REVIEW

Reconstituting the kinetochore–microtubule interface:what, why, and how

Bungo Akiyoshi & Sue Biggins

Received: 23 November 2011 /Revised: 15 January 2012 /Accepted: 16 January 2012 /Published online: 31 January 2012# Springer-Verlag 2012

Abstract The kinetochore is the proteinaceous complexthat governs the movement of duplicated chromosomes byinteracting with spindle microtubules during mitosis andmeiosis. Faithful chromosome segregation requires thatkinetochores form robust load-bearing attachments to thetips of dynamic spindle microtubules, correct microtubuleattachment errors, and delay the onset of anaphase until allchromosomes have made proper attachments. To understandhow this macromolecular machine operates to segregateduplicated chromosomes with exquisite accuracy, it is crit-ical to reconstitute and study kinetochore–microtubule inter-actions in vitro using defined components. Here, we reviewthe current status of reconstitution as well as recent progressin understanding the microtubule-binding functions ofkinetochores in vivo.

Introduction

Accurate partitioning of genetic material during mitosis andmeiosis is essential for all organisms to proliferate. Defects inthis process result in aneuploidy that can lead to cell death,cancer, or birth defects (Holland and Cleveland 2009;Schvartzman et al. 2010). In eukaryotes, chromosome segre-gation is directed by the kinetochore that assembles onto the

centromeric region of each chromosome and by dynamicmicrotubules that grow and shrink by incorporating or disso-ciating α/β tubulin subunits at the ends (Nicklas 1997;Cheeseman and Desai 2008; Santaguida and Musacchio2009; Lampert and Westermann 2011). Microtubules contin-uously switch from growth to shortening (catastrophe) andfrom shortening to growth (rescue), a property known asdynamic instability (Mitchison and Kirschner 1984). Kineto-chores govern chromosome movement by coupling to thesegrowing and shrinking microtubule tips. Faithful chromosomesegregation requires that sister kinetochores form bi-orientedattachments to spindle microtubules emanating from oppositepoles. In addition, attachment errors must be corrected toavoid mis-segregation. The spindle checkpoint monitors at-tachment status and delays the onset of anaphase until allchromosomes form correct bi-oriented attachments.

The simplest characterized kinetochore is found in bud-ding yeast where each kinetochore assembles onto a defined125 bp of centromeric DNA and binds a single microtubule(Winey et al. 1995; Westermann et al. 2007). In contrast,most organisms have much larger centromeres and theirkinetochores bind to multiple microtubules (Cleveland etal. 2003). Yet, the conservation of kinetochore proteinssuggests that the larger kinetochores of other eukaryotesmay be assembled from the repetition of the buddingyeast-type kinetochores called the repeat subunit model(Zinkowski et al. 1991; Joglekar et al. 2008). The core ofthe budding yeast kinetochore is composed of ∼40 proteins,most of which are present in multiple copies within eachkinetochore. It has been estimated that anywhere from ap-proximately 200 or more (Joglekar et al. 2006) to as many as500 (Coffman et al. 2011; Lawrimore et al. 2011) totalproteins constitute the core of the budding yeast kineto-chore. In addition, >20 additional regulatory proteins mod-ulate kinetochore functions, such as kinases and motor

Communicated by Erich Nigg

B. Akiyoshi (*)Sir William Dunn School of Pathology, University of Oxford,Oxford OX1 3RE, UKe-mail: bungo.akiyoshi@path.ox.ac.uk

S. BigginsDivision of Basic Sciences,Fred Hutchinson Cancer Research Center,1100 Fairview Ave N, PO Box 19024, Seattle, WA 98109, USA

Chromosoma (2012) 121:235–250DOI 10.1007/s00412-012-0362-0

proteins (Cheeseman and Desai 2008). The complexity anddynamic nature of the kinetochore makes it a daunting taskto understand how it accurately segregates duplicated chro-mosomes. The most basic kinetochore function is to mediatethe interaction between centromeric DNA and spindlemicrotubules. In this regard, kinetochores can be classifiedinto two functional parts: the inner kinetochore that bindscentromeric DNA and the outer kinetochore that bindsmicrotubules. Here, we focus on the kinetochore–microtu-bule interface. We refer readers to recent reviews on innerkinetochores (Buscaino et al. 2010; Black and Cleveland2011; Gascoigne and Cheeseman 2011; Perpelescu andFukagawa 2011; Verdaasdonk and Bloom 2011).

Kinetochore constituent lists

Extensive efforts over the last 30 years have led to the iden-tification of most kinetochore proteins in major model organ-isms (for detailed reviews, see (Westermann et al. 2007;Cheeseman and Desai 2008; Santaguida and Musacchio2009; Przewloka and Glover 2009; Perpelescu and Fukagawa2011). The following picture has emerged from these studies.Kinetochores consist of several subcomplexes that representfunctional units. Overall kinetochore architecture appears con-served from yeast to humans based on the conservation ofkinetochore proteins, as well as the similar stoichiometry andposition of subcomplexes across species (Joglekar et al. 2006;Schittenhelm et al. 2007; Joglekar et al. 2008; 2009;Wan et al.2009; Johnston et al. 2010). At the base of the kinetochore liesthe centromere-specific histone H3 variant, CENP-A (alsocalled CenH3) (Allshire and Karpen 2008; Mendiburo et al.2011). Although CENP-A forms specialized chromatin that isessential for kinetochore assembly, the exact composition andnature of CENP-A containing nucleosomes in vivo remainsunclear (Henikoff and Furuyama 2010; Black and Cleveland2011). The so-called constitutive centromere-associated net-work (CCAN: at least 16 components in human, 13 in bud-ding yeast) loads onto centromeric chromatin and is importantfor outer kinetochore assembly (Perpelescu and Fukagawa2011). The outer kinetochore consists of the conservedKMN network (KNL1, Mis12, Ndc80 subcomplexes) thatserves as a core microtubule-binding module, as well as othermicrotubule-binding subcomplexes (e.g., Dam1 complex infungi, Ska1 complex in mammals) (Cheeseman and Desai2008). It is estimated that there are ∼7 copies of KMN permicrotubule-binding unit per single CENP-A nucleosome(Joglekar et al. 2006; Furuyama and Biggins 2007; Joglekaret al. 2008; 2009; Wan et al. 2009; Johnston et al. 2010;Coffman et al. 2011; Lawrimore et al. 2011). In addition tothese core kinetochore components, many regulatory proteinsalso localize to kinetochores, such as spindle checkpoint pro-teins, microtubule-associated proteins (MAPs), motor pro-teins, mitotic kinases, and phosphatases (Cheeseman and

Desai 2008). As the list of kinetochore components is becom-ing complete, the next challenge is to understand how thesenumerous proteins function to form the macromolecular ma-chine that segregates chromosomes with exquisite fidelity. Invitro reconstitution of kinetochore functions using individualsubcomplexes as well as their ensembles is critical to achievethis goal.

