Molecular composition and function of the kinetochore-microtubule
interface in chromosome segregation.
Stable kinetochore-microtubule attachments are essential for equal division of chromosomes
among daughter cells. To correctly separate chromosomes in mitosis, kinetochores must capture
and bind spindle microtubules, stably attach to these dynamic structures, and release a
microtubule when wrongly attached. In the past ten years, elaborate research has been done to
explore and examine the key players that form the direct coupling between chromosomes and
spindle microtubules. The KMN network, Dam1 complex in yeast, and Ska complex in mammalian
cells are three main kinetochore complexes that compose the kinetochore-microtubule interface,
and are required for proper chromosome segregation. Function and interplay of these complexes
will be discussed in this review to gain more insight into how the dynamic process of chromosome
segregation is regulated and to find gaps that need to be filled to fully understand the process.
Introduction
During mitotic cell division, proper segregation of sister chromatids is an important step in
the formation of two viable daughter cells. Each chromosome contains a centromere region which
directs the assembly of a protein structure named the kinetochore. The mammalian kinetochore
consists of more than 80 different proteins and coordinates attachment of chromosomes to the
mitotic spindle, the spindle assembly checkpoint, and chromosome movements1. Conventional
fixation electron microscopy of vertebrate kinetochores revealed that the kinetochore has three
layers; the inner kinetochore, which interacts with chromatin of the centromere; the outer
kinetochore, which forms the binding site for spindle microtubules; and a less dense region, which
separates the inner and outer kinetochore. When not attached to spindle microtubules, a fibrous
corona protrudes from the outer kinetochore2.
Dynamic spindle microtubule plus-ends are captured by the outer kinetochore to align all
chromosomes in a metaphase plate. Initial kinetochore-microtubule attachments are frequently
erroneous and need to be released to prevent chromosome segregation errors. Only if all
kinetochores are stably attached to the mitotic spindle and forces can be generated for chromosome
segregation, the spindle assembly checkpoint will be silenced and mitosis can proceed to anaphase.
Segregation errors occur when misattached or unattached kinetochores are not detected by the
spindle assembly checkpoint. This can result in cell death or aneuploidy, which is found in many
tumors. It is therefore of great importance for the kinetochore to correctly execute three main
functions in chromosome segregation: capturing and attachment of spindle microtubules, staying
attached during microtubule dynamics and force generation, and releasing erroneous attachments to
allow error correction. In this review, I will discuss current knowledge and recent findings on the role
of kinetochore proteins in these processes.
Microtubule binding activities in the kinetochore
Extensive research has been done to elucidate the physical connection between the outer
kinetochore and spindle microtubules. The core complex essential for kinetochore-microtubule
interactions is the conserved KMN network. KNL-1, the Mis12 complex, and the Ndc80 complex
together form this network which contains two distinct microtubule-binding activities (Figure 1A).
The Ndc80 complex directly interacts with stabilized microtubules in vitro, however with low affinity.
KNL-1 also binds microtubules directly, in a concentration dependent and oligomeric manner. The
affinity of KNL-1 for microtubule binding is increased when stabilized by co-expression of the Mis12
complex3. Mis12 complex member NSL1 has a scaffold function, essential for KNL-1 binding and
localization at kinetochores. Also Ndc80 complex kinetochore localization is mediated by NSL1
binding4. Reconstitution of the KMN network by mixing KNL-1, Mis12 complex, and Ndc80 complex
dramatically increases microtubule binding activity compared to Ndc80 complex or KNL-1 alone3.
Within the KMN network, microtubule binding by the Ndc80 complex is studied most
elaborately and is considered to be the most important microtubule binding activity of the
kinetochore. The Ndc80 complex is composed of four subunits, and forms a long rod with globular
heads linked by α-helical coiled-coils5,6. The coiled-coil region provides the complex with its extended
shape to span the distance between the kinetochore and microtubule plus-end. In human cells, HEC1
and NUF2 form the microtubule interacting site of the Ndc80 complex7, whereas SPC24/SPC25
interacts with other KMN network components Mis12 complex and KNL-13 (Figure 1A).
Tetramerization of the Ndc80 complex occurs through binding of the C-terminal ends of the
HEC1/NUF2 dimer and the N-terminal ends of the SPC24/SPC25 dimer. Interacting ends of both
dimers have a coiled-coil conformation5,6.
Microtubule binding by the Ndc80 complex is accomplished by the HEC/NUF2 head regions.
The microtubule binding heads of HEC1 and NUF2 fold into calponin-homology (CH) domains8, which
contain two distinct microtubule binding regions9. CH-domains are also found in other microtubule
binding proteins such as the microtubule plus-end tracking protein EB17. The human Ndc80 complex
interacts with both α- and β-tubulin9. This binding is predominantly electrostatic by interactions
between the positive charges of the CH-domains and negative charges of the C-terminal tubulin
tails8. Charge reversal point mutations in the CH-domain of HEC1 severely disrupted the interaction
with microtubules, indicating the importance of the CH-domain’s positive charge for stable
microtubule binding10. Furthermore, kinetochores containing HEC1 CH-domain mutants fail to form
stable interactions with microtubules in vivo, resulting in failure of stable microtubule attachment
formation or chromosome alignment9.
Besides microtubule binding by KMN network components KNL-1 and Ndc80, three other
microtubule binding activities are found in mammalian kinetochores. (1) Kinesin-like motor protein
CENP-E localizes at kinetochores from prophase to early anaphase, to capture and bind spindle
microtubules. The extended CENP-E coiled-coil dimer protrudes from the outer kinetochore and
interacts with microtubules with its C-terminal microtubule-binding domain22 (Figure 1A). CENP-E
microtubule binding activity is important for maintaining stable kinetochore attachments and the
generation of tension23. (2) Cytoplasmic linker protein (CLIP)-170 is involved in the formation of
kinetochore-microtubule attachments and localizes at the outer kinetochore and at microtubule
A
B
Dam1 ring
C
Ska complex
dimer
Figure 1. Composition of the kinetochore-microtubule interface. (A) The KMN network consisting of KNL-1, Mis12
complex, and Ndc80 complex forms the core attachment site for kinetochore-microtubule binding. KNL-1 and the
HEC1/NUF2 dimer of Ndc80 contain microtubule binding activity. Kinesin-like motor protein CENP-E mediates the
capturing and subsequent binding of spindle microtubules. (B) The budding yeast Dam1 complex forms rings to encircle
spindle microtubules, thereby contributing to kinetochore-microtubule attachments. (C) In mammalian cells, the Ska
complex forms W-shaped dimers to stably bind spindle microtubules. Ska complex microtubule binding is essential for
stable kinetochore-microtubule attachments. (Adapted from Cheeseman and Desai 20081).
plus-ends during mitosis. Since CLIP-170 is only found at unattached kinetochores, it is likely that it
mediates initial kinetochore-microtubule interactions. CLIP-170 is also important for chromosome
alignment24. (3) The Ska complex also localizes at kinetochores and contains microtubule binding
activity, which is essential for stable kinetochore-microtubule interactions (Figure 1C)14,16,17.
