Chromosome congression and segregation require robust yet dynamic attachment of the kinetochore with the spindle microtubules. Force generated at the kinetochore–microtubule interface plays a vital role to drive the attachment, as it is required to move chromosomes and to provide signal to sense correct attachments. To understand the mechanisms underlying these processes, it is critical to describe how the force is generated and how the molecules at the kinetochore–microtubule interface are organized and assembled to withstand the force and respond to it. Research in the past few years or so has revealed interesting insights into the structural organization and architecture of kinetochore proteins that couple kinetochore attachment to the spindle microtubules. Interestingly, despite diversities in the molecular players and their modes of action, there appears to be architectural similarity of the kinetochore-coupling machines in lower to higher eukaryotes. The present review focuses on the most recent advances in understanding of the molecular and structural aspects of kinetochore–microtubule interaction based on the studies in yeast and vertebrate cells.

Introduction

Eukaryotic organisms duplicate and segregate their genetic materials in the form of chromosomes during each cell division, ensuring that their progeny inherits an exact copy of the parental genome. Prior to segregation, each pair of sister chromatids of chromosomes becomes tethered to kinetochore (KT), a supra-molecular protein complex assembled at the centromere, and then bi-orients to establish bipolar attachments with the kinetochore–microtubule (k-MT) plus ends [17]. Once the correct attachment is established, segregation is triggered, followed by movement of the segregated chromatids to the poles. A major decision-making step prior to activation of chromosome segregation is proper congression and alignment of the chromosomes to the metaphase plate. KT plays crucial roles in both these processes by establishing and maintaining stable attachment with the k-MT plus ends and also by providing signals to the cells to prevent cells to proceed to anaphase until all of the chromosomes are correctly congressed and aligned. Defective KT-MT attachment compromises fidelity of chromosome segregation leading to chromosomal instability and tumorigenesis.

MT plus ends are intrinsically dynamic in nature as these ends continually switch between polymerization and depolymerization. During MT polymerization, the GTP-bound tubulin subunits are incorporated into the growing MT plus ends. Eventually, the GTP in the tubulin hydrolyzes to GDP, and those GDP-bound subunits remain associated as a straight structure via intra- and inter-protofilament interactions [8,9]. When the plus ends switch to depolymerization, the GDP–tubulin dimers lose these stabilizing interactions, resulting in dissociation of the tubulin subunits in the form of curved protofilaments that peel back outward from the MT lattice. The polymerization–depolymerization dynamics of MTs is increased several fold when cells transit from interphase to mitosis involving actions of many MT-associating proteins. It is, therefore, a challenging task for the KT to maintain persistent attachment with the MTs and then to couple the chromosome movement to the dynamics of their plus ends. A functionally efficient coupling also requires critical balance between the strength of KT attachment and the dynamicity of the plus ends. Attachment must be strong enough to keep the KT tethered to the plus ends, but at the same time, it also has to allow addition and removal of tubulin subunits to and from the ends. How do KTs couple chromosome movement to the polymerization–depolymerization dynamics of MT ends? What are the factors that stabilize KT attachment to the depolymerizing ends where the MT protofilaments are continually dissociating? How do KTs differentiate a correct vs incorrect attachment and ensure the correct attachment? These fundamental questions have been challenging cell biologists for many years. An array of different approaches, namely, biochemical, ultra-resolution microscopic, genetic and cell biological, has helped, during the past few years, to partly answer these questions. In the present review, we have focused on the latest advances in the molecular details of KT-MT interaction, with a special emphasis on the mechanistic basis of KT-MT attachments based on the latest models.

Microtubule–kinetochore attachment: force is crucial

Bipolar spindle attachment of sister chromatids requires establishment of physical linkage of the KT with the MT plus ends [1,5,10]. Force plays a vital role in establishing and sustaining this linkage. Using force-calibrated micro-needles on grasshopper spermatocytes, it was demonstrated that the force needed to physically move a chromosome is ∼700 pN [11]. Mitotic spindle machinery must evolve a mechanism that can generate this force to move each single chromosome. During depolymerization, each single depolymerizing protofilament generates ∼5–6 pN of force. Considering 13 protofilaments in an MT lattice, a single depolymerizing MT can generate force up to ∼65 pN [12,13]. During polymerization, an opposite polymerization-driven force is also likely to generate [14]. It is thought that the force generated by MT polymerization and depolymerization is utilized by the KT to drive chromosome movement. A critical question is how KT harnesses this force to generate chromosome motion.

