the spindle checkpoint: structural insights into dynamic signalling

REVIEWS

It is crucial for cells to maintain the integrity of theirgenomes. DNA replication and chromosome segrega-tion are extremely complex, error-prone processes, andmany cell-cycle controls have evolved to regulate them(BOX 1). Here, we review one such control, the spindlecheckpoint (FIG. 1). This checkpoint has had severalnames (spindle assembly, KINETOCHORE attachment,metaphase), but we use ‘spindle checkpoint’ for simplic-ity. There are other, less well-characterized checkpointsthat act later in mitosis and that seem to monitor spin-dle-pole positioning and spindle orientation (BOX 1).

Components of the spindle checkpoint were firstidentified through genetic screens in budding yeast1,2.These led to the discovery of the Mad and Bub proteins(TABLE 1). Over a decade later, these are still viewed as thecore components of the spindle checkpoint and have allbeen found to be conserved from yeast to human.When problems arise in the chromosome-segregationmachinery, the combined action of Mad and Bub delaysthe onset of anaphase by maintaining sister-chromatidcohesion. More recently, other checkpoint components,such as Zw10 (Zeste-white 10) and Rod (Rough-deal),have been identified in higher eukaryotes (TABLE 1).

After DNA replication, the chromosomes consist ofpairs of sister chromatids held together by complexes ofproteins known as cohesins3. During mitosis, the sisterchromatids attach to spindle microtubules throughtheir kinetochores4, which are highly complex protein

machines that assemble at the centromere5 of eachchromosome. To ensure equal segregation of thegenetic material, the two kinetochores in each sister-chromatid pair must interact with microtubules thatemanate from opposite spindle poles to one another(this is known as bipolar attachment; FIG. 2). Entry intoanaphase depends on all chromosomes being attachedin a bipolar manner. How bipolar attachment isensured is discussed below, but it seems that cells moni-tor both the attachment of microtubules to kineto-chores and the tension that is exerted at kinetochoresupon bipolar attachment.

Molecular details of the regulation of mitotic pro-gression are now well understood (FIG. 1). In the absenceof bipolar attachment, the spindle-checkpoint proteinsemit a global signal throughout the mitotic machineryto inhibit the onset of anaphase. The kinetochore isthought to act as a catalytic site for the production ofthis ‘wait anaphase’ signal. It was initially shown in bothbudding6 and fission7 yeasts that the target of the spin-dle checkpoint is an accessory subunit of the anaphase-promoting complex (APC) (REF. 8) that is typically referredto as CDC20 (also known as Fizzy, Slp1 or p55). Mad2 wasshown to bind directly to Cdc20, and cdc20 mutantsthat no longer interact with Mad2 were found to beinsensitive to checkpoint inhibition6,7. The precise invivo form of the Cdc20–APC inhibitor is the subject ofintense study and some controversy. However, it is clear

THE SPINDLE CHECKPOINT:STRUCTURAL INSIGHTS INTODYNAMIC SIGNALLINGAndrea Musacchio* and Kevin G. Hardwick‡

Chromosome segregation is a complex and astonishingly accurate process whose inner workingis beginning to be understood at the molecular level. The spindle checkpoint plays a key role inensuring the fidelity of this process. It monitors the interactions between chromosomes andmicrotubules, and delays mitotic progression to allow extra time to correct defects. Here, wereview and integrate findings on the dynamics of checkpoint proteins at kinetochores withstructural information about signalling complexes.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 3 | OCTOBER 2002 | 731

*Department ofExperimental Oncology,European Institute ofOncology, Via Ripamonti435, 20141 Milan, Italy.‡Wellcome Trust Centre forCell Biology, ICMB,University of Edinburgh,King’s Buildings,Mayfield Road,Edinburgh EH9 3JR, UK.e-mails: [email protected];[email protected]:10.1038/nrm929

KINETOCHORE

Complex protein assembly thatlinks chromosomes to themitotic spindle. Kinetochoresassemble around specializedchromosomal regions known ascentromeres, whose sequenceand overall structures varyconsiderably between differentorganisms.

APC

Anaphase-promoting complex.A multiprotein complex withubiquitin-ligase activity that isresponsible for theubiquitylation of several keycell-cycle regulators. Also knownas the cyclosome.

© 2002 Nature Publishing Group

CDC20

Positive regulator of the APCand a target of the spindlecheckpoint. Also known asp55Cdc20, Fizzy and Slp1.

CONGRESSION

The microtubule-dependentmovement of paired sisterchromatids, whereby theybecome aligned at themetaphase plate.

3F3/2 PHOSPHOANTIGENS

Unknown antigens that arephosphorylated inprometaphase, and becomedephosphorylated when tensionis exerted on sister chromatids.There are probably severaldistinct antigens, including APCsubunits126.

732 | OCTOBER 2002 | VOLUME 3 www.nature.com/reviews/molcellbio

R E V I E W S

Sensory machineryAddressing the temporal correlation between the localiza-tion of checkpoint proteins at kinetochores and the pro-gression of chromosome attachment and congressionsheds light on the signals that trigger the checkpoint. Twoconditions are associated with the bipolar orientation ofsister chromatids to the spindle: attachment, resultingfrom end-on docking of microtubules to the kinetochoresurface; and tension, arising after bipolar attachment asan equilibrium between poleward and anti-polewardforces including sister-chromatid cohesion (FIG. 2).

Attachment. Three microtubule motors — CENP-E,dynein and MCAK/XKCM1 — and several microtubule-binding proteins have been localized to metazoan kineto-chores during mitosis14–16. The function of MCAK doesnot seem to be essential for chromosome CONGRESSION butis required at anaphase. CENP-E, a kinesin-like plus-end-directed motor, has been implicated in chromosome con-gression and metaphase alignment, and, more recently, incheckpoint signalling17–21. CENP-E forms a stoichiomet-ric complex with BubR1 in HeLa cells, which providedthe first direct link between microtubule attachment andthe spindle-checkpoint machinery17–20.