The Ska complex consists of Ska1, Ska2, and Ska314,15,16,17,19. Ska1-Ska2 and Ska1-Ska3
interactions are present throughout the complex structure whereas Ska2 and Ska3 only interact in a
small area, indicating that Ska1 is essential for Ska complex formation. Ska complex dimerization is
required for correct cell division and occurs when the N-terminal ends of two Ska complexes interact
to form a W-shaped dimer16,20. The complex localizes at kinetochores and spindle microtubules
throughout mitosis, and is recruited to kinetochores by Ndc80 complex member HEC114,16,17.
Furthermore, the C-terminal domains of both Ska1 and Ska3 are essential for Ska complex
localization and function at the kinetochore-microtubule interface. Co-depletion of Ska1 and Ska3
results in chromosome congression failure, chromosome segregation defects and subsequent cell
death because of destabilization of kinetochore-microtubule interactions14,18. It is proposed that Ska3
also functions in spindle assembly checkpoint silencing. Ska3 depletion causes accumulation of
checkpoint proteins at kinetochores and metaphase arrest41. Taken together, besides the KMN
network, dimeric Ska complex localization at kinetochores during mitosis is essential for proper cell
division.
An important property for Ska complex function in kinetochore-microtubule attachments is
its microtubule binding activity. The conserved C-terminal microtubule binding domain of Ska1 is
essential for the formation of stable kinetochore-microtubule interactions. Structural analysis
revealed that the Ska1 microtubule-binding domain is a variation of a winged-helix domain. Wingedhelix domains are primarily implicated in DNA binding, but can also function in protein-protein
interactions. The Ska1 variation of the winged-helix domain has the same length as tubulin
monomers. It is therefore likely that the Ska1 microtubule-binding domain interacts with tubulin
monomers and not dimers such as in HEC1-microtubule binding, which is important for dynamic
microtubule tracking (see below)21,26. Both C-terminal microtubule binding domains of Ska1 and Ska3
are required, but not sufficient for the decoration of microtubules by Ska in vitro. This points towards
a role for the Ska complex in proper positioning of the microtubule binding domains of Ska1 and
Ska320. Mutations in the microtubule binding domains of Ska1 and Ska3 in human cells phenocopy
depletion of the complete Ska complex resulting in increased mitotic delay, and showing the
importance of microtubule binding by the Ska complex21.
In budding yeast, besides in the KMN network, microtubule binding activity is found in the
kinetochore localized Dam1 complex11. The Dam1 complex consists of ten proteins and oligomerizes
into a ring structure which encircles the microtubule (Figure 1B). Various measurements has been
done to calculate Dam1 complex numbers in the Dam1 ring, resulting in various outcomes. The ring
contains between 16 and 30 Dam1 complexes, however the exact number remains unknown 12,13.
Loss of functional Dam1 complex results in unequal chromosome segregation indicating that the
Dam1 ring is essential for chromosome attachment to spindle microtubules13. Because of the
indispensable nature of Dam1 in kinetochore-microtubule attachments in budding yeast, extensive
searching for a Dam1 complex homolog in vertebrates has been done. A structural Dam1 homolog
has never been found, however, the Ska complex functionally fulfills this role. Both complexes are
essential in maintaining stable kinetochore-microtubule attachments and are localized and regulated
by the same kinetochore proteins and mechanisms, which will be discussed later in this review.
Distinct proteins and protein complexes have been described to localize at the outer
kinetochore and function in formation of stable kinetochore-microtubule attachments. To elucidate
the molecular mechanisms of chromosome segregation, it is of great importance to understand how
all microtubule-binding activities at the kinetochore cooperate in this process.
Dynamic microtubule tracking by the kinetochore
Microtubules are highly dynamic structures which continuously polymerize and
depolymerize. In mitosis, the dynamic nature of spindle microtubules is essential for chromosome
congression into the metaphase plate, and subsequent chromosome segregation during anaphase.
Microtubule dynamics provoke a complicated task for kinetochores, namely to stably attach and
track the dynamic spindle microtubules in various stages of mitosis. Different models have been
proposed and tested for how kinetochores achieve stable microtubule attachments and tracking.
Basically, three models for microtubule tracking can be distinguished: multiple low affinity
interactions with the microtubule, capturing of microtubules with ring structures, and cooperative
binding of microtubules with multiple protein complexes. Detailed findings supporting each of these
models and recent data on molecular function of kinetochore proteins in dynamic microtubule
tracking will be discussed below.
Multiple low-affinity interactions with microtubules through protein clustering
The direct interaction of the Ndc80 complex with microtubules was found to be a low-affinity
3
binding . Therefore, binding of a microtubule by a single Ndc80 complex would not allow for stable
kinetochore-microtubule attachment in chromosome segregation. In budding yeast, approximately
eight Ndc80 complexes are present around a kinetochore-bound spindle microtubule25. Microtubule
binding by multiple Ndc80 complexes potentially allows microtubule dynamics to continue while
simultaneously contributing to stable a kinetochore-microtubule interaction.
To achieve kinetochore-microtubule binding with multiple low-affinity interactions of Ndc80,
oligomerization of Ndc80 complexes into clusters is a critical step. Different in vitro binding assays
using recombinant Ndc80 complex showed microtubule binding by Ndc80 complex oligomers8,25,26.