Kinetochore: structure and composition

KT is a highly complex yet organized assembly of supramolecular protein complexes at the centromere. The simplest KT, identified in budding yeast (Saccharomyces cerevisiae), is made of ∼38 core structural proteins associated with a 125-bp long centromere [1517]. Each KT is attached to a single MT in this organism. In higher eukaryotes, the complexities of KT structure and composition, and the number of MTs attached per KT, are increased several fold. For example, each single human KT is consists of ∼150–200 proteins that wrap around a large centromeric DNA molecule 10 Mb long and it can attach to ∼15–20 MTs [1821]. Negatively stained electron microscopy (EM) of vertebrate chromosomes has shown that the KT is assembled in three apparently distinguishable layers (Figure 1) [2224]. While the inner layer forms the interaction interface of centromeric chromatin, the outer layer mainly provides the interface for k-MT binding. The inner and the outer layers are separated by a relatively less dense layer, called the central layer. A large network of interactions, with specific hierarchy between the proteins within the individual layers as well as across the layers, is crucially involved in the assembly of a functional KT. A major structural framework to assemble KT in these different layers is provided by an evolutionarily conserved and structurally unique group of proteins, called constitutive centromere-associated network (CCAN) proteins [17,25,26]. CCAN proteins, on one hand, constitute the integral structural part of the inner KT architecture; on the other hand, they orchestrate recruitment of the key protein complexes to assemble the outer layer [18,27,28]. The outer layer is mainly involved in establishing the end-on attachment with the k-MT plus ends. During this process, this layer also dynamically reorganizes itself [3,29]. Although the molecular mechanism of k-MT end-on attachment to the outer KT and the structural organization of the proteins at the site are yet to be fully elucidated, research during past decade or so has identified several key proteins at this site that are crucial for the end-on attachment. One such group of proteins is the KMN network complex proteins. KMN is a nine-subunit mega-complex comprising three subcomplexes, KNL1, Mis12 and Ndc80 [1,7,29]. These multi-subunit complexes constitute the core part of the outer KT and establish direct linkage between the KT and k-MTs [30,31]. Almost all of the components of the KMN complexes are conserved across eukaryotes (Figure 2) [3234]

Schematic view of chromosome–microtubule attachment interface.

Figure 1.
Schematic view of chromosome–microtubule attachment interface.

(A) Organization of the mitotic spindles and the chromosomes attached to the kinetochore microtubules (K-fibers). (B) Representation of the kinetochore holding the sister chromatids and its attachment with the microtubules on both sides. (C) Schematic representation of three layers of the kinetochore: inner, central and outer layers. (D) Schematic representation of the components of the protein complex, KMN (KNL1, Ndc80 and Mis12), at the outer kinetochore layer that interacts with the microtubule plus end.

Figure 1.
Schematic view of chromosome–microtubule attachment interface.

(A) Organization of the mitotic spindles and the chromosomes attached to the kinetochore microtubules (K-fibers). (B) Representation of the kinetochore holding the sister chromatids and its attachment with the microtubules on both sides. (C) Schematic representation of three layers of the kinetochore: inner, central and outer layers. (D) Schematic representation of the components of the protein complex, KMN (KNL1, Ndc80 and Mis12), at the outer kinetochore layer that interacts with the microtubule plus end.

Structural and functional domains/motifs of human Ndc80 complex proteins, Mis12 complex proteins, Ska complex proteins and EB1 and their sequence conservations.

Figure 2.
Structural and functional domains/motifs of human Ndc80 complex proteins, Mis12 complex proteins, Ska complex proteins and EB1 and their sequence conservations.