The only known minus-end-directed motor at thekinetochore, dynein is a candidate for driving the pole-ward movement of sister chromatids after attachmentand at anaphase22. Zw10 and Rod are required in meta-zoan cells for localizing cytoplasmic dynein to kineto-chores23, and their inactivation reduces the rate of pole-ward chromosome movement24,25. They have recentlybeen reported to be required for spindle-checkpointfunction26,27, and this could provide a second link withthe process of microtubule attachment.

that both Mad2 and BubR1/Mad3 have key roles9.Inhibition of Cdc20–APC prevents the destruction ofsecurins and thereby delays sister-chromatid separationand anaphase (FIG. 1).

The recent use of live-cell fluorescence microscopyand structural biology have clarified aspects of spindle-checkpoint function and led to the formulation of newtestable hypotheses that will influence future research inthis field. Here, we focus on recent work that describesthe dynamic behaviour of checkpoint proteins withinthe mitotic apparatus, along with insights gained fromthe genetic, biochemical and structural analyses ofcheckpoint complexes.

Kinetochores host the spindle checkpointElegant cell-biological studies have shown that a singleunattached kinetochore is enough to inhibit the onset ofanaphase throughout the cell. Laser ablation of thiskinetochore relieves the mitotic delay10. Equally impres-sive manipulations of chromosomes in insect spermato-cytes showed that tension exerted across kinetochoresduring mitosis or unresolved crossovers during meiosiswere enough to satisfy the spindle checkpoint11. It wastherefore no surprise that first Mad2 (REFS 12,13) andthen all of the vertebrate Mad, Bub and Mps1 proteins(with the exception of Bub2) were localized to kine-tochores. What they are actually doing there is lessclear. Kinetochores are the site at which the chromo-some–microtubule interactions are monitored and arethe source of checkpoint signals. In addition, kineto-chores are a key effector site, because sister-chromatidseparation must be prevented both locally and globally.Kinetochore localization of a checkpoint protein there-fore reveals little about its function.

Box 1 | Checkpoints

Checkpoint controls (also known as surveillance mechanisms)ensure the dependency of cell-cycle transitions on thecompletion of earlier events. They consist of three distinct setsof functions: sensors (which look out for defects and emit asignal); signal-transduction cascades (checkpoint signals needto be transmitted throughout the nucleus or cell); andeffectors (a target is regulated to delay cell-cycle progression).

Checkpoints were first defined genetically by Weinert andHartwell, with the isolation of rad9 mutants that are defectivefor the DNA-damage checkpoint97,98. Other checkpointsmonitor the completion of DNA replication, spindle-poleposition and spindle orientation. In the spindle checkpoint,cells monitor the interaction between chromosomes andmicrotubules, which takes places at highly specialized regionsof chromosomes known as kinetochores4. Unattachedkinetochores emit a signal that is transmitted throughout themitotic machinery to inhibit the onset of anaphase.

Other checkpoints act during mitosis. In budding yeast, aBub2-dependent pathway monitors the position of spindle-pole bodies99–102 and only when the mother spindle pole103 hasentered the bud is the completion of anaphase permitted. Thispathway clearly regulates the initiation of septation in fission yeast104 but its conservation and function in vertebrate cellsremain to be clearly shown. In fission yeast, a checkpoint that monitors spindle orientation has recently been described.This is activated if the actin cytoskeleton is perturbed during mitosis105.

Cell-biologicaldefect

Cell-cycledelay

Metaphase Anaphase

Sensors

Signal transduction

Effector



R E V I E W S

systems supports the idea that Mad2 localization tokinetochores correlates mainly with tension duringmeiosis and with attachment during mitosis. Inmaize, a correlation has been observed betweenmicrotubule attachment and Mad2 disappearanceduring mitosis, but the dissociation of Mad2 frommeiotic kinetochores occurs at the same time as theloss of the 3F3/2 PHOSPHOANTIGENS, indicating that tensionrather than occupancy is important28. In the absenceof tension, Mad2 disappears from attached kineto-chores in PtK1 and HeLa cells29–31, whereas the ten-sion-sensitive 3F3/2 phosphoantigen32 remains on allkinetochores29. Low concentrations of vinblastin pre-serve kinetochore–microtubule connections but activatea Mad2-independent spindle checkpoint in HeLa cells,with no Mad2 at kinetochores30. By contrast, Bub1 andBubR1 remain at kinetochores in vinblastin- or noscap-ine-treated cells, suggesting that these proteins might beinvolved in the pathway that senses tension30,31,33.

Attachment versus tension. The view that occupancy isenough to inactivate the Mad2 checkpoint might be toosimplistic. Syntelic chromosomes (chromosomes withboth sisters attached to the same pole) often retainMad2 at their kinetochores34, suggesting that attach-ment is not the end of the story in Mad2 localization. Inaddition, although Mad2 is not found on attached kine-tochores in taxol-treated cells, it is still required to main-tain a mitotic arrest. This, however, might be due to thepresence of a small proportion of unattached kineto-chores29. In budding yeast, it has been shown that Mad2is required for cells to respond to lack of attachment ortension during both meiosis35 and mitosis36. Mad1, Rodand Zw10 (as well as Mad2) are removed from kineto-chores on attachment during mitosis, whereas Bub1,Bub3, BubR1 and CENP-E are only moderatelydepleted17,29,30,33,37–42.

Although this has led to the proposition that this lastgroup of proteins might be involved in tension sensing,the application of quantitative approaches to fluores-cence measurements reveals a more complex picture.For instance, although Bub1 is actively recruited tokinetochores when tension is released using taxol orvinblastin30, it is asymmetrically distributed at kineto-chores of mono-oriented sister-chromatid pairs, withdecreased levels on the attached kinetochore33. Theseresults imply that Bub1 starts to dissociate from kineto-chores upon attachment and that its kinetochore levelsare regulated by both tension and attachment.Significantly, the distribution of BubR1 is not affectedby attachment, consistent with its possible involvementin sensing tension33.

