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The Kinetochore and the
Centromere: A Working
Long Distance Relationship
Marcin R. Przewloka and David M. Glover
University of Cambridge, Department of Genetics, Cambridge, CB2 3EH,
United Kingdom; email: [email protected], [email protected]
Annu. Rev. Genet. 2009. 43:439–65
Key Words
First published online as a Review in Advance on
August 24, 2009
cell cycle, mitosis, microtubule, aneuploidy, protein complex
The Annual Review of Genetics is online at
genet.annualreviews.org
This article’s doi:
10.1146/annurev-genet-102108-134310
c 2009 by Annual Reviews.
Copyright All rights reserved
0066-4197/09/1201-0439$20.00
Abstract
Accurate chromosome segregation is a prerequisite for the maintenance
of the genomic stability. Consequently, elaborate molecular machineries and mechanisms emerged during the course of evolution in order
to ensure proper division of the genetic material. The kinetochore,
an essential multiprotein complex assembled on mitotic or meiotic
centromeres, is an example of such machinery. Recently considerable
progress has been made in understanding their composition, the recruitment hierarchy of their components, and the principles of their
regulation. However, these advances are accompanied by a growing
number of unanswered questions about the function of the individual
subunits and of how the structure of the different subcomplexes relates
to function. Here we review our rapidly growing knowledge on interacting networks of structural and regulatory proteins of the metazoan
mitotic kinetochore: its centromeric foundations, its structural core,
its components that interact with spindle microtubules and the spindle
assembly checkpoint.
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INTRODUCTION
The kinetochore is a complex structure inexorably associated with the centromeric part of
the chromosome to carry out a common shared
duty in ensuring the equitable sharing of chromosomes upon cell division. In most organisms,
or at least in most of those used as experimental
models, the centromere occupies a distinct
a
position on each chromosome. This arrangement is characteristic for so-called monocentric
chromosomes (Figure 1a). Caenorhabditis elegans is one notable exception in which the
centromere is said to be holocentric: instead
of occupying one particular region, it is spread
out along the length of each chromosome.
On monocentric chromosomes, it is possible
b
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Chromosome arm
Kinetochore
Microtubule
Centromere (CCAN)
Inner centromere
c
200 nm
Without microtubules
With microtubules
Figure 1
Kinetochores as seen through the microscope. (a) Scanning electron micrograph of a metaphase
chromosome; sister chromatids are easily distinguishable and still attached at the centromere (arrow).
(b) Schematic of a bi-oriented chromosome; labeled are structures discussed in this review and they comprise
inner centromere, centromere (including CENP-A containing chromatin and CCAN) and kinetochore.
(c) Electron micrographs of kinetochores from PtK1 (rat kangaroo kidney epithelial) cells. Kinetochores
without microtubules exhibit a trilaminar structure (arrow head ) consisting of two dense plates and an
external fibrous corona. The binding of microtubules does not alter the trilaminar configuration, although
the corona is no longer conspicuous. (a) Courtesy of Terry D. Allen from “Molecular Biology of the Cell” by
Alberts et al. 2008 (b) Courtesy of Helder Maiato from Maiato et al. 2006, Chromosoma 115:469–480 (100).
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for centromeres to be built at ectopic sites,
something that happens at extremely low
frequency and leads to the creation of a
neocentromere. However, even when a second
bone fide centromere is introduced onto a
chromosome, e.g., as a result of a chromosome
translocation, only one of the two centromeres
is functional. Otherwise, the presence of an
extra centromere leads to aberrant segregation
and either cell death or mutation in the future
generations. This is an extremely important
feature that ensures each chromosome has only
one kinetochore and so is correctly segregated
to the daughter cells at cell division. One important property of the centromere is therefore
the ability to propagate its single site from one
generation of cells to the next, which is achieved
through the properties of its constituent and
associated proteins (3). A second property is
that in mitosis the centromere usually provides
the last attachment between sister chromatids, a
link that must finally be broken to allow the segregation of the sisters at anaphase. The favored
mechanism for achieving this end is through
the breakage of ring-like complexes of proteins
(cohesins) that bind the sisters together, initially along the length of mitotic chromosomes
and finally at the centromere alone. We do not
discuss this aspect of centromeric function in
detail as it is covered elsewhere in this volume
(122). Finally, and of particular significance
in respect to this review, the centromere acts
as a loading platform on which to assemble
the kinetochore upon entry into M phase
(22). We examine the essential centromeric
components necessary for this function and
see how they cooperate with true kinetochore
proteins to mediate several of the kinetochore’s
functions.
The main function of the kinetochore is to
interact with the centromere on the one hand
and spindle microtubules on the other. The correct association of the kinetochore with just a
single bundle of kinetochore microtubules is
both monitored and corrected by the spindle
assembly checkpoint (SAC). We examine the
organization of each of these complexes; how
they assemble sequentially to form a functional
kinetochore; and how they also interact with
SAC proteins to regulate the fidelity of the
metaphase to anaphase transition.
STRUCTURAL BASIS
OF THE CENTROMERE
Centromeres generally appear as constricted
regions of mitotic chromosomes (Figure 1a)
and serve as the foundations for the kinetochores, which assemble just before and during the very early stages of mitosis (Figure 1b
and c) (3, 22, 162). In the monocentric chromosomes of budding yeast and its close evolutionary relatives, centromeres are built upon
DNA of a defined sequence. This kind of centromere is called a point centromere (107,
114). Centromeres built on tandem repeats
of highly repetitive (satellite) elements, socalled regional centromeres, are characteristic
of higher eukaryotes (3). However, satellite sequences are neither sufficient nor essential for
the functional centromere to form and different fragments of the chromosome may develop
into neocentromeres. It is well established that
epigenetic marks, and not the DNA sequence,
are responsible for the identity of regional centromeres (31, 43, 113, 118). Despite this revelation, it is still not well understood how centromeric properties are transmitted from one
generation to another.
The development of proteomic techniques
has led to the identification of many (but
perhaps still not all) centromere proteins in
yeast and higher eukaryotes (24, 48, 67, 70)
(Table 1). RNA has also been reported to play
a crucial role in the proper formation of centromeres, the significance of which still has
to be fully explored (3, 9, 16, 181). Similarly,
condensins have been found to influence the
centromere structure and function (see sidebar,
Condensins). Although we now know the key
components of the centromere-kinetochore interface, we still know very little about the
regulatory mechanisms that control assembly
and disassembly of the kinetochore on centromeres. Here we summarize our knowledge
of centromeric components and of how the
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Table 1
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Centromere network proteins identified in model organisms1
S. cerevisiae
CTF19 complex
Cse4
S. pombe
SIM4 complex
Cnp1
C. elegans
HCP-3
D. melanogaster
CID
Abp1, Cbh1, Cbh2
Human CCAN
CENP-A
Alternative name
CenH3
CENP-B
CENP-D
RCC1; RanGEF
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CENP-G
Mif2
Cnp3
Mcm16
Fta3
HCP-4
CENP-C
CENP-C
CENP-H
Ctf3
Mis6
CENP-I
Sim4
CENP-K
Solt; Sim4R
Fta1
CENP-L
Fta1R
Iml3
Mis17
CENP-M
PANE1
Chl4
Mis15
CENP-N
Chl4R
Mcm21
Mal2
CENP-O
Mcm21R
Ctf19
Fta2
CENP-P
Fta7
CENP-Q
CENP-R
YOL0W86-A
CENP-S
SPBC800
CENP-T
Fta4
CENP-U
CENP-50; BPIP1
CENP-W
CUG2
1
CENP-A, CENP-B, CENP-D (82) and CENP-G (55, 63) are not formally members of CTF19, SIM4 or CCAN complexes, but they are
included here for completeness. Subcomplexes of the CCAN comprise CENP-O complex (CENP-O, -P, -Q, -R), CENP-H complex
(CENP-H, -I, -K) and CENP-T/W complex. CENP-E (188), CENP-F (164), and CENP-V (155), although their names suggest a
centromeric identity, are in fact kinetochore proteins and therefore are not listed.
centromere is assembled as the foundation upon
which the kinetochore rests.
