskeleton

Among the most fascinating proteins that associate with the cytoskeleton are the motor proteins. Tese proteins bind to a polarized cytoskeletal flament and use the energy derived from repeated cycles of ATP hydrolysis to move along it. Dozens of diferent motor proteins coexist in every eukaryotic cell. Tey difer in the type of flament they bind to (either actin or microtubules), the direction in which they move along the flament, and the “cargo” they carry. Many motor pro- teins carry membrane-enclosed organelles—such as mitochondria, Golgi stacks, or secretory vesicles—to their appropriate locations in the cell. Other motor pro- teins cause cytoskeletal flaments to exert tension or to slide against each other, generating the force that drives such phenomena as muscle contraction, ciliary beating, and cell division. Cytoskeletal motor proteins that move unidirectionally along an oriented polymer track are reminiscent of some other proteins and protein complexes dis- cussed elsewhere in this book, such as DNA and RNA polymerases, helicases, and ribosomes. All of these proteins have the ability to use chemical energy to propel themselves along a linear track, with the direction of sliding dependent on the structural polarity of the track. All of them generate motion by coupling nucleo- side triphosphate hydrolysis to a large-scale conformational change (see Figure 3–75).

Bacterial Cell Organization and Division Depend on Homologs of Eukaryotic Cytoskeletal Proteins While eukaryotic cells are typically large and morphologically complex, bacterial cells are usually only a few micrometers long and assume simple shapes such as spheres or rods. Bacteria also lack elaborate networks of intracellular mem- brane-enclosed organelles. Historically, biologists assumed that a cytoskeleton was not necessary in such simple cells. We now know, however, that bacteria con- tain homologs of all three of the eukaryotic cytoskeletal flaments. Furthermore, Contraction of FtsZ-ring bacterial actins and tubulins are more diverse than their eukaryotic versions, both divides bacterial cell in two in the types of assemblies they form and in the functions they carry out. Nearly all bacteria and many archaea contain a homolog of tubulin called FtsZ, which can polymerize into flaments and assemble into a ring (called the Z-ring) at the site where the septum forms during cell division (Figure 16–7). Although FtsZ is analogous to tubulin the Z-ring persists for many minutes, the individual flaments within it are highly (assembly by GTP hydrolysis) dynamic, with an average flament half-life of about thirty seconds. As the bacte- rium divides, the Z-ring becomes smaller until it has completely disassembled. Bacterial division is extremely FtsZ flaments in the Z-ring are thought to generate a bending force that drives precise. FtsZ forms at the membrane invagination necessary to complete cell division. Te Z-ring may (0.50 0.01) L also serve as a site for localization of enzymes required for building the septum ± between the two daughter cells. (A) Many bacteria also contain homologs of actin. Two of these, MreB and Mbl, 1 µm How does bacteria know where are found primarily in rod-shaped or spiral-shaped cells where they assemble to place the contractile ring? to form dynamic patches that move circumferentially along the length of the cell (Figure 16–8A). Tese proteins contribute to cell shape by serving as a scaf- fold to direct the synthesis of the peptidoglycan cell wall, in much the same way that microtubules help organize the synthesis of the cellulose cell wall in higher 25

Figure 16–7 The bacterial FtsZ protein, a tubulin homolog in prokaryotes. (A) A band of FtsZ protein forms a ring in a dividing bacterial cell. This ring has been labeled by fusing the FtsZ protein to green fluorescent (B) protein (GFP), which allows it to be observed in living E. coli cells with a 100 nm fluorescence microscope. (B) FtsZ filaments and circles, formed in vitro, as visualized using electron microscopy. (C) Dividing chloroplasts (red) from a red alga also cleave using a protein ring made from FtsZ (yellow). (A, from X. Ma, D.W. Ehrhardt and W. Margolin, Proc. Natl Acad. Sci. USA 93:12998–13003, 1996; B, from H.P. Erickson et al., Proc. Natl Acad. Sci. USA 93:519–523, 1996. Both with permission from National Academy of Sciences; C, from S. Miyagishima et al., Plant Cell 13:2257–2268, 2001, with permission from (C) American Society of Plant Biologists.) 1 µm