The Aurora kinases are conserved regulators ofmitosis and cytokinesis43. Recent studies identified theIpl1–Sli15 complex (the budding-yeast homologue ofthe Aurora-B–INCENP complex) as a tension-sensi-tive activity that acts by severing kinetochore–spindleinteractions until proper bipolar orientation is estab-lished44,45. Apparently, Ipl1 corrects improper attach-ment by increasing the turnover of microtubule–kine-tochore interactions. The effects of Ipl1 on the spindle

Tension. There have been several attempts to distinguishthe relative effects of kinetochore tension and occupancyon checkpoint silencing, but there is as yet no definitiveview of how the checkpoint is regulated by these phe-nomena.Analysis of Mad2 localization in several cellular

Figure 1 | Key steps in the regulation of mitotic progression. After replication, the two copiesof the genome are held together by complexes of proteins known as cohesins. In highereukaryotes, cohesin is first removed from chromosome arms. Residual cohesion at thecentromere region is enough to prevent sister-chromatid separation. At the onset of anaphase,these complexes must be disrupted through the proteolytic cleavage of a cohesin subunit(Scc1)94. This is carried out by a caspase-related activity known as separase. The timing ofseparase action is the key control point for the onset of anaphase, so how is this regulated?Perhaps the major mode of regulation is that, for most of the cell cycle, separase is inhibitedthrough direct association with a protein called securin. Securin levels are themselves regulatedby proteolysis. Securin destruction is carried out by the proteasome and is therefore ubiquitinmediated. Polyubiquitin chains are added to securin by an E3 ubiquitin ligase known as theanaphase-promoting complex or cyclosome (APC). An accessory factor, typically known asCdc20, is required for this and is a key target of the spindle checkpoint6,7. In addition, Scc1 onlybecomes a good substrate for separase-dependent cleavage once it has been phosphorylatedby Polo kinase95. An additional pathway regulates separase activity — it must be phosphorylated,probably directly by cyclin-dependent kinases, before it can efficiently cleave cohesin96. Whetherthis is also regulated by a checkpoint remains to be shown. An important regulatory mechanismis provided by the spindle checkpoint. During prometaphase, unattached kinetochores arebelieved to generate a ‘wait anaphase’ signal that results in the formation of Cdc20 complexeswith Mad2 and BubR1–Bub3. A single quaternary complex containing Mad2, BubR1–Bub3 andCdc20 might be formed. The interaction of Mad2 and BubR1–Bub3 with Cdc20 prevents theAPC from ubiquitylating securin, thus ultimately preventing the activation of separase and loss ofcohesion. The attainment of bipolar attachment at metaphase extinguishes the ‘wait anaphase’signal, triggering the attachment of polyubiquitin chains onto securin. This leads to separaseactivation, proteolytic degradation of cohesin and, finally, to anaphase.

P

Mad2

Mad2P P P

P P

P

Separase

Securin

Separase

Separase

Securin

APC

P P

P

P

APC

Cdc20

Cdc20

BubR1Bub3

BubR1Bub3SecurinUb

UbUb

Ub

Wait anaphase!

Prometaphase

Metaphase

Anaphase

Cohesin

Attached kinetochore

Unattached kinetochore

Microtubules

Ubiquitin

Phosphorylation



R E V I E W S

microtubule–kinetochore interactions, occupancy isexpected to be lost at the same time. Because ipl1mutants cannot clear faulty contacts that do not pro-duce tension, continued microtubule occupancy mightprevent checkpoint activation.

Budding yeast is characterized by point centromereswhose kinetochores attach to a single microtubule48, andso bipolar attachment and tension will occur at the sametime. Is an attachment-severing machine similar to Ipl1’s

checkpoint have also been investigated46. In buddingyeast, lack of tension in the presence of attachment canbe introduced by completely preventing DNA replica-tion47, and this condition activates the spindle check-point36. In ipl1 mutants, the spindle checkpoint doesnot respond to this condition, implicating Ipl1 as partof the tension-sensing machinery of the spindle check-point46. However, there is another possible explanation.If Ipl1–Sli15 reacts to a lack of tension by severing

Table 1 | Checkpoint components and complexes

Component Main structural features Comments

Mad1 Coiled-coil Recruits Mad2 at kinetochoresIts levels are not cell-cycle regulated

Mad2 Horma domain122 Binds Mad1 and Cdc20Its levels are not cell-cycle regulated

BubR1 (Mad3) Serine/threonine kinase Binds Bub3, capable of autophosphorylationIts levels are not cell-cycle regulatedYeast Mad3 lacks the kinase domain

Bub1 Serine/threonine kinase Reported substrates include Bub3 (REF. 107), Mad1 (REF. 81) andadenomatous polyposis coli (REF. 121)Its levels are cell-cycle regulated33

Bub3 Seven WD40 repeats* Interacts with Bub3-binding domains in Bub1 and BubR1Its levels are not cell-cycle regulated

Mps1 Dual-specificity kinase Also known as T-cell tyrosine kinase (TTK)Role in the recruitment of checkpoint proteins at kinetochoresReported substrates include Mad1, Spc110 (also required for spindle-pole-body duplication in budding yeast)

CENP-E Kinesin-like plus-end- Substrate of mitogen-activated protein kinase123

directed motor Interacts with microtubules and BubR1Yeast kinesin homologues unclear

Ipl1 Aurora kinase Clear role in ensuring bipolar orientation44

Proposed to sense tension at kinetochores46

Zw10 None described Probably complexed to RodImportant for localizing dynein at kinetochoreNo clear yeast homologue

Rod None described Possibly complexes to Zw10Important for localizing dynein to kinetochoreNo clear yeast homologue

Mitogen- Kinase Important in many signal-transduction pathways, but role in activated protein checkpoint unclearkinase Activates p90Rsk, which in turn activates Bub1 during Xenopus

oocyte maturation124

Complex Comments

Mad1–Mad2 Possibly located at nuclear pore complex in interphase82. Very stable except at kinetochores, where it might be turned into a high-turnover complex