CENP-A
At the very core of the centromere lies a histone
H3 variant, centromeric protein A (CENPA, also known as CenH3) that is essential for
centromere and kinetochore formation in all
organisms studied to date (3). CENP-A has a histone fold domain, characteristic of all core histones, but also contains an extended N-terminal
tail whose length differs significantly between
species (12). Very little is known about the function of the CENP-A tail, but it appears to be
necessary for proper kinetochore function and
cytokinesis (87, 151, 191). However, thus far
there are no proteins known to bind directly
and specifically to the tail. Interestingly, the
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motif responsible for the specific binding of
CENP-A to the centromeric nucleosomes lies
not in the tail, but between helices α1 and α2
of the histone fold (13, 154, 165). This is called
the CENP-A targeting domain (CATD) and
was shown to be sufficient for targeting of not
only CENP-A to centromeric nucleosomes, but
even canonical histone H3 with the CATD artificially inserted in its histone fold domain (14).
Nucleosomes containing CENP-A seem to
be quite unusual and their composition may
vary between species (3). Surprisingly, it was
found that centromeric nucleosomes induce
positive DNA supercoils (52). Dalal and colleagues have reported that in Drosophila they
are tetrameric rather than octameric (32, 33).
In budding yeast, the Scm3 protein was proposed to replace histone dimers H2A/H2B
in centromeric nucleosomes (116) although it
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has been suggested that this may be only a
loading intermediate. Indeed, in fission yeast
Scm3 plays the role of the CENP-A recruitment factor (135, 179). Centromeric chromatin is also enriched in a histone H2A variant called H2A.Z in vertebrates and histone
H2A.v in Drosophila (48). In both Drosophila
and human cells, histone modifications in centromeric chromatin differ from those found in
both euchromatic and heterochromatic chromatin, suggesting an epigenetic mechanism for
creating a special environment for proper centromere establishment (148, 153).
CENP-A is deposited onto centromeric
chromatin in two steps (reviewed in References
3, 43, 113). The first takes place in S phase,
when nucleosomes are removed from template
DNA and loaded onto replicated DNA. During
this process CENP-A, along with all other histones, is transferred onto the new chromatid.
New canonical histones are added in this step,
but there is no addition of new CENP-A. Thus,
the number of CENP-A containing nucleosomes per chromatid decreases two fold after
DNA replication. The second step involves deposition of newly synthesized CENP-A after
mitosis, in early G1 (73, 149). This process requires CENP-A specific factors that include the
Mis18 (KNL2)-like group of proteins in C. elegans and vertebrates (50, 62, 96). In Drosophila, a
genome-wide screen identified a protein called
Cal1, which together with CENP-C is required for loading CENP-A (CID in flies) (45).
In vertebrate cells, HJURP (holliday junction
recognition protein) directly binds CATD of
CENP-A and together with its cofactor, Nucleophosmin 1, participates in CENP-A loading
(39, 49). This timing of CENP-A deposition
means that for a long period in the cell cycle centromeres contain 50% of the maximal amount of CENP-A. At this time, we
do not understand the biological relevance of
this, but we speculate that it is possible that
reduced CENP-A contributes to the flexibility of the centromeric heterochromatin during
mitosis.
Other factors found to be necessary for
the establishment of the centromere identity
CONDENSINS
A growing body of evidence points to the condesins having a crucial role at centromeric heterochromatin. In both the budding
and fission yeasts, the accumulation of condensin at nucleoli (at
rDNA) and at mitotic centromeres is essential for the correct
chromosome segregation (6, 121, 189). In Drosophila, depletion
of the CAP-H/Barren subunit of condensin results in a dramatic
failure of proper chromosome congression and of sister chromatids to resolve (11). These defects seem not to be due to incorrect microtubule attachment but rather to abnormal centromeric
chromatin structure (129). A recent study found that the ATPase
activity of condensin affects the stiffness and function of the centromere (141). It was also shown previously that the geometry of
centromeres is important for the error-free mitosis (95). Taken
together, these results suggest that the mechanical properties of
the centromere play a crucial role during cell division and that
condensins are essential for achieving correct architecture of centromeres. Furthermore, a genetic interaction between Drosophila
CENP-A and the condensin subunit CAP-G has been reported
(71), and so perhaps the condensin complex binds directly to the
CENP-A–containing centromeric chromatin. However, the exact nature of the interaction between condensins and centromeres
still remains to be characterized.
and efficient CENP-A incorporation include
protein complexes CAF (51), FACT (91), and
NASP (38) as well as the transcription of certain retroviral repeats from the centromeric sequences (19).
There are several models for how centromeric nucleosomes become positioned in
chromatin. Stretched centromeric chromatin
fibers show the existence of subdomains, in
which several CENP-A-containing nucleosomes are interspersed with canonical H3containing nucleosomes (15). It is believed that
such patches of CENP-A nucleosomes in correctly organized chromatin form a plate or
small territory in the primary constriction that
faces the outer part of the chromosome and
thus points toward kinetochores. In this model,
patches of canonical H3-nucleosomes create
another territory, which faces the other chromatid and is positioned inside the mitotic chromosome (also called inner centromere) (148).
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At present it is not clear how this arrangement
of centromeric chromatin could accommodate
the major structural changes required for the
centromere to behave like a flexible spring, as
observed in several systems and now thought
essential for the silencing of the spindle checkpoint (see below) (110, 141).
The Constitutive Centromere
Associated Network
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Centromeric proteins localize to centromeres
in both interphase and mitosis. They form a
network, which in vertebrates is called CCAN
(constitutive centromere associated network)
(22) or CENP-A NAC/CAD (CENP-A nucleosome associated complex and CENP-A distal)
complex (48), that comprises proteins copurifying and colocalizing with CENP-A. Whether
these proteins bind to CENP-A containing
nucleosomes directly or indirectly is not well
established at present (also see References 20,
67). In higher eukaryotes kinetochore proteins
are largely recruited only for mitosis and
thereafter are removed from centromeres. Recently identified exceptions to this rule include
the human Mis18 protein, which becomes
centromeric only between telophase and early
G1 (50); human BMI-1 protein, which binds
centromeres in interphase but not during mitosis (126); and the Drosophila Mis12 kinetochore
protein, which behaves more like a centromeric
than a kinetochore protein because in most
(but not all) interphase cells it colocalizes with
CENP-A (Z. Venkei, M.R. Przewloka, D.M.
Glover, unpublished results). Nevertheless,
there is a general pattern by which components
of the centromere and kinetochore are separately recruited in space and time in a manner
that reflects their physical separation.
Whereas the regional centromeres of fission
yeast and vertebrates comprise an alphabet
soup of CENPs (Table 1), the protein complement of Drosophila and C. elegans centromeres
appears much simpler and comprises only
two key proteins, CENP-A and CENP-C,
although it cannot be discounted that other
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centromeric proteins still remain to be discovered in these organisms (22, 48, 67, 127). The
CENP-A loading machinery also seems to be
different and simpler in these organisms than
in other systems (3, 22, 45, 96). The apparent
simplicity of the C. elegans and Drosophila
systems argues that they will be very good
models to investigate the central relationships
between centromeres and kinetochores.