The unstructured N-terminal tail of HEC1 mediates Ndc80 complex oligomerization along
microtubule protofilaments and is therefore essential for stable microtubule binding9,26. The exact
A
B
Figure 2. Ndc80 complex interacts with spindle microtubules in clusters to track microtubule dynamics. (A) Role for
the unstructured N-terminal tail of HEC1 in microtubule binding by Ndc80 clusters. Figure shows two models of
cluster formation of Ndc80 complexes. The HEC1 tail could directly interact with the microtubule, thereby aligning
CH-domains of HEC1 and NUF2 for microtubule binding and oligomerization (drawn above the microtubule). On the
other hand, the tail could also interact with the neighboring NUF2 head. In this situation, the tail would directly
mediate oligomerization of Ndc80 complexes on the spindle microtubule (drawn under the microtubule). It is under
debate whether the NUF2 domain of the Ndc80 complex is involved in microtubule binding or clustering of Ndc80
complexes. (B) The HEC1 tail is also essential for tracking of microtubule dynamics. By binding with the CH-domain
and the N-terminal tail, HEC1 forms a bipartite binding with the spindle microtubule. Upon microtubule
protofilament curvature, the CH-domain will lose the affinity for the microtubule but the tail remains attached to the
microtubule. It is suggested that the tail can slide over the microtubule to reattach stabilized tubulin for rebinding of
the HEC1 CH-domain. Ndc80 clustering at the microtubule lattice would contribute to tail sliding by retaining
interactions with the sliding Ndc80 complex. (Adapted from Alushin et al., 201026; Tooley and Stukunberg 201127).
role for the HEC1 N-terminal tail in this process is still unclear. The tail could directly contribute to
microtubule binding by interactions with tubulin, thereby aligning CH-domains of HEC1 and NUF2 for
microtubule binding. Properly aligned HEC1 would then be able to interact with the neighboring
NUF2 molecule for microtubule binding in clusters. Alternatively, instead of interacting with the
microtubule, the N-terminal tail of HEC1 could interact with an acidic patch on the neighboring NUF2
molecule to form Ndc80 complex clusters8,9(Figure 2A).
NUF2 could also play a role in Ndc80 complex microtubule binding and oligomerization. In
vitro co-sedimentation assays showed that the HEC1/NUF2 dimer and not HEC1 alone interacts with
stabilized microtubules7. Because of the presence of a CH-domain, this initially pointed towards
contribution of NUF2 in direct microtubule binding26. However, NUF2 CH-domain mutants were able
to form stable kinetochore-microtubule attachments in vivo, indicating that the NUF2 CH-domain is
not essential for microtubule binding by Ndc80 complex clusters10. Possibly, NUF2 plays a role in
Ndc80 complex oligomerization rather than microtubule binding, thereby contributing to highaffinity and stable kinetochore-microtubule interactions. That would also explain the differences of
NUF2 CH-domain mutants in microtubule binding assays in vitro and in vivo. The effect of NUF2
dysfunction in Ndc80 complex positioning might be less severe when other kinetochore proteins are
present to position the Ndc80 complexes for microtubule binding in clusters10.
Ndc80 cluster formation is induced by initial microtubule binding of single Ndc80 complexes,
which induces the growth of larger areas of Ndc80 binding8. This observation was confirmed by
visualization of Ndc80 binding showing heterogeneous Ndc80 binding with saturated and
undecorated microtubule patches, strongly pointing towards microtubule binding by Ndc80 complex
clusters. Furthermore, in the absence of microtubules Ndc80 cluster formation was not observed,
demonstrating that Ndc80 oligomerization is microtubule dependent. Ndc80 cluster size on
microtubules was also analyzed. Formation of four Ndc80 complexes per cluster was found most
likely to occurs. Given that 6-8 Ndc80 complexes bind one microtubule25, this would mean that two
or three clusters of Ndc80 complexes per kinetochore-microtubule attachment will bind26.
By binding microtubules in clusters of Ndc80 complexes, the kinetochore allows tracking of
depolymerizing microtubules during anaphase. With multiple Ndc80 complexes associating with the
microtubule, it can stay attached to the kinetochore when several Ndc80 complexes are released as a
result of microtubule depolymerization. Also the bipartite interaction of HEC1, binding microtubules
with the CH-domain and the N-terminal tail, could play a role in tracking of dynamic microtubules by
the Ndc80 complex. The HEC1 CH-domain only interacts with straight tubulin protofilaments,
whereas microtubule binding by the N-terminal tail is insensitive to tubulin conformation26. Upon
microtubule depolymerization, protofilaments curve and subsequently fall apart into tubulin dimers.
It is suggested that during depolymerization and protofilament curvature, HEC1 releases CH-domain
binding from the microtubule but retains attached to it by interactions with the N-terminal tail. The
tail slides over the microtubule to find a stabilized tubulin subunit and rebinds, thereby tracking the
depolymerizing microtubule. Binding of Ndc80 complexes in clusters might contribute to N-terminal
tail sliding by preventing complete release of the sliding Ndc80 complex27(Figure 2B).
Other KMN network components KNL-1/Mis12 are also involved in cluster binding of
microtubules. Microtubule binding efficiency of KNL-1/Mis12 is concentration dependent, indicating
that -like Ndc80- they bind microtubules as oligomers. Oligomerization of KNL-1/Mis12 on the
microtubule lattice even more dramatically depends on protein concentration than Ndc80
oligomerization on microtubules, demonstrating a strong advantage of cluster binding for KNL1/Mis123. When mixing all KMN network proteins, concentration dependent microtubule binding is
also present. It is therefore proposed that microtubule binding in KMN network clusters is mainly
driven by KNL-1 oligomeric binding and increased by Ndc80 clustering.
In budding yeast, besides the KMN network also the ten proteins of the Dam1 complex
oligomerize to stably bind spindle microtubules and track microtubule dynamics. The interaction of
the Dam1 complex with microtubules was found to be formed by electrostatic interactions. In vitro
binding assays showed that the positively charged C-terminus of the Dam1p subunit and the
negatively charged C-termini of α- and β-tubulin are essential for strong microtubule binding. These
electrostatic interactions enable the Dam1 complex to diffuse along microtubules, which would be
more complicated when other interactions such as covalent bonds would be formed28. However,
contrary results were found by Miranda and co-workers. They observed that after removal of the Cterminal tails of α- and β-tubulin, Dam1 complexes could still bind to and form rings around
microtubules. In their model, binding of Dam1 to microtubules is mediated by extensions from the
Dam1 complex that protrude and dock into the microtubule wall13. For definitive conclusions about
how the interaction between the Dam1 ring and microtubules is established and maintained during
sliding, more research needs to be done.