Mad2–Cdc20 Forms in vitro using purified components but will not form in cells unless Mad1 is presentInhibits the ability of Cdc20 to activate APC

Bub3–Bub1, Bub3–BubR1/Mad3 Bub3 might form constitutive interactions with Bub1 andBubR1/Mad3. Bub3 purifies with BubR1, suggesting that thecomplex is stable

Mad2–Cdc20–BubR1/Mad3–Bub3 Has been observed in budding yeast and HeLa cells. Might be crucial for checkpoint inhibition of APC

BubR1–CENP-E Stoichiometric complex. Possible link between attachmentmachinery to the spindle checkpoint

BubR1–Cdc20 Forms in vitro using purified components. Inhibits the ability of Cdc20to activate APC

Zw10–Rod Direct interaction has not been shown, but the two proteins purify in a complex125

*The crystal structure of budding yeast Bub3 has recently been solved (David Wilson, University of California, Davis, personalcommunication) and shown to consist of the classic arrangement of seven blades, forming a propeller structure. APC, anaphase-promoting complex.



R E V I E W S

In cells with regional centromeres, whose kinetochorescapture multiple microtubules, the attachment processcould proceed through many steps, with a weak-attach-ment phase followed by a tight-attachment phase. Weakattachment might be lost often, whereas strong attach-ment would ensue only after bipolar orientation hadbeen attained51,52.

Dynamic checkpoint pathwaysMad–Bub dependencies at kinetochores. The picture thatemerges is that the kinetochore serves as a site at whichdifferent checkpoint complexes are brought into closeproximity,perhaps stimulating post-translational modifi-cation and even the exchange of partners (see BOX 2 andbelow). The nature of the direct molecular interaction(s)between checkpoint proteins and kinetochores is, how-ever, poorly understood. To date, the only well-docu-mented interaction is between the outer-plate kinetochoreprotein CENP-E (a kinesin-like protein for which there isno clear yeast counterpart) and the BubR1 checkpointprotein17–20. However, kinetochore recruitment of BubR1is a relatively late event40 and is therefore unlikely to beabsolutely required for the localization of other check-point proteins.

The network of interactions and interdependen-cies of checkpoint proteins is better defined for kine-tochore localization. Mad2 requires Mad1 (REF. 53),mammalian Bub1 and BubR1 require Bub3 (REF. 39),and fission yeast Mad3 requires Bub1, Bub3 andMph1 (the Mps1 homologue)54. Xenopus Bub1 isrequired for Bub3, Mad1, Mad2 and CENP-E localiza-tion55; Xenopus Mps1 is required for CENP-E, Mad1and Mad2 localization56; and it has recently beenshown that immunodepletion of Xenopus BubR1reduces the levels of Bub1, Bub3, Mad1, Mad2 andCENP-E at kinetochores57. These observations indi-cate that checkpoint components are dependent onone another, albeit to varying extents, for robust kine-tochore localization. We think that one or morecheckpoint proteins (probably Bub1 and/or Mad1)probably act as a scaffold for the recruitment of oth-ers. Bub1 is required for the kinetochore recruitmentof many other proteins55 and it has also been shownbiochemically to associate with Bub3 and Mad1 (REF. 58).The Bub1–Mad1 interaction is observed only whenthe checkpoint is active. The amino terminus ofXenopus Mad1 (residues 1–445) has recently beenshown to be sufficient for kinetochore targeting59.This part of Mad1 does not bind to Mad2 or Bub1,and it is a good candidate for direct interaction with akinetochore protein.

Mad2 exchange at kinetochores. The association ofMad2 with kinetochores is extremely dynamic. Usingfusion constructs between Mad2 and green fluorescentprotein (GFP), and microinjected Alexa-labelled Mad2conjugates, it was shown by fluorescence recovery afterphotobleaching that the half-life of Mad2 on an unat-tached kinetochore is ~24 s (REF. 60). Turnover rates forother checkpoint proteins (BubR1, Bub3 and Cdc20)are of a similar magnitude, if not higher61.

conserved in vertebrate cells? When a kinase-inactive,dominant-negative Aurora-B mutant was expressed innormal rat kidney cells, kinetochore–microtubule inter-actions were profoundly affected and Mad2 was prema-turely released from kinetochores49. Injection of anti-Xenopus Aurora B antibodies into Xenopus tissue culture(XTC) cells perturbed kinetochore–microtubule inter-actions and chromosome congression. Antibody injec-tion into XTC cells or antibody addition to Xenopus eggextracts perturbed checkpoint signalling50. However,once again it is unclear how direct these effects ofAurora B inhibition are on the checkpoint because bothdynein and CENP-E were depleted from kinetochores49.

Figure 2 | Kinetochores and checkpoint signalling. a Electron microscopy reveals distinctplates within the kinetochore, and immunoelectron microscopy has localized molecules to theregions indicated. The dynamic plus ends of kinetochore microtubules interact with the outercorona and outer plate. b | There is a dynamic exchange of checkpoint proteins at unattachedkinetochores. Mad2, and other checkpoint proteins, are recruited from soluble pools and interactwith a scaffold, of which Bub1 and Mad1 could form an important part. The checkpoint proteinsare then released from kinetochores, perhaps in a quaternary complex(Mad2–Cdc20–BubR1–Bub3), to act as an APC inhibitor. c | Bipolar attachment of sisterkinetochores leads to stretching of the centromeric DNA and tension. How this is monitoredremains unclear but the Ipl1 (Aurora) kinase has been proposed to sense this tension. d | Checkpoint silencing. Several checkpoint proteins have been shown to move fromkinetochores along microtubules to the spindle poles, where they are released. This pathwaydepends on dynein, and might also require the functions of Rod, Zw10 and CENP-E.

a Kinetochore

b Dynamic exchange c Tension

d Checkpoint silencing

Unattachedkinetochore

Centromericheterochromatin(INCENP/Aurora)

Fibrous corona(CENP-E, dynein)

Inner plateOuter plate (BubR1)

Microtubules

Bub1Mad1

Mad2Cdc20BubR1–Bub3APC inhibitor

Mad2–Mad1

BubR1–Bub3

Cdc20

Bub1–Bub3

Bipolarattachment

Tension

Dynein-dependent movementof checkpoint proteins

Spindle pole

Mad2 (Rod/Zw10/CENP-E)



R E V I E W S

in checkpoint silencing, by turning off the spindlecheckpoint once all chromosomes are attached to thespindle (FIG. 2d). Because kinetochores are catalytic gen-erators of the ‘wait anaphase’ signal, a dynamic cycle ofrecruitment and release might remodel their biochemi-cal composition step by step during the attachmentprocess, finally leading to checkpoint inactivation.