All components of the CCAN with the
exception of CENP-B protein and CENP-O
complex (Table 1) are essential and necessary
for the proper chromosome congression and
segregation. CENP-B is a DNA-binding protein (42, 119), but appears not to be a crucial component, given that it is absent from
fully functional neocentromeres (159, 166) and
CENP-B knockout mice survive (78, 133). It
likely participates in preventing de novo centromere formation in cells that already contain
active centromeres by ensuring that chromatin
in the active centromere can be distinguished
from other heterochromatin (128, 162). It is
also possible that CENP-B may be associated
with pericentromeric retrotransposons rather
than with the centromere itself (18).
CENP-C is a key centromeric player whose
depletion leads to severe defects emphasizing
its importance in chromosome segregation, mitotic checkpoint function, and kinetochore assembly (64, 67, 88, 117, 139). Recently, it was
revealed that in Drosophila embryos CENP-C is
loaded onto centromeric chromatin along with
CENP-A late in mitosis (149).
Several studies have been undertaken to
examine the interdependence of binding of
different components of the CCAN (see examples in References 22, 23, 67, 139). As might
be expected, the resulting picture is complex.
Given that the function of most of these
components is not fully understood, we do not
propose to discuss this aspect of centromeric
function further. To add to the complexity
of the whole system, it has been noticed that
centromeric components interact differentially
with the kinetochore proteins. For instance,
vertebrate CENP-K centromeric protein and
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KNL1/Spc105 kinetochore protein seem to cooperate to achieve fully functional and properly
assembled kinetochores. This may be facilitated by a direct interaction between Ndc80
complex and the centromeric subcomplex of
CENP-H, -I, and -K (23). It is becoming
clear that single centromeric proteins and
centromeric subcomplexes differentially influence kinetochore assembly and function. One
example is the possible physical link made by a
complex of CENP-T and CENP-W between
the centromere and kinetochore that differs
from the link provided by CENP-C. These
two distinct connections of the centromere and
kinetochore govern different functions (e.g., in
respect to the spindle checkpoint signaling) and
manifest in different phenotypes in knockdown
experiments (67) (see also Reference 109).
How Do Centromeres
Become Kinetochores?
Centromeric proteins effectively serve as beacons that mark locations on chromosomes to
which kinetochore proteins recruit. At a certain
moment, centromeres become kinetochores.
They first acquire the ability to recruit key kinetochore components that in turn attract proteins responsible for the microtubule binding
and sequential aspects of mitotic regulation.
We can only speculate that the series of these
temporal events needs to be controlled by the
general cell cycle regulatory mechanisms and
that most likely phosphorylation or other posttranslational modification of one or more centromeric proteins must take place to mark sites
for the recruitment of new kinetochore components. Posttranslational modifications may
translate into structural changes of one protein or groups (complexes) of proteins modifying a scaffold to allow new proteins to be
recruited. It is also possible that kinetochore
proteins that are about to bind centromeres
are posttranslationally modified. Knowledge of
the recruitment hierarchy among the centromere and kinetochore subunits may give us
clues about these regulatory mechanisms in relation to kinetochore function.
MOLECULAR COMPONENTS
OF THE CORE KINETOCHORE
Centromeres and kinetochores are dynamic
structures whose composition changes to reflect functions at specific stages of the cell cycle.
Despite this dynamism, centromere and kinetochore proteins form biochemically stable complexes. Below we would like briefly summarize
what is presently known about how complexes
build the structural core of the kinetochore.
Mis12 Complex
The Mis12 complex is first to be recruited to
centromeres by associating directly with the
proteins of CCAN or with the centromeric
chromatin. It is composed of four proteins:
Mis12, Nnf1, Nsl1, and Dsn1 (46, 123), all
of which are required for the complex to assemble properly (86). If one or more of these
four subunits are missing, other components of
the Mis12 complex become mislocalized. This
means that loss of any single component results
in a similar phenotype. One exception is seen
in C. elegans where depletion of Dsn1 leads to
a stronger phenotype than depletion of Mis12,
Nsl1, or Nnf1, hence the other name for the
Dsn1 in this organism is KNL-3 (kinetochore
null 3) (24).
The Mis12 complex in Drosophila
melanogaster is exceptional in its organization: it differs from the canonical four-subunit
complexes found in other species as no Dsn1
protein has yet been identified (139, 147). Possibly Dsn1 does not exist at all in Drosophila,
leading to the suggestion that its function
could have been adopted by the Spc105-related
protein (Spc105R) that influences recruitment
of Mis12 complex components (138).
Depletion of Mis12 complex components
results in defects in chromosome alignment,
orientation, and segregation. The structure
of the whole kinetochore is affected, because
other complexes, whose recruitment depends
on Mis12 complex, are either mislocalized completely or severely reduced (24, 56, 86, 139).
Loss of the Mis12 complex is reported not
only to lead to decreased accumulation of
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kinetochore proteins such as Ndc80, BubR1,
and CENP-E, but also to lower levels of centromeric CENP-H and even CENP-A (86).
Chromosomes still attach to spindles in Mis12
depleted cells, but they show signs of decreased
tension between sister kinetochores; mitotic
progression is delayed, and there are lagging
chromosomes in anaphase (86). The defective
kinetochores result in abnormal associations of
chromosomes with microtubules; spindles become very long, and the lagging chromosomes
in cells that do pass through mitosis develop
into micronuclei during interphase (56, 139).
The precise phenotype depends on the extent
of the depletion and on the organism being
studied but the mitotic defects are invariably
severe.
The timing of recruitment of Mis12 complex components is not clearly established. It
seems that the Mis12 protein itself localizes
to centromeres not only in mitosis, but also
in interphase (56, 65, 86, 139). However, the
presence of some interphase cells not stained
with anti-Mis12 antibodies suggests cell cycle–
specific recruitment of Mis12. Other studies
suggest the remaining members of the Mis12
complex localize to the kinetochore some time
in G2, although human Nnf1 has been described to recruit to kinetochores only during mitosis (106). Our own preliminary studies
suggest that the Mis12 complex of Drosophila
has been assembled at the nascent kinetochore
by the time of nuclear envelope breakdown
(Z. Venkei, M.R. Przewloka, D.M. Glover,
unpublished results).
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Spc105
The Spc105 protein owes its name to the founding member of the family first identified as a
budding yeast spindle pole body component. In
Saccharomyces cerevisiae, it is complexed with the
protein Ydr532 (123). However, in all higher
eukaryotes, it exists separately as a single protein. It is a large protein (2342 amino acids in
human; 1959 in Drosophila) that is essential for
kinetochore function in all species studied (23,
24, 123, 139). It appears to serve as a scaffold to
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which other kinetochore components bind. Its
sequence and size have diverged between yeasts
and vertebrates, but in most species it retains
short conserved motifs in the form of S/GILK,
RRVSF, and MELT repeats and coiled-coil domains identifying regions of the molecule potentially important for function (24, 123).
In C. elegans, depletion of Spc105 results
in a complete loss of the kinetochore, giving
the characteristic kinetochore-null phenotype
and hence the name KNL1 (24). Similarly, in
other species the depletion of Spc105 causes severe kinetochore defects, resulting in the missegregation of chromosomes (23, 139). In some
cases, phenotypes are similar to those appearing
following depletion of CENP-A or CENP-C,
although these centromeric proteins are located
upstream in the kinetochore building pathway
(24, 139).
