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The KMN protein network – chief conductors of thekinetochore orchestra

Dileep Varma* and E. D. SalmonDepartment of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA

*Author for correspondence (dileep@email.unc.edu)

Journal of Cell Science 125, 5927–5936� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.093724

SummarySuccessful completion of mitosis requires that sister kinetochores become attached end-on to the plus ends of spindle microtubules

(MTs) in prometaphase, thereby forming kinetochore microtubules (kMTs) that tether one sister to one spindle pole and the other sisterto the opposite pole. Sites for kMT attachment provide at least four key functions: robust and dynamic kMT anchorage; force generationthat can be coupled to kMT plus-end dynamics; correction of errors in kMT attachment; and control of the spindle assembly checkpoint

(SAC). The SAC typically delays anaphase until chromosomes achieve metaphase alignment with each sister kinetochore acquiring afull complement of kMTs. Although it has been known for over 30 years that MT motor proteins reside at kinetochores, a highlyconserved network of protein complexes, called the KMN network, has emerged in recent years as the primary interface between thekinetochore and kMTs. This Commentary will summarize recent advances in our understanding of the role of the KMN network for the

key kinetochore functions, with a focus on human cells.

Key words: Checkpoint, Kinetochore, KNL1, MIS12, Mitosis, NDC80

IntroductionKinetochores are mega-molecular assemblies that are formed at

the centromeres of chromosomes at the onset of mitosis (Fig. 1A).

Extensive research over the past two decades has identified over 80

different proteins, in ten or more major complexes, that localize to

human kinetochores (Fig. 1B). The KMN (named for the Knl1

complex, the Mis12 complex and the Ndc80 complex; see below)

network is part of the protein architecture within kinetochores that

links centromeric DNA to the plus ends of spindle microtubules

(MTs) (Cheeseman and Desai, 2008; Santaguida and Musacchio,

2009). The site where kinetochores are assembled is determined by

the presence of a modified histone H3, CENP-A in humans (Cse4

in budding yeast), within nucleosomes at the periphery of each

sister centromere (Fig. 1B). In addition to CENP-A-containing

chromatin, the ‘inner’ domain of the kinetochore (Fig. 1B)

contains a complex of peripheral centromeric proteins consisting

of CENP-C, -H, -I, -K, -L, -M, -N, -O, -P, -S, -T, -U, -W and -X,

called the ‘constitutive centromere-associated network’ (CCAN).

A major function of CCAN is to link the MT-binding KMN

network in the kinetochore ‘outer’ domain to centromeric DNA

within the inner domain (Fig. 1B) (Hori et al., 2008; Hori and

Fukagawa, 2012; Perpelescu and Fukagawa, 2011; Nishino et al.,

2012; Saitoh et al., 1992; Takeuchi and Fukagawa, 2012). The

kinase Aurora B, which localizes to centromeric chromatin before

anaphase at high concentrations (Fig. 1B), has a central role in

regulating the stability of kMT attachments and attachment error

correction in concert with the KMN network (Cheeseman and

Desai, 2008; Santaguida and Musacchio, 2009; Maresca and

Salmon, 2010).

The highly conserved KMN network includes the Knl1

complex, which in vertebrates consists of KNL1 (also known

as CASC5 or blinkin in humans, Spc105 in yeast and Spc105R in

fly) and ZWINT; the Mis12 complex consisting of the four

proteins MIS12 (Mtw1 or MIND in yeast), NSL1 (also called

DC31 or MIS14), NNF1 (also known as PMF1) and DSN1 (also

known as MIS13 in humans; KNL-3 in Caenorhabditis elegans);

and the four-subunit Ndc80 complex comprising NDC80 (also

known as HEC1), NUF2, SPC24 and SPC25 (Cheeseman et al.,

2006; Cheeseman et al., 2004; DeLuca et al., 2006; Kops et al.,

2005). The KMN network associates with kinetochores in

prophase and disappears from kinetochores in telophase

(Santaguida and Musacchio, 2009). The outer end of the Ndc80

complex is the primary binding site for the plus ends of spindle

MTs (DeLuca and Musacchio, 2012; Gascoigne and Cheeseman,

2011; Joglekar et al., 2010; Santaguida and Musacchio, 2009;

Tooley and Stukenberg, 2011), whereas the inner end is anchored

either to the CCAN protein CENP-T (Hori and Fukagawa, 2012)

or to the Mis12 complex (Petrovic et al., 2010). KNL1 also binds,

at its outer end, to kMTs and, at its inner end, to the Mis12

complex (Fig. 1B) (Petrovic et al., 2010). The Mis12 complex is

linked through CENP-C to CENP-A-containing chromatin

(Fig. 1B) (Hori and Fukagawa, 2012). Both the Ndc80 complex

and KNL1 have an elongated shape, extending outwards from

their junctions with CENP-T or the Mis12 complex, and are

oriented along the kMT axis at metaphase (DeLuca et al., 2006;

Joglekar et al., 2009; Przewloka et al., 2011; Schittenhelm et al.,

2009; Schittenhelm et al., 2007; Wan et al., 2009).

A number of other proteins within and at the periphery of the

kinetochore outer domain depend on the presence of members of

the KMN network for their kinetochore localization. These

include MT-associated proteins in the proximity of kinetochore

MT (kMT) plus-ends and members of the spindle assembly

checkpoint (SAC) (Fig. 1B) (Musacchio and Salmon, 2007; Kops

and Shah, 2012). The KMN network is also thought to associate

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with proteins that form the peripheral region of the outer

kinetochore, including fibrous proteins, such as CENP-F, and MT

motor proteins, such as the kinesin CENP-E and the cytoplasmic

dynein–dynactin complex (Fig. 1B) (Cheeseman and Desai,

2008). The precise nature of the interactions between KMN

network and other outer domain kinetochore proteins are only

just being unraveled. In this Commentary, we will discuss recent

advances in our knowledge of the structure and function of the

proteins associated with KMN network and their potential roles

in achieving the key functions of the kinetochore to ensure

accurate chromosome segregation. We begin with the inner-most

member of the KMN network, the Mis12 complex, then continue

to describe the structure and function of the other two

components, the Knl1 and Ndc80 complexes, and close with a

discussion on other proteins and mechanisms involved in

controlling the function of the KMN network.

The Mis12 complexThe Mis12 complex has been referred to as the kinetochore

‘keystone’ complex, as it serves as a major platform for outer

kinetochore assembly (Cheeseman and Desai, 2008). The human

Mis12 complex is 22-nm long and rod shaped, with the subunits

linearly tethered to each other in the order NNF1, MIS12, DSN1

and NSL1, from inside to outside, within vertebrate kinetochores

(Fig. 2) (Maskell et al., 2010; Petrovic et al., 2010). Electron

microscopy analysis of the homologous yeast Mtw1 complex

assembled in vitro essentially yields a similar elongated structure,

although the yeast complex has been proposed to have a ‘comma’

or bi-lobbed shape (Hornung et al., 2011; Maskell et al., 2010).

Both the yeast Dsn1 and Nsl1 subunits have been found to be

elongated, with them spanning much of the length of the entire

yeast complex. On the basis of in vitro protein assembly assays

using purified components of the human KMN network, it has

been found that the C-terminal tail of the NSL1 subunit is able to

attach to both SPC24 and SPC25, which are located at the inner

end of the Ndc80 complex, and to the C-terminus of KNL1

(Fig. 2) (Petrovic et al., 2010). A similar analysis of the yeast

Mtw1 complex suggests that the complex has a direct link to

Spc24 and Spc25 of the Ndc80 complex (Hornung et al., 2011).

Hence, the current evidence suggests that even though the human

and yeast Mis12 complexes might show variations in their

structure, they essentially perform very similar functions.

Although it has been shown that the inner end of the Mis12

complex interacts directly with CENP-C, it is not clear which of the

Mis12 complex subunits are involved in these interactions in

vertebrate cells (Screpanti et al., 2011). In Drosophila melanogaster,

it has been shown that the centromere-proximal Nnf1 subunit is

instrumental in the interaction between the Mis12 complex and

CENP-C (Przewloka et al., 2011). The yeast Mtw1 complex has also

been shown to associate directly with the yeast functional

homologue of CCAN, a Ctf19-containing complex called COMA

(Hornung et al., 2011). As CCAN in turn directly associates with

CENP-A-containing chromatin (Foltz et al., 2006; Okada et al.,

2006), the Mis12 complex is an integral part of the machinery that

connects the outer kinetochore to the inner centromeric DNA.

Initially isolated as a key component of the core MT attachment site,

Mis12 has not been shown to bind to purified MTs, even though it

has been reported to enhance the MT-binding affinities of the Ndc80

complex and of KNL1 (Cheeseman et al., 2006), and recent

evidence indicates that it interacts with the microtubule-associated

protein (MAP) complex Ska (Chan et al., 2012).