Capturing microtubules with ring structures
To mediate chromosome segregation during mitosis, the kinetochore has to track
depolymerizing microtubules while retaining the interaction with them. The Dam1 complex captures
microtubules by the formation of a ring around the end of the spindle microtubule lattice. Dam1
rings have the capacity to track dynamic microtubules without turnover, also when coupled to cargo,
which is important for chromosome congression and segregation during mitosis. In vitro, microbeads
coupled to Dam1 rings can be translocated by microtubule filament shortening12,29. Microtubule
dynamics-driven movement of microbeads was observed over a distance of approximately 3
micrometers, which corresponds with chromosome movements in vivo. The microbead-bearing
attachments of Dam1 could furthermore withstand relevant forces and were stable for a relevant
time to mediate chromosome segregation during mitosis in yeast29. The Dam1 ring may directly
mediate chromosome segregation by translating force generation during microtubule
depolymerization to movement of chromosomes.
The importance of Dam1 ring formation for the ability of kinetochores to track dynamic
microtubules is uncertain, because Dam1-coated microbeads can also be transported by
depolymerizing microtubules without formation of complete Dam1 rings. When using a Dam1
mutant complex with reduced capacity to oligomerize into heterodecamers, tracking of shortening
microtubules still took place30,31. Mutant Dam1-coated microbeads roll over the depolymerizing
microtubule rather than sliding, observed with Dam1 rings. Rotations found in microbead rolling in
the absence of rings indicates that this kind of movement is unlikely to transport mitotic
chromosomes, because they do not rotate while moving. However, nonencircling Dam1 patches can
translocate with depolymerizing microtubule ends and diffuse faster than Dam1 rings31. It would be
important to translate these findings to the in vivo situation to gain more information about the
conformation of Dam1 that forms the functional unit in chromosome segregation. It is thereby
interesting to further investigate the function of Dam1 patches in microtubule tracking and
chromosome segregation.
To explain the role of the Dam1 complex in transport of chromosomes during mitosis in
yeast, two hypothesizes are proposed in which Dam1 forms a ring to slide along microtubules, or
forms patches to diffuse along microtubules. The Dam1 ring conformation can diffuse along the
microtubule and is driven towards the microtubule minus-end by protofilament curving during
depolymerization. Thereby the ring tracks the depolymerizing microtubule tip to facilitate
chromosome translocation towards the spindle pole. Alternatively, Dam1 patches diffuse along
microtubules in a biased way that results in tip tracking of depolymerizing microtubules. Because the
association and diffusion rates of single Dam1 complexes with microtubules are higher than the
dissociation rate, they diffuse away from the depolymerizing microtubule end. This biased-diffusion
model explains stable kinetochore attachment to both polymerizing and depolymerizing
microtubules, and questions the role of Dam1 rings in chromosome segregation in vivo32.
Cooperative microtubule binding with multiple protein complexes
It is commonly accepted that the KMN network alone does not have the capacity to stably
bind and track dynamic microtubules, and that additional kinetochore localized microtubule binding
proteins mediate this process. Microtubule binding and tracking by the kinetochore during
(de)polymerization could be mediated by the cooperative function of several kinetochore-
microtubule interacting proteins and protein complexes. The main microtubule binding activity
within the KMN network accomplished by the Ndc80 complex, is supported by binding of the
previously mentioned Dam1 complex or Ska complex in yeast and mammalian cells, respectively.
Dam1 complex and Ndc80, the yeast homolog of HEC1, interact directly33. This interaction is
essential for stabilization of microtubule binding by Ndc80. Free Ndc80 complexes bind stabilized
microtubules only in a diffuse and transient manner. However, Ndc80 dissociation from the
microtubule is decreased in the presence of the Dam1 complex in a Dam1 concentration dependent
manner. This indicates that the Ndc80 and Dam1 complexes function cooperatively at the
kinetochore-microtubule interface, to stably bind dynamic spindle microtubules34.
In mammalian cells, the Ndc80 complex and Ska complex interact to function together. Like
Dam1 in yeast, the Ska complex is essential for stabilization of kinetochore-microtubule attachments.
Loss of Ska results in chromosome congression and segregation defects14,15,16,17,18. Ndc80 complex
subunits HEC1 and NUF2 recruit the Ska complex to the outer kinetochore and spindle microtubules.
Also KNL-1 and DSN1 are involved in Ska recruitment to kinetochores during mitosis, indicating that
the KMN network directly regulates Ska complex localization18. Presence of the Ndc80 complex at
kinetochores also promotes the affinity of the Ska complex for microtubules in a dose-dependent
manner, pointing towards a direct Ndc80-Ska interaction when microtubules are present21.
Interestingly, binding of both Dam1 complex in yeast and Ska complex in mammalian cells to
the Ndc80 complex is mediated by the HEC1/Ndc80
loop region. Within the HEC1/NUF2 coiled-coil shaft,
A
an unstructured loop consisting of 50-60 amino acids
protrudes out of the structure8,33. Proteins that bind
the HEC1/Ndc80 loop region when microtubules are
end-on attached, are in close proximity to the
dynamic microtubule terminus. Interactions of the
Ska1/Dam1 complex with the HEC1/Ndc80 loop
perfectly positions the complexes to perform their
role of tracking depolymerizing microtubule
protofilaments in chromosome segregation35 (Figure
3).
Ndc80 loop mutants showed little interaction
B
with Dam1 indicating that the loop region mediates
the Ndc80-Dam1 interaction. In cells expressing
Ndc80 loop mutants, initial kinetochore-microtubule
interactions are formed but sister chromatid biorientation is defective, possibly by defective end-on
attachment. Also Dam1 localization at kinetochores is
mediated by the Ndc80 loop. Taken together, the
interaction of Ndc80 and Dam1 seems to be essential
for kinetochore-microtubule attachments. However,
contradictory results were found in a yeast colony
growth experiment expressing Ndc80 depleted for the
Figure 3. Dam1 and Ska complex microtubule
N-terminal tail. Viability was not affected by depletion
binding is mediated by the Ndc80/HEC1
of the Ndc80 tail whereas mutations in Dam1 did
unstructured loop region. (A/B) Dam1 and Ska
cause reduced colony growth. Ndc80 and Dam1
complex are important for dynamic microtubule
double mutants were impaired in growth, compared
tracking in yeast and mammalian cells
to single mutants, indicating that the N-terminal tail of
respectively.