Several components of the kinetochore outerdomain, including Mad2, BubR1, CENP-E, the 3F3/2phosphoantigens and Rod, undergo a dynamic redistri-bution cycle during checkpoint activation60,65. Theseproteins are continuously removed from kinetochoresby a dynein-dependent mechanism. At the same time,the kinetochore pool is replenished by an ATP-depen-dent step (at least while the checkpoint is active). Ifdynein is inhibited, Mad2 accumulates at kinetochoresand keeps cycling with a high turnover rate, showingthat distinct mechanisms regulate the interaction ofMad2 with kinetochores60,65,66. Furthermore, the newobservations argue that attachment per se is not enoughto arrest or silence checkpoint signalling, and that thedynein-mediated redistribution of Mad2 and othercomponents away from kinetochores as attachmentproceeds is essential. Indeed, in PtK1 cells, there is a pre-cise delay between the depletion of Mad2 from the kine-tochore of the last-congressing chromosome and theonset of anaphase (10.7±1.2 min)13,29,60,67,68. This timecorrelation does not imply that the removal of Mad2from kinetochores sets the timing of anaphase, becausethis is also determined by other molecular eventsincluding securin destruction and cohesin cleavage.

As reported above, Zw10 and Rod are required tolocalize dynein at kinetochores, which in turn isrequired for checkpoint inactivation. Thus, a pro-longed activation of the Mad2 checkpoint might beexpected upon Zw10 and Rod inactivation, but theinactivation of Zw10 and Rod prevents cells from acti-vating the spindle checkpoint26,27. As yet, there is noknown interaction between the Zw10–Rod andMad–Bub pathways. Localization of Mad1, Mad2,BubR1, Bub3 or CENP-E at kinetochores is notaffected by depletion of Zw10–Rod, and Bub1 is notrequired for the interaction of Zw10 and Rod withkinetochores26,41,69. It is therefore possible that Zw10and Rod belong to a parallel branch of the checkpointmachinery that co-operates with the Mad2 pathway togenerate the ‘wait anaphase’ signal. However, the mole-cular nature of the checkpoint signal that is generatedby the Zw10–Rod pathway is unknown.

CENP-E could also deplete Mad2 from attachedkinetochores. When human CENP-E is depleted usingantisense oligonucleotides, or when anti-CENP-Eantibodies are microinjected, chromosomes do notalign efficiently. Even though kinetochores areattached to microtubules in these cells, the cells havestrong Mad2 staining and the spindle checkpoint isactivated19,21. Paradoxically, in Xenopus extracts,CENP-E was shown to be required for checkpointactivation and maintenance, and for the localizationof several checkpoint proteins (including Mad2) atkinetochores20. The reasons for these discrepancies are

These studies of Mad2 dynamics led to the proposalthat the kinetochore acts as a catalytic site for the pro-duction of the checkpoint signal. It is thought thatMad2 is ‘activated’ at kinetochores to enable it to effi-ciently inhibit Cdc20–APC function. It will be impor-tant to determine which checkpoint proteins binddirectly to Mad2 at kinetochores and whether they leaveseparately or as a complex. Bub1, Mad1 and Mps1 are allknown to be required for Mad2 kinetochorelocalization53,55,56, but there is no evidence that any ofthose proteins have a direct interaction with Cdc20 or adirect role in APC inhibition. We favour the hypothesisthat these checkpoint components have signalling func-tions and aid in the transfer of Mad2 onto Cdc20.

The other checkpoint protein that has been shown toinhibit Cdc20–APC function directly in vitro is BubR1(REFS 62,63). Does BubR1–Mad3 leave kinetochores alongwith Mad2? Although it has been found in direct associ-ation with both Mad2 and Cdc20 in extracts, there issome doubt about the source of this complex andwhether kinetochores are really involved in its produc-tion. For example, it was found that a humanMad2–Cdc20–BubR1–Bub3 complex could be purifiedfrom interphase extracts62, and that the budding yeastMad2–Cdc20–Mad3–Bub3 complex can still be gener-ated in ndc10-1 mutants64, which are thought to lackkinetochore structures entirely.

However, the kinetochore could be the main catalyticsite for the production of such complexes and/or itcould modify them in some undefined way. It will beimportant to determine whether the APC cyclesthrough unattached kinetochores and, if so, how neces-sary this is for checkpoint function.

Mad–Bub dynamics and checkpoint silencing. In addi-tion to the rapid turnover of Mad2 described above,there is a microtubule-dependent pathway involved inthe movement of checkpoint proteins away from kine-tochores. This pathway has been proposed to have a role

Box 2 | Checkpoint kinases and phosphorylation

Genetic studies in budding yeast have implied that the Mps1 (REF. 106) and Bub1 (REF. 107) kinases act at or near the top of the checkpoint pathway, because theoverexpression of either wild-type (MPS1) or mutant (BUB1–5) forms of their genesrequires all other Mad and Bub proteins to induce a metaphase arrest80,108.