Consistent with a role of the Mis12 complex in the recruitment

of the Ndc80 and Knl1 complexes, depleting its various subunits

results in defects in metaphase chromosome alignment, stable

kMT formation, kinetochore bi-orientation and chromosome

segregation (Kline et al., 2006). Seminal studies of the Mis12

homologue in yeast have demonstrated that the Mis12 complex

performs important functions in controlling spindle structure,

Mitotic cell

kMTs

Chromosome

Kinetochore assembledat the centromere

BACENP-A

Aurora B

CCAN(innerkinetochore)

KMN network(outerkinetochore)

kMTs

Outer kinetochore (unattached)[contains dynein, CENP-F, CENP-E

and the SAC proteins]

Sister chromatids

Fig. 1. Protein composition and domain structure of the vertebrate kinetochore in mitotic cells. (A) Kinetochores are assembled at the periphery of sister

centromeres on mitotic chromosomes and attach to spindle microtubules. (B) The site of kinetochore assembly is specified by the presence of the modified histone H3

CENP-A. The CCAN protein network within the inner kinetochore domain links CENP-A-containing chromatin to the KMN network within the outer kinetochore

domain. The KMN network is the major interface between kMTs and their associated proteins. The peripheral region of the outer domain contains long fibrous proteins,

such as CENP-F, the microtubule motors CENP-E and dynein, as well as their associated proteins, particularly in the absence of kMT attachments at prometaphase.

Protein components of the SAC are also found in the outer domain. Although primarily within the peripheral region, these proteins probably also extend into the inner

parts of the outer domain because many of these are connected to the KMN network. See text for details.

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accurate chromosome segregation, sister centromere biorientation

and separation (Goshima et al., 1999; Goshima and Yanagida,

2000). Drosophila embryos with mutations in Mis12 complex

subunits also exhibit similar defects in chromosome segregation

(Venkei et al., 2011). Interestingly, extensive investigations have

failed to identify a Dsn1 homologue in Drosophila, and it has been

proposed that this subunit has been functionally replaced in part by

the fly Knl1 homologue, Spc105R, on which the other three

subunits of the Mis12 complex depend for their localization to the

kinetochore (Przewloka and Glover, 2009). In budding yeast, the

V-shaped heterotetrameric monopolin complex, consisting of

Mam1, Csm1, Lrs4 and Hrr25, has been shown to bridge the

Dsn1 subunit of Mis12 with centromere-localized CENP-C and

influence normal meiotic chromosome segregation (Corbett et al.,

-

Inner end

Outer end

Mis12 complex

Ndc80 complex

KNL1–ZWINT complex

CCAN network

CN

N

(MT binding domains)

N-terminal tail

Mis12 complex KNL1 (CASC5 or blinkin)

Centromeric DNA

MT

MT bindingMIS12 bindingPP1 binding

Coiled-coilregion

ZWINTbinding

AurB sites

Bub1 TPR

BubR1 TPR

(177–186) (213–222)

KI1 KI2

(11–14) (38–42)

Coiled-coilregion

ZWINT Ndc80 complexSPC24

SPC25

Loop (kink)NDC80 (HEC1)

NUF2

CH domains

7721

250 500 1500 1833 2316

21061904

S/GILK RRVSF

Molecular distribution along the axis of kMTs

(Distance in nm from CENP-I)

0 10 20 30 40 50 60 70

NNF1

MIS12

DSN1NSL1

C

Fig. 2. Structure and composition of the KMN network. The molecular distribution of the components of the KMN network is shown in the centre of the figure

and is projected along the axis of kMTs (shown in light blue). The CCAN (shown in yellow) forms the key connection between the KMN network at outer

kinetochores and the centromeric chromatin. The human Mis12 complex (shown on the top left) consists of four subunits NNF1, MIS12, DSN1 and NSL1 that are

apparently arranged linearly along the inside–outside axis of kinetochores in the sequence indicated (Petrovic et al., 2010). The C-terminal tail of the NSL1 subunit at

the kinetochore-proximal outer end of the complex is important for interacting with the Ndc80 complex and KNL1, whereas the centromeric DNA-proximal inner end

binds to the CCAN network. High-resolution two-color fluorescent imaging has shown that the Mis12 complex is located ,50 nm inside of the outer end of the

Ndc80 complex and ,15 nm outside of CENP-I (part of the CCAN), and that MIS12 is probably oriented at an angle along the inner–outer kinetochore axis (Wan et

al., 2009). Human KNL1 (top right) is a large multi-domain protein with the known functional domains and motifs indicated. KNL1 is elongated with its N-terminal

region, which contains the proposed MT-binding site, located ,40 nm away from CENP-I, and its C-terminal region, which is required for its kinetochore targeting

(where it binds to MIS12 and its partner ZWINT), ,15 nm away from CENP-I (Kiyomitsu et al., 2007; Petrovic et al., 2010; Wan et al., 2009). ZWINT (bottom left)

is a relatively small coiled-coil rich kinetochore protein that binds the C-terminal coiled-coil domain of KNL1 and forms a tight complex with KNL1 (Petrovic et al.,

2010). It has also been shown that ZWINT interacts with the Mis12 complex and the Ndc80 complex. The human Ndc80 complex (bottom right) is the major

component of the core MT attachment module at kinetochores and consist of four subunits NDC80 (HEC1), NUF2, SPC24 and SPC25, which heterotetramerize

through their coiled-coil regions as indicated. The N-terminal CH domain and charged unstructured tail regions of the NDC80 subunit form a bipartite MT-binding

interface, whereas the globular C-terminal domains of SPC24 and SPC25 bind to CENP-T or the Mis12 complex to link the Ndc80 complex to the inner kinetochore.

The CH domain of NDC80 has also been shown to be important for the retention of checkpoint proteins MAD1 and MAD2 at unattached kinetochores (Guimaraes et

al., 2008; Martin-Lluesma et al., 2002; Miller et al., 2008). The conserved internal loop region of the Ndc80 complex is highly flexible, allowing the a-helical coiled-

coil rod that ends in the MT-binding CH or head domains to rotate or twist at angles between 0 and 120 , with the loop region serving as a hinge. The N-terminal CH

domain of NDC80 is ,100 nm outside of CENP-A, which is bound to the centromere (not depicted), and about 65 nm outside of CENP-I within MT-bound

metaphase kinetochores (Wan et al., 2009). It has also been observed that the distance between the MT-proximal NDC80 CH domain and the head domain of the

centromere-proximal SPC24–SPC25 complex is only about 45 nm in human kinetochores (as compared to the 57-nm-long extended confirmation of the Ndc80

complex reported in yeast) suggesting that the MT-bound confirmation of the Ndc80 complex in human cells is slightly bent during metaphase (Wan et al., 2009).

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2010), but it is not known if a similar complex exists in highervertebrates. In addition to centromere-proximal CENP-A andCCAN, Mis12 has also been shown to require the chaperone

complex Hsp90–Sgt1 for its appropriate targeting to kinetochores(Davies and Kaplan, 2010). Several studies have found that theNsl1 subunit interacts with the heterochromatic protein HP1, and

that this association is important for the formation of the innerkinetochore (Kiyomitsu et al., 2010; Kiyomitsu et al., 2007; Obuseet al., 2004). However, many of the mitotic defects observed by the

perturbation of HP1 could be attributed to improper kinetochorerecruitment of the Ndc80 complex, as both HP1 and the Spc24 orSpc25 subunits of the Ndc80 complex have been shown to bind thesame site in the C-terminal of Nsl1 and that their binding to Nsl1 is

competitive in nature (Petrovic et al., 2010). A recent study hasalso shown that the Dsn1 subunit of the Mis12 complex and Knl1are targets for Aurora B kinase phosphorylation that reduces the

MT-binding affinity of the KMN network (Welburn et al., 2010).This study provides evidence that the phosphorylation of Dsn1sensitizes the KMN network towards dramatic changes in MT-

binding activity.

In summary, the Mis12 complex is a key component of theKMN network that is required to properly target the other twoMT-binding components, the Ndc80 complex and KNL1 tokinetochores. The main function of this complex is to connect the

outer kinetochore region to the inner centromeric DNA inassociation with the proteins of the CCAN network.

The Knl1 complexThe Knl1 complex is a heterodimer of KNL1 (also known as

CASC5 or blinkin in humans, Spc105 in yeast and Spc105R infly) with ZWINT (Petrovic et al., 2010). Human KNL1 is a large300-kDa protein that is recruited to kinetochores by the Mis12

complex (Fig. 2).

KNL1

KNL1 has been shown to have a role in the recruitment of severalouter-domain kinetochore proteins, including its binding partnerZWINT, CENP-F and the mitotic checkpoint proteins BUB1 and

BUBR1 (also known as BUB1B) (Cheeseman et al., 2008;Kiyomitsu et al., 2007). X-ray crystallographic and other studieshave revealed that helical motifs, called the KI motifs (,10–12

amino acids long), in the N-terminal region of KNL1 interactwith the TPR domains of BUBR1 and BUB1 and are importantfor maintaining normal SAC activity in human cells (Bolanos-

Garcia et al., 2011; Kiyomitsu et al., 2011; Krenn et al., 2012).One of these studies has also demonstrated that the interactionbetween the KI motif of KNL1 and the TPR motif of BUB1 doesnot control the targeting of BUB1 to the kinetochore, but instead

might influence the ability of BUB1 to interact with BUBR1,suggesting that KNL1 serves as a scaffold that brings togetherBUB1 and BUBR1 to help generate the kinetochore SAC signal

(assembly of the mitotic checkpoint complex, Cdc20, Mad1,BubR1 and Bub3; Krenn et al., 2012).