What are the functions of kinetochores?

Lateral and end-on attachments, error correction,and the spindle checkpoint

A fundamental kinetochore function is to interact with dy-namic microtubules. Because the microtubule lattice offers amuch larger contact surface than its tips, kinetochores firstform lateral attachments, which are then converted to end-onattachments (Fig. 1) (Rieder and Alexander 1990; Tanaka etal. 2005). The fact that microtubule polymerization/depoly-merization occurs only at the tips means that kinetochoresmust stay bound to dynamic microtubule tips while allowingthe incorporation and dissociation of thousands of tubulinsubunits at the ends. This end-on attachment allows kineto-chores to translate the energy stored in dynamic microtubulesto move chromosomes upon depolymerization (Mandelkowet al. 1991; Grishchuk et al. 2005; Wang and Nogales 2005).End-on attachments also allow kinetochores to modulate mi-crotubule dynamics, which may be critical to regulate attach-ment stability (see below). The observation that tip-boundkinetochores appear to be under tension in vivo means thatkinetochores must form load-bearing attachments (Khodjakovand Rieder 1996; Waters et al. 1996). It is estimated that 0.4 to8 pN of force is transmitted to kinetochores per attachedmicrotubule in vivo (Nicklas 1988; Powers et al. 2009). Theinteraction between kinetochores and microtubules must bestable enough to maintain attachments throughout mitosis(which can last for more than 1 h), yet flexible enough toallow destabilization of erroneous attachments. Although aback-to-back positioning of sister kinetochores increases thechance of bi-orientation (Östergren 1951; Indjeian andMurray 2007; Sakuno et al. 2009), the formation oferroneous attachments is somewhat inevitable becausekinetochores are unable to tell from which pole anygiven microtubules emanate. For example, both kineto-chores can attach to microtubules from the same pole(called mono-oriented attachment, as shown in Fig. 2b),or one kinetochore can attach to microtubules from bothpoles (called merotelic attachment). Although it remainsunclear how often erroneous attachments are formed duringmitosis, many erroneous kinetochore–microtubule attachmentsare observed during prometaphase of meiosis I, suggesting thaterror correction is essential to achieve correct bi-orientation

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(Kitajima et al. 2011; Sakuno et al. 2011). How do cellsdistinguish bi-oriented attachments from erroneous ones?One distinct feature of bi-oriented attachment is the presenceof robust tension due to microtubule pulling forces that areopposed by the linkage between sister chromatids (Fig. 2a). Incontrast, less tension is produced when sister kinetochores aremono-oriented (Fig. 2b). Therefore, by destabilizing tension-less attachments and/or stabilizing attachments under full ten-sion, all chromosomes should be able to achieve bi-orientation.Classic micromanipulation studies in insect spermatocytes aswell as chromosome engineering experiments in budding yeastdemonstrated that tension indeed stabilizes attachments in vivo(Nicklas and Koch 1969; Nicklas andWard 1994; Dewar et al.2004). However, because protein–protein interactions are

typically destabilized by tension (Bell 1978; Merkel et al.1999), it is intriguing that cells rely on tension to stabilizekinetochore–microtubule attachments. As discussed later, theprevailing model for the underlying molecular mechanism thatleads to the destabilization of attachments is phospho-regulation by the Aurora B kinase. In addition, our recentreconstitution studies discovered another mechanism bywhichtension directly stabilizes attachments by modulating microtu-bule tip dynamics (Akiyoshi et al. 2010).

Cells must also coordinate the timing of anaphaseonset with chromosome alignment so that segregationoccurs only after all chromosomes have achieved bi-orientation. A surveillance mechanism, known as thespindle checkpoint, evolved for this purpose. Spindlecheckpoint proteins are recruited to kinetochores priorto bi-orientation, activating the signaling cascade thatinhibits the activation of the anaphase promoting com-plex (for reviews, see (Peters 2006; Musacchio andSalmon 2007; Nezi and Musacchio 2009; Murray 2011;Pines 2011)). These proteins dissociate from kinetochoresonce correct attachments are formed, suggesting that theymonitor attachment status and/or tension at kinetochores.However, it is still unclear how many of the spindlecheckpoint proteins interact with kinetochores and howthis interaction is regulated.

Fig. 1 Lateral and end-on attachments. a Lateral attachments. Kinet-ochores initially encounter the lateral side of spindle microtubulesduring prometaphase. b End-on attachments during metaphase. Kinet-ochores interact with the tips of spindle microtubules where incorpo-ration and dissociation of tubulin subunits occur. In this example,microtubules emanating from the left pole grow and those from theright pole shorten (top), leading to rightward movement of the attachedchromosomes (bottom). c End-on attachments during anaphase. Link-age between sister chromatids is lost and microtubules from both sidesshorten, resulting in the separation of sister chromatids to oppositepoles

Fig. 2 Different types of kinetochore–microtubule attachments. a Bi-oriented attachments in which sister kinetochores attach to microtu-bules emanating from opposite poles. Kinetochores are under robusttension due to microtubule pulling forces that are opposed by linkagebetween sister chromatids. Tension stabilizes these proper bi-orientedattachments. b Mono-oriented attachment in which sister kinetochoresare attached to microtubules emanating from the same pole (top). Lackof tension destabilizes these improper attachments, giving cells anotherchance to achieve correct bi-oriented attachments (bottom)

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Regulation of microtubule attachments by mitotic kinasesand phosphatases