By
interacting
with
the
Ndc80 is essential, but only when Dam1 function is
Ndc80/HEC1 loop, these complexes are in
absent. This points towards a redundant function for
proximity of the dynamic microtubule terminus
the N-terminal tail of Ndc80 and Dam1 in
and thereby perfectly positioned to track
chromosome segregation in yeast36. The internal loop
curving microtubule protofilaments. (Adapted
of HEC1 also has a significant role in Ska complex
from Schmidt et al., 201221).
localization and function at kinetochores. HEC1 loop-depleted cells formed normal Ndc80 complexes
which localized at kinetochores but could not function to mediate chromosome alignment and
segregation. Furthermore, the HEC1 loop sequence is important for Ska complex binding and
function in chromosome segregation35. So, the HEC1/Ndc80 unstructured loop region is an essential
structure for cooperative microtubule binding by kinetochore localized protein complexes in both
yeast and mammalian cells.
Besides stabilizing the interaction of Ndc80 with microtubules, the Dam1 complex also
mediates Ndc80 tracking of microtubule dynamics. Bead-bearing Ndc80-microtubule interactions and
traveling along microtubules increases in the presence of Dam1. Together with the Ndc80 stabilizing
function of Dam1, this indicates that Dam1 is a processivity factor for Ndc80 complex-microtubule
interactions in yeast34. In mammalian cells, a comparable role is performed by the Ska complex. Ska
functions synergistically with the Ndc80 complex in tracking dynamic microtubules. Ndc80 complex
alone is unable to associate with microtubule tips when they depolymerize. In contrast, when the Ska
complex is present Ndc80 complex remains associated with depolymerizing microtubules, suggesting
that this is promoted by the Ska complex. Addition of the Ska complex results in processive transport
of Ndc80 complexes along depolymerizing microtubules21.
The capacity of the mammalian kinetochore to track dynamic microtubules seems to be
mainly mediated by the Ska complex. Total internal reflection fluorescence microscopy experiments
showed that the Ska complex localizes at and tracks depolymerizing microtubule tips. In contrast,
Ndc80 complex failed to track depolymerizing microtubules in these experiments. These
observations were confirmed by experiments demonstrating that the binding affinity of the Ska
complex for the microtubule lattice and curved protofilaments is similar, whereas lattice binding by
the Ndc80 complex was significantly stronger than its binding to curved microtubules21. It is
therefore hypothesized that the Ska and Ndc80 complexes interact at the kinetochore-microtubule
interface to form a complex that cooperatively binds and tracks depolymerizing microtubules to
separates chromosomes in mitosis. The Ndc80 complex integrates the Ska complex into the
kinetochore and positions it for stable interactions with both the microtubule lattice and curved
protofilaments, whereas the Ska complex is essential for Ndc80 to remain attached to
depolymerizing microtubules21. Although the Ndc80 and Ska complexes together attach to and track
depolymerizing microtubules, studies adding load to the complex has not been performed yet. It will
be a future goal to investigate the behavior of microtubule tracking by Ndc80/Ska when they carry
cargo, as they do during chromosome segregation.
To achieve stable interactions between multiple protein complexes at the kinetochore and dynamic
spindle microtubules, mechanisms described above have to cooperate in microtubule tracking. The
dynamic nature of microtubules and various movements of chromosomes during mitosis makes the
kinetochore-microtubule interaction a very complex one. Several models for microtubule tracking
have been proposed, but the question remains which proteins and mechanisms are required for this
tracking during various stages of mitosis.
Error-correction of erroneous kinetochore-microtubule interactions
Bipolar, amphitelic spindle attachment of a pair of sister chromatids before chromosome
segregation occurs, is essential for correct cell division and viability of daughter cells. Monotelic and
syntelic attachments will result in daughter cells with an incorrect number of chromosomes, and
merotelic attachments will even cause rupture of chromosomes during anaphase. Cell division with
misattached kinetochores induces cell death or aneuploidy, which can be the cause for cancer.
Therefore, it is of great importance that kinetochore-microtubule interactions are tightly regulated
and checked before division takes place. Regulation of microtubule binding by KMN network
components, Dam1 complex, and Ska complex are all regulated by Aurora B mediated
phosphorylation3,18,34.
A model for regulation of outer kinetochore proteins inclusing the KMN network, Ska
complex, and Dam1 complex by Aurora B phosphorylation is proposed, in which Aurora B is located
at the inner centromere. By this localization, Aurora B has a spatial phosphorylation range, in which
phosphorylation activity towards outer kinetochore proteins depends on kinetochore tension. When
microtubules are attached correctly, tension will be present to stretch the kinetochore and Aurora B
will be spatially separated from outer kinetochore proteins and not be able to phosphorylate them
(Figure 4B). When microtubules are wrongly attached, no tension will be present resulting in
phosphorylation of outer kinetochore proteins, which are now in close proximity to Aurora B.
Subsequently, the wrongly attached microtubule is released and reformation of correct bipolar
attachment can occur38 (Figure 4A).
To eliminate incorrect Ndc80 complex mediated kinetochore-microtubule attachments,
Ndc80 becomes phosphorylated by Aurora kinase B. Positively charged residues on the N-terminal
tail of HEC1 become phosphorylated, resulting in loss of electrostatic interactions with negatively
charged C-terminal tubulin tails3,8,37. KMN network proteins KNL-1 and DSN1 (part of the Mis12
complex) are also conserved Aurora B targets. Phosphorylation of HEC1, KNL-1, or DSN1 alone is not
sufficient to disrupt kinetochore-microtubule attachments, whereas phosphorylation of all three
proteins completely abolishes microtubule binding. Besides that, through fine-tuned phosphorylation
of the KMN network by Aurora B, microtubule binding can also be modulated to destabilize an
attachment without eliminating it38.
Regulation of Dam1 complex mediated kinetochore-microtubule interactions is also
accomplished by phosphorylation. The mechanism of regulation of Dam1-microtubule interactions is
comparable to regulation of Ndc80-microtubule interactions. Ipl1 kinase, the Aurora B homolog in
yeast, facilitates bi-orientation of sister chromatids by promoting microtubule release from the
kinetochore. When tension at the kinetochore is achieved by attachment to both spindle poles, Ilp1
becomes inactive towards kinetochore-micortubule attaching proteins39. Mutations that disrupt
phosphorylation of Dam1p causes chromosome segregation defects as a result of hyper-stable
microtubule attachments, proving Dam1p to be a critical Ipl1 target40. Besides that, Ipl1 mediated
phosphorylation of the Dam1 complex abolishes the interaction between Dam1 and Ndc80
complexes. When not bound to Ndc80, Dam1 complex can no longer execute its stabilizing and
processivity functions towards Ndc80 anymore, resulting in microtubule release by the kinetochore34.