Human mitotic cells that have been depleted of KNL1 areseverely compromised in forming stable kMTs, and cells that

enter anaphase often exhibit sister chromosome missegregation(Cheeseman et al., 2008; Kiyomitsu et al., 2007; Schittenhelmet al., 2009). In agreement with a role for KNL1 in the

recruitment of BUB1 and BUBR1, both proteins are found to bedisplaced from kinetochores after KNL1 depletion (Kiyomitsuet al., 2007). Live imaging has revealed that these cells undergo

accelerated mitosis and premature chromosome decondensation

prior to the onset of anaphase (Kiyomitsu et al., 2007). Apartfrom recruiting BUB1 and BUBR1 to the kinetochore, KNL-1has been shown to have a role in recruiting the Ndc80 complex

and the RZZ checkpoint complex in C. elegans (Cheeseman et al.,2008; Essex et al., 2009; Gassmann et al., 2008). However, inhuman cells, NDC80 recruitment to kinetochores does not appearto require the function of KNL1 (Cheeseman et al., 2008;

Kiyomitsu et al., 2007). KNL1 has also been shown to have MT-binding activity at its extreme N-terminus (Cheeseman et al.,2006; Espeut et al., 2012), and it has been shown in C. elegans

that this is necessary for SAC activity but dispensable for theformation of proper kMT attachments and other aspects ofchromosome segregation (Espeut et al., 2012). In that study, the

authors expressed a mutant KNL-1 that lacked the MT-bindingactivity in C. elegans embryos, and found that they exhibit delaysin anaphase onset and persistent checkpoint activation. Even

though the amino acids at positions 1–500 are required for MTbinding, the crucial sequences were narrowed down to a basicstretch of nine amino acid residues at the extreme N-terminus ofthe protein (Espeut et al., 2012).

The extreme N-terminus of KNL1 has also been shown to beimportant for the recruitment of protein phosphatase 1 (PP1) toouter kinetochores. PP1 activity counteracts the activity of the

kinase Aurora B, which is involved in the destabilization ofkMTs (by phosphorylating multiple sites within the KMNnetwork), and consequently assists in accurate chromosomebiorientation and alignment (Liu et al., 2010; Meadows et al.,

2011; Rosenberg et al., 2011; Welburn et al., 2010). A recentstudy has suggested that the recruitment of PP1 to thekinetochore by KNL1 also contributes to SAC activity in an

independent and possibly additive manner to that of its MT-binding activity (Espeut et al., 2012). This study further proposesthat the MT-binding activity of KNL1 acts as a sensor of kMT

attachment and relays this information to the checkpointmachinery for silencing of the SAC. More recently, threeseparate studies have shown that the yeast Knl1 homologue

Spc105 is a target for the Mps1 (Mph1) checkpoint kinase andthat this activity in turn is responsible for the recruitment of theBub1–Bub3 checkpoint complex to kinetochores (London et al.,2012; Shepperd et al., 2012; Yamagishi et al., 2012). One of these

studies also suggests that this phosphorylation activity of Mps1 isopposed by the phosphatase activity of Knl1-recruited PP1(London et al., 2012).

In summary, KNL1 is a large multi-domain and multi-functional scaffold protein that is required for kinetochoretargeting of several other outer-domain kinetochore proteins,including its binding partner ZWINT, the SAC proteins BUB1

and BUBR1, CENP-F and possibly, the RZZ complex. In concertwith the other constituents of the KMN network, KNL1 alsoserves important roles in kMT attachment and silencing the SAC.

ZWINT

ZWINT (KBP-5 in C. elegans) is a 277-amino-acid kinetochoreprotein that initially has been implicated in the targeting of ZW10

to kinetochores (Starr et al., 2000) (Fig. 2). ZW10 is part ofanother outer kinetochore complex, the RZZ complex, which alsocontains the two proteins ROD (also known as KNTC1) and

ZWILCH (Karess, 2005). The RZZ complex in turn recruits theadaptor protein Spindly that serves to attach the dynein–dynactinmotor complexes to checkpoint proteins such as the MAD1–MAD2

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complex (Gassmann et al., 2008; Griffis et al., 2007). A role forZWINT in the recruitment of the RZZ complex to kinetochores

has been confirmed in subsequent studies (Kops et al., 2005; Linet al., 2006; Wang et al., 2004), Surprisingly, ZWINT was foundto co-purify with the components of the KMN network, but notwith the RZZ complex (Cheeseman et al., 2006; Cheeseman et al.,

2004; Kops et al., 2005). Recently, ZWINT, whose structure hascoiled-coil motives, has been demonstrated to form a tightcomplex with the C-terminal coiled-coil domain of KNL1

(Petrovic et al., 2010), and this might explain reports ofinteractions between ZWINT and MIS12 (Obuse et al., 2004)and the NDC80 (HEC1) subunit of the Ndc80 complex (Lin et al.,

2006; Vos et al., 2011). Similar to the other constituents of theKMN network (Santaguida and Musacchio, 2009), it has alsobeen shown that ZWINT is a stable component of thekinetochore, whereas the RZZ complex is more dynamic in its

residency at metaphase kinetochores (Famulski et al., 2008).ZWINT is also known to be a substrate for phosphorylationby the Aurora B kinase. It has been proposed that this

phosphorylation is required for recruitment of the RZZcomplex and the dynein motor to kinetochores, which in turn isimportant for chromosome motility and SAC signaling (Famulski

and Chan, 2007; Kasuboski et al., 2011). Very recently, apotential functional homologue of ZWINT has been identified infission yeast as a binding partner for the yeast KNL1 homologue

Spc105 (Jakopec et al., 2012).

The Ndc80 complexA number of extensive reviews have discussed the structure and

function of the Ndc80 complex (see Alushin and Nogales, 2011;DeLuca and Musacchio, 2012; Joglekar et al., 2010; Lampert andWestermann, 2011; Takeuchi and Fukagawa, 2012), and this

Commentary will thus only attempt to summarize the recentadvances in our understanding of this complex. The Ndc80complex is a 57-nm-long heterotetrameric complex consisting of

two dimers, NDC80 (HEC1) and NUF2, and SPC24 and SPC25,that are held together by overlapping a-helical coiled coildomains located at the C-terminus of the NDC80–NUF2 dimerand the N-terminus of the SPC24–SPC25 dimer (Cheeseman and

Desai, 2008; Ciferri et al., 2008; Wei et al., 2007). The C-terminus of SPC24–SPC25 interacts with both CENP-T inhumans (Gascoigne et al., 2011) and budding yeast (Bock et al.,

2012; Schleiffer et al., 2012), or with the NSL1 and DSN1components of the Mis12 complex in humans and Drosophila

(see above).

Studies that have analyzed the various predicted MT-bindingdomains within the Ndc80 complex during mitosis suggest thatboth the NDC80 CH domain and its charged N-terminal tail areimportant for the formation of stable kMT attachments and for

chromosome alignment, whereas the CH domain of NUF2,although it is non-essential for these functions, is required forproducing normal MT-dependent kinetochore force and timely

mitotic progression (Sundin et al., 2011; Tooley et al., 2011).Furthermore, it has been demonstrated that the N-terminal tail ofNDC80 is required to interact with the negatively charged C-

terminal tails (the so-called E-hooks) of tubulin monomers(Alushin et al., 2010; Ciferri et al., 2008; Tooley et al., 2011).These interactions are weakened by phosphorylation of the nine

Aurora B target sites within the N-terminal tail of NDC80(Cheeseman et al., 2006; DeLuca et al., 2006; Santaguida andMusacchio, 2009; Wei et al., 2007). It has been demonstrated in

vivo that Aurora B kinase activity is important for destabilizingkMT attachment and correction of MT-attachment errors, which

is highly relevant for the fidelity of mitotic chromosomesegregation (Cheeseman et al., 2006; Cimini et al., 2006;DeLuca et al., 2006; Sandall et al., 2006; Welburn et al.,2010). Studies with reconstituted protein preparations in vitro

show that dephosphorylated NDC80 bound to beads can track,stabilize and promote rescue of the ends of depolymerizing MTs,whereas Aurora B phosphomimetic versions of the complex are

unable to do so, further emphasizing the importance of Aurora Bphosphorylation for the function of the Ndc80 complex (Umbreitet al., 2012).