The Aurora B kinase, a catalytic subunit of the chromosom-al passenger complex (CPC), plays a critical role in desta-bilizing erroneous attachments (Ruchaud et al. 2007; Kellyand Funabiki 2009). Because it localizes to the inner cen-tromere where it overlaps with kinetochores before bi-orientation but not after, it was proposed that this spatialarrangement of Aurora B allows selective destabilization oftension-less attachments (Tanaka et al. 2002). More recently,based on previous observations that Aurora B rapidly turnsover at inner centromeres (Murata-Hori and Wang 2002), adiffusion-based intra-cellular phosphorylation gradient wasproposed and experimentally observed in vivo (Fuller et al.2008; Liu et al. 2009; Welburn et al. 2010; Wang et al. 2011;Tan and Kapoor 2011). However, Aurora B can efficientlyphosphorylate its targets even if they localize at a microm-eter rather than nanometer distance scale from the innercentromere, suggesting that counteracting phosphatasesmust play critical roles to balance local phosphorylation onkinetochores (Tan and Kapoor 2011; Wang et al. 2011). Animportant phosphatase that counteracts Aurora B is theprotein phosphatase 1 (PP1) (for reviews, see (De Wulf etal. 2009; Wurzenberger and Gerlich 2011)). PP1 is found atouter kinetochores, showing enrichment at bi-oriented kinet-ochores (Trinkle-Mulcahy et al. 2003; Liu et al. 2010).Several kinetochore proteins recruit PP1 onto kinetochores,including KNL1, CENP-E, and Fin1 (Akiyoshi et al. 2009a;Kim et al. 2010; Liu et al. 2010; Meadows et al. 2011;Rosenberg et al. 2011). It remains to be determined if PP1is recruited by different kinetochore proteins to dephosphor-ylate distinct substrates. Recent work suggests that PP2Amay also promote attachment stability by counteractingAurora B and Plk1 (Foley et al. 2011). In addition to AuroraB, the Mps1 kinase is also implicated in regulating kineto-chore–microtubule attachments, although its mechanism ofaction remains to be determined (Maure et al. 2007; Jellumaet al. 2008; Kemmler et al. 2009; Hewitt et al. 2010; Lan andCleveland 2010; Maciejowski et al. 2010; Santaguida et al.2010; Dou et al. 2011).

Numerous microtubule-binding kinetochore proteins arephosphorylated by Aurora B. For example, the Ndc80 proteinis regulated byAurora B and the non-phosphorylatablemutantshows hyper-stable attachments and severe segregationdefects in human and PtK1 cells, demonstrating the impor-tance of phospho-regulation (DeLuca et al. 2006; 2011). Incontrast, the non-phosphorylatable mutant is viable in bud-ding yeast and chicken cells, implying that the regulation ofadditional targets is sufficient for faithful chromosome segre-gation in these organisms (Akiyoshi et al. 2009b; Welburn etal. 2010). KNL1 and Dsn1 are also phosphorylated by AuroraB, and their combinatorial phosphorylation appears important

to regulate the microtubule-binding activity of the KMNnetwork (Welburn et al. 2010). Dam1 is a key Aurora targetin budding yeast, and non-phosphorylatable mutants showchromosome segregation defects while phospho-mimickingmutants can suppress temperature sensitivity of a kinase mu-tant (Cheeseman et al. 2002). Although many kinetochoreproteins are regulated by kinases, it is less clear how phos-phorylation affects their activity or behavior at molecularlevels. Phosphorylation can regulate attachment stability byaffecting various parameters, such as detachment rate, micro-tubule dynamics, and interaction strength with associatingkinetochore proteins. To understand how these kinases/phos-phatases regulate kinetochore–microtubule attachments, it isessential to identify their targets as well as to reveal theparameters that are modulated by phosphorylation.

Why do we need to reconstitutethe kinetochore–microtubule interface?

Reconstitution of the kinetochore–microtubule interface iscritical to understanding aspects of kinetochore function thathave not been easily answered by in vivo studies. Within thecell, numerous factors affect kinetochore–microtubuleattachments either directly or indirectly. For example, inac-tivation of proteins important for microtubule dynamics canlead to kinetochore–microtubule attachment defects even ifthey have no direct roles at kinetochores. Also, kinetochoresare thought to assemble in a hierarchical manner such thatthe localization of outer kinetochore proteins depends oninner kinetochores (De Wulf et al. 2003; Nekrasov et al.2003; Pinsky et al. 2003). Therefore, the inactivation of onekinetochore protein can indirectly affect many others, com-plicating the interpretation of the results. Reconstitutingkinetochore functions in vitro with pure components allowsresearchers to observe a system in a controlled manner aswell as to interrogate specific proteins or change otherparameters to see how the system responds. One of theearliest reconstitution studies using isolated chromosomesled to the important finding that depolymerizing microtu-bules can do mechanical work to move chromosomes in theabsence of any molecular motor (Mitchison and Kirschner1985; Koshland et al. 1988; Coue et al. 1991). This was latercorroborated by the finding that minus end directed motorsare not required for poleward chromosome movement inyeast (Grishchuk and McIntosh 2006; Tanaka et al. 2007).

In vitro studies are also crucial to reveal the structure of thekinetochore–microtubule interface, which is essential to ulti-mately understanding how kinetochores couple chromosomesto dynamic microtubule ends (Welburn and Cheeseman2008). Although electron microscopy (EM) studies on cellshave pictured overall kinetochore structure (Brinkley andStubblefield 1966; McEwen et al. 1998; Dong et al. 2007;

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McIntosh et al. 2008; McEwen and Dong 2010), higherresolution images are required to reveal the nature of individ-ual kinetochore–microtubule attachments. Another limitationof cellular EM is that it is difficult to reveal the identity ofobserved structures. For example, although EM tomographicreconstitution ledMcIntosh and colleagues to discover slenderstructures (called “fibrils”) that appear to connect kinetochoresto the inner face of microtubule plus ends, it remains unknownwhich proteins make up the fibrils (McIntosh et al. 2008).Reconstituting kinetochore functions in vitro for biochemicaland structural analyses is therefore key to understanding howthe dynamic kinetochore–microtubule interface is formed andregulated.