In cultured Drosophila cells, RNAi-based depletion of Spc105R leads first to a scattered
distribution of chromosomes on the spindle as
a result of incorrect chromosome congression,
alignment, and segregation. This is followed by
a severe block to cell proliferation and a dramatic decrease in mitotic index. RNAi of other
Drosophila core kinetochore components does
not lead to such a strong mitotic arrest, but it
is not clear whether this is due to differences in
protein function or the efficiency of knockdown
(139).
In human cells, Spc105 is known as hKNL1
or Blinkin, and the effects of its depletion are
not as strong as in Caenorhabditis and Drosophila
where recruitment of Ndc80 complex depends
on the presence of Spc105 (23, 85, 139). This
dependency of Ndc80 recruitment on Spc105
is also not seen in budding and fission yeast
(81). In Drosophila and in fission yeast, the correct formation of the Mis12/MIND complex
formation also depends on Spc105 (81, 139),
but this dependency has not been described in
other organisms. Human Spc105/Blinkin has
been shown to bind directly the checkpoint proteins Bub1 and BubR1 via its N-terminal region (85). Consistently, Blinkin depletion by
RNAi resembles the consequences of Bub1 or
BubR1 depletion. The protein interacts with
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the kinetochore by binding to Nsl1 and Dsn1
components of the Mis12 complex (21, 85).
It remains to be determined whether in other
species Spc105 also behaves as a potential center for kinetochore checkpoint signaling.
Finally, Spc105 also shows concentrationdependent microtubule binding capacity (21).
This is not a particularly strong interaction with
the isolated protein. However, when combined
with the Mis12 and Ndc80 complexes, an array
of double-site binding units is formed that is
responsible for the microtubule attachment to
the kinetochore (see below).
Ndc80 Complex
The Ndc80 complex comprises the four proteins, Spc24, Spc25, Nuf2, and Ndc80, each of
which has extensive coiled-coil content within
its secondary structure (25, 26, 177). Atomic
force and electron microscopy have revealed the
complex to have an elongated rod-like structure with globular domains at both ends (174).
A smaller version of the complex has been created by deleting extreme parts of the coiledcoil to generate a so-called bonsai form that
has been crystallized and studied at high resolution (27). The structure reveals a microtubulebinding interface containing a pair of tightly
interacting calponin-homology (CH) domains
that make a cooperative, predominantly electrostatic interaction with microtubules.
The tetrameric Ndc80 complex is effectively formed by binding two stable dimers:
Spc24/Spc25 and Nuf2/Ndc80. Spc24 and
Spc25 are considerably smaller than the other
two components. They have C-terminal globular domains, which after the assembly face
the centromeric side of the kinetochore and
long N-terminal coiled-coils that are directly
bound to Nuf2/Ndc80 dimer (72, 108, 173).
A similar principle, but with opposite polarity, applies to the Nuf2/Ndc80 dimer. These
two proteins have globular domains at their
N-terminal regions and long coiled-coil structures on their C termini. The globular domains of Ndc80/Nuf2 are thought to bind directly to microtubules, whereas the coiled-coils
are responsible for binding to the Spc24/Spc25
dimer (25, 174). The polarized positioning of
the complex with respect to the centromere implies that getting rid of the Spc24/Spc25 dimer
should affect Nuf2/Ndc80 localization, but not
vice versa. Indeed, such a relationship has been
observed in several systems (10, 26, 139, 178).
In the course of these experiments, it was also
realized that the Nuf2/Ndc80 dimer was essential for the function of the entire complex.
Thus, the binding of microtubules appears to be
the most important role played by the complex
in vivo.
The localization of the Ndc80 complex
components to mitotic kinetochores has been
observed in all organisms studied. Because
the complex is located in the outer part of
the structural kinetochore core, its depletion
usually does not disrupt the inner kinetochore.
Lack of the Ndc80 complex consistently results
in severe spindle attachment defects (reviewed
in Reference 26). For example, in C. elegans depletion of Ndc80 or Nuf2 (here called HIM-10)
causes disorganized metaphase plates, delayed
sister chromatid separation, and extensive missegregation (68). In cultured Drosophila cells,
the loss of any Ndc80 complex component
leads to the formation of an elongated mitotic
spindle with a scattered distribution of chromosomes and extensive mis-segregation defects
(139). Thus, in all systems, loss of the complex
leads to attachment defects, loss of checkpoint
control, and chromosome mis-segregation.
Recently, the structural (Figure 2a and
Reference 112) and molecular basis for the interaction of the Ndc80 complex with microtubules has been intensively studied, providing
an understanding of how the binding occurs
and how it is regulated (21, 27, 59, 115, 172,
180). The N-terminal regions of both proteins,
Ndc80 and Nuf2, contain Calponin-homology
(CH) domains that interact with microtubules
and are similar to the microtubule-binding domain of the protein EB1. However, the Ndc80
protein has a unique feature that is crucial for
microtubule binding by the whole complex.
Closer to the N-terminus than the CH domain is an unstructured tail that is essential for
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b
a
i
ii
iii
Centromeric
chromatin
CENP-H/I/K
RZZ
Fibril
MT
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c
CEN
Ndc80 complex
Bub1
Protofilaments
Zwint-1
Mis12
complex
Spc105
BubR1
Ndc80
complex
Spc105
KMN
network
Mis12
complex
CCAN
complexes
Centromeric
chromatin
Model 1
Model 2
Figure 2
Kinetochores and their attachment to centromeres and microtubules. (a) The structural basis of kinetochore and microtubule
interactions. Electron micrograph (i ) and its interpretations based on biochemical data (ii and iii ): single microtubule protofilaments
are attached to centromeric chromatin via fibrils. These fibrils may be composed of, at least in part, Ndc80 complexes. (b) Schematic
detailing the KMN supercomplex and its direct interactors. Microtubules (MT) associate with the Ndc80 complex portion of the
KMN. Ndc80 has also been suggested to directly bind the centromeric complex CENP-H, which contains CENP-H, -I, and -K.
Closer to the centromere (CEN), Bub1 and BubR1 bind directly to Spc105. Zwint1 bridges the RZZ complex to Spc105. However,
Zwint-1 has yet to be identified in Drosophila and Caenorhabditis elegans suggesting that perhaps this intermediate is not crucial for
interaction between RZZ and KMN. (c) Two models for the mode of binding of the KMN network to centromeres. Centromeric
chromatin, which contains both CENP-A and H3 nucleosomes, may be associated with KMN complexes via proteins from the CCAN,
e.g., CENP-C (Model 1) or more directly, e.g., via HP1 or other chromatin proteins (Model 2). Interactions between CCAN and
KMN are also possible in Model 2 ( yellow arrow). (a) is courtesy of J. Richard McIntosh from McIntosh et al. 2008, Cell 135: 322–333
(112).
interaction with microtubules. Thus, CH domains of the Ndc80/Nuf2 dimer and the N-tail
of Ndc80 protein together interact electrostatically with microtubules and are both required
to achieve the proper binding characteristics.
Moreover, the binding surfaces of Ndc80 have
been shown to be targets for the Aurora B
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kinase that facilitates the correction of erroneous kinetochore-microtubule attachments
(21, 27, 36) (see below). Residues of the Ndc80
protein found to be phosphorylated both in vivo
and in vitro are located within the unstructured
tail. This means that although CH domains are
important for the microtubule interactions, it is
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likely that the N-terminal tail regulates binding
depending upon its phosphorylation status.