Distributions of α-helices and β-strands were predicted using JPRED, and coiled-coil domains using COILS in congression with the PDB database. The functional domains identified as major interaction sites are specified. Enzymatic/phosphorylation sites are also shown. The sequence conservation plots of the human proteins with respect to their homologs/orthologs in other eukaryotic species based on multiple sequence alignments (CLUSTAL OMEGA) are shown at the top. Bars with lighter yellow coloration refer to the highest conservation and bars with darker colors denote decreasing conservation. Accession numbers of the proteins analysed are as follows: Hec1 [NP_006092.1 (human), NP_001252831.1 (monkey), NP_075783.2 (mouse), NP_001003863.1 (zebrafish)]; Nuf2 [NP_663735.2 (human), XP_011758637.1 (monkey), NP_956604.1 (zebrafish)]; Spc24 [NP_872319.1 (human), XP_001104117.1 (monkey), NP_080558.1 (mouse), XP_017209818.1 (zebrafish)]; Spc25 [CAG33478.1 (human), XP_011742374.1, NP_001292729.1 (mouse), NP_001116529.1 (zebrafish)]; Nnf1 [NP_001186583.1 (human), XP_014967494.1 (monkey), NP_080204.1 (mouse), XP_692676.2 (zebrafish)]; Mis12 [CAG38491.1 (human), NP_001181118.1 (monkey), NP_080269.1 (mouse), NP_956672.1 (zebrafish)]; Dsn1 [NP_079194.3 (human), NP_001245047.1 (monkey), NP_080129.2 (mouse), XP_001923648.1 (zebrafish)]; Nsl1 [sp|Q96IY1.3| (human), XP_001107619.1 (monkey), NP_941056.3 (mouse), NP_001020692.1 (zebrafish)]; Ska1 [NP_001034624.1 (human), NP_001181746.1 (monkey), NP_079857.3 (mouse), NP_001002592.1 (zebrafish)]; Ska2 [NP_872426.1 (human), NP_001252983.1 (monkey), NP_079653.1 (mouse), NP_001186604.1 (zebrafish)]; Ska3 [NP_659498.4 (human), NP_001248159.1 (monkey), NP_941007.1 (mouse), NP_001189366.1 (zebrafish)]; and EB1 [NP_036457.1 (human), NP_001253729.1 (monkey), NP_031922.1 (mouse), NP_998805.1 (zebrafish)].

Figure 2.
Structural and functional domains/motifs of human Ndc80 complex proteins, Mis12 complex proteins, Ska complex proteins and EB1 and their sequence conservations.

Distributions of α-helices and β-strands were predicted using JPRED, and coiled-coil domains using COILS in congression with the PDB database. The functional domains identified as major interaction sites are specified. Enzymatic/phosphorylation sites are also shown. The sequence conservation plots of the human proteins with respect to their homologs/orthologs in other eukaryotic species based on multiple sequence alignments (CLUSTAL OMEGA) are shown at the top. Bars with lighter yellow coloration refer to the highest conservation and bars with darker colors denote decreasing conservation. Accession numbers of the proteins analysed are as follows: Hec1 [NP_006092.1 (human), NP_001252831.1 (monkey), NP_075783.2 (mouse), NP_001003863.1 (zebrafish)]; Nuf2 [NP_663735.2 (human), XP_011758637.1 (monkey), NP_956604.1 (zebrafish)]; Spc24 [NP_872319.1 (human), XP_001104117.1 (monkey), NP_080558.1 (mouse), XP_017209818.1 (zebrafish)]; Spc25 [CAG33478.1 (human), XP_011742374.1, NP_001292729.1 (mouse), NP_001116529.1 (zebrafish)]; Nnf1 [NP_001186583.1 (human), XP_014967494.1 (monkey), NP_080204.1 (mouse), XP_692676.2 (zebrafish)]; Mis12 [CAG38491.1 (human), NP_001181118.1 (monkey), NP_080269.1 (mouse), NP_956672.1 (zebrafish)]; Dsn1 [NP_079194.3 (human), NP_001245047.1 (monkey), NP_080129.2 (mouse), XP_001923648.1 (zebrafish)]; Nsl1 [sp|Q96IY1.3| (human), XP_001107619.1 (monkey), NP_941056.3 (mouse), NP_001020692.1 (zebrafish)]; Ska1 [NP_001034624.1 (human), NP_001181746.1 (monkey), NP_079857.3 (mouse), NP_001002592.1 (zebrafish)]; Ska2 [NP_872426.1 (human), NP_001252983.1 (monkey), NP_079653.1 (mouse), NP_001186604.1 (zebrafish)]; Ska3 [NP_659498.4 (human), NP_001248159.1 (monkey), NP_941007.1 (mouse), NP_001189366.1 (zebrafish)]; and EB1 [NP_036457.1 (human), NP_001253729.1 (monkey), NP_031922.1 (mouse), NP_998805.1 (zebrafish)].