Structural studies have provided important details of MTbinding by the Ndc80 complex (Wilson-Kubalek et al., 2008;Alushin et al., 2010). A key MT-binding region is located withinthe CH domain of NDC80 at a site termed ‘toe print’. The ‘toe’

serves as a sensor of the tubulin conformation that enables theNdc80 complex to bind to straight MT protofilaments with highaffinity and to curled protofilaments at the ends of

depolymerizing MTs with low affinity. The Alushin et al. studyand two other studies have found that the Ndc80 subunit NUF2,which also contains a CH domain, is not involved in the

interaction with MTs (Sundin et al., 2011; Alushin et al., 2010;Wilson-Kubalek et al., 2008), despite a previous report showingthat mutations within the NUF2 CH domain interfere with the

binding of the Ndc80 complex to MTs (Ciferri et al., 2008).Additionally, Alushin et al. provide evidence that theunstructured N-terminal tail of NDC80 aids in tetheringtogether adjacent Ndc80 complexes upon MT binding, an idea

that has been supported by several other studies (Cheesemanet al., 2006; Ciferri et al., 2008; Powers et al., 2009; Tooley et al.,2011). Another recent study dissected the role of the tail domain

in MT binding and Ndc80 clustering in even more detail (Alushinet al., 2012). This study shows that Aurora B sites are organizedas two separate segments or zones in the tail region, one at the

tubulin-binding interface and the other at the interface with anadjacent MT-bound Ndc80 complex; phosphorylation at the twodifferent sites serves to fine tune the interaction of the Ndc80complex with tubulin monomers and with neighboring Ndc80

complexes.

The CH domain of NDC80 is also important for recruitingspindle checkpoint proteins such as the MAD1–MAD2 complex

(Guimaraes et al., 2008, Martin-Lluesma et al., 2002, Milleret al., 2008). In addition, serine 165 within the NDC80 CHdomain is a target for the kinase NEK2, and this phosphorylation

is important for chromosome alignment and the SAC (Wei et al.,2011). Cells expressing a non-phosphorylatable mutation ofNDC80 at serine 165 are unable to recruit MAD1–MAD2 to

kinetochores and exhibit premature anaphase onset witherroneous chromosome segregation. Phosphorylation ofNDC80 and the Ndc80 complex by Aurora B have also beenshown to be important for the recruitment of the checkpoint

kinase MPS1 (also known as TTK in vertebrates) tokinetochores, leading to its activation, which is essential forthe SAC to prevent anaphase (Vigneron et al., 2004; Hewitt et al.,

2010; Santaguida et al., 2010; Maciejowski et al., 2010).

Taken together, the heterotetrameric Ndc80 complex emerges asthe major MT-binding interface at kinetochores and uses both the

N-terminal CH domain and tail domains of its NDC80 subunit tobind MTs. Phosphorylation of key amino acid residues at the N-terminal tail by Aurora B kinase negatively influences MT-binding

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affinity and serves as an effective mechanism to regulate the

strength of kMT attachment and the process of attachment error

correction. The N-terminal CH domain also plays a role in

recruiting MAD1, MAD2 and Mps1 to kinetochores.

Further insights into KMN network functionDam/Dash and Ska MAP complexes

Another active area of research has been to address the pivotal

question of how the activity of the KMN network is coupled to

the polymerization and depolymerization dynamics of kMT plus-

ends by MAPS that potentially interact with the Ndc80 complex

or the KMN network and are required for effective kMT

attachments and force generation (Joglekar et al., 2010). In yeast,

the Dam complex (also known as the Dash complex) is a MT-

binding protein that depends on the kinetochore-bound Ndc80

complex for its localization to kinetochores. The Dam/Dash

complex is composed of 10 subunits that assemble into oligomers

and form either closed stable ring-like structures or open spirals

around the MT lattice (Lampert and Westermann, 2011). Dam/

Dash has been shown to increase the processivity of the Ndc80

complex when it is attached to depolymerizing MTs, and is also a

prominent target for the yeast homologue of Aurora B kinase,

Ipl1 (Alushin and Nogales, 2011, Grishchuk et al., 2008; Lampert

and Westermann, 2011). Structural homologs of the Dam/Dash

complex components have not been discovered in higher

eukaryotes, but recent studies are beginning to shed light on

other MAPs that interact with the Ndc80 complex or the KMN

network. In human cells, the kinetochore-localized Ska complex

has emerged as one of the major modes of these regulatory

functions as discussed below.

The Ska complex was initially identified in human cells as a

complex comprising two proteins SKA1 and SKA2. SKA1 and

SKA2 were found to localize to spindle MTs and to concentrate

at kinetochores in a manner that is dependent on kinetochore-

bound NDC80 (Hanisch et al., 2006). Subsequently, a third

subunit of the complex, SKA3, also called RAMA1, was

discovered that localizes to kinetochores and spindle MTs, and

knockdown of this protein results in identical phenotypes to those

upon knockdowns of SKA1 and SKA2 (Daum et al., 2009;

Gaitanos et al., 2009; Raaijmakers et al., 2009; Theis et al., 2009;

Welburn et al., 2009). SKA3 is required for the proper stability

and functioning of the Ska complex as a whole (Gaitanos et al.,

2009). In human cells, the Ska complex is reported to be

important for normal kMT formation, metaphase alignment, SAC

silencing and anaphase chromosome segregation (Daum et al.,

2009; Gaitanos et al., 2009; Hanisch et al., 2006; Raaijmakers

et al., 2009; Theis et al., 2009; Welburn et al., 2009). The human

Ska complex has been reconstituted in vitro and shown to bind

MTs in a cooperative manner (Welburn et al., 2009). Moreover,

that study, as well as more recent data (Schmidt et al., 2012),

have shown that the Ska complex can form oligomeric

assemblies that diffuse along MTs and that it binds to

Box 1. Further insights from the structure of the Ska complex

The crystal structure of the dimerization domains of the human Ska complex has been recently solved, which sheds light into the molecular basis

of its attachment to MTs (Jeyaprakash et al., 2012). A major finding of that study is that the Ska complex is a ,35-nm-wide W-shaped dimer,

consisting of two copies of each of the SKA1, SKA2 and SKA3 subunits, and this complex can be envisaged to possess four MT-binding sites

(see upper panel of the figure), one each in the SKA1 and SKA3 subunits (see lower panel of the figure).

The crystal structure of the dimerization domain of the Ska complex suggests that the SKA1 subunit functions as a structural framework to

bridge the interaction between SKA2 and SKA3. The structural core of the Ska monomer consists of the N-terminal helical domains of SKA1 and

SKA3 and the entire SKA2, which is also completely helical. These helical structures interact with each other at two separate regions to form two

intertwined helical bundles. The N-terminal ends of the short helical

bundles from each of the subunit interact face-to-face to form the

dimerization interphase, whereas the C-terminal ends of the longer

helical bindles of each of the three subunits intertwine with each other

and point outwards into the solvent to form the two arms of the W-shaped

structure. The N-terminal ends of the short helices that form the central

hydrophobic dimerization interface has an open confirmation that enables

key molecular interactions which are utilized to accomplish effective

dimerization. The globular C-terminal domains of both the SKA1 and SKA3

subunits project from the ends of the two arms of the ‘W’ after dimerization

and contain the four putative MT-binding sites of the Ska complex.

The authors speculate that this proposed geometry of the Ska complex

might help it to bind transversely across the curvature of MTs. This lateral

linkage, in concert with the longitudinal protofilament interaction geometry

of the Ndc80 complex, is proposed to enhance kinetochore attachment and

processivity at depolymerizing MT ends. Cells containing Ska complexes

that either lack the MT-binding domains or have mutations in key residues

in the central hydrophobic dimerization core exhibit severe delays in mitotic

progression or mitotic arrest (Jeyaprakash et al., 2012). Another recent

study has determined that the SKA1 MT-binding site within the C-terminal

domain is a winged-helix tertiary structure that is characteristic of DNA-

binding proteins, and that phosphorylation of sites within this C-terminal

domain inhibit MT binding. The MT-binding activity of the SKA3 C-terminal

domain remains unclear (Schmidt et al., 2012). The diagram of the crystal

structure was kindly provided by A. Arockia Jeyaprakash and Elena Conti

(Max-Planck Institute of Biochemistry, Martinsried, Germany).

SKA3 SKA1

MT-binding

SKA2

~35 nm

~ 18 nm

SKA1ΔCSKA2ΔCSKA3ΔC

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depolymerizing MT plus-ends along both straight and curved MT

protofilaments (the Ndc80 complex binds only straight

protofilaments near MT ends). The purified Ska complex

exhibits processive motion at the ends of depolymerizing MTs

and strengthens plus-end tracking of the purified Ndc80 complex.

Moreover, the Ska complex enhances the capacity of the Ndc80

complex to bind MTs in a cooperative manner. These properties

of the Ska complex, together with the requirement of MTs and

the Ndc80 complex for its localization to kinetochores, suggests

that the Ska complex could be a functional (but possibly not

structural) homologue in vertebrates of the Dam/Dash complex in

yeast (Gaitanos et al., 2009; Hanisch et al., 2006; Welburn et al.,

2009; Schmidt et al., 2012).

There is also evidence that the Ska complex interacts with

KNL1 and the Mis12 complex, in addition to the Ndc80 complex,

and that the recruitment of Ska to kinetochores depends on all three

members of the KMN network (Chan et al., 2012). That study also

showed that SKA1 and SKA3 are targets for phosphorylation by

Aurora B and that their phosphorylation negatively regulates the

association of the Ska complex with the KMN network, which in

turn inhibits the recruitment of the Ska complex to kinetochores

and impairs its role in stabilizing kMT attachments.