Reconstitution should also be invaluable to understand-ing the spindle checkpoint. Although checkpoint proteinslocalize to unattached kinetochores, the molecular require-ments to displace checkpoint proteins from kinetochoresare unclear. Do they dissociate upon formation of lateralor end-on attachment? Or, if attachment is not enough,how much tension is required to displace them fromkinetochores? Although the extent of inter-kinetochorestretch (distance between sister kinetochores) is oftenused as a read-out for tension, work from several labssuggests that it is rather the intra-kinetochore stretch thatis monitored by the checkpoint (Maresca and Salmon2009; Uchida et al. 2009; Wan et al. 2009; Maresca andSalmon 2010; Suzuki et al. 2011). Further complicatingthe matter, recent high-resolution 3D imaging of humancells showed that even laterally attached kinetochoresmay also be under some tension (Magidson et al. 2011).Difficulties in understanding the molecular details of thespindle checkpoint derive from a lack of robust reconsti-tution assays that would allow researchers to directlyexamine the effect of microtubule binding and tensionon the behavior of checkpoint proteins at kinetochores(Murray 2011).

How can the kinetochore–microtubule interfacebe reconstituted?

Preparation of pure kinetochores and their functional assays

To reconstitute and study the kinetochore–microtubule inter-face in vitro, one needs kinetochores, microtubules, andassays. There are two major approaches to prepare pure kinet-ochores: expression of recombinant proteins and purificationfrom cells. Due to difficulties in purifying native kinetochoresor obtaining large quantities of kinetochore proteins fromcells, most studies have utilized recombinant expression toreconstitute kinetochore subcomplexes. Althoughmany kinet-ochore proteins are insoluble when expressed individually,solubility can often be achieved by co-expressing interacting

partners (for example (Ciferri et al. 2005; Wei et al. 2005)).Polycistronic vectors that allow the simultaneous expressionof multiple genes from a single vector facilitate the formationand purification of stable subcomplexes (Tan 2001). Althoughwhole kinetochores have not yet been reconstituted, studies ofsubcomplexes have led to significant progress in understand-ing the molecular mechanism of kinetochore functions. Whilerecombinant expression represents a bottom-up approach,purification of native kinetochores from cells is a top-downone. Both approaches are critical to understanding the func-tions of individual complexes as well as their ensemble prop-erties. Table 1 summarizes the complexes reconstituted orpurified and used in microtubule-binding assays in vitro todate.

There are a variety of microtubule-binding assays. So far,most experiments have used stabilized microtubules and con-ventional microtubule sedimentation assays (Fig. 3a). Althoughthese assays are useful to determine if proteins possessmicrotubule-binding activity, they can't distinguish the precisetype of interaction (for example, do they bind the microtubulelattice or the tip? How many contact points do they make?).Therefore, it is essential to directly study the interface betweenkinetochore proteins and individual microtubules. EM is apowerful technique that can visualize the kinetochore–micro-tubule interface at the nanometer level. It is also critical to usedynamic microtubules to understand how kinetochores coupleto polymerizing and depolymerizing microtubule tips. Single-molecule techniques enable studies of individual interactionsbetween microtubules and kinetochore proteins/subcomplexes.Total internal reflection fluorescence (TIRF)microscopy allowsvisualization of fluorescently labeled proteins and microtubulesat a single-molecule sensitivity by restricting the imaging areawithin ∼100 nm of the coverslip (Axelrod et al. 1984) (Fig. 3b).Optical trapping is a technique that can apply mechanical forceto protein–protein linkages (Block et al. 1990). In this assay,polystyrene beads that are decorated with kinetochore subcom-plexes (or larger assemblies) are attached to microtubules soone can assess their biophysical characteristics, such as howmuch force they can bear (Franck et al. 2010) (Fig. 3c). Below,we highlight some reconstituted subcomplexes and the insightsgained from their study.

Reconstitution using recombinant individual kinetochoresubcomplexes: Ndc80, Dam1, and more

Despite the identification of numerous kinetochore proteins,the identity of the factors that directly bind to microtubulesremained unknown for a long time. Although unattachedkinetochores were observed in mutational studies of manykinetochore proteins, one could not tell if they have directmicrotubule-binding activity or general structural roles thatlead to defective microtubule attachments when inactivated(Pinsky et al. 2006). A breakthrough came when the

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Table 1 List of characterized microtubule-binding kinetochore proteins and complexes

Name Assays Comments References

Ndc80 complex Sedimentation —Formation of oligomeric arrays in vitro (Ciferri et al. 2005; Wei et al. 2005;Cheeseman et al. 2006;Wei et al. 2007; Ciferri et al. 2008;McIntosh et al. 2008; Wilson-Kubaleket al. 2008; Powers et al. 2009; Alushinet al. 2010)

TIRF —Higher affinity to lattice than tips

Optical trap

EM

—Diffusion-based motility

—Load-bearing attachment to growing andshrinking tips

—Rupture force ~3 pN

—Mean attachment duration and traveldistance to dynamic microtubules, <1 min,0.4 μm (under 1.8 pN of force, 2,000:1Ndc80:bead ratio)

KMN network Sedimentation —Cooperative binding, showing highermicrotubule binding than the sum ofindividual complexes

(Cheeseman et al. 2004; Cheesemanet al. 2006; Welburn et al. 2010)

Dam1 complex Sedimentation —Formation of rings and spirals in vitro (Hofmann et al. 1998; Miranda et al. 2005;Westermann et al. 2005; Asbury et al. 2006;Westermann et al. 2006; Franck et al. 2007;Gestaut et al. 2008; Grishchuk et al. 2008;Gao et al. 2010; Lampert et al. 2010)

TIRF —Autonomous plus end tracker

Optical trap

EM

—Load-bearing attachment to growing andshrinking tips

—Rupture force ~3 pN

—Mean attachment duration and traveldistance: to growing microtubules, 3 min,1.1 μm (under 0.5 pN of force). 2 min,0.8 μm (under 2.0 pN of force): toshortening microtubules, 17 s, 2.7 μm(under 0.5 pN of force). 9 s, 0.5 μm(under 2.0 pN of force)

Ndc80 complex+Dam1complex

Sedimentation —Dam1 confers plus-end tracking activity toNdc80, enhancing Ndc80's load-bearingattachments