Structural identity of KT-MT interface: KT-fiber and KT-fibril models

Electron tomography analyses of high pressure frozen kinetochores from cultured PtK1 cells revealed that the outer KT plate forms long fibers oriented in the plane of the plate in the absence of MT attachments [29]. Interestingly, when bound to MTs, a more shrub-like network of short fibers is formed. The fibers appear to link to MTs radially to the plus end tip of MTs orienting in the plane of the outer plate and also extend towards the distal part of the k-MTs from the outer plate by binding along the sides of MTs. It is believed that upon MT binding, the KT outer plate proteins make additional fibers to entangle the k-MTs to extra-stabilize KT-MT attachment. Although it is technically challenging, comparison of these structures with the EM data of reconstituted proteins bound to MTs largely supported that KMN complex proteins are the major players in the formation of these fibers and also helped to identify the specific outer KT components that form these fibers [7,30,3538]. The fibers are believed to use the MT lattice as an array of binding sites to generate the MT disassembly-coupled force. According to this model, KT-MT attachment is stabilized by multiple weak linkages that are spread over a stretch on the MT lattice behind the plus end. Since the individual interactions are weak in nature, the linkages constantly break and reform under normal thermal fluctuations and such processes allow the attachment site to diffuse along the MT lattice. During MT depolymerization, loss of tubulin dimers creates a kind of moving boundary near these attachment sites and biases the diffusion towards the minus end of the MTs. The flared arrangements of the MT protofilaments during depolymerization are thought to further facilitate the diffusion, because the attachment of the KT proteins is pushed further away from the plus end due to such conformational strain. In this model, however, the energy of individual interactions and the number of linkages are critical for effective force generation and maintaining persistent attachment with the k-MTs. Both of these parameters should depend on the types of molecules on the MT lattice that the fibers interact with, because the lattice is likely to be bound with various MT-associated proteins. The fiber model, however, does not explain this aspect. Interestingly, EM analysis of KTs of PtK1 cells in another study with a slightly modified imaging approach showed the presence of fibril structures, termed KT-fibrils, rather than fibers [39]. In that study, the morphology of the plus end of individual k-MT protofilaments was obtained by ultrathin sectioning. Averaging over many such protofilaments revealed the presence of fibril-like structures that bind to the inner layer of the curved MT protofilaments, i.e. the lumen-facing surface of the MT protofilaments. It was also observed that the curvature of the protofilaments in the k-MTs undergoing depolymerization is relatively less than the same displayed by the protofilaments of unattached MTs, suggesting that the strain energy in the MT lattice during depolymerization is less relaxed in k-MTs than in other free-end MTs. It was proposed that binding of the fibrils to the inner lumen of each of the curved k-MT protofilaments can covert the strain into a minus-end-directed force and couple KT movement. However, the effective force generation by this mechanism would depend on the ability of the fibrils to precisely confine to the curling k-MT protofilaments, synchronization of multiple fibril–protofilament attachments and regulation of break and re-establishment of the attachments. None of these are clearly understood.