Taken together, these studies show that the Ska complex works

in close concert with the components of the KMN network, and

the Ndc80 complex in particular, to provide stability to kMT

attachments unless it is phosphorylated by Aurora B. Recent

evidence also indicates that Ska is indeed a vertebrate functional

homologue of the yeast Dam/Dash complex, but the finer details

of how Ska associates with the KMN network and the MT lattice

to fulfill this function are far from being resolved. Also

unresolved is whether there are additional activities for the Ska

complex in controlling mitotic progression, such as has been

proposed by Daum et al. (Daum et al., 2009), for preventing the

premature loss of sister chromatid cohesion. Further insights that

can be gained from the Ska structure are presented in Box 1.

The role of the NDC80 loop domain

There is a prominent kink or bend in the structure of the Ndc80

complex ,16 nm away from the MT-binding CH domain of

NDC80. The location of the kink coincides with a break in

registry of the central coiled-coil region within NDC80, which

likely produces a loop in the secondary structure at this site

(Wang et al., 2008). The sequence encompassing the loop was

found to be evolutionarily conserved from yeast to human

Box 2. Function of the human NDC80 loop domain

Recent studies in human cells on the role of the KMN network in kMT attachment have found that the loop domain of NDC80 (HEC1) is

instrumental in stabilizing kMTs attachments (see figure) (Zhang et al., 2012; Varma et al., 2012; Matson and Stukenberg, 2012). Several lines of

evidence suggest that the Ndc80 complex is slightly bent with a centrally located loop region that provides flexibility in complex confirmation.

Both the CH domain and the N-terminal tail of NDC80 (see figure, shown in red) and the extreme N-terminus of KNL1 bind to MTs. In addition,

the central loop region of NDC80 provides a third MT-binding site within the Ndc80 complex, albeit indirectly. Linkage from the loop domain to

kMTs requires MAPs, such as Dis1 (fission yeast), the Dam/Dash complex (budding yeast), or possibly the Ska complex (human cells). In

human cells, the DNA replication licensing protein CDT1 also binds to the loop region and is required for robust kMT attachment (see below). We

only have limited knowledge about the exact location and orientation of

these proteins relative to kMTs, and their relative positions within the

kinetochore are not depicted accurately in the figure.

Sequence modifications in the loop region have shown that this

region is important for the recruitment of other proteins that are

required for normal kMT attachments. Zhang et al. found that the

Ndc80 complexes assembled in the absence of the loop region are

unable to associate properly with the Ska complex (Zhang et al., 2012).

They proposed that the loop domain directly recruits the Ska complex

to kinetochores, and that the absence of the loop-recruited Ska

complex led to the observed defects in MT attachments to

kinetochores in mitotic cells with loop mutations. A second study, by

our group, presented a new mechanism for the stabilization of kMT

attachments (Varma et al., 2012). We identified the DNA replication

licensing protein CDT1 as a novel kinetochore protein that interacts

with the Ndc80 complex. Inhibition of CDT1 function specifically during

mitosis by either small interfering RNA-based approaches or

microinjection of function-blocking antibodies induces a SAC-

dependent late prometaphase arrest with severe defects in kMT

attachments (Varma et al., 2012). The localization of CDT1 to

kinetochores is dependent on the loop region of NDC80, and

scrambling the loop region sequence induces a mitotic arrest that is

similar to that observed when the loop is missing (Zhang et al., 2012)

or when CDT1 is inhibited (Varma et al., 2012). Detailed analysis of the

structure of the Ndc80 complex suggests that the binding of CDT1 to

the loop region of kinetochore-bound NDC80 helps to maintain an

extended confirmation of the Ndc80 complex in the presence of kMTs,

which aids in more robust kMT attachments. Intriguingly, our study did

not observe a significant decrease in the concentration of the Ska

complex at kinetochores when the NDC80 loop region was mutated.

Human NDC80 loop-binding protein, CDT1

MAPs binding the loop in yeast (e.g. Dis1 and Dam1)

Human KMN network and the Ndc80 loop-interacting complex (Ska)

CENP-C

CENP-T

KNL1

CENP-A

Mis12 complex

(View normal to the axis of kMT)

Ndc80 complex

Key

NDC80 loop motif

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(Wang et al., 2008), but until very recently its function was not

clear.

Two studies in yeast determined that the loop region isimportant for stable kMT attachment. The first study, inSaccharomyces cerevisiae, showed that the loop region is

important for the recruitment of the Dam/Dash complex tokinetochores and possibly for its loading on to kMTs (Maureet al., 2011). Those authors observed that, when the loop region is

deleted or mutated, there is a severe defect in the conversion oflateral to load-bearing (i.e. end-on) kMT attachments andkinetochore bi-orientation. The other study in the fission yeast

Saccharomyces pombe, which also using deletion or directedmutation of key residues or sequences within the loop region,showed that this region is important for the recruitment of theMAP Dis1 (TOG or XMAP215) to kinetochores with stable end-

on attachments to spindle MT ends (Hsu and Toda, 2011).Consequently, the SAC remains persistently activated in cellswith mutated loop domains and they undergo prolonged mitotic

arrest. Deletion of the Dis1 gene phenocopies the defects seenwith loop mutations and was rescued by the artificial targeting ofDis1 to the Ndc80 complex.

These studies, along with recent studies in human cells (see

Box 2), support the notion that in addition to the CH domain andN-terminal tail of NDC80, its loop domain also constitutes a MT-binding interface, thus providing a total of three different MT-

binding domains within the Ndc80 complex.

Concluding remarks and future directionsFuture work will help to delineate the connections between the

different components of the KMN network and the SAC(including the Mads, the Bubs and the RZZ). We already knowthat the CH domain of NDC80 is involved in recruiting MAD1,

and that KNL1 is required for recruiting ZWINT, BUBR1 andBUB1, as well as the RZZ complex in C. elegans (Gassmannet al., 2008). Both ZWINT and the Ndc80 complex recruit the

RZZ complex, which in turn appears to be a prerequisite for therecruitment of MAD1 and MAD2 (Fig. 3). However, we still lackprecise knowledge of how MT attachments trigger dynein-mediated removal of SAC components (‘stripping’) and silencing

of the checkpoint, or how the checkpoint feeds back into theattachment machinery of the KMN network. Part of the problemlies in the fact that the association of checkpoint proteins with the

KMN network are possibly highly transient, which makes theirbiochemical analysis exceedingly difficult. We are slowlybeginning to understand some of these mechanisms as shown

by recent work that suggests that MT-binding by Knl1 is likely tobe a key sensor for controlling checkpoint activity (Espeut et al.,2012), but more work is mandatory to elucidate the intricacies ofthe interplay between MT attachment and SAC and control

mechanisms governed by Aurora B, MPS1 and PP1. As Knl1 is alarge multi-domain protein, progress in our understanding of itsstructure and function has been relatively slow and more research

in this direction is likely to be worthwhile. Although we know tosome extent the functions of the N- and the C-termini of Knl1, itscentral region remains a ‘black box’ and analysis of this region is

likely to yield valuable information of its role in the SAC andkMT attachment. An area, in which there is a high chance forimmediate progress is the further study of the function of the loop

in the Ndc80 complex and the proteins that interact with it.Research efforts in this direction will help us to understand if theSka complex is indeed an orthologue of Dam/Dash and how

exactly the Ska complex and the KMN network coordinate kMTattachment, force generation, attachment error correction andcontrol of the SAC. Future studies should also shed light on the

exact molecular mechanism by which the replication licensingprotein CDT1 is able to influence the conformational change ofNdc80 complex in the presence of kMTs (see Box 2), and alsoelucidate whether different Ndc80 loop-binding proteins act in

concert to accomplish stable kMT attachments.

AcknowledgementsWe would like to acknowledge A. Arochia Jeyaprakash and ElenaConti for providing us with the high-resolution structure of the Skacomplex dimerization domains. We would also like to thank JeanCook, Jenifer Deluca, and several past and present members of theSalmon laboratory for insightful discussions.

FundingThe work of our laboratory is supported by the National Institutes ofHealth [grant number R37GM024364 and supplement to E.D.S.].Deposited in PMC for release after 12 months.

Note added in proofAfter the acceptance of this manuscript, a review written by JakobNilsson was published in the journal Bioessays, describing indetailed the function of the Ndc80 loop domain (Nilsson, 2012).

Fig. 3. Role of the KMN network in the SAC. The schematic illustration

shows the known function of KMN network components in the recruitment of

SAC proteins (red) and their recruitment of each other (indicated by arrows).

ZWINT (KBP-5) and KNL-1 in C. elegans are required for the recruitment of

the RZZ checkpoint complex. Both the Ndc80 complex and the RZZ complex

have been shown to be required for the proper localization of the MAD1–

MAD2 checkpoint complex to kinetochores that are unattached in

prometaphase. KNL1 is important for the kinetochore recruitment of ZWINT

and the BUB1–BUBR1 checkpoint complex. Spindly (recruited by the RZZ

complex) serves as an adaptor between the dynein–dynactin motor complex

and the MAD1–MAD2 and RZZ complexes, and is required for the dynein

motor-driven stripping of these checkpoint complexes during SAC silencing.