Surprisingly, the importance of the kinase domains for Bub1 and BubR1 checkpointfunction has been brought into doubt. In Xenopus laevis egg extracts, kinase-deadproteins can efficiently rescue immunodepleted extracts55,57; kinase-dead humanBubR1 efficiently inhibits the anaphase-promoting complex (APC) in vitro63; and, inbudding yeast, a kinase-dead bub1 mutant is capable of robust checkpoint function109.Yeast Mad3 proteins lack the carboxy-terminal kinase domain found in their vertebrateBubR1 homologue54,71. It remains to be shown whether the Bub/BubR1 kinase activitiesdo have checkpoint roles, perhaps in the amplification of subtle defects or maintenanceof prolonged checkpoint arrests. Mps1 kinase activity is clearly required for spindle-checkpoint function56,110.

Most of the checkpoint proteins are phosphorylated (Mad1 (REF. 111), Mad2 (REF. 79),BubR1 (REFS 33,57), Bub1 (REFS 55,58) and Mps1 (REF. 110)) but, in general, it is not knownwhere this takes place or whether it is important for checkpoint function. It has beenshown that the hyperphosphorylated forms of Bub1 and BubR1 are enriched onchromosomes57 and that this modification depends on Mad1 but not on Mad2 (REF. 57).



R E V I E W S

unclear and more studies will be needed to assess thepossible role(s) of CENP-E in the checkpoint. Mad2has recently been reported to move onto the spindle infission yeast cells late in mitosis70, but the proteins thatare required for this are currently unknown.

Mad behaviour and interactions with Cdc20What are the molecular bases for the mechanisms thatunderlie the dynamic behaviour of spindle-checkpointproteins? A first step towards such an understandingcomes from the observation that Mad2 is a componentof both the ‘wait anaphase’ signal and the catalyticmachinery that is required to generate it. Two relatedinteractions of Mad2 — with Cdc20 and with Mad1 —are central to the checkpoint (FIG. 3).

The Mad2–Cdc20 complex is regarded as a promi-nent component of the ‘wait anaphase’ signal. FastMad2 kinetics observed at the kinetochore might repre-sent its binding to Cdc20 and subsequent release fromkinetochores in the form of a Mad2–Cdc20 complex.The interaction of Mad2 with Mad1 is absolutelyrequired to form a Mad2–Cdc20 complex in vivo6,59,64,71.Mad2 recruitment to kinetochores depends on Mad1,supporting models in which the kinetochore is the siteat which the Mad2–Cdc20 complex forms. Duringcheckpoint silencing, it is possible that the dynein-dependent mechanism acts directly on the Mad1–Mad2complex, to reduce its concentration at attaching kine-tochores and so progressively inhibit their ability to gen-erate the ‘wait anaphase’ signal. Mad1-binding sitesseem to be absent from attached kinetochores. Whetherthey are simply masked by bound microtubules or areremoved, perhaps via the dynein-dependent pathway,remains to be seen.

The mechanism of Mad2 ligand binding wasrevealed by structures of the Mad2–MBP1 (Mad2-bind-ing peptide 1) complex72 and of the Mad1–Mad2tetramer73. Ligand binding elicits a striking conforma-tional change in the Mad2 carboxy-terminal tail, remi-niscent of the buckling–unbuckling cycle of a safetybelt. Building on previous work6,7,74–76, it was shown thatCdc20 and Mad1 share a conserved ten-residue Mad2-binding motif that binds the same Mad2 pocket72,73.

These findings establish that Mad1 and Cdc20 arecompetitive Mad2 ligands and identify Mad1 as both apositive regulator and a competitive inhibitor of theMad2–Cdc20 complex. This contradictory role might beexplained by the hypothesis that Mad1-bound Mad2 isthe only source of Mad2 that is available for Cdc20 bind-ing, despite the presence of cytosolic pools of Mad2 (seebelow). The remarkable stability of unperturbedMad1–Mad2 might prevent Mad2 from reaching Cdc20when the checkpoint is inactive53,74,77,78. Mad1-mediatedlocalization of Mad2 at the spindle might increase thelocal concentration of Mad2 to push complex forma-tion, possibly explaining the positive role of Mad1 in theinteraction59,63. Kinetochore-localized assembly factorscould recognize the Mad1–Mad2 complex and favoursubsequent Mad2 exchange onto Cdc20. TheBub1–Bub3 complex, for instance, forms a complex withMad1, disruption of which abrogates the checkpoint58.

Figure 3 | Structural insights into Mad2 function. The carboxy-terminal tail of Mad2 behaves likea molecular safety belt. A | Alignment of Mad2-binding motifs found in Mad1 and Cdc20. Ba | Ribbon diagram of unbound Mad2. The carboxy-terminal region is shown in red and theposition of the amino and carboxyl termini are indicated. b | The carboxy-terminal tail opens to let theMad1 ligand (blue) occupy the Mad2-binding site. c | After entering the site, the carboxy-terminal tailstarts closing and finally achieves the locked state (d). The structures in a and d have beendetermined experimentally73,75; the intermediate has not been structurally characterized. e | Thestructure of Cdc20 (cyan) is unknown but, as shown in the inset, Mad1 and Cdc20 share a Mad2-binding motif, which is expected to be structurally equivalent to that present in Mad1. The unknownparts of the structure are indicated by dots. To let Cdc20 in, the carboxy-terminal tail must open topromote the release of Mad1. f | After the exchange reaction, the carboxy-terminal tail can lockagain (g). Although the relative affinities of Mad1 and Cdc20 for Mad2 are determined only by theircontacts in the complex, the opening of the carboxy-terminal tail that is required for the exchangereaction slows down the reaction73. We suggest that there are mechanisms that regulate thetransition of the carboxy-terminal tail of Mad2 to favour the exchange reaction. In these pictures, onlyhalf of the tetrameric 2:2 Mad1–Mad2 complex is shown. Figure modified with permission from REF. 73. +, positively charged residue; φ, hydrophobic residue; X, any amino acid.