A
B
Figure 4. Regulation of kinetochore-microtubule attachment by Aurora B phosphorylation. (A) Aurora kinase B
localizes at the inner centromere to phosphorylate kinetochore proteins. At unattached or wrongly attached
kinetochores, no tension is present. Aurora B is in close proximity of its substrates including KNL-1, DNS1, and HEC1
which will become phosphorylated (red dots) and lose their affinity for microtubules. Via this mechanism, monotelic,
synthelic, and merotelic attachments will be released to allow reformation of correct bipolar microtubule attachment.
(B). Upon correct kinetochore-microtubule attachment, tension will stretch the kinetochore to spatially separate
Aurora B form its targets. The microtubule binding can stabilize for subsequent chromosome segregation. (Adapted
from Welburn et al., 201038). Regulation of microtubule binding by kinetochore localized Dam1 and Ska complexes in
yeast and mammalian cells respectively, occurs in the same tension-dependent way as KMN network regulation.
Kinetochore localization of the Ska complex in mammalian cells is also tightly regulated by
Aurora B mediated phosphorylation, which negatively regulates the association of the complex with
kinetochores. Phosphomimic mutants of Ska1 and Ska3 fail to localize at kinetochores whereas
nonphosphorylatable Ska1 and Ska3 mutants cause hyper-stabilization of kinetochore-microtubule
attachments. Hyper-stabilization of microtubule binding by the kinetochore interferes with error
correction resulting in mitotic defects. Therefore, precise temporal phosphorylation and
dehosphorylation of Ska proteins is of significant importance for mitotic progression18. It is suggested
that Aurora B mediated phosphorylation of kinetochore bound Ska is regulated in the same tensiondependent manner as KMN network phosphorylation (see Figure 4). At unattached tensionless
kinetochores, the Ska complex is spatially close to Aurora B localized at the inner centromere and is
therefore phosphorylated. After bi-orientation of sister chromatids, tension is established and
dephosphorylation of the Ska complex takes place. This results in stabilization of Ska complex
localization at kinetochores and subsequent stabilization of kinetochore-microtubule attachments.
The fine-tuned phosphorylation of KMN network proteins by Aurora B might have a second,
more sophisticated function in chromosome segregation. Besides facilitating the correction of
erroneous kinetochore-microtubule interactions, Aurora B regulation might also mediate
microtubule tracking by oligomeric Ndc80 complexes. Combining oligomeric Ndc80 complex binding
of microtubules, fine-tuned KMN network regulation by Aurora B, and HEC1 N-terminal tail sliding on
depolymerizing microtubules (all discussed above) results in a speculative model for Ndc80 complex
tracking depolymerizing microtubules during anaphase. Differential phosphorylation of the KMN
network and especially the HEC1 N-terminal unstructured tail would allow for either release or
tracking of the microtubule when it depolymerizes. Upon initial capture of the microtubule by Ndc80,
the KMN network will be partially phosphorylated by Aurora B because of lack of tension. When the
kinetochore-microtubule interaction matures, tension increases and dephosphorylation of the KMN
network results in strong Ndc80 binding of the microtubule in clusters of Ndc80 complexes. During
microtubule depolymerization, Ndc80 complexes slide over the microtubule with the N-terminal tail
while interactions with other Ndc80 complexes may prevent release from the microtubule. If
kinetochore-microtubule attachments are erroneous, no tension is present and the HEC1 tail
becomes highly phosphorylated by Aurora B. It thereby loses the affinity for the microtubule and is
not able to mediate Ndc80 cluster formation and binding27. This model might also be important for
kinetochore-microtubule interactions in metaphase, which must be highly dynamic as well.
Chromosomes oscillate in the metaphase plate, but remain attached to spindle microtubules. Partial
phosphorylation of KMN network proteins by Aurora B could mediate continuous release and
rebinding of microtubules to allow tracking of microtubule dynamics. However, this process might
also be achieved by another mechanism, independent of Aurora B phosphorylation.
Aurora B kinase is the key regulator of kinetochore-microtubule attachments. By regulating
error-correction of wrongly attached microtubules Aurora B is essential for correct chromosome
segregation, but also more complex regulation mechanisms at the kinetochore-microtubule interface
could be mediated by Aurora B.
Concluding remarks and future goals
Much effort have been put into unravelling regulation of the complex mechanism of chromosome
segregation in mitosis. In this review, I have attempted to assemble general knowledge and recent
findings on the main complexes that physically compose the kinetochore-microtubule interactions in
yeast and mammalian cells. Our understanding of how kinetochore-microtubule interactions are
formed, retained, controlled, and corrected has been expanding tremendously. However, still many
gaps in our knowledge about chromosome segregation exist, and need to be filled to fully
understand one of the most fundamental cellular mechanisms of life. It is therefore important to
uncover remaining questions about how chromosome segregation is regulated.
It seems that the Ndc80 complex in both yeast and mammalian cells is essential but not sufficient to
bind and track dynamic spindle microtubules. The role of Ndc80 clustering in tracking of
depolymerizing microtubules is important and the HEC1 N-terminal tail and NUF2 CH-domain
mediate cluster formation. It is unclear whether the HEC1 tail interacts with microtubules to align
Ndc80 complexes for clustering, or directly mediates Ndc80 clustering by binding of an acidic patch
on the neighbouring NUF2 head domain. Also the function of the NUF2 CH-domain in Ndc80 complex
oligomerization remains elusive and should be explored in more detail. In budding yeast, the Dam1
complex assists the KMN network in microtubule binding and tracking. Rings and patches of Dam1
complexes are formed on microtubules, both capable of tracking microtubule dynamics while
coupled to cargo. Many questions remain in relation to Dam1 function in microtubule binding and
chromosome segregation. How does Dam1 complex interact with the microtubule lattice? Does this
interaction differ in ring conformation compared to nonencircling patches? Which Dam1
conformation is the most important for chromosome segregation? How many Dam1 complexes are
incorporated in a Dam1 ring? Is Dam1 microtubule tracking generated by protofilament curvature
induced movement or by biased diffusion? In mammalian cells, the Ska complex appears to be the
functional homolog of Dam1 in yeast, by mediating KMN network induced microtubule tracking.