See text for details.

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ReferencesAlushin, G. and Nogales, E. (2011). Visualizing kinetochore architecture. Curr. Opin.

Struct. Biol. 21, 661-669.

Alushin, G. M., Ramey, V. H., Pasqualato, S., Ball, D. A., Grigorieff, N., Musacchio,

A. and Nogales, E. (2010). The Ndc80 kinetochore complex forms oligomeric arraysalong microtubules. Nature 467, 805-810.

Alushin, G. M., Musinipally, V., Matson, D., Tooley, J., Stukenberg, P. T. andNogales, E. (2012). Multimodal microtubule binding by the Ndc80 kinetochorecomplex. Nat. Struct. Mol. Biol. 19, 1161-1167.

Bock, L. J., Pagliuca, C., Kobayashi, N., Grove, R. A., Oku, Y., Shrestha, K., Alfieri,

C., Golfieri, C., Oldani, A., Dal Maschio, M. et al. (2012). Cnn1 inhibits theinteractions between the KMN complexes of the yeast kinetochore. Nat. Cell Biol. 14,614-624.

Bolanos-Garcia, V. M., Lischetti, T., Matak-Vinkovic, D., Cota, E., Simpson, P. J.,Chirgadze, D. Y., Spring, D. R., Robinson, C. V., Nilsson, J. and Blundell, T. L.

(2011). Structure of a Blinkin-BUBR1 complex reveals an interaction crucial forkinetochore-mitotic checkpoint regulation via an unanticipated binding Site. Structure

19, 1691-1700.

Chan, Y. W., Jeyaprakash, A. A., Nigg, E. A. and Santamaria, A. (2012). Aurora Bcontrols kinetochore-microtubule attachments by inhibiting Ska complex-KMNnetwork interaction. J. Cell Biol. 196, 563-571.

Cheeseman, I. M. and Desai, A. (2008). Molecular architecture of the kinetochore-microtubule interface. Nat. Rev. Mol. Cell Biol. 9, 33-46.

Cheeseman, I. M., Niessen, S., Anderson, S., Hyndman, F., Yates, J. R., 3rd,Oegema, K. and Desai, A. (2004). A conserved protein network controls assembly ofthe outer kinetochore and its ability to sustain tension. Genes Dev. 18, 2255-2268.

Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M. and Desai, A. (2006). Theconserved KMN network constitutes the core microtubule-binding site of thekinetochore. Cell 127, 983-997.

Cheeseman, I. M., Hori, T., Fukagawa, T. and Desai, A. (2008). KNL1 and theCENP-H/I/K complex coordinately direct kinetochore assembly in vertebrates. Mol.

Biol. Cell 19, 587-594.

Ciferri, C., Pasqualato, S., Screpanti, E., Varetti, G., Santaguida, S., Dos Reis, G.,Maiolica, A., Polka, J., De Luca, J. G., De Wulf, P. et al. (2008). Implications forkinetochore-microtubule attachment from the structure of an engineered Ndc80complex. Cell 133, 427-439.

Cimini, D., Wan, X., Hirel, C. B. and Salmon, E. D. (2006). Aurora kinase promotesturnover of kinetochore microtubules to reduce chromosome segregation errors. Curr.

Biol. 16, 1711-1718.

Corbett, K. D., Yip, C. K., Ee, L. S., Walz, T., Amon, A. and Harrison, S. C. (2010).The monopolin complex crosslinks kinetochore components to regulate chromosome-microtubule attachments. Cell 142, 556-567.

Daum, J. R., Wren, J. D., Daniel, J. J., Sivakumar, S., McAvoy, J. N., Potapova,

T. A. and Gorbsky, G. J. (2009). Ska3 is required for spindle checkpoint silencingand the maintenance of chromosome cohesion in mitosis. Curr. Biol. 19, 1467-1472.

Davies, A. E. and Kaplan, K. B. (2010). Hsp90-Sgt1 and Skp1 target human Mis12complexes to ensure efficient formation of kinetochore-microtubule binding sites.J. Cell Biol. 189, 261-274.

DeLuca, J. G. and Musacchio, A. (2012). Structural organization of the kinetochore-microtubule interface. Curr. Opin. Cell Biol. 24, 48-56.

DeLuca, J. G., Gall, W. E., Ciferri, C., Cimini, D., Musacchio, A. and Salmon, E. D.

(2006). Kinetochore microtubule dynamics and attachment stability are regulated byHec1. Cell 127, 969-982.

Espeut, J., Cheerambathur, D. K., Krenning, L., Oegema, K. and Desai, A. (2012).Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at thekinetochore. J. Cell Biol. 196, 469-482.

Essex, A., Dammermann, A., Lewellyn, L., Oegema, K. and Desai, A. (2009).Systematic analysis in Caenorhabditis elegans reveals that the spindle checkpoint iscomposed of two largely independent branches. Mol. Biol. Cell 20, 1252-1267.

Famulski, J. K. and Chan, G. K. (2007). Aurora B kinase-dependent recruitment ofhZW10 and hROD to tensionless kinetochores. Curr. Biol. 17, 2143-2149.

Famulski, J. K., Vos, L., Sun, X. and Chan, G. (2008). Stable hZW10 kinetochoreresidency, mediated by hZwint-1 interaction, is essential for the mitotic checkpoint.J. Cell Biol. 180, 507-520.

Foltz, D. R., Jansen, L. E., Black, B. E., Bailey, A. O., Yates, J. R., 3rd and

Cleveland, D. W. (2006). The human CENP-A centromeric nucleosome-associatedcomplex. Nat. Cell Biol. 8, 458-469.

Gaitanos, T. N., Santamaria, A., Jeyaprakash, A. A., Wang, B., Conti, E. and Nigg,

E. A. (2009). Stable kinetochore-microtubule interactions depend on the Ska complexand its new component Ska3/C13Orf3. EMBO J. 28, 1442-1452.

Gascoigne, K. E. and Cheeseman, I. M. (2011). Kinetochore assembly: if you build it,they will come. Curr. Opin. Cell Biol. 23, 102-108.

Gascoigne, K. E., Takeuchi, K., Suzuki, A., Hori, T., Fukagawa, T. and Cheeseman,I. M. (2011). Induced ectopic kinetochore assembly bypasses the requirement forCENP-A nucleosomes. Cell 145, 410-422.

Gassmann, R., Essex, A., Hu, J. S., Maddox, P. S., Motegi, F., Sugimoto, A.,

O’Rourke, S. M., Bowerman, B., McLeod, I., Yates, J. R., 3rd et al. (2008). A newmechanism controlling kinetochore-microtubule interactions revealed by comparisonof two dynein-targeting components: SPDL-1 and the Rod/Zwilch/Zw10 complex.Genes Dev. 22, 2385-2399.

Goshima, G. and Yanagida, M. (2000). Establishing biorientation occurs withprecocious separation of the sister kinetochores, but not the arms, in the early spindleof budding yeast. Cell 100, 619-633.

Goshima, G., Saitoh, S. and Yanagida, M. (1999). Proper metaphase spindle length isdetermined by centromere proteins Mis12 and Mis6 required for faithful chromosomesegregation. Genes Dev. 13, 1664-1677.

Griffis, E. R., Stuurman, N. and Vale, R. D. (2007). Spindly, a novel protein essentialfor silencing the spindle assembly checkpoint, recruits dynein to the kinetochore.J. Cell Biol. 177, 1005-1015.

Grishchuk, E. L., Efremov, A. K., Volkov, V. A., Spiridonov, I. S., Gudimchuk, N.,

Westermann, S., Drubin, D., Barnes, G., McIntosh, J. R. and Ataullakhanov, F. I.(2008). The Dam1 ring binds microtubules strongly enough to be a processive as wellas energy-efficient coupler for chromosome motion. Proc. Natl. Acad. Sci. USA 105,15423-15428.

Guimaraes, G. J., Dong, Y., McEwen, B. F. and Deluca, J. G. (2008). Kinetochore-microtubule attachment relies on the disordered N-terminal tail domain of Hec1.Curr. Biol. 18, 1778-1784.

Hanisch, A., Sillje, H. H. and Nigg, E. A. (2006). Timely anaphase onset requires anovel spindle and kinetochore complex comprising Ska1 and Ska2. EMBO J. 25,5504-5515.

Hewitt, L., Tighe, A., Santaguida, S., White, A. M., Jones, C. D., Musacchio, A.,Green, S. and Taylor, S. S. (2010). Sustained Mps1 activity is required in mitosis torecruit O-Mad2 to the Mad1-C-Mad2 core complex. J. Cell Biol. 190, 25-34.

Hori, T. and Fukagawa, T. (2012). Establishment of the vertebrate kinetochores.Chromosome Res. 20, 547-561.

Hori, T., Amano, M., Suzuki, A., Backer, C. B., Welburn, J. P., Dong, Y., McEwen,B. F., Shang, W. H., Suzuki, E., Okawa, K. et al. (2008). CCAN makes multiplecontacts with centromeric DNA to provide distinct pathways to the outer kinetochore.Cell 135, 1039-1052.