MBoC6 m16.24/16.07 VOLUME 87, NUMBER 27 P H Y S I C A L R E V I E W L E T T E R S 31 DECEMBER 2001

regulate cell division throughout the division cycle of the cell. In Fig. 2, we plot the time-averaged MinD and MinE densities as a function of position. MinD shows a pro- nounced dip in concentration close to midcell, which al- lows for the removal of division inhibition at midcell. This is in qualitative agreement with the experimental data of Ref. [8]. MinE peaks at midcell, with a minimum at the cell extremities. We also investigated longer filamentous bacteria and FIG. 1. Space-time plots of the total MinD (left) and MinE found a multiple MinE band structure (not shown). Multi- (right) densities. The grey scale runs from 0.0 to 2.0 times ple MinE bands always combined into a single MinE band the average density of MinD or MinE, respectively. The MinD in cell lengths shorter than the natural wavelength indi- depletion from midcell and the MinE enhancement at midcell are immediately evident. Time increases from top to bottom, cated by linear stability analysis. and the pattern repeats indefinitely as time increases. The grey- The oscillation period as a function of the average MinD scale reference bar spans 100 s. The horizontal scale spans the concentration is shown in Fig. 3 (left). We find a linear bacterial length (2 mm). relationship indicated by the best-fit line, where the pe- riod approximately doubles as the MinD concentration is model (not shown). The physical origin of this instability quadrupled. A linear relationship has also been suggested Minlies in the system disparity between the oscillations membrane and cytoplas- experimentally provide [3]. The period of oscillation as a function mic diffusion rates, and also in the slower rate at which of cell length is shown in Fig. 3 (right). Below lengths cuesMinE disassociates for the from the membrane.formation This ensures that ofof 1.2FtsZmm the bacterium ring does not sustain oscillating pat- the MinE dynamics lags that of the MinD, setting up the terns. For lengths above this minimum, the oscillation pat- oscillating patterns. The existence of the linear instability terns are stable and the period increases with length— as in Eqs. (1)–(4) is crucial, since it means that the oscillat- observed experimentally [7]. The periods measured from ing pattern will spontaneously generate itself from a vari- our numerics for cell lengths of 2 mm are around 100 s, ety ofPredator-prey initial conditions—including like nearly dynamics homogeneous in good agreement with experiments, where periods from FtsZ ones. In our simulations, we used random initial condi- 30 120 s0s have been found [3]. A single MinE band state tions, althoughbetween identical MinD patterns were and also MinE observed with is stable over a wide range of lengths for a given density asymmetricproteins initial distributionsproduce of MinD oscillations and MinE. The of min proteins. This provides strong evidence that the eventual oscillating state is stabilized by the nonlinearities min system is capable of regulating accurate cell division in Eqs.on (1)a –minute(4). At the midcell, time this scale, oscillating patternwhich has over normally occurring cell lengths as the cell grows be- a minimum of the time-averaged MinD concentration — an tween division events. At longer lengths of around 6 mm, MinC essentialis much feature of divisionshorter regulation than— and typical a maximum we observe long-lived metastable states with two MinE of the time-averageddivision MinE time concentratio. (~20 min). bands. These multiple bands can survive for a thousand Space-time plots of the MinD and MinE concentrations seconds or more before decaying into a single band. At for a cell length of 2 mm are shown in Fig. 1. In excellent still longer lengths the two band state appears stable; this agreement with the experimental results, the MinE sponta- occurs around 8.4 mm—twice the dominant wavelength MinD neouslyOn forms average a single band at MinC/MinD midcell which then sweeps given by thetime linear stability analysis. This explains why towards a cell pole, displacing the MinD, which then re- the characteristic wavelength of linear stability analysis formsproteins at the opposite pole. are Once depleted the MinE band reaches is rather longer than a normal E. Coli bacterium—if the the cell pole it disappears into the cytoplasm, only to re- length scale were smaller, then multiple MinE bands might form at midcellnear where the the processcell repeats, center, but in the other MinE half of the cell. These patterns are stable over at least 109 iterationswhere (104 s)FtsZ—long enoughring for forms! the min system to 120 1500 100 1000

1.00 1.00 80 500

max 50s period (sec) ρ MinD MinE ⇠ 60 0 0.75 0.75 500 1000 1500 2000 1 2 3 4 5 67 (x)>/