Ndc80 complex and its interaction with MTs

The major component of KMN that is predominantly and more directly involved in k-MT attachment is Ndc80 [30,4044]. It is a tetrameric complex of four proteins, Ndc80 (also known as Hec1 in human), Nuf2, Spc24 and Spc25 [30,40,45]. All four components are highly conserved across eukaryotes [32]. The complex is organized as a rod-like structure with two dumb-bell-like heads at opposite sides. One pair of heads formed by the N-terminal globular domains of Hec1 and Nuf2 constitute the interface suitable for MT binding, whereas the C-terminal globular domains of Spc24 and Spc25 form the heads at the other side that interact with the inner KT components. The two globular ends of the complex are connected by a central shaft of long coiled-coil domains contributed by all of the four Ndc80 components (Figure 1). The N-termini of Hec1 and Nuf2 can fold into a calponin homology (CH) domain. CH domains generally serve as binding interfaces for many microtubule- and actin-binding proteins [4648]. The MT-binding ability of Ndc80 complex has been studied extensively in recent years. Both the CH domain and its 80-residue N-terminal unstructured tail of Hec1 (human Ndc80) are the binding sites for MTs [27,30,40,4951]. Cryo-electron reconstitution analysis of MTs decorated with Ndc80 complex showed that the complex binds to the tubulin subunits both at the intra- and inter-dimer interface [36]. The purified complex also tends to cluster on the MT lattice, forming heterogeneous fibril-like arrangements, similar to those observed in the case of isolated mammalian KTs [52]. Fitting the crystal structures of the functional unit of Ndc80 complex and αβ-tubulin dimers revealed the presence of a unique ‘toe’-like print of the Hec1 MT-interacting site [27,53,54]. Experiments with Ndc80 complex-coated beads along the reconstituted MTs have shown that the complex has the ability to track on the depolymerizing MTs. Ndc80 exhibits a diffusion-based movement along the MT lattice following MT depolymerization. As Ndc80 complex can form fiber structures similar to the outer-KT fibers observed in KTs of mammalian cells [24,29], it has been proposed that many Ndc80 complex units can mediate multiple weak interactions in the form of fibers, which can induce a biased diffusion of the KT on the MT.

Although Ndc80 plays an important role in coupling KT to depolymerizing MTs, it alone does not seem to be sufficient for a number of reasons. First of all, the results of in vitro load-bearing experiments indicated that Ndc80 can sustain its MT attachment and MT depolymerization-driven movement against an opposing force of up to 3 pN [55,56]. This is much less than the force of a single MT depolymerization [12,13]. Therefore, even for a single-MT–single-KT attachment, as in the case of budding yeast, the Ndc80 attachment is less likely to withstand the force of MT depolymerization. Secondly, Ndc80 attachment to the MTs is predominantly favored to straight MT protofilaments; as the depolymerizing plus end primarily consists of curved protofilaments, force coupling to the curved protofilaments by Ndc80 is less likely to be favored. Furthermore, purified Ndc80/Hec1 binds poorly to the MTs with minimal residence time on the MT lattice [5658]. Although Ndc80 diffusion along the MT lengths away from the depolymerizing end can, in principle, harness some force, the rate of diffusion is much slower than the MT depolymerization rate [59]. Therefore, KT coupling via Ndc80 diffusion is also likely to be less effective.

Dam1 complex: stabilizer of KT-MT attachment in budding yeast

In budding yeast, a multi-protein complex, Dam1/DASH, has emerged as a critical regulator of KT-MT attachment. Dam1 is a hetero-decamer complex and is composed of ten different outer KT proteins. The initially identified ones are Duo1p and Dam1p, which exist in a complex and contribute to spindle function that is sensed by the spindle assembly checkpoint machinery [60]. Related studies by Winey and co-workers demonstrated a major functional role of Dam1p in spindle microtubule integrity in yeast [61]. Later, four new components of the Duo1p–Dam1p complex were shown to exert novel functions in sister chromatid bi-orientation [62]. Subsequent studies by yeast two-hybrid screens and affinity purification led to identification of other components of the complex, resulting in the number of subunits of the complex totalling ten. The Dam1 complex is essential for end-on attachment of KTs in yeast and, furthermore, it is targeted by the yeast Aurora kinase lpl1 during KT-MT attachment [59,6266]. Dam1 also regulates mitotic spindle length as it increases MT rescue and suppresses catastrophe [67]. Force-coupling experiments with Dam1 complex have shown that it acts as a highly efficient coupler to strain energy into work generating ∼40–60 pN of force per MT [13,68]. Detailed analyses in these studies have further demonstrated that Dam1 interacts with and recruits Ndc80 to the plus ends by increasing its residence time on the MTs and stabilizing Ndc80 binding to the MTs [58,6971]. These results suggest that the Dam1–Ndc80 interaction could play a synergistic role in force harnessing and chromosome coupling to the depolymerizing MTs.