Hornung, P., Maier, M., Alushin, G. M., Lander, G. C., Nogales, E. and

Westermann, S. (2011). Molecular architecture and connectivity of the buddingyeast Mtw1 kinetochore complex. J. Mol. Biol. 405, 548-559.

Hsu, K. S. and Toda, T. (2011). Ndc80 internal loop interacts with Dis1/TOG to ensureproper kinetochore-spindle attachment in fission yeast. Curr. Biol. 21, 214-220.

Jakopec, V., Topolski, B. and Fleig, U. (2012). Sos7, an essential component of theconserved Schizosaccharomyces pombe Ndc80-MIND-Spc7 complex, identifies anew family of fungal kinetochore proteins. Mol. Cell. Biol. 32, 3308-3320.

Jeyaprakash, A. A., Santamaria, A., Jayachandran, U., Chan, Y. W., Benda, C.,

Nigg, E. A. and Conti, E. (2012). Structural and functional organization of the Skacomplex, a key component of the kinetochore-microtubule interface. Mol. Cell 46,274-286.

Joglekar, A. P., Bloom, K. and Salmon, E. D. (2009). In vivo protein architecture ofthe eukaryotic kinetochore with nanometer scale accuracy. Curr. Biol. 19, 694-699.

Joglekar, A. P., Bloom, K. S. and Salmon, E. D. (2010). Mechanisms of forcegeneration by end-on kinetochore-microtubule attachments. Curr. Opin. Cell Biol. 22,57-67.

Karess, R. (2005). Rod-Zw10-Zwilch: a key player in the spindle checkpoint. Trends

Cell Biol. 15, 386-392.

Kasuboski, J. M., Bader, J. R., Vaughan, P. S., Tauhata, S. B., Winding, M.,

Morrissey, M. A., Joyce, M. V., Boggess, W., Vos, L., Chan, G. K. et al. (2011).Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-bindingplatform on kinetochores. Mol. Biol. Cell 22, 3318-3330.

Kiyomitsu, T., Obuse, C. and Yanagida, M. (2007). Human Blinkin/AF15q14 isrequired for chromosome alignment and the mitotic checkpoint through directinteraction with Bub1 and BubR1. Dev. Cell 13, 663-676.

Kiyomitsu, T., Iwasaki, O., Obuse, C. and Yanagida, M. (2010). Inner centromereformation requires hMis14, a trident kinetochore protein that specifically recruits HP1to human chromosomes. J. Cell Biol. 188, 791-807.

Kiyomitsu, T., Murakami, H. and Yanagida, M. (2011). Protein interaction domainmapping of human kinetochore protein Blinkin reveals a consensus motif for bindingof spindle assembly checkpoint proteins Bub1 and BubR1. Mol. Cell. Biol. 31, 998-1011.

Kline, S. L., Cheeseman, I. M., Hori, T., Fukagawa, T. and Desai, A. (2006). Thehuman Mis12 complex is required for kinetochore assembly and proper chromosomesegregation. J. Cell Biol. 173, 9-17.

Kops, G. J. and Shah, J. V. (2012). Connecting up and clearing out: how kinetochoreattachment silences the spindle assembly checkpoint. Chromosoma 121, 509-525.

Kops, G. J., Kim, Y., Weaver, B. A., Mao, Y., McLeod, I., Yates, J. R., 3rd, Tagaya,M. and Cleveland, D. W. (2005). ZW10 links mitotic checkpoint signaling to thestructural kinetochore. J. Cell Biol. 169, 49-60.

Krenn, V., Wehenkel, A., Li, X., Santaguida, S. and Musacchio, A. (2012). Structuralanalysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunitKnl1 interaction. J. Cell Biol. 196, 451-467.

Lampert, F. and Westermann, S. (2011). A blueprint for kinetochores - new insightsinto the molecular mechanics of cell division. Nat. Rev. Mol. Cell Biol. 12, 407-412.

Lin, Y. T., Chen, Y., Wu, G. and Lee, W. H. (2006). Hec1 sequentially recruits Zwint-1 and ZW10 to kinetochores for faithful chromosome segregation and spindlecheckpoint control. Oncogene 25, 6901-6914.

Liu, D., Vleugel, M., Backer, C. B., Hori, T., Fukagawa, T., Cheeseman, I. M. andLampson, M. A. (2010). Regulated targeting of protein phosphatase 1 to the outerkinetochore by KNL1 opposes Aurora B kinase. J. Cell Biol. 188, 809-820.

London, N., Ceto, S., Ranish, J. A. and Biggins, S. (2012). Phosphoregulation ofSpc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores. Curr. Biol. 22,900-906.

Maciejowski, J., George, K. A., Terret, M. E., Zhang, C., Shokat, K. M. and

Jallepalli, P. V. (2010). Mps1 directs the assembly of Cdc20 inhibitory complexes

The KMN kinetochore network 5935

Journ

alof

Cell

Scie

nce

during interphase and mitosis to control M phase timing and spindle checkpointsignaling. J. Cell Biol. 190, 89-100.

Maresca, T. J. and Salmon, E. D. (2010). Welcome to a new kind of tension:translating kinetochore mechanics into a wait-anaphase signal. J Cell Sci. 123, 825-835.

Martin-Lluesma, S., Stucke, V. M. and Nigg, E. A. (2002). Role of Hec1 in spindlecheckpoint signaling and kinetochore recruitment of Mad1/Mad2. Science 297, 2267-2270.

Maskell, D. P., Hu, X. W. and Singleton, M. R. (2010). Molecular architecture andassembly of the yeast kinetochore MIND complex. J. Cell Biol. 190, 823-834.

Matson, D. R. and Stukenberg, P. T. (2012). Cdt1 throws kinetochore-microtubuleattachments for a loop. Nat. Cell Biol. 14, 561-563.

Maure, J. F., Komoto, S., Oku, Y., Mino, A., Pasqualato, S., Natsume, K., Clayton,L., Musacchio, A. and Tanaka, T. U. (2011). The Ndc80 loop region facilitatesformation of kinetochore attachment to the dynamic microtubule plus end. Curr. Biol.

21, 207-213.Meadows, J. C., Shepperd, L. A., Vanoosthuyse, V., Lancaster, T. C., Sochaj, A. M.,

Buttrick, G. J., Hardwick, K. G. and Millar, J. B. (2011). Spindle checkpointsilencing requires association of PP1 to both Spc7 and kinesin-8 motors. Dev. Cell 20,739-750.

Miller, S. A., Johnson, M. L. and Stukenberg, P. T. (2008). Kinetochore attachmentsrequire an interaction between unstructured tails on microtubules and Ndc80(Hec1).Curr. Biol. 18, 1785-1791.

Musacchio, A. and Salmon, E. D. (2007). The spindle-assembly checkpoint in spaceand time. Nat. Rev. Mol. Cell Biol. 8, 379-393.

Nilsson, J. (2012). Looping in on Ndc80 - how does a protein loop at the kinetochorecontrol chromosome segregation? Bioessays 34, 1070-1077.

Nishino, T., Takeuchi, K., Gascoigne, K. E., Suzuki, A., Hori, T., Oyama,

T., Morikawa, K., Cheeseman, I. M. and Fukagawa, T. (2012). CENP-T-W-S-Xforms a unique centromeric chromatin structure with a histone-like fold. Cell 148,487-501.

Obuse, C., Iwasaki, O., Kiyomitsu, T., Goshima, G., Toyoda, Y. and Yanagida,M. (2004). A conserved Mis12 centromere complex is linked to heterochromatic HP1and outer kinetochore protein Zwint-1. Nat. Cell Biol. 6, 1135-1141.

Okada, M., Cheeseman, I. M., Hori, T., Okawa, K., McLeod, I. X., Yates, J. R., 3rd,Desai, A. and Fukagawa, T. (2006). The CENP-H-I complex is required for theefficient incorporation of newly synthesized CENP-A into centromeres. Nat. Cell

Biol. 8, 446-457.Perpelescu, M. and Fukagawa, T. (2011). The ABCs of CENPs. Chromosoma 120,

425-446.Petrovic, A., Pasqualato, S., Dube, P., Krenn, V., Santaguida, S., Cittaro,

D., Monzani, S., Massimiliano, L., Keller, J., Tarricone, A. et al. (2010). TheMIS12 complex is a protein interaction hub for outer kinetochore assembly. J. Cell

Biol. 190, 835-852.Powers, A. F., Franck, A. D., Gestaut, D. R., Cooper, J., Gracyzk, B., Wei, R. R.,

Wordeman, L., Davis, T. N. and Asbury, C. L. (2009). The Ndc80 kinetochorecomplex forms load-bearing attachments to dynamic microtubule tips via biaseddiffusion. Cell 136, 865-875.

Przewloka, M. R. and Glover, D. M. (2009). The kinetochore and the centromere: aworking long distance relationship. Annu. Rev. Genet. 43, 439-465.