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KMN Network
The structural core of the kinetochore comprises a supercomplex of proteins, the KMN
network, named after its constituents: the protein KNL-1/Spc105/Blinkin, and the Mis12
and the Ndc80 subcomplexes (21). This network of interacting proteins and complexes was
isolated from several different organisms, including yeast (123). In C. elegans, it was first
called KNL-1/3 interacting proteins (24); and
in fission yeast, NMS for Ndc80-MIND-Spc7
(93). Recently, the expression KMN network
has begun to be widely used, and so we will
adopt this acronym.
This supercomplex appears to be more
elaborate in budding yeast, where it has to
interact with the point centromere and a single
microtubule, than it is in higher eukaryotes
with their regional centromeres and microtubule bundles (176). In budding yeast, the
members of the four complexes, COMA,
MIND, NDC80, and SPC105, have been
referred to as linker proteins to emphasize
the fact that they mediate the interaction
between DNA-bound centromeric complexes
and microtubule-bound proteins (4, 107, 114).
In higher eukaryotes, the SPC105 complex
has been diminished to the Spc105 protein
itself and two members of COMA complex
cannot be identified. The other two members
of COMA, CENP-P (Ctf19) and CENP-O
(Mcm21), are part of the CCAN: they copurify
with other components of the centromeric
complexes and bind centromeres throughout
the cell cycle, and so they may be considered
as centromeric, not kinetochore proteins.
Thus, organisms bearing regional centromeres
have only 3 linker complexes, which include
the Mis12/MIND complex (Mis12, Nnf1,
Nsl1, and Dsn1), the Ndc80 complex (Spc24,
Spc25, Nuf2, and Ndc80) and the big scaffold
protein Spc105 (as the single component of the
Spc105 complex) (22, 114). Importantly, all the
components of the metazoan linker complexes
share the same biochemical characteristics and
easily copurify, reflecting the high affinity of
members of each subcomplex, the feature that
is evolutionary conserved.
The link made by the KMN network to connect the centromere to the microtubule fiber of
the mitotic spindle must be strong enough to
sustain the pulling forces during anaphase. At
the same time, it must be sufficiently dynamic
to permit the polymerization-depolymerization
of the plus ends of microtubules. Additionally,
it has to respond efficiently to the regulatory
mechanisms that enable chromosomes to align
at the metaphase plate prior to anaphase. Thus,
it has to have a number of key properties: to
signal both correct and incorrect attachment
of chromosomes; to enable a weakening of microtubule binding when there is a need to correct the attachment; and to delay anaphase until
proper attachment has occurred.
The KMN network must also bind proteins
that transiently interact with the kinetochore
to provide auxiliary functions (Figure 2b). One
well-studied example is the direct binding of
Bub1 and BubR1, proteins essential for the activity of the spindle assembly checkpoint (see
below), by the human Spc105 (85). Thus, the
KMN fulfills structural functions as a scaffold and binding platform; regulatory functions
through both direct and indirect protein interactions; and effector functions related to its
interactions with microtubules and their regulatory proteins.
One of the most important questions still
remaining unanswered is how the KMN
network is attached to the CCAN and how this
binding is regulated (Figure 2c). We know that
the CCAN is more complex and not as stable
as the KMN. The CCAN is also not as well
evolutionarily conserved as the KMN, bringing
us back to the question previously posed by us
as to whether CENP-A and CENP-C might be
the major candidates for direct CCAN-KMN
interactions. Several lines of evidence indicate
that CENP-C could provide the link between
the centromere and the kinetochore. CENP-C
is relatively large and so could form a good
landing pad for the recruitment of kinetochore
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proteins. Indeed, the kinetochore will not
assemble once CENP-C is depleted (88, 139).
Moreover, it copurifies with the KMN network
components, as shown in C. elegans (24). On
the kinetochore side, the Mis12 complex could
be a potential candidate for interaction with
CENP-C as it resides in the inner plate of the
kinetochore. Furthermore, Mis12 protein also
localizes to interphase centromeres at least
for part of the cell cycle, giving it a partially
centromeric character (56, 139). The Mis12
complex itself is also crucial for kinetochore
formation and lies high in the hierarchy of
kinetochore protein recruitment. Thus, a
CENP-C and Mis12 complex interaction
seems likely to mediate the assembly of the
KMN network on the centromere (88).
However, another line of evidence suggests
that the Mis12 complex might also interact
with a different centromeric partner, heterochromatic protein 1 (HP1) (Figure 2c and
Figure 3). HP1 is enriched in affinity pulldowns in which members of the Mis12 complex
were used as bait (125). New data from two
26 nm
KMN
network
KMN
network
Aur
INCENP
CCAN
HP1
HP1
Me
Me
Me P
Me P
Me P
Aur
HP1
HP1
Me P
Me P
Me P
Me
Me
H3
H3
H3
H3
H3
CENP-A
H3
H3
H3
H3
H3
Bor Sur
10 nm
Figure 3
Model of the molecular interactions between major components of the centromere and kinetochore.
The majority of nucleosomes containing histone H3 are methylated on Lys9 and phosphorylated on Ser10, a
double-modification that evicts HP1 from mitotic chromatin. However, a small number of H3 nucleosomes
still bind HP1 and these might serve as the foundation or could regulate the KMN network’s association with
the centromere. This interaction likely results from the direct binding of the Mis12 complex’s Nsl1 protein
and HP1. In this model, CENP-A-containing nucleosomes are located in the center of the centromere, close
to the chromosomal passenger complex, and they do not participate in KMN interactions. CCAN complexes
may either bind directly to CENP-A nucleosomes, e.g., via CENP-N (20) or may be located close to CENP-A
but not interact directly. Localization of the chromosomal passenger complex restricts the Aurora B activity
to a certain area (dashed line). The model is adapted from an Andrea Musacchio presentation at the EMBO
workshop Chromosome Segregation: Centromeres and Kinetochores, Arcachon, France; October 2008.
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different laboratories support such interactions
(A. Musacchio and M. Yanagida, personal
communication). The human Mis12 complex
appears capable of interacting directly with
HP1 both in vitro and in vivo. This is likely to
be mediated by Nsl1 which binds HP1 in twohybrid assays. The interaction of HP1 with
chromosomes is itself controlled during cell cycle progression. HP1 binds to histone H3 when
its lysine 9 is methylated (7). Phosphorylation
of histone H3 at the adjacent serine 10 by Aurora B in mitosis leads to HP1 being displaced
from the chromatin. This happens along the
chromosome arms as they are condensing during prophase but HP1 remains associated with
the centromeric regions where it is thought
to bind through a mechanism resistant to H3
phosphorylation (47). Thus, there are two
pools of HP1; the majority of HP1 is excluded
from DNA during mitosis and the smaller pool
binds to centromeres. That centromeric pool
could be responsible for the Mis12 complex
binding. The relative roles played by CENP-C
and HP1 in interacting with the Mis12 complex are at the moment unclear. However, it is
possible that at different moments of the cell
cycle, the Mis12 complex may be binding to
different partners. In this sense, perhaps both
interactions may be important for the proper
binding of the Mis12 complex to centromeres
and the later recruitment of other KMN
network members during mitosis.