(Lampert et al. 2010; Tien et al. 2010)TIRF

—Rupture force ~5 pNOptical trap

—Mean attachment duration and traveldistance to dynamic microtubules, 3 min,1.6 μm (under 1.8 pN of force, 2000:1Ndc80:bead ratio, in the presence of freeDam1 complexes in solution)

Ska1 complex Sedimentation —Formation of oligomers in vitro (Welburn et al. 2009)Beads assay —Tracking with depolymerizing microtubule tips

EM —Coupling duration to shortening microtubules:18 s, 3.5 μm (in the absence of force)

Kinetochore particlespurified from buddingyeast

TIRF —Largest kinetochore assemblies characterized to date (Akiyoshi et al. 2010)Optical trap —Robust load-bearing attachments to growing and

shrinking tips

—Rupture force ~9 pN

—Mean attachment duration and travel distance todynamic microtubules, 17 min, 5 μm (under 1.9 pNof force, 200:1 Dsn1:bead ratio). 50 min (under 5 pNof force, 200:1 Dsn1:bead ratio)

The following proteins show microtubule binding activity as well as localization to kinetochores: SKAP-Astrin/Kinastrin complex (Mack andCompton 2001; Manning et al. 2010; Schmidt et al. 2010; Dunsch et al. 2011), CENP-Q (Amaro et al. 2010), MCAK (Wordeman and Mitchison1995; Desai et al. 1999; Cooper et al. 2010; Oguchi et al. 2011), CENP-E, (Kim et al. 2008; Maffini et al. 2009), Fin1 (Woodbury and Morgan2007), Kar3 (Tanaka et al. 2005; Tytell and Sorger 2006), INCENP/Survivin (Wheatley et al. 2001; Sandall et al. 2006), XMAP215 (Garcia et al.2001; He et al. 2001; Brouhard et al. 2008; Kitamura et al. 2010; Al-Bassam and Chang 2011), CLASP (Maiato et al. 2003; Cheeseman et al. 2005;Ortiz et al. 2009), CENP-F (Feng et al. 2006) that recruits NDE1, NDEL1 (Vergnolle and Taylor 2007), RZZ complex (Karess 2005) and Spindly(Griffis et al. 2007; Gassmann et al. 2008) that recruit dynein/dynactin to kinetochores (Howell et al. 2001; Yang et al. 2007). Other proteins thatmay directly bind microtubules or contribute to microtubule binding by recruiting other factors include: Cep57 (Emanuele and Stukenberg 2007),Bod1 (Porter et al. 2007), Shugoshin (Salic et al. 2004; Huang et al. 2007; Tanno et al. 2010), mDia3 (Yasuda et al. 2004; Cheng et al. 2011), Kebab(Meireles et al. 2011), and CAMP (Itoh et al. 2011)

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conserved Ndc80 complex (composed of Ndc80, Nuf2,Spc24, and Spc25) was reconstituted and shown to havemicrotubule-binding activity in vitro (Cheeseman et al.2006; Wei et al. 2007). Structural studies showed that theNdc80 complex is a 57-nm elongated structure with globu-lar domains at both ends (Ciferri et al. 2005; Wei et al.2005). The Ndc80–Nuf2 globular domain binds microtu-bules while the Spc24–Spc25 globular domain faces theinner kinetochore (Cheeseman et al. 2006; DeLuca et al.2006). Initial EM analysis revealed that the Ndc80 complexbinds microtubules at a defined angle (Cheeseman et al.2006). X-ray crystallography studies show that bothNdc80 and Nuf2 possess a calponin-homology (CH) do-main (Wei et al. 2007; Ciferri et al. 2008), which is foundin the EB1 microtubule tip-tracking protein (Hayashi andIkura 2003), although the Ndc80 complex does not exhibit

high affinity to microtubule tips (Alushin et al. 2010). Fur-thermore, higher-resolution images of the Ndc80 complexbinding to taxol-stabilized microtubules revealed that thecomplex contacts microtubules at the interface betweentubulin monomers via the CH domain of Ndc80, not Nuf2(Wilson-Kubalek et al. 2008; Alushin et al. 2010). Consis-tent with these results, mutations of the Ndc80 CH domaincause severe defects in vivo, while those of Nuf2 CHdomain result in only minor phenotypes (Guimaraes et al.2008; Miller et al. 2008; Sundin et al. 2011). Furthermore,Ndc80 complexes self-associate to form oligomeric arraysalong the microtubule lattice, which are mediated by its N-terminal extension (Alushin et al. 2010). Aurora B phos-phorylates this N-terminal extension at multiple sites(Cheeseman et al. 2006; DeLuca et al. 2006; Ciferri et al.2008) and inhibits the oligomerization of Ndc80 complexes(Alushin et al. 2010), reducing its microtubule-binding ac-tivity. We note that the oligomeric arrays have so far onlybeen observed in vitro and their physiological relevanceremains unclear. It is likely that phosphorylation also direct-ly reduces attachment stability by adding negative chargesto Ndc80. Therefore, Aurora B may regulate Ndc80'smicrotubule-binding activity by multiple mechanisms.

In contrast to EM techniques that reveal static images of thekinetochore–microtubule interface at sub-nanometer levels,single-molecule techniques can reveal dynamic pictures. TheAsbury lab carried out a detailed characterization of theNdc80 complex using TIRF microscopy and optical trapassays to reveal biophysical insights into their functions(Powers et al. 2009). They found that individual Ndc80 com-plexes bind to the microtubule lattice with a weak affinity andrapidly diffuse along the lattice. Oligomers of Ndc80 com-plexes can track with depolymerizing tips as microtubulesshorten, while individual complexes cannot. Based on theseresults, they proposed that a biased-diffusion mechanism (Hill1985) underlies Ndc80's motility such that the movement ofNdc80 complexes is biased by depolymerizing microtubuletips. Using an optical trap, they further show that Ndc80complexes can form load-bearing attachments (up to 3 pN offorce) to dynamic microtubule tips as long as 6 to 30 com-plexes are present (Powers et al. 2009). Although the esti-mates of Ndc80 complexes per microtubule-binding site varybetween studies (∼7 (Joglekar et al. 2006; Johnston et al.2010), ∼20 (Lawrimore et al. 2011), or ∼40 (Coffman et al.2011)), the Ndc80 complexes clearly play a significant role inmicrotubule binding in vivo. Taken together, reconstitution ofthe Ndc80 complex and analyses of its mode of microtubulebinding facilitated an in-depth characterization of this impor-tant kinetochore subcomplex, demonstrating the power of theapproach.