However, to my knowledge, no Ska complex load-bearing experiments have been done yet. It is
therefore interesting to examine behaviour of the combined Ndc80 and Ska complexes when
coupled to cargo, which simulates the process of chromosome segregation.
So, many steps have already been made in elucidating chromosome segregation, but many will
follow to ultimately understand the entire process. We know which players are involved, how they
function independently, and some of their regulation mechanisms. The main challenge will be to
explain how all the kinetochore-localized microtubule-binding complexes function together to cause
dynamic chromosome movements required in various stages of mitosis.
References
1. Cheeseman, I.M., Desai, A. (2008). Molecular architecture of the kinetochore-microtubule
interface. Nat. Rev. Mol. Cell Biol. 9, 33-46.
2. McEwen, B.F., Dong, Y., Van den Beldt, K.J. (2007). Using electron microscopy to understand
functional mechanisms of chromosome alignment on the mitotic spindle. Methods Cell Biol.
79, 259-293.
3. Cheeseman, I.M., Chappie, J.S., Wilson-Kubalek, E.M., Desai, A. (2006). The conserved KMN
network constitutes the core microtubule-binding site of the kinetochore. Cell. 127, 983-997.
4. Petrovic, A., Pasqualato, S., Dube, P., Krenn, V., Santaguida, S., Cittaro, D., Monzani, S.,
Massimiliano, L., Keller, J., Tarricone, A., Maiolica, A., Stark, H., Musacchio, A. (2010). The
MIS12 complex is a protein interaction hub for outer kinetochore assembly. J. Cell Biol. 190,
835-852.
5. Ciferri, C., DeLuca, J., Monzani, S., Ferrrari, K.J., Ristic, D., Wyman, C., Stark, H., Kilmartin, J.,
Salmon, E.D., Musacchio, A. (2005). Architecture of the human ndc80-hec1 complex, a critical
constituent of the outer kinetochore. J. Biol. Chem. 280, 29088-29095.
6. Wei, R.R., Sorger, P.K., Harrison, S.C. (2005). Molecular organization of the Ndc80 complex,
an essential kinetochore component. Proc. Natl. Acad. Sci. 102, 5363-5367.
7. Wei, R.R., Al-Bassam, J., Harrison, S.C. (2007). The Ndc80/HEC1 complex is a contact point for
kinetochore-microtubule attachment. Nat. Struct. Mol. Biol. 14, 54-59.
8. Ciferri, C., Pasqualato, S., Screpanti, E., Varetti, G., Santaguida, S., Dos Reis, G., Maiolica, A.,
Polka, J., DeLuca, J.G., De Wulf, P., Salek, M., Rappsilber, J., Moores, C.A., Salmon, E.D.,
Musacchio, A. (2008). Implications for kinetochore-microtubule attachment from the
structure of an engineered Ndc80 complex. Cell. 133, 427-439.
9. Tooley, J.G., Miller, S.A., Stukenberg P.T. (2011). The Ndc80 complex uses a tripartite
attachment point to couple microtubule depolymerization to chromosome movement. Mol.
Biol. Cell. 22, 1217-1226.
10. Sundin, L.J., Guimaraes, G.J., DeLuca, J.G. (2011). The NDC80 complex proteins Nuf2 and
Hec1 make distinct contributions to kinetochore-microtubule attachment in mitosis. Mol.
Biol. Cell. 22, 759-768.
11. Cheeseman, I.M., Brew, C., Wolyniak, M., Desai, A., Anderson, S., Muster, N., Yates, J.R.,
Huffakar, T.C., Drubin, D.G., Barnes, G. (2001). Implication of a novel multiprotein Dam1p
complex in outer kinetochore function. J. Cell Biol. 155, 1137-1145.
12. Westermann, S., Wang, H.W., Avila-Sakar, A., Drubin, D.G., Nogales, E., Barnes, G. (2006). The
Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends.
Nature. 440, 565-569.
13. Miranda, J.J., King, D.S., Harrison, S.C. (2007). Protein arms in the kinetochore-microtubule
interface of the yeast DASH complex. Mol. Biol. Cell. 18,2503-2510.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
1
Gaitanos, T.N., Santamaria, A., Jeyaprakash, A.A., Wang, B.,
Conti, E., Nigg, E.A. (2009). Stable kinetochore-microtubule interactions depend on the Ska
complex and its new component Ska3/C13Orf3. EMBO J. 28, 1442-1452.
Theis, M., Slabicki, M., Junqueira, M., Paszkowski-Rogacz, M., Sontheimer, J., Kittler, R.,
Heniger, A.K., Glatter, T., Krussmaa, K., Poser, I., Hyman, A.A., Pisabarro, M.T., Gstaiger, M.,
Aebersold, R., Shevchenko, A., Buchholz, F. (2009). Comparative profiling identifies C13orf3as
a component of the Ska complex required for mammalian cell division. EMBO J. 28, 14531465.
Welburn, J.P., Grishchuk, E.L., Backer, C.B., Wilson-Kubalek, E.M., Yates, J.R. 3rd., Cheeseman,
I.M. (2009). The human kinetochore Ska1 complex facilitates microtubule depolymerisationcoupled motility. Dev. Cell. 16, 374-385.
Raaijmakers, J.A., Tanenbaum, M.E., Maia, A.F., Medema, R.H. (2009). RAMA1 is a novel
kinetochore protein involved in kinetochore-microtubule attachment. J. Cell Sci. 122, 24362445.
Chan, Y.W., Jeyaprakash, A.A., Nigg, E.A., Santamaria, A. (2012). Aurora B controls
kinetochore-microtubule attachments by inhibiting Ska complex-KMN network interaction. J.
Cell Biol. 196, 563-571.
Hanisch, A., Silljé, H.H., Niggs, E.A. (2006). Timely anaphase onset requires a novel spindle
and kinetochore complex comprising Ska1 and Ska2. EMBO J. 25, 5504-5515.
Jeyaprakash, A.A., Santamaria, A., Jayachandran, U., Chan, Y.W., Benda, C., Niggs, E.A., Conti,
E. (2012). Structural and functional organization of the Ska complex, a key component of the
kinetochore-microtubule interface. Mol. Cell. 46, 274-286.