The Dsn1 subunit of the Mis12 complex
may play another particularly important role
in early kinetochore formation at the interface
between the complex and other KMN components. Dsn1 is quite unstable and only becomes stabilized after being incorporated into
the Mis12 complex. This observation led to
the idea that the presence of the CCAN platform induces local stabilization of Dsn1 by the
assembly of the Mis12 complex and restricting formation of the KMN network to the
centromere (22).
In summary, the KMN network is the essential core of the kinetochore: it directly binds
to centromeres by yet unidentified means and
to microtubules through a better understood
mechanism, and serves to enable a variety of
proteins to interact with kinetochores in a regulated manner.
LINKING STRUCTURE
TO FUNCTION
An abundance of regulatory proteins, which are
located either at kinetochores or microtubule
plus-ends, reflects the dynamism of the mitotic spindle and the complexity of its control.
The list of these proteins is too extensive to
cover here in full (reviewed in References 2,
98, 120, 184). We therefore briefly describe the
properties of only some of them in relation to
main kinetochore functions. We will concentrate upon the mechanisms that function at the
kinetochore to monitor correct attachment to
the spindle microtubules and trigger the onset
of anaphase.
Establishing Connections
with Microtubules
Research data accumulated within recent years
allowed for the proposal of models for spindle assembly that explained how spindle microtubules attach to chromosomes.
Search and capture. This widely accepted
mechanism describes how centrosomally nucleated microtubules search the prometaphase
cytoplasm in all directions until being captured
by kinetochores (124). Initially, kinetochores
make only lateral contact with microtubules,
and this is followed by poleward movement
of the chromosome, powered by the minus
end–directed dynein motor protein complex.
Dynein requires the so-called RZZ complex in
order to bind to kinetochores when they are
laterally attached to microtubules immediately
after capture (146, 150, 163). This complex
comprises Rough-deal (ROD), Zeste-white
10 (ZW10), and Zwilch. It was first identified
in Drosophila and subsequently found in all
metazoans but not in the yeasts (reviewed
in Reference 80). The proteins of the KMN
network associate with the RZZ complex
via an intermediate known as Zwint-1 (a
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protein curiously lacking in Drosophila and
C. elegans) (168). The protein Spindly (SPDL-1
in C. elegans) also requires RZZ to localize
to the kinetochore, and it participates with
the RZZ in dynein recruitment (58). Studies
in C. elegans indicate that RZZ complex and
protein Spindly/SPDL-1 might regulate the
conversion of lateral to end-on microtubule attachments (28, 54) (the bundle of microtubules
attached to a kinetochore in a end-on orientation is called a k-fiber). In vertebrates, stable
end-on kinetochore-microtubule attachment
seems to be also dependent on the recently
discovered complex of 3 proteins, Ska1, Ska2,
and Ska3/RAMA1 (53, 60). Functional similarities between the eukaryotic Ska complex and
the 10 subunit-large Dam1/DASH complex
present only in yeast were suggested (175).
Monotelic orientation, when the kinetochore of only one chromatid is connected
to k-fibers and the kinetochore on the other
chromatid remains unattached, usually soon
changes to the bi-orientation (amphitelic orientation), when microtubules searching from
the opposite pole are captured by the free sister
kinetochore positioned on the other chromatid
of the same chromosome. Chromosomes then
start to move or congress toward the middle of
the cell to participate in forming the metaphase
plate as a result of both shrinking (depolymerization) and elongation (polymerization) of kfiber microtubules. The bipolar attachment of
sister kinetochores then permits further spindle elongation and contributes to the separation of centrosomes (157). This may not be
the only possible mechanism for congression.
Kapoor and colleagues (79) have found that
monooriented chromosomes could glide toward the spindle equator alongside kinetochore
fibers attached to other already bioriented chromosomes in a process dependent upon the plus
end-directed microtubule motor CENP-E.
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Chromatin–kinetochore mediated microtubule attachment. Besides the search and
capture pathway, microtubule attachment can
also occur through a centrosome independent
process whose contribution varies depending
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upon the organism and the cell type. The
acentrosomal pathway relies on the nucleation
of microtubules around chromatin and their
accumulation at kinetochores. RanGEF (also
known as RCC1 or CENP-D) (Table 1), the
exchange factor for the small G-protein Ran,
is a major nuclear protein that is distributed
throughout chromosomes but also accumulates
at centromeres. It is responsible for a gradient of RanGTP extending from the chromosomes required to promote release of spindle regulatory proteins and to facilitate mitotic spindle assembly (reviewed in Reference
76). Components of the nuclear pore complex,
which help facilitate Ran’s role in nuclear import/export, become associated with the kinetochore during mitosis. These include RanGAP1 and RanBP2/Nup358, which are important for kinetochore composition, chromosome
alignment, and the recruitment of factors such
as CENP-E, CENP-F, and some checkpoint
proteins; additionally, Nup358 appears to have
other roles at the inner centromere (see below)
(34, 75, 130, 145).
MAPs. Microtubule
associated
proteins
(MAPs) of the CLASP family localize near
the plus-end of microtubules where they
promote polymerization and stabilization
(167). They also bind to kinetochores independently of microtubules (and, in interphase,
to microtubule plus ends independently
of kinetochores; see Reference 152). The
CLASPs are able to stimulate growth of k-fiber
microtubules and are important for proper
chromosome congression and the maintenance
of the spindle bipolarity (99, 102, 132). The
Drosophila CLASP protein, Mast/Orbit, has
been shown to play a significant part in the
centrosome-independent pathway of spindle
assembly by facilitating the stabilization and
thereby elongation of kinetochore attached
microtubules (89, 101). The kinetochore roles
of other plus end–binding proteins such as
CLIP-170 (CLIP-190 in Drosophila), the
adenomatous polyposis coli protein (APC),
and its binding partner, EB1, are less certain.
Depletions of APC and EB1 are unusual in
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affecting the movement and orientation of
paired sister chromatids at the metaphase
plate without perturbing kinetochore-MT
attachment per se and cause poor activation of
the checkpoint (37). CLIP170 binds to those
kinetochores to which microtubules are not
attached and after attachment has taken place
it leaves the kinetochores (40).
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Monitoring Metaphase
Sensing improper attachments by SAC is not
understood at the molecular level, but one can
try to explain the mechanism of the error detection by monitoring the tension within the
kinetochore after the establishment of the endon microtubule attachment.
Monitoring tension. Kinetochore tension
arises because of the pulling forces exerted by
microtubules and associated motors and the resistance offered by the physically attached sister centromeres. Sister chromatid cohesion is
maintained through the actions of the cohesin
complex and topoisomerase II.
The cohesin complex comprises four proteins: two of the structural maintenance of chromosomes family, SMC1 and SMC3, and two
non-SMC subunits, Rad21/Scc1 (α-kleisin)
and SA/Scc3. Cohesin complexes are believed
to form a ring-like structure around the paired
sister chromatids. In higher eukaryotes, cohesin
is lost from mitotic chromosomes in two waves.
Loss of cohesin from the arms occurs concurrently with chromosome condensation during
prophase and is promoted by the phosphorylation of the SA subunit (Polo family kinases
are involved at this step) (61). Another pool of
cohesin is associated with the centromere and
remains there until the metaphase-anaphase
transition (134). At anaphase onset, separase,
activated by the anaphase promoting complex/cyclosome (APC/C)-mediated degradation of its securin inhibitor, cleaves the cohesin
subunit Rad21/Scc1 and by doing so allows sister chromatids to fully separate. Until that time,
centromeric cohesin is protected from removal
by the protein Shugoshin and by the presence of
protein phosphatase PP2A, which antagonizes
the activity of centromeric kinases (84, 111,
171). The separase-independent mode of the
cohesin elimination, dependent upon Polo-like
kinase 1, occurs in the same manner during
prophase of mitotic and meiotic cells. However, the second, separase-dependent wave of
the cohesin removal from centromeres is regulated differently in meiosis (83, 170).