Although the Ndc80 complex is essential for microtubuleattachments, it is not sufficient to achieve bi-orientation. Inbudding yeast, cells fail to form bi-oriented attachments when

Fig. 3 Examples of microtubule-binding assays. a A microtubule co-sedimentation assay analyzes whether proteins of interest stay associ-ated with stabilized (non-dynamic) microtubules that are pelleted bycentrifugation. The level of co-sedimentation is typically determinedby immunoblots. b A fluorescence-based assay observes binding be-tween fluorescent microtubules attached to a coverslip and fluores-cently labeled proteins. TIRF microscopy enables visualization ofmolecules only in the evanescent field (within ∼100 nm of cover slip),providing single-molecule sensitivity (i.e., observation of bindingevents between individual microtubules and individual kinetochoreproteins or complexes). Dynamically growing and shortening micro-tubules can also be used, allowing studies of lateral versus end-onattachments. c Optical trap assays use a focused laser beam to applyforce to the interaction between bead-bound proteins and dynamicmicrotubule tips. Growth and shrinkage of individual microtubulescan be monitored by video-enhanced microscopy and the number ofbead-bound proteins can be diluted low enough to allow studies ofsingle molecules or complexes

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any component of the ten-subunit Dam1 complex is inacti-vated (Hofmann et al. 1998; Jones et al. 2001; Cheeseman etal. 2002; Janke et al. 2002). To understand how the Dam1complex binds to microtubules, the Harrison lab constructedan elegant polycistronic vector containing all ten subunits(Miranda et al. 2005). Initial characterization showed thatDam1 complexes form rings and spirals around microtubules(Miranda et al. 2005; Westermann et al. 2005). This was anexciting finding because rings had previously been proposedto function as a coupler to track on depolymerizing microtu-bule tips (Margolis and Wilson 1981; Mitchison et al. 1986).Indeed, Dam1 complexes can be induced to move by depoly-merizing tips (Westermann et al. 2005; 2006), and they canform attachments even in the presence of external force ashigh as 3 pN (Asbury et al. 2006; Franck et al. 2007). Theestimate of at least 16 copies per kinetochore is sufficient toform a ring (Joglekar et al. 2006; Coffman et al. 2011;Lawrimore et al. 2011), implying that this complex mightform a ring or similar structure in vivo. However, this modelwas challenged by the finding that ring formation is notrequired for its processive movements in vitro (Gestaut et al.2008; Grishchuk et al. 2008; Gao et al. 2010) and the failure sofar to identify ring-like structures in vivo. Furthermore, theDam1 complex is not essential for viability in Schizosacchar-omyces pombe and is not found outside of fungi (Liu et al.2005; Sanchez-Perez et al. 2005). One critical differencebetween the kinetochores of budding yeast and fission yeastis that the former bind a single microtubule while the latterbind multiple microtubules. Therefore, it is possible that bud-ding yeast heavily relies on Dam1 to avoid complete loss ofattachments, an idea supported by recent studies in Candidaalbicans (Burrack et al. 2011; Thakur and Sanyal 2011). TheSka1 complex has been proposed to be a functional homologof Dam1 because it shows the most similar biophysical prop-erties (Welburn et al. 2009). Ska1 localizes to kinetochoresand spindles in vivo, possesses direct microtubule-bindingactivities, can track on depolymerizing microtubules, and isphosphorylated by Aurora B (Hanisch et al. 2006; Daum et al.2009; Gaitanos et al. 2009; Raaijmakers et al. 2009; Welburnet al. 2009; McIntosh et al. 2010). Ska1 complexes also formoligomers around microtubules in vitro, although rings havenot been detected (Welburn et al. 2009). Additional biophys-ical assays should reveal whether the Ska1 complex can alsoform load-bearing attachments and is a functional equivalentof the yeast Dam1 complex.

Besides these proteins, there are many others that affectkinetochore–microtubule attachment either directly or indi-rectly. For example, microtubule sedimentation assaysshowed that KNL1/Blinkin/Spc105 has weak microtubule-binding activity (Cheeseman et al. 2006; Pagliuca et al.2009). Its depletion phenotype is very severe even thoughNdc80 still localizes to kinetochores (Kiyomitsu et al.2007). This result also shows that Ndc80 is not sufficient

for kinetochore–microtubule interaction, consistent with thecooperative behavior of the KMN complex (see below).Other proteins that have microtubule-binding activities areshown in Table 1, but because these proteins have not beenanalyzed in detail in vitro, we will not discuss them furtherin this review.

Multiple subcomplexes

Once individual subcomplexes are reconstituted, the next goalis to reconstitute larger assemblies (ultimately whole kineto-chores) to understand how the numerous kinetochore compo-nents function as a single macromolecular unit. This is alsouseful to infer which subcomplexes interact with each otherwithin kinetochores. For example, the CENP-C inner kineto-chore protein directly interacts with the reconstituted Mis12complex (composed of Mis12, Dsn1, Nsl1, and Nnf1 pro-teins), revealing an important linkage between inner and outerkinetochores (Gascoigne et al. 2011; Przewloka et al. 2011;Screpanti et al. 2011). Interestingly, the Mis12 complexchanges its shape upon CENP-C binding (Screpanti et al.2011), which might be important for the regulation of kinet-ochore assembly. The Mis12 complex also directly binds toKNL1 (Maskell et al. 2010; Petrovic et al. 2010) as well as tothe Ndc80 complex (Petrovic et al. 2010; Hornung et al.2011). These results suggest that the Mis12 complex alsoplays a key role in linking inner and outer kinetochore com-ponents. Similarly, characterization of a mixture of Ndc80 andDam1 complexes suggested that Dam1 interacts with Ndc80in the presence of microtubules and that the interaction enhan-ces the microtubule-binding activity of the Ndc80 complex(Lampert et al. 2010; Tien et al. 2010). Like Ndc80, the Dam1complex is targeted by Aurora B (Cheeseman et al. 2002),resulting in reduced microtubule-binding activity as well asweakened interaction with the Ndc80 complex (Shang et al.2003; Wang et al. 2007; Gestaut et al. 2008; Lampert et al.2010; Tien et al. 2010).