KT-MT attachment via Dam1 complex oligomers: the ring model

EM analyses of reconstituted MTs bound with purified Dam1 complex proteins have shown that the Dam1 complex can form ring structures around the microtubules (Figure 3) [66,7274]. Each ring consists of between 16 and 23 Dam1 complex units and is assembled on the MTs through interaction with the underlying tubulin subunits. The rings also impart stabilization to the MT lattice [66,75]. Although assembly of the rings requires MT surface, they appear to have a much wider diameter (50 nm) compared with the average diameter (∼25 nm) of the MTs [72,73], suggesting involvement of more unknown factors. More interestingly, Dam1 complex rings can move or slide along the MT lattice as the MT ends depolymerize [66,76], suggesting that interaction of the ring components with the MTs are dynamic in nature in order to allow ring mobility. EM analyses of purified KT particles of S. cerevisiae bound to taxol-stabilized MTs have evidenced formation of rod-like extended structures connecting the KT particles with the Dam1 rings. Specifically, the Ndc80 subcomplexes of KMN make additional extensions to contact the Dam1 rings [70]. Taken together, these observations led to the proposal that MT depolymerization force can be harnessed more efficiently by the extended Dam1 ring–Ndc80 linkages and the ability of the Dam1 rings to move, and can facilitate persistent attachment of the KTs during MT depolymerization. Although the presence of Dam1 rings in vivo is yet to be established, the findings from the reconstituted systems have put forward an attractive possibility that Dam1 complex rings can couple KT movement to the depolymerizing MTs by providing a stable yet mobile support to stabilize Ndc80 attachment to the MTs [66,72]. Surprisingly, no homologs of any components of the Dam1 complex are found in higher eukaryotes. This alludes to the probable contribution of other factors in establishing functional KT-MT linkage in higher organisms.

Schematic view of the oligomeric ring structures formed by yeast Dam1 complex vs human EB1–Ska complex on the microtubules.

Figure 3.
Schematic view of the oligomeric ring structures formed by yeast Dam1 complex vs human EB1–Ska complex on the microtubules.

The diameter of the Dam1 rings is much wider than the microtubules with the possibility of allowing the rings to slide along the microtubule lengths; whereas the EB1–Ska ring structures appear as microtubule-bound structures that closely wrap the microtubule lattice.

Figure 3.
Schematic view of the oligomeric ring structures formed by yeast Dam1 complex vs human EB1–Ska complex on the microtubules.

The diameter of the Dam1 rings is much wider than the microtubules with the possibility of allowing the rings to slide along the microtubule lengths; whereas the EB1–Ska ring structures appear as microtubule-bound structures that closely wrap the microtubule lattice.

Ska complex: stabilizer of KT-MT attachment in higher eukaryotes

Studies in humans and Caenorhabditis elegans have identified a protein complex called spindle and kinetochore associated (Ska), that plays essential roles in stabilizing KT-MT attachment. Ska is composed of three components, Ska1, Ska2 and Ska3, that localize to the spindle microtubules and are recruited to the kinetochore. Ska1 was first identified by mass-spectrometry-based proteomics screening [77], and later, Ska2 [78] and Ska3 [79,80] were identified as the binding partners of Ska1. Ska is present in all vertebrates, but not in fungi and Drosophila, although it is present in other insects and also in C. elegans (Figure 2). Ska plays an essential role in establishing KT-spindle MT linkage during mitosis in human cells. Ska depletion results in severe KT-MT attachment defects and unstable K-fibers [7782]. Subsequent studies have shown that abrogation of MT binding to only Ska1 is enough to induce these defects [83,84].