Schmidt, J.C., Arthanari, H., Boeszoermenyi, A., Daskevich, N.M., Wilson-Kubalek, E.M.,
Monnier, N., Markus, M., Oberer, M., Milligan, R.A., Bathe, M., Wagner, G., Grishchuk, E.L.,
Cheeseman, I.M. (2012). The kinetochore-bound Ska1 complex tracks depolymerizing
microtubules and binds to curved protofilaments. Dev. Cell. Epub ahead of print.
Grancell, A., Sorger, P.K. (1998). Chromosome movement: kinetochores motor along. Curr.
Biol. 8, R382-R385.
McEwen, B.F., Chan, G.K., Zubrowski, B., Savoian, M.S., Sauer, M.T., Yen, T.J. (2001). CENP-E
is essential for reliable bioriented spindle attachment, but chromosomealignment can be
achieved via redundant mechanisms in mammalian cells. Mol. Biol. Cell. 12, 2776-2789.
Tanenbaum, M.E., Galjart, N., Van Vugt, M.A., Medema, R.H. (2006). CLIP-170 facilitates the
formation of kinetochore-microtubule attachments. EMBO J. 25, 45-57.
Joglekar, A.P., Bouck, D.C., Molk, J.N., Bloom, K.S., Salmon, E.D. (2006). Molecular
architecture of a kinetochore-microtubule attachment site. Nat. Cell Biol. 8, 581-585.
Alushin, G.M., Ramey, V.H., Pasqualato, S., Ball, D.A., Grigorieff, N., Musacchio, A., Nogales,
E. (2010). The Ndc80 kinetochore complex forms oligomeric arrays along microtubules.
Nature. 467, 805-810.
Tooley, J.G., Stukenberg, P.T. (2011). The Ndc80 complex: integrating the kinetochore’s many
movements. Chromosome Res. 19, 377-391.
28. Westermann, S., Avila-Sakar, A., Wang, H.W., Niederstrasser, H., Wong, J., Drubin, D.G.,
Nogales, E., Barnes, G. (2005). Formation of a dynamic kinetochore-microtubule interface
through assembly of the Dam1 ring complex. Mol. Cell. 17, 227-290.
29. Asbury, C.L., Gestaut, D.R., Powers, A.F., Franck, A.D., Davis, T.N. (2006). The Dam1
kinetochore harnesses microtubule dynamics to produce force and movement. Proc. Natl.
Acad. Sci. 103, 9873-9878.
30. Gestaut, D.R., Graczyk, B., Cooper, J., Widlund, P.O., Zelter, A., Wordeman, L., Asbury, C.L.,
Davis, T.N. (2008). Phosphoregulation and depolymerisation-driven movement of the Dam1
complex do not require ring formation. Nat. Cell Biol. 10, 407-414.
31. Grishchuk E.L., Spiridonov, I.S., Volkov, V.A., Efremov, A., Westermann, S., Drubin, D., Barnes,
G., Ataullakhanov, F.I., McIntosh, J.R. (2008). Different assemblies of the DAM1 complex
follow shortening microtubules by distinct mechanisms. Proc. Natl. Acad. Sci. 105, 69186923.
32. Gardner, M.K., Odde, D.J. (2008). Dam1 complexes go it alone on disassembling
microtubules. Nat. Cell Biol. 10, 379-381.
33. Maure, J.F., Komoto, S., Oku, Y., Mino, A., Pasqualato, S., Natsume, K., Clayton, L.,
Musacchio, A., Tanaka, T.U. (2011). The Ndc80 loop region facilitates formation of
kinetochore attachment to the dynamic microtubule plus end. Curr. Biol. 21, 207-213.
34. Tien, J.F., Umbreit, N.T., Gestaut, D.R., Franck, A.D., Cooper, J., Wordeman, L., Gonen, T.,
Asbury, C.L., Davis, T.N. (2010). Cooperation of the Dam1 and Ndc80 kinetochore complexes
enhances microtubule coupling and is regulated by aurora B. J. Cell Biol. 189, 713-723.
35. Zhang, G., Kelstrup, C.D., Hu, X.W., Kaas Hansen, M.J, Singleton, M.R., Olsen, J.V., Nilsson, J.
(2012). The Ndc80 internal loop is required for recruitment of the Ska complex to establish
end-on microtubule attachment to kinetochores. J. Cell Sci. 125, 3243-3253.
36. Demirel, P.B., Keyes, B.E., Chaterjee, M., Remington, C.E., Burke, D.J. (2012). A redundant
function for the N-terminal tail of Ndc80 in kinetochore-microtubule interaction in
Saccharomyces cerevisiae. Genetics. 192, 753-756.
37. DeLuca, J.G., Gall, W.E., Ciferri, C., Cimini, D., Musacchio, A., Salmon, E.D. (2006). Kinetochore
microtubule dynamics and attachment stability are regulated by Hec1. Cell. 127, 969-982.
38. Welburn, J.P., Vleugel, M., Liu, D., Yates, J.R. 3rd., Lampson, M.A., Fukagawa, T., Cheeseman,
I.M. (2010). Aurora B phosphorylates spatially distinct targets to differentially regulate the
kinetochore-microtubule interface. Mol. Cell. 38, 383-392.
39. Tanaka, T.U., Rachidi, N., Janke, C., Pereira, G., Galova, M., Schiebel, E., Stark, M.J., Nasmyth,
K. (2002). Evidence that the Ipl1-Sli5 (Aurora kinase-INCENP) complex promotes
chromosome bi-orientation by altering kinetochore-spindle pole connections. Cell. 108, 317329.
40. Cheeseman, I.M., Anderson, S., Jwa, M., Green, E.M., Kang, J.S., Yates, J.R. 3rd., Chan, C.S.,
Drubin, D.G., Barnes, G. (2002). Phospho-regulation of kinetochore-microtubule attachments
by the Aurora kinase Ipl1p. Cell. 111, 163-172.
41. Daum, J.R., Wren, J.D., Daniel, J.J., Sivakumar, S., McAvoy, J.N., Potapova, T.A., Gorbsky, G.J.
(2009). Ska3 is required for spindle checkpoint silencing and the maintenance of
chormosome cohesion in mitosis. Curr. Biol. 19, 1467-1472.