By resisting the forces acting on sister kinetochore microtubules, the cohesin complex is
essential for the correct alignment of chromosomes on the metaphase plate and for silencing of the spindle assembly checkpoint (SAC).
Lack of tension, indicating incorrect or absent
microtubule attachment, activates the SAC, delaying the activation of the anaphase promoting
complex/cyclosome (APC/C) ubiquitin protein
ligase (120). Depletion of cohesin reduces tension and so activates the SAC leading to mitotic
delay. Inhibition of Topoisomerase II (Topo II),
overrides such a delay, presumably because increased chromosome catenation substitutes for
cohesin in counteracting spindle forces to generate tension (158, 161).
Topoisomerase II is an abundant component
of the structural scaffold of mitotic chromosomes (41). Mitotic centromeres are also enriched in Topo II (140), but it has not been clear
whether this centromeric pool plays a purely
structural role or whether its enzymatic activity is also important during mitosis (136). In
vertebrates, RanBP2/Nup358, a component of
the interphase nuclear pore complex and also
the mitotic kinetochore protein, is responsible for Topo II sumoylation. This is critical
for the proper localization of Topo II to inner
centromeres (35). Recently, it has been shown
that Topo II is essential not only for the sister
chromatid separation and the centromere resolution, but also for the proper activation and
localization of Aurora B kinase (29).
The Aurora B protein kinase stands a little
apart from the other spindle assembly checkpoint kinases in that it has a specific role
in correcting misattachments of kinetochore
microtubules (see sidebar, Spindle Assembly
Checkpoint Kinases). Aurora B kinase together
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SPINDLE ASSEMBLY CHECKPOINT KINASES
Apart from Aurora B, numerous protein kinases populate the
centromere-kinetochore and participate in the SAC. In addition,
the roles of Nek2a or p38, better known outside mitosis, are
being acknowledged to have a SAC function as well. There is
also emerging evidence that Chk1, critical to monitor correct
DNA repair and replication, in addition participates in the SAC
(131, 190). The following kinases have a somewhat clearer SAC
role:
• Bub1: enriched at unattached kinetochores; critical for the
centromere-kinetochore localization of the BubR1 and Mps1 kinases; able to phosphorylate Cdc20 but the significance of this is
unclear (94, 156).
• BubR1: a component of the MCC; its phosphorylation of
CENP-E is inhibited when CENP-E binds microtubules (69, 97,
103).
• Mps1: contributes to kinetochore recruitment of Mad1 and
Mad2; also phosphorylates Borealin that in turn regulates Aurora
B (74).
• Polo (Plk1 in vertebrates): controls multiple mitotic events
(reviewed in Reference 5); centromere/kinetochore localization
depends on INCENP (57), Shugoshin 1 (137), and CENPU, which it phosphorylates (77, 90); phosphorylates also Bub1,
BubR1, and PICH; not essential for SAC.
• CDK1: targets INCENP and BubR1 to create a binding site
for Plk1 (44, 57, 183).
with INCENP (inner centromere protein),
survivin, and borealin comprise the chromosome passenger complex (CPC), so called because it relocates from the inner centromere to
the central spindle at the metaphase anaphase
transition (reviewed in Reference 144). The
proper localization and activation of the CPC
is regulated by another passenger-like protein,
TD60 (Telophase Disc-60 kD protein) (143)
and Haspin kinase (30). Aurora B corrects misattachments by phosphorylating kinetochore
proteins responsible for microtubule binding
(Ndc80 and MCAK in higher eukaryotes), and
this leads to the release of erroneously attached
microtubules (144). Indeed, the kinase may
have a broader role in promoting microtubule
turnover at kinetochores during prometaphase,
which is particularly important in correcting
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merotelic orientation (when one kinetochore is
attached to opposite poles, the other to a single
pole). To understand how an enzyme complex
located at the inner centromere might influence kinetochore-microtubule attachments, it
is useful first to consider the roles played by
flexibility in structures at the centromere and
kinetochore.
The flexible and expandable properties of
the centromeric regions of chromosomes liken
them to springs, and indeed their behavior
can be described using the appropriate physical equations applicable to springs (141). The
entire structure connecting sister kinetochores
may be considered as having three spring-like
components: two extending from the outer
kinetochore plates to their linked inner centromeres (the kinetochore springs) and another
connecting the sister centromeres (the centromeric spring). Pulling forces applied by the
spindle and resisted by sister chromatid cohesion will result in the extension of kinetochore
and centromeric springs. Observations on the
relative positions of centromeric and kinetochore markers in situations when tension is
present or absent have revealed that to satisfy
the spindle assembly checkpoint, proper tension must be applied to the kinetochore springs,
but the status of the centromeric spring does
not affect the checkpoint activity (104, 160).
In other words, forces within, but not between
kinetochores, are monitored by the SAC (110).
The unbalanced tension resulting from
merotelic orientation is hypothesized to pull
one of the two kinetochores more toward a peak
of Aurora B activity in the inner centromere that
is compressible owing to its spring-like properties. The result would be that the misattached
kinetochores (which are not under proper tension) encounter high kinase activity, and consequently, microtubules are released, mainly due
to the phosphorylation of the Ndc80 protein
(92). The unattached kinetochore then would
immediately trigger the SAC response. In other
words, the sensing of the improper tension
might be first translated into improper attachment and only then could the checkpoint be
activated (120).
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Intriguingly, a CPC component INCENP
interacts with Plk1 (57), which has shown
to be responsible for generating the tensiondependent phosphoepitope 3F3/2 (1, 182, 183).
Here the functions of Plk1 may involve the
PICH protein (Plk1 interacting checkpoint helicase), which is recruited in a Plk1-dependent
manner to inner centromeres and kinetochores
during mitosis (187). In early anaphase, PICH
decorates threads of DNA that connect sister
centromeres (8). Most likely, these threads are
catenated centromeric DNA molecules that are
resolved and separated in a Topoisomerase IIdependent fashion in anaphase. Depletion of
PICH results in chromosome mis-segregation
and loss of Mad2 from kinetochores, symptomatic of checkpoint abrogation (8). It has
been suggested that PICH may also participate
in the monitoring of tension, but a mechanism
for this is not clear (169).
Association of checkpoint proteins with the
kinetochore. The metaphase-anaphase transition is tightly controlled by the ubiquitin
protein ligase APC/C (anaphase promoting
complex or cyclosome) that primarily targets
the destruction of securin and cyclin B among
other cell cycle regulatory molecules. The spindle assembly checkpoint (SAC) prevents these
targets from being cleaved prematurely, i.e., until all chromosomes are correctly aligned on the
metaphase plate (120). Even a single unattached
kinetochore is sufficient to trigger this checkpoint response and maintain the APC/C in an
inhibited state (142). The checkpoint proteins
comprise Bub3, Mad1, Mad2, Mad3/BubR1,
and the Bub1 and Mps1 kinases (see sidebar, Spindle Assembly Checkpoint Kinases).