The Ska complex can directly bind to MTs [82], specifically, the C-terminus (residues 133–255 in human) of Ska1 [85]. Ska1 consists of a conserved coiled-coil domain in its N-terminus and an unstructured linker region that connects the N- and the C-terminus. Ska1 devoid of the C-terminal MT-binding domain localizes poorly to the spindle MTs, but it can still be recruited to the KT [86], implicating that the N-terminal coiled-coil and the flexible linker regions have additional roles in KT regulation independent of the C-terminus and its MT-binding activity. Ska can also mediate interaction with Ndc80. Although a direct interaction could not be detected in solution, both of the protein complexes exhibit a synergy in interacting with MTs in vitro, indicating that one stabilizes the MT-binding of the other [78,82,83]. Detailed molecular studies have further shown that Ska is recruited to the KT through the loop region of Ndc80 located in its central coiled-coil domain [87]. In vitro motility assays with microsphere beads coated with recombinant Ska complex on the reconstituted MTs have demonstrated that Ska can processively move the microspheres along the depolymerizing MT end [82]. This has been shown both with human and with C. elegans Ska proteins, supporting it as a conserved property. These observations also led to the proposal that Ska may play a functionally similar role to the Dam1 complex in the coupling of KTs to the depolymerizing K-fibers in vertebrate cells. However, electron tomography revealed that Ska does not form rings on MTs like the Dam1 complex; rather it forms oligomeric hair-like extensions on the MT surface [83].

MT-binding experiments demonstrated that Ska complex cannot persistently maintain its attachment with the plus end, rather its exhibits a cooperative microtubule-binding behavior like Ndc80, and it also diffuses along the MT surface [82,83]. Although the rate of Ska diffusion appears to be 2–3 folds higher than Ndc80, it is not clear how efficiently Ska-diffusion is synchronized with plus end depolymerization. EM analysis also evidenced that Ska induces more curvature to the depolymerizing protofilaments [83]. Such an action is likely to further destabilize the depolymerizing end and, hence, disfavor KT attachment. Stabilization of MT lattice proximal to the depolymerizing end is likely to be necessary for maintaining a persistent and stable Ska attachment to the MTs. In a recent study, we have demonstrated that the MT plus end tracking protein (+TIP) end binding 1 (EB1) plays a critical role in this process [88].

+TIPs

+TIPs are a structurally and functionally diverse group of proteins that selectively localize to and track the growing plus ends of microtubules. +TIPs are implicated in controlling almost all MT-driven cellular processes. These proteins can bind to the plus ends either autonomously or through accessory proteins that facilitate their localization by hitchhiking them to the plus ends [89,90]. EB family proteins (EB1, EB2 and EB3) are the extensively studied +TIPs that can autonomously track the plus ends and master-control recruitment of several other +TIPs to the ends. Among the EB family members, EB1 is a highly conserved (Figure 2) and the most ubiquitously expressed +TIP in eukaryotes.

EB1 regulates Ska-mediated KT-MT attachment

Numerous studies have evidenced crucial roles of EB1 in mitotic spindles and chromosome regulation during mitosis [9196]. It regulates microtubule dynamics and modulates plus end structure [89,97100]. Down-regulation or depletion of EB1 induces chromosome congressional defects and mis-segregation in cultured mammalian cells [94,101103]. Antibody-based inhibition of EB1 leads to prolonged mitosis with defects in mitotic spindle symmetry [104]. EB1 also influences activities of Aurora B kinase and PP2A phosphatase, which play crucial roles in error correction in KT-MT attachments [105,106]. EB1 has been shown to associate with the KTs via its attachment to the plus ends of mitotic spindles [98,105]. EB1 is essential for plus end localization of TIP150, SKAP, CLIP170, and chTOG and modulates their functions in mitotic spindle assembly and/or KT-MT attachments (Table 1). Although all of these studies point towards a regulatory function of EB1 in MT-chromosome interaction, a direct role of EB1 in KT-MT attachment and the molecular details of its interactions at the KT-MT interface were not demonstrated until recently [88]. We have shown that EB1 regulates Ska complex recruitment to the KT in cultured human cells [88]. EB1 depletion disrupts localization of Ska complex components at the KT, resulting in characteristic chromosome congression defects [88]. The majority of the chromosomes in the EB1-depleted cells appear to be dispersed or loosely congressed around the metaphase plate, while a fraction of them fail to align to the plate [94,104,107]. This phenotype, though not exactly identical, bears significant similarity with the Ska1-depletion phenotypes reported in earlier studies [82,83]. Interestingly, EB1 and Ska1 co-depletion results in complete loss of chromosome congression, reflecting a massive loss of KT-MT attachment. This suggests that EB1 and Ska complex together have a crucial role in KT-MT attachment. Detailed biochemical data have shown that EB1 stabilizes Ska attachment to the MTs at the KT through a direct and robust interaction with Ska1 [88].