Hs_Mad1 (541) KVLHMSLNP (549)Dm_Mad1 (551) KVVHFSENP (560)Sc_Mad1 (581) RILQLRDGP (589)Hs_Cdc20 (129) KILRLSGKP (137)Dm_Cdc20 (176) KVVYSIKTP (184)Sc_Cdc20 (201) RILQYMPEP (209)Consensus +φφXφXXXP

A Ba

Mad2unbound

Lockopen

Lockopen

Mad1

N

N

N

NN

NN

C

C

CC

C

C

C

Mad1IN

Lockclosed

Lockclosed

Cdc20

Cdc20IN

Mad1OUT

b c

d e

f g



R E V I E W S

and Mad2 HAPLOINSUFFICIENCY in mice could causetumours (BOX 3)84. The fact that the balance betweenMad1 and Mad2 is crucial for the proper working of thecheckpoint might also explain the contradictory effectsof Mad1 or Mad2 overproduction on cell-cycle progres-sion72,78,85. For instance, Mad1 might promote cell-cyclearrest at low concentrations, but impair the checkpointat high concentrations.

Deconstructing the ‘wait anaphase’ signalWhat is the destiny of the ‘wait anaphase’ signal that isgenerated at unattached kinetochores? As it diffusesaway from unattached or partially attached kineto-chores, this signal could simply encounter and inhibitAPC. Alternatively, it might be captured by spindlemicrotubules and be transported to an ‘integration cen-tre’ that controls anaphase progression, possibly the cen-trosome. This hypothesis was suggested by het-erokaryon experiments, which showed that acheckpoint signal cannot diffuse away from the spindlefrom which it originated86.

If the conditions for the formation of theMad2–Cdc20 complex directly from cytosolic pools donot exist, what prevents its falling apart after releasefrom kinetochores? One possibility is that the ‘safetybelt’ binding mechanism acts as a kinetic trap to prolongthe half-life of the complex, despite the relatively lowaffinity of the interaction73. It is also possible thatMad2–Cdc20 is stabilized by incorporation into a largercomplex. When submitted to size-exclusion chromatog-raphy, most APC-inhibitory activity in HeLa cells frac-tionates in a 450–700-kDa peak that contains most ofthe BubR1 (REFS 62,63,87). Attempts to define the compo-sition of this fraction have yielded conflicting results,possibly as a result of significant differences in thepurification procedures. In one case, BubR1–Bub3 wasidentified as its predominant component, with sub-stoi-chiometric amounts of Cdc20 (REF. 63); in another,Mad2, Cdc20, BubR1 and Bub3 were detected asapproximately stoichiometric components of the APCinhibitor, and attributed to this complex (named MCCfor mitotic checkpoint complex) a substantially strongerAPC inhibition activity than to Mad2 alone62. An equiv-alent quaternary complex containing Mad2, Cdc20,Mad3 and Bub3 has been observed in budding yeast64,71.

It has recently been shown54 that fission-yeastMad3 is required for Mad2 to inhibit APC in vivo andthat immunodepletion of XBubR1 from egg extractsimpairs the interaction between Mad2, Bub3 andCdc20 (REF. 57). However, the question of whetherMad2 and BubR1–Bub3 enter a quaternary complexwith Cdc20 is open, because other groups havereported their inability to co-precipitate Mad2 andBubR1 from mammalian cells19,30,87. Although recom-binant BubR1 inhibits Cdc20-mediated APC activa-tion with a higher specific activity than Mad2 in vitro,these proteins show co-operative Cdc20 binding andAPC inhibitory effects, and they both prevent directbinding of Cdc20 to the APC, suggesting that they actby titrating out Cdc20 (REFS 63,87). This is difficult toreconcile with reports showing that Mad2 and

Full-length Cdc20 binds Mad2 with a lower affinity thanfragments that contain only the Mad2-binding site63,76,suggesting that Cdc20 might change its conformation tobind Mad2. Furthermore, the bulk of Cdc20 is bound tocellular chaperones, hinting that it might be partly orcompletely unfolded79.

Thus, a Mad1–Mad2 complex might have differ-ent fates depending on the state of the checkpoint. Alow-turnover cytosolic Mad1–Mad2 complex couldbe recruited to kinetochores and turned into a high-turnover complex that releases Mad2 to Cdc20 whilereplenishing itself with Mad2 from a cytosolic pool. Aconformational change in the tetrameric assemblymight be required for Mad2 release73, and it is possi-ble that a phosphorylation–dephosphorylationswitch regulates this transition. Human Mad2 isphosphorylated during mitosis79. Mps1 phosphory-lates Mad1 in budding yeast80, whereas human Mad1is a substrate of Bub1 in vitro81. Mitotic phosphoryla-tion of human and Xenopus Mad1 has not beenobserved53,82, but this might be due to the transientnature of the modification. As an alternative mecha-nism, it has been suggested that oligomerization ofMad2 is important for checkpoint function83, butrecent evidence has questioned this73.

As already mentioned, there is a cytosolic pool of freeMad2. This might be important to maintain a highturnover rate of Mad2 at the spindle, because itsdecrease has been shown to impair the checkpoint59.Consistent with this, Mad2+/− cells become ANEUPLOID

ANEUPLOID

Cell in which one or morechromosomes are missing orpresent in more than their usualcopy number.

HAPLOINSUFFICIENCY

Loss of a functional allele from adiploid cell results inhaploinsufficiency if the productof the remaining allele isinsufficient to perform itsfunction correctly.

Box 3 | Spindle-checkpoint defects and disease

Genetic defects in the spindle checkpoint lead to chromosome loss during mitosis andmeiosis112,113, and could therefore be a cause of aneuploidy and birth defects. In buddingyeast, the Mad and Bub proteins do not contribute equally to chromosome segregationfidelity. Deletion of either Bub1 or Bub3 has the greatest effect, and these two proteinsmight have roles in chromosome segregation in addition to their checkpoint function109.