In metazoans, additional checkpoint functions
are also provided by the previously mentioned
RZZ complex. RZZ functions both in assembling other checkpoint proteins at the kinetochore and in facilitating their removal once
the checkpoint is satisfied (reviewed in Reference 80). The three proteins Mad3/BubR1,
Mad2, and Bub3 associate with the APC/C activating molecule Cdc20 to form the mitotic
checkpoint complex (MCC), which acts as the
effector of APC/C inhibition (120). The direct
binding of the MCC induces a conformation
change to the APC/C thought to prevent its
binding to and ubiquitination of its substrates
(66). A widely accepted model is that kinetochores of unattached chromosomes mediate
the assembly and release of the MCC that diffuses away to inhibit the APC/C. However, the
MCC is able to assemble and prevent mitosis
even in the absence of kinetochores. How can
these facts be reconciled? Current models assume there are two phases in the SAC response:
a kinetochore independent response that controls the overall timing of mitosis (when MCC
can be formed); and a kinetochore dependent
mechanism that delays anaphase until all the
correct microtubule attachments are in place
(also dependent on MCC) (120). Nevertheless,
the ability of the MCC to form independently
of kinetochores does question somewhat the
central role of the KMN in checkpoint signaling and has led Burke & Stukenberg (17) to
suggest that more attention should be paid to
the potential role of the KMN in coordinating
the activity of the multiple protein kinases in
potentially generating a signal through a phosphorylation cascade.
The KMN does serve as a binding site for
many of the checkpoint proteins. The Ndc80
complex is required to bind Mad1/Mad2 and
Mps1 kinase recruitment (105); KNL1/Blinkin
directly associates with Bub1 and BubR1 (85).
In C. elegans, SPDL-1, which along with the
RZZ complex is involved in the regulation of
dynein recruitment to kinetochores, was shown
to be a receptor for Mad1 (186). This underscores the importance of Spindly/SPDL-1 because the recruitment of Mad1 is crucial in assembling other checkpoint proteins onto unaligned kinetochores. It is also possible that
Mad1 interacts with KNL1/Spc105 through
Bub1 as well as with Ndc80 and RZZ, although these interactions may not be direct.
A simple model is that kinetochores can either bind to Mad1 or to microtubules; thus,
when microtubules make contact with the kinetochore, Mad1 is displaced, allowing dynein
to transport away Mad1, Mad2, and the RZZ
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to the spindle poles, and this silences the
checkpoint.
The characteristic of the diffusible signal
that informs the whole APC/C-regulated mitotic apparatus about attached or not attached
kinetochores remains unknown. In order to
fully understand how the SAC works, we first
need to describe on a molecular level how
exactly the lack of a single attachment generates a signal that is amplified to inhibit
the entire APC/C virtually instantaneously.
Conversely, how exactly is the checkpoint silenced? The p31comet protein is known to bind
Mad2 and antagonize its ability to inhibit
APC/C(Cdc20). By counteracting the function
of Mad2, p31comet is required for the silencing of the spindle checkpoint (120, 185). The
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picture that currently emerges from the accumulated data is complex; it suggests high
levels of redundancy, the existence of parallel
networks, and extensive feedback loops (28).
The KMN network must play a central role
through its direct interactions with the ancillary proteins that regulate kinetochore microtubules and the SAC. Our knowledge of the
details of that binding and its mode of regulation is currently very patchy. This identifies
a key area in which more work is needed to
gain fuller understanding. Such an understanding may well open new doors leading to potential ways of influencing and manipulating cells
in which chromosome segregation goes awry
as it does, for example, in many cases of human
cancer.
SUMMARY POINTS
1. The metazoan kinetochore is a very complicated multiprotein structure, which ensures
proper distribution of genetic material to daughter cells.
2. Formation of kinetochores on mitotic chromosomes depends on the existence of centromeres, which need to be propagated correctly to new generations of cells and this
propagation is controlled epigenetically.
3. Proper centromere structure and composition is essential for building functional kinetochores.
4. The KMN network, which is the structural foundation of the kinetochore, is responsible
for the microtubule binding and for the interaction with the auxiliary proteins, which
influence association of the kinetochore with the mitotic spindle and the activity of the
spindle assembly checkpoint.
5. Binding of the kinetochore to the microtubules of the mitotic spindle has to be very dynamic and responsive to changing properties of the microtubule attachment and therefore
is affected and modulated by many regulatory proteins.
6. Members of the spindle assembly checkpoint signal lack or improper attachments of
microtubules to kinetochores and are responsible for introducing corrections before
cells progress to anaphase and exit mitosis.
7. Integration and regulation of all structural and functional components of the kinetochore
ensure its proper activity.
FUTURE ISSUES
1. What is the mechanism of the centromere propagation to the next generation of cells
and how is it regulated?
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2. What is the real function of the centromere in respect to the kinetochore formation?
3. What are the structure and function of single subunits of the CCAN and KMN networks?
4. What are the key components of the centromere-kinetochore interface?
5. What are the specific functions of proteins involved in the interaction of the kinetochore
with the spindle?
6. What is the molecular mechanism of the spindle assembly checkpoint activation and
silencing and how is the function of the spindle assembly checkpoint integrated?
7. How, structurally and functionally, are components of the inner centromere involved in
the overall function of the kinetochore?
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8. How is the assembly and disassembly of the kinetochore regulated in time and space?
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
Work in the authors’ laboratory on kinetochores is supported by Cancer Research UK and the
BBSRC. Authors would like to thank Mitsuhiro Yanagida, Andrea Musacchio, Genevieve Almouzni, and Don Cleveland for the communication of unpublished results. We are very grateful
to Matthew Savoian and Viji Draviam for their helpful comments on the manuscript. We apologize to all molecules that at some stage associate with the kinetochore whose function we have
not mentioned because of space limitations. We also regret not being able to refer to the work of
everyone in the field.
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Genetic and Epigenetic Mechanisms Underlying Cell-Surface
Variability in Protozoa and Fungi
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Regressive Evolution in Astyanax Cavefish
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Mimivirus and its Virophage
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Regulation Mechanisms and Signaling Pathways of Autophagy
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The Role of Mitochondria in Apoptosis
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Biomineralization in Humans: Making the Hard Choices in Life
Kenneth M. Weiss, Kazuhiko Kawasaki, and Anne V. Buchanan p p p p p p p p p p p p p p p p p p p p p p p p 119
Active DNA Demethylation Mediated by DNA Glycosylases
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Gene Amplification and Adaptive Evolution in Bacteria
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Bacterial Quorum-Sensing Network Architectures
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How the Fanconi Anemia Pathway Guards the Genome
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Mechanism of Auxin-Regulated Gene Expression in Plants
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Steve D.M. Brown, Wolfgang Wurst, Ralf Kühn, and John Hancock p p p p p p p p p p p p p p p p p p p 305
Thioredoxins and Glutaredoxins: Unifying Elements in Redox Biology
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Roles for BMP4 and CAM1 in Shaping the Jaw: Evo-Devo and Beyond
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Regulation of Tissue Growth through Nutrient Sensing
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Hearing Loss: Mechanisms Revealed by Genetics and Cell Biology
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The Kinetochore and the Centromere: A Working Long Distance
Relationship
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Multiple Roles for Heterochromatin Protein 1 Genes in Drosophila
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Genetic Control of Programmed Cell Death During Animal
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Cohesin: Its Roles and Mechanisms
Kim Nasmyth and Christian H. Haering p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 525
Histones: Annotating Chromatin
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Systematic Mapping of Genetic Interaction Networks
Scott J. Dixon, Michael Costanzo, Charles Boone, Brenda Andrews,
and Anastasia Baryshnikova p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 601
Errata
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Contents
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