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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 ([email protected])
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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