Pioneering work by Cheeseman et al. (2006) succeededin reconstituting a complex composed of KNL1, Mis12, andNdc80, called the KMN network. This study showed thatKNL1 and Ndc80 complex synergize to bind to microtu-bules in the presence of the Mis12 complex that does notdirectly bind microtubules. Because of the conservation ofKMN components and the severity of their knockdownphenotype, it is now widely accepted that KMN forms acore microtubule-binding module across eukaryotes. How-ever, although a KMN complex containing 1 copy each ofK, M, and N can be readily reconstituted in vitro (at leastusing worm proteins), it is not clear if larger kinetochoreassemblies can be reconstituted without additional post-translational modifications. For example, the Aurora B ki-nase is implicated in promoting outer kinetochore assemblyonto inner kinetochore proteins by phosphorylating Dsn1

242 Chromosoma (2012) 121:235–250

(Emanuele et al. 2008; Yang et al. 2008), and Cdk1 phos-phorylates the CENP-T component of the CCAN to promotekinetochore assembly (Gascoigne et al. 2011).

Kinetochore particles isolated from cells

As an alternative approach to reconstituting kinetochoreswith recombinant proteins, researchers tried to purify kinet-ochores from cells. However, this approach was initially notvery successful due to several challenges, including theirlow abundance and unknown biochemical properties. Byutilizing artificial minichromosomes that possessmicrotubule-binding activity (Kingsbury and Koshland1993) and divide faithfully during cell division (Clarkeand Carbon 1980), we established a method to purifycentromere-bound kinetochores from budding yeast(Akiyoshi et al. 2009a). Taking advantage of the informa-tion we obtained to maintain kinetochores throughout thepurification process (e.g., buffer containing a physiologicalsalt concentration was important to maintain kinetochoreintegrity), we then succeeded in isolating a complex inhigher purity and quantity that contains almost all corekinetochore proteins via affinity-purification of the Dsn1protein (a component of the Mis12 complex) (Akiyoshi etal. 2010). Using TIRF microscopy, we found that fluores-cently labeled kinetochore particles stably bind to the mi-crotubule lattice. This lateral binding depends on the Ndc80and KNL1/Spc105 complexes, but not on the Dam1 com-plex, consistent with in vivo studies that showed the Dam1complex is dispensable for initial lateral attachments(Tanaka et al. 2005). Unlike the Ndc80 complex, individualkinetochore particles do not diffuse on the lattice, yet theytrack on depolymerizing microtubule tips, consistent withbiased-diffusion based motility. Using an optical trap, weshowed that the individual kinetochore particles stay boundto the tip of dynamically growing and shrinking microtu-bules in the presence of external force. Compared to theattachments mediated by Ndc80 or Dam1 complexes, kinet-ochore particle-mediated attachments persisted for a muchlonger time (>20 min), comparable to the duration of mitosisin budding yeast. Purified kinetochores form robust load-bearing attachments, persisting even under 11 pN of force.Although not required for lateral attachment, Dam1 is es-sential to form robust tip attachments in these assays, againconsistent with previous in vivo studies. Therefore, kineto-chore particles purified from budding yeast reconstitutefundamental kinetochore functions.

Our in vitro reconstitution of kinetochore–microtubuleattachments allowed us to directly study the effect of tipattachments and mechanical tension on the dynamics of indi-vidual microtubules, as well as the stability of the attachmentdepending on the state of the microtubule (Fig. 4a). It has longbeen known that the dynamics of kinetochore microtubules

are different from non-kinetochore microtubules in vivo(Nicklas and Kubai 1985; Mitchison et al. 1986; Tanaka etal. 2007), although the significance of this to the state ofkinetochore–microtubule attachments was unclear. It was alsounknown whether kinetochores bind more stably to growingor shortening microtubules. We therefore measured the fol-lowing four parameters in the presence of a variable level oftension: catastrophe rate, rescue rate, and the detachment ratefrom growing versus shortening microtubules. Strikingly, ten-sion increases the rescue rate and decreases the catastropherate, causing microtubules to spend more time in the assemblyphase (Fig. 4b). In addition, kinetochores bind more stably togrowing microtubules than to shortening microtubules(Fig. 4b). Changes in these four parameters result in a netincrease in total attachment time as tension is increased withina certain force range (0.5–5 pN). In the presence of higherforce (>5 pN), total attachment time then decreases. Thisbehavior resembles the “catch bonds” that occur betweenreceptor–ligand interactions to enhance cell adhesion in thepresence of mechanical tension (Marshall et al. 2003; Thomaset al. 2008; McEver and Zhu 2010). Therefore, reconstitutingkinetochore–microtubule attachments in vitro demonstratedthat tension directly stabilizes kinetochore–attachments, andthe properties that regulate this behavior can now be furtherexplored.

One major advantage of this top-down approach is thatpurified native kinetochore particles may retain importantpost-translational modifications and structural integrity,which might be difficult to achieve from the bottom-up

Fig. 4 Effects of tension on microtubule tip dynamics and attachmentstability. a End-on attachments allow kinetochores and tension todirectly regulate microtubule tip dynamics. b Tension stabilizes recon-stituted kinetochore–microtubule interactions. Left: Tension increasesrescue rate and decreases catastrophe rates, resulting in a state wheremicrotubules spend more time in the assembly phase. Right: There isalso a tendency for the detachment rate from shortening microtubulesto decrease in the presence of higher tension whereas that from grow-ing microtubules increases. Changes in these four parameters result in anet increase in total attachment time as tension is increased within acertain force range (0.5–5 pN) (Akiyoshi et al. 2010)

Chromosoma (2012) 121:235–250 243

approach. It will be important to identify and characterizethese modifications to facilitate the reconstitution of largerkinetochore assemblies from recombinant proteins. In addi-tion, the availability of conditional mutants that can beinactivated prior to purification allows particles lackingessential proteins to be analyzed. However, a limitation ofthe approach is that the purified kinetochore particles areunlikely to be bound to centromeric DNA. Therefore, al-though kinetochore particles show robust microtubule-binding activity, it is not clear if they truly represent thestate of kinetochores within the cell. It will be important todetermine whether kinetochore particles possess DNA-binding activity and, if so, how centromeric DNA affectstheir behavior. Regardless of the nature of kinetochore par-ticles, they are the most complete kinetochore assembliesobtained thus far and provide excellent opportunities toanalyze the ensemble properties of kinetochores in vitro.For example, we are actively using the system to furthercharacterize the kinetochore–microtubule interface by EMand other techniques. These efforts should synergize withthe bottom-up approach that reveals the function of individ-ual kinetochore subcomplexes and lead to better understand-ing of kinetochore functions.