ρ ρD (#/µm) length (µm) < 0.50 0.50 0 120 12 FIG. 3. Left: Plot of the period of oscillation (in seconds) 21 x (µm) x (µm) against MinD density (in mm ), at fixed average MinE con- centrationH. Meinhardt of 85 mm21. The and solid P.A.J. line is a linearde Boer best fit., Right: FIG. 2. The time-average MinD (left) and MinE (right) densi- Plot of oscillation period against cell length, for fixed MinD and ties, ͗r͑x͒͘͞rmax, relative to their respective time-average max- MinE concentrations.PNAS 98, Below 14202 bacterial lengths(2001) of 1.2 mm, oscil- ima, as a function of position x (in mm) along26 the bacterium. lation is not observed.

278102-3 278102-3 is lowest right at the middle of the cell. In considering wave equations in the context of fluid mechanics or electromagnetism, it is well appre- ciated that the same governing dynamical equations that generate propagating waves in an extended medium will lead to standing waves when certain kinds of boundary conditions are imposed. Inspection of the dynamics of MinD localization inside of living bacterial cells (see Figure 20.16) suggests that its behavior is consistent with what might be expected when a traveling-wave system is confined to the small oblong box of the bacterial cell. As shown in Figure 20.16(A), MinD oscillates from one pole to another of a pre-divisional bacterial cell, with a period T of about 40 s, which is much shorter than the cell’s division time (of the order of about 30 minutes). As the oscil- lating MinD carries MinC with it, the time-averaged concentration of the MinC protein will necessarily be lowest right at the cell’s center, exactly where the FtsZ ring is supposed to assemble, and preventing FtsZ ring assembly near the cell poles, which would result in forma- tion of minicells devoid of DNA. The nature of the MinD standing wave is illustrated more dramatically in long cells where division has been inhibited, as shown in Figure 20.16(B). For these filamentous cells, the standing-wave pattern shows a spatial periodicity that is about twice the normal length of an individual cell. We can use the physical dimensions of the standing wave to estimate whether the Min traveling waves observed for the purified system in vitro really are a plausible physical underpinning for the center- finding mechanism in living cells. Mathematically, a standing wave can be described as two traveling waves, with the same amplitude, wave- length, and propagation speed, but moving in opposite directions. The wavelength λ of the in vivo standing wave is about 4–6 µm, which is twice the length of the pre-divisional cell shown in Figure 20.16(A), or the spacing between peaks shown in Figure 20.16(B). The period T is about 40 s. Setting v λ/T ,wecanestimatev to be around 0.1 µm/s. = This is the same order of magnitude as the wave propagation speed for the in vitro system, v 0.3–0.8 µm/s, and the estimate becomes = even closer if we recall that rates of diffusion for small molecules in cytoplasm are typically about fourfold slower than rates in water (see Figure 14.4, p. 549), which, according to our toy model, would decreaseMin the propagation system speed oscillationsin vivo by a factor in of large four. cells Overall the Min system in E. coli is a remarkable fulfillment of the biological promise of reaction–diffusion mechanisms as envisioned by Turing for establishing biological patterns. Certainly, there are a numberMinD of details oscillations in the in system thatMinD he had oscillations not imagined in E. (and Coli, we normal E. Coli where division is prevented

Figure 20.16: Min oscillations and (A) (B) how the bacterium finds its middle. (A) Fluorescently tagged MinD can be seen to oscillate rapidly from one pole 0 s of the cell to another with a period of only about 40 s, which is much less 0 s 20 s 40 s 60 s than the cell’s division time. (B) In cells 20 s that are prevented from physically 1 mm dividing, the Min oscillations continue, forming an oscillating stripe pattern 40 s throughout the entire length of the filamentous cell. The total size of the cell is shown in the transmitted-light micrograph in the bottom panel. (Adapted from D. M. Raskin and 5 mm P. A . J . d e B o e r, Proc. Natl Acad. Sci. USA 96:4971, 1999.) R. Phillips et al., Physical Biology of the Cell 924 Chapter 20 BIOLOGICAL PATTERNS: ORDER IN SPACE AND TIME 27

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