Although the spindle checkpoint is not essential for growth in yeast, the two reportedmouse knockouts (Mad2 and Bub3) have revealed that it is required for embryonicviability in mammals114,115. After embryonic day 5 or 6, cells of these embryos accumulatemitotic errors and undergo apoptosis. Why is the spindle checkpoint essential inmammals? Disrupting Bub1 and Mad2 function (with dominant alleles or antibodies)has been shown to accelerate mitotic exit in vertebrate cells38,67. Such shortened mitosesprobably have increased error rates and lead to aneuploidy. In Caenorhabditis elegans,embryogenesis and larval development can proceed in the absence of spindle-checkpointfunction. However, there are so many segregational errors that the worms are sterile andshort-lived116. Similarly, in Drosophila, loss of bub1 function results in an accumulationof mitotic abnormalities and lethality at the larva–pupa transition69.

Although checkpoint nulls are unlikely to be observed in humans, haploinsufficiency ofone checkpoint component (Mad2) has been reported84, with very interestingphenotypes. Deletion of one Mad2 allele results in a defective checkpoint in humancancer cells and primary mouse embryonic fibroblasts, and Mad2+/− mice are susceptibleto lung cancer in later life. Bub1 and BubR1 mutations have also been reported to co-operate with BRCA2 deficiency in the pathogenesis of breast cancer117. In addition,dominant Bub1 mutants were suggested to be responsible for the chromosome-instability phenotype of certain colorectal cancer cell lines118, although this finding hasrecently been disputed because the cell lines that harbour these mutations have beenfound to be capable of quite a robust checkpoint response119. It is likely that additionalmutations within these cell lines, such as the commonly found truncations of theadenomatous polyposis coli protein, also contribute to chromosomal instability120,121.



R E V I E W S

assays with purified components should help to clarifythese issues.

ConclusionA great deal remains to be understood of the molecu-lar basis of the dynamic signalling that is carried outby spindle-checkpoint proteins. However, the struc-tural information that is currently available has alreadyshed significant light on the molecular mechanism ofMad2 function. In the future, this will no doubt becombined with the powerful techniques of fluores-cence microscopy and biochemical reconstitution togive a much deeper understanding of this importantcell-cycle control.

Note added in proofRecent findings indicate that Mad2 and BubR1 act in asingle, common pathway, which produces a mitoticdelay in PtK cells in hypothermic conditions127. Suchcells have normal numbers of kinetochore micro-tubules, but with decreased tension. This finding sup-ports REFS 29,36 in arguing that Mad2 is required for cellsto respond to the lack of tension. In addition, it has nowbeen shown that when human Hec1 (Ndc80) isdepleted from cells by siRNA, neither the Mad1/Mad2complex nor the Mps1 kinase can be detected at kineto-chores, yet the spindle checkpoint is activated128.

Bub3–BubR1 are incorporated into a complex withCdc20–APC during mitosis18,76,78,83,88–91.

Increased affinity of mitotic APC for Cdc20, possiblyas a consequence of APC or Cdc20 phosphorylation,might also be important for the recruitment step8,92.Consistent with this, it was found that interphase MCCcan inhibit APC that is purified from mitotic — but notinterphase — cells62. Most checkpoint studies haveassumed that APC complexes in the cell are homoge-nous, but recent work in Drosophila has questioned thisassumption93. GFP–Cdc16 and GFP–Cdc27 are bothincorporated into the endogenous APC, yet they arelocalized differently, and RNA INTERFERENCE experimentsindicate that they might even perform distinct func-tions. Clearly, such ideas need to be confirmed in othersystems, ideally with non-tagged APC subunits, but itraises the possibility that the spindle checkpoint onlyhas to inhibit a subpopulation of the total APC in cells.

Finally, how is inhibition of the APC relieved? Asdiscussed above, the dynein-mediated pathway — bywhich checkpoint proteins move from kinetochores tospindle poles — is one possible mechanism of pre-venting the production of APC inhibitory complexes.In addition, several Mad2 complexes have beenreported to be disassembled before mitotic exit70,90, andirreversible APC activation might ensue after therelease of BubR1–Bub3 and Mad2. Reconstitution

RNA INTERFERENCE

The process by which anintroduced double-strandedRNA specifically silences theexpression of genes throughdegradation of their cognatemRNAs.

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63. Tang, Z., Bharadwaj, R., Li, B. & Yu, H. Mad2-independentinhibition of APC–Cdc20 by the mitotic checkpoint proteinBubR1. Dev. Cell 1, 227–237 (2001).A checkpoint complex containing BubR1 and Bub3 ispurified from mitotic human cells and found tointeract with Cdc20 in the absence of other proteinsand to block the binding of Cdc20 to APC. Togetherwith ref. 62 this paper establishes a direct role ofBubR1 in Cdc20 binding and checkpoint activity.

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65. Wojcik, E. et al. Kinetochore dynein: its dynamics and role inthe transport of the Rough deal checkpoint protein. NatureCell Biol. 3, 1001–1007 (2001).Together with ref. 66, this paper shows that dyneinmight contribute to shutting off the metaphasecheckpoint, allowing anaphase to ensue. Acomplementary set of kinetochore proteins ismonitored relative to those described in ref. 66,revealing a general mechanism of checkpointinactivation.

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AcknowledgementsWe thank S. Piatti, A. Pidoux and V. Vanoosthuyse for critical read-ing of the manuscript. A.M. is an EMBO Young Investigator and aScholar of the Italian Foundation for Cancer Research. K.G.H. is aSenior Research Fellow of the Wellcome Trust.

Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/Aurora-B | BRCA2 | INCENP | MCAK | Zw10Saccharomyces genome database: http://genome-www.stanford.edu/Saccharomyces/Bub1 | Bub2 | Bub3 | Cdc20 | Ipl1 | Mad1 | Mad2 | Mad3 | Mph1 |ndc10-1 | rad9 | Sli15Flybase: http://flybase.bio.indiana.edu/Cdc16 | Cdc27 | Polo | RodAccess to this interactive links box is free online.


the spindle checkpoint: structural insights into dynamic signalling

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