Other approaches to understanding kinetochores

Reconstitution of kinetochores on exogenous DNAtemplates in vitro

In vitro reconstitution of kinetochore proteins on exogenousDNA sequences is another approach to study kinetochorefunctions. Because of its simplicity and sequence specificity,the budding yeast centromere was utilized for this purpose.In the presence of yeast extracts, fluorescent beads coatedwith centromere DNA can bind microtubules (Sorger et al.1994). However, this activity does not depend on CENP-Aor the KMN core-microtubule-binding factor (Sandall et al.2006). Instead, the DNA binding CBF3 complex and Sli15/Bir1 complex connect centromeric DNA and microtubules(Sandall et al. 2006), suggesting that this reconstitutionsystem is unlikely to represent a core microtubule-bindingevent in vivo.

Recently, the Straight lab achieved the significant mile-stone of assembling kinetochores on reconstituted CENP-Achromatin (Guse et al. 2011). The reconstituted kinetochorescontain key kinetochore components (including the KMNnetwork) and stabilize microtubules. They can, albeit at alow efficiency, activate the spindle checkpoint when treatedwith microtubule drugs. To date, this is the most successfulreconstitution of kinetochore assembly in vitro, and it will beimportant to further test functionality with biophysical andstructural assays. This cell-free system also provides a unique

opportunity to study how the underlying chromatin environ-ment affects kinetochore assembly. Because there is muchcontroversy about the composition of centromeric chromatin(Henikoff and Furuyama 2010; Black and Cleveland 2011), itwill be interesting to test whether kinetochores can be assem-bled onto various types of CENP-A nucleosomes (e.g., right-handed nucleosomes, hemisomes) and to examine how theunderlying chromatin environment affects kinetochore assem-bly and function.

Construction of minimal kinetochores

It is not clear why there are so many constituents in even thesimplest eukaryotic kinetochore. In striking contrast, elegantreconstitution of the bacterial R1 plasmid segregation systemshowed that just two proteins are sufficient to segregate theplasmid DNA in vitro (Garner et al. 2007). In this system, oneprotein binds DNAwhile the other protein forms a polymer thatis stabilized by the DNA-binding protein, promoting DNAsegregation. To understand the design and working principlesof kinetochores that contain numerous components, one ap-proach is thus to dissect their minimal requirements. Artificiallytethering the Dam1 complex to non-centromeric DNA wasfound to promote chromosome segregation in budding yeast(Lacefield et al. 2009; Kiermaier et al. 2009). Although theDNA-binding CBF3 complex is dispensable for its function,the majority of other kinetochore proteins appear to be recruitedonto this Dam1-based machinery. Recently, tethering theCENP-C protein to the spindle pole was shown to be sufficientto assemble kinetochore proteins including KMN and spindlecheckpoint proteins in the fly (Przewloka et al. 2011), andsimilar results were obtained by tethering CENP-C/CENP-Tto the chromosome arm region in human and chicken cells(Gascoigne et al. 2011). Although CENP-A is dispensable inthese systems, the CENP-T-based system cannot replace en-dogenous kinetochore functions (Gascoigne et al. 2011) and theDam1-based segregation system is not as efficient as endoge-nous kinetochores (Lacefield et al. 2009; Kiermaier et al. 2009).It remains to be determined whether the other systems lackingCENP-A are functional. Interestingly, meiotic chromosomesegregation in Caenorhabditis elegans appears to occur in aCENP-A-independent manner (Monen et al. 2005). It is note-worthy that trypanosomatids (such as Trypanosoma brucei thatcauses African sleeping sickness) lack CENP-A (Malik andHenikoff 2003; Lowell and Cross 2004; Berriman et al. 2005).Thus far, no kinetochore proteins have been identifiedin these organisms and their mechanism of chromosomesegregation is a black box. It will be interesting toreveal how these organisms assemble DNA-segregationmachinery without CENP-A, which may also revealwhy CENP-A is used to assemble kinetochores in mosteukaryotes.

244 Chromosoma (2012) 121:235–250

Conclusions and perspectives

Although reconstituting kinetochore functions in vitro hasrevealed key mechanistic details about kinetochore–micro-tubule interactions, there are a number of challenges for thefuture. Higher resolution structural analyses of larger assem-blies such as the KMN network and purified kinetochoreparticles are a critical next step to understanding how indi-vidual kinetochore subcomplexes cooperate to interact withmicrotubules. These studies may also shed light on howkinetochores that possess multiple microtubule-binding sitesare arranged, and how attachments to individual bindingsites are coordinated as a whole. In vitro studies will alsolead to a more complete understanding of how kinases andphosphatases control kinetochore–microtubule interactions,as well as determine whether phosphorylation solely affectsattachment stability or also regulates tip dynamics and otherparameters. Finally, in vitro reconstitution will be essentialfor fully dissecting the spindle checkpoint. Purified chromo-somes have been used to recapitulate the wait-anaphasesignal from kinetochores in vitro (Kulukian et al. 2009),but the efficiency of the checkpoint was low and it wasnever tested whether microtubule attachment silences thesignal. Kinetochore particles purified from budding yeastprovide a novel substrate for this approach because manycheckpoint proteins co-purify (Akiyoshi et al. 2010).Addressing these and other questions should lead to betterunderstanding of kinetochores, the intricate chromosomesegregation machinery in eukaryotes.

Acknowledgments We thank Chip Asbury for critically reading themanuscript. B.A. was supported by postdoctoral fellowships from theEMBO and Human Frontier Science Program. S.B. was supported bygrants from the National Institutes of Health (GM078069 andGM064386).

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