Przewloka, M. R., Venkei, Z., Bolanos-Garcia, V. M., Debski, J., Dadlez, M. and

Glover, D. M. (2011). CENP-C is a structural platform for kinetochore assembly.Curr. Biol. 21, 399-405.

Raaijmakers, J. A., Tanenbaum, M. E., Maia, A. F. and Medema, R. H. (2009).RAMA1 is a novel kinetochore protein involved in kinetochore-microtubuleattachment. J. Cell Sci. 122, 2436-2445.

Rosenberg, J. S., Cross, F. R. and Funabiki, H. (2011). KNL1/Spc105 recruits PP1 tosilence the spindle assembly checkpoint. Curr. Biol. 21, 942-947.

Saitoh, H., Tomkiel, J., Cooke, C. A., Ratrie, H., 3rd, Maurer, M., Rothfield, N. F.and Earnshaw, W. C. (1992). CENP-C, an autoantigen in scleroderma, is acomponent of the human inner kinetochore plate. Cell 70, 115-125.

Sandall, S., Severin, F., McLeod, I. X., Yates, J. R., 3rd, Oegema, K., Hyman,

A. and Desai, A. (2006). A Bir1-Sli15 complex connects centromeres to microtubulesand is required to sense kinetochore tension. Cell 127, 1179-1191.

Santaguida, S. and Musacchio, A. (2009). The life and miracles of kinetochores.EMBO J. 28, 2511-2531.

Santaguida, S., Tighe, A., D’Alise, A. M., Taylor, S. S. and Musacchio, A. (2010).Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpointthrough the small molecule inhibitor reversine. J. Cell Biol. 190, 73-87.

Schittenhelm, R. B., Heeger, S., Althoff, F., Walter, A., Heidmann, S., Mechtler,

K. and Lehner, C. F. (2007). Spatial organization of a ubiquitous eukaryotickinetochore protein network in Drosophila chromosomes. Chromosoma 116, 385-402.

Schittenhelm, R. B., Chaleckis, R. and Lehner, C. F. (2009). Intrakinetochorelocalization and essential functional domains of Drosophila Spc105. EMBO J. 28,2374-2386.

Schleiffer, A., Maier, M., Litos, G., Lampert, F., Hornung, P., Mechtler, K. andWestermann, S. (2012). CENP-T proteins are conserved centromere receptors of theNdc80 complex. Nat. Cell Biol. 14, 604-613.

Schmidt, J. C., Arthanari, H., Boeszoermenyi, A., Dashkevich, N. M., Wilson-

Kubalek, E. M., Monnier, N., Markus, M., Oberer, M., Milligan, R. A., Bathe,

M. et al. (2012). The kinetochore-bound ska1 complex tracks depolymerizing

microtubules and binds to curved protofilaments. Dev. Cell 23, 968-980.

Screpanti, E., De Antoni, A., Alushin, G. M., Petrovic, A., Melis, T., Nogales, E. and

Musacchio, A. (2011). Direct binding of Cenp-C to the Mis12 complex joins the

inner and outer kinetochore. Curr. Biol. 21, 391-398.

Shepperd, L. A., Meadows, J. C., Sochaj, A. M., Lancaster, T. C., Zou, J., Buttrick,

G. J., Rappsilber, J., Hardwick, K. G. and Millar, J. B. (2012). Phosphodependent

recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1 kinase maintains the spindle

checkpoint. Curr. Biol. 22, 891-899.

Starr, D. A., Saffery, R., Li, Z., Simpson, A. E., Choo, K. H., Yen, T. J. and

Goldberg, M. L. (2000). HZwint-1, a novel human kinetochore component that

interacts with HZW10. J. Cell Sci. 113, 1939-1950.

Sundin, L. J., Guimaraes, G. J. and Deluca, J. G. (2011). The NDC80 complex

proteins Nuf2 and Hec1 make distinct contributions to kinetochore-microtubule

attachment in mitosis. Mol. Biol. Cell 22, 759-768.

Takeuchi, K. and Fukagawa, T. (2012). Molecular architecture of vertebrate

kinetochores. Exp. Cell Res. 318, 1367-1374.

Theis, M., Slabicki, M., Junqueira, M., Paszkowski-Rogacz, M., Sontheimer,

J., Kittler, R., Heninger, A. K., Glatter, T., Kruusmaa, K., Poser, I. et al. (2009).

Comparative profiling identifies C13orf3 as a component of the Ska complex required

for mammalian cell division. EMBO J. 28, 1453-1465.

Tooley, J. and Stukenberg, P. T. (2011). The Ndc80 complex: integrating the

kinetochore’s many movements. Chromosome Res. 19, 377-391.

Tooley, J. G., Miller, S. A. and Stukenberg, P. T. (2011). The Ndc80 complex uses a

tripartite attachment point to couple microtubule depolymerization to chromosome

movement. Mol. Biol. Cell 22, 1217-1226.

Umbreit, N. T., Gestaut, D. R., Tien, J. F., Vollmar, B. S., Gonen, T., Asbury, C. L.

and Davis, T. N. (2012). The Ndc80 kinetochore complex directly modulates

microtubule dynamics. Proc. Natl. Acad. Sci. USA 109, 16113-16118.

Varma, D., Chandrasekaran, S., Sundin, L. J., Reidy, K. T., Wan, X., Chasse, D. A.,

Nevis, K. R., DeLuca, J. G., Salmon, E. D. and Cook, J. G. (2012). Recruitment of

the human Cdt1 replication licensing protein by the loop domain of Hec1 is required

for stable kinetochore-microtubule attachment. Nat. Cell Biol. 14, 593-603.

Venkei, Z., Przewloka, M. R. and Glover, D. M. (2011). Drosophila Mis12 complex

acts as a single functional unit essential for anaphase chromosome movement and a

robust spindle assembly checkpoint. Genetics 187, 131-140.

Vigneron, S., Prieto, S., Bernis, C., Labbe, J. C., Castro, A. and Lorca, T. (2004).

Kinetochore localization of spindle checkpoint proteins: who controls whom? Mol.

Biol. Cell 15, 4584-4596.

Vos, L. J., Famulski, J. K. and Chan, G. K. (2011). hZwint-1 bridges the inner and

outer kinetochore: identification of the kinetochore localization domain and the

hZw10-interaction domain. Biochem. J. 436, 157-168.

Wan, X., O’Quinn, R. P., Pierce, H. L., Joglekar, A. P., Gall, W. E., DeLuca, J. G.,

Carroll, C. W., Liu, S. T., Yen, T. J., McEwen, B. F. et al. (2009). Protein

architecture of the human kinetochore microtubule attachment site. Cell 137, 672-684.

Wang, H., Hu, X., Ding, X., Dou, Z., Yang, Z., Shaw, A. W., Teng, M., Cleveland,

D. W., Goldberg, M. L., Niu, L. et al. (2004). Human Zwint-1 specifies localization

of Zeste White 10 to kinetochores and is essential for mitotic checkpoint signaling.

J. Biol. Chem. 279, 54590-54598.

Wang, H. W., Long, S., Ciferri, C., Westermann, S., Drubin, D., Barnes, G. and

Nogales, E. (2008). Architecture and flexibility of the yeast Ndc80 kinetochore

complex. J. Mol. Biol. 383, 894-903.

Wei, R. R., Al-Bassam, J. and Harrison, S. C. (2007). The Ndc80/HEC1 complex is a

contact point for kinetochore-microtubule attachment. Nat. Struct. Mol. Biol. 14, 54-59.

Wei, R., Ngo, B., Wu, G. and Lee, W. H. (2011). Phosphorylation of the Ndc80

complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and

signaling of the spindle assembly checkpoint. Mol. Biol. Cell 22, 3584-3594.

Welburn, J. P., Grishchuk, E. L., Backer, C. B., Wilson-Kubalek, E. M., Yates,

J. R., 3rd and Cheeseman, I. M. (2009). The human kinetochore Ska1 complex

facilitates microtubule depolymerization-coupled motility. Dev. Cell 16, 374-385.

Welburn, J. P., Vleugel, M., Liu, D., Yates, J. R., 3rd, Lampson, M. A., Fukagawa, T.

and Cheeseman, I. M. (2010). Aurora B phosphorylates spatially distinct targets to

differentially regulate the kinetochore-microtubule interface. Mol. Cell 38, 383-392.

Wilson-Kubalek, E. M., Cheeseman, I. M., Yoshioka, C., Desai, A. and Milligan,

R. A. (2008). Orientation and structure of the Ndc80 complex on the microtubule

lattice. J. Cell Biol. 182, 1055-1061.

Yamagishi, Y., Yang, C. H., Tanno, Y. and Watanabe, Y. (2012). MPS1/Mph1

phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components. Nat.

Cell Biol. 14, 746-752.

Zhang, G., Kelstrup, C. D., Hu, X. W., Kaas Hansen, M. J., Singleton, M. R., Olsen,

J. V. and Nilsson, J. (2012). The Ndc80 internal loop is required for recruitment of

the Ska complex to establish end-on microtubule attachment to kinetochores. J. Cell

Sci. 125, 3243-3253.

Journal of Cell Science 125 (24)5936