Table 1
Functions of EB1-interacting +TIPs in mitosis
Protein Localization Role in mitosis Interaction motifs binding to EB1 References 
APC MT +end, kinetochore, centrosome Spindle organization, and positioning, astral MT attachment, chromatin compaction, KT capture, cell polarity SXIP [94,102,108111
CLIP 170 MT +end, kinetochore, centrosome Chromosome poleward movement, KT-MT attachment, recruiting PLK1 to KT CAP-Gly, SXIP [112,113
P150glued MT +end, centrosome Anaphase astral MT elongation, stimulation of cytokinesis CAP-Gly, SXIP [96
CLASP1/2 MT +end, centrosome Astral elongation, anaphase MT force generation, chromosome alignment, tubulin subunit addition at KT SXIP [114117
MCAK MT +end, centrosome Spindle formation, KT orientation, end-on attachment of KT, K-fiber depolymerization SXIP [118120
TIP150 MT +end, kinetochore, centrosome Chromosome bi-orientation, KT-MT attachment SXIP [121,122
CDK5RAP2 MT +end, centrosome Spindle orientation, centriole engagement, centrosomal MT assembly SXIP [123,124
DDA3 MT +end, kinetochore, centrosome KT-MT attachment, chromosome movement SXIP [125127
Ch-TOG MT +end, kinetochore, centrosome Sensing tension of KT-MT attachment, spindle assembly SXIP [128,129
LIS1 MT +end, centrosome Spindle organization, astral MT attachment, KT-MT attachment SXIP [130,131
Protein Localization Role in mitosis Interaction motifs binding to EB1 References 
APC MT +end, kinetochore, centrosome Spindle organization, and positioning, astral MT attachment, chromatin compaction, KT capture, cell polarity SXIP [94,102,108111
CLIP 170 MT +end, kinetochore, centrosome Chromosome poleward movement, KT-MT attachment, recruiting PLK1 to KT CAP-Gly, SXIP [112,113
P150glued MT +end, centrosome Anaphase astral MT elongation, stimulation of cytokinesis CAP-Gly, SXIP [96
CLASP1/2 MT +end, centrosome Astral elongation, anaphase MT force generation, chromosome alignment, tubulin subunit addition at KT SXIP [114117
MCAK MT +end, centrosome Spindle formation, KT orientation, end-on attachment of KT, K-fiber depolymerization SXIP [118120
TIP150 MT +end, kinetochore, centrosome Chromosome bi-orientation, KT-MT attachment SXIP [121,122
CDK5RAP2 MT +end, centrosome Spindle orientation, centriole engagement, centrosomal MT assembly SXIP [123,124
DDA3 MT +end, kinetochore, centrosome KT-MT attachment, chromosome movement SXIP [125127
Ch-TOG MT +end, kinetochore, centrosome Sensing tension of KT-MT attachment, spindle assembly SXIP [128,129
LIS1 MT +end, centrosome Spindle organization, astral MT attachment, KT-MT attachment SXIP [130,131

Role of EB1–Ska interaction on MT stabilization

Biochemical analyses with purified reconstituted proteins have revealed that EB1 and Ska1 or Ska complex (Ska1, Ska2 and Ska3 together) in solution, form large oligomers and further, the complex imparts stabilization on the MTs against depolymerization [88]. Although Ska complex alone can also stabilize depolymerization-induced MTs, the effect is enhanced by ∼2 fold in the presence of EB1–Ska1 complex than Ska1 or Ska complex alone, suggesting that Ska attachment to the MTs is potentiated by its prior binding to EB1. This has also been supported by MT-binding results, which show increased Ska1 association with the MTs in the presence of EB1. Together, these results support that EB1 acts as a stabilizing factor for the Ska complex attachment with the MTs. It is yet to be understood though whether the EB1-mediated Ska stabilization on to MTs is specific to the depolymerizing curved protofilaments or to the straight protofilaments near the depolymerizing end or both.

Enzymatic activities in KT-MT attachment

Enzymatic activities such as kinase and phosphatase activities have been shown to play crucial roles in the regulation of KT-MT interactions and fidelity of chromosome segregation. The enzymes modulate the affinity of KT-MT attachments by targeting multiple substrates at centromere, KT and k-MTs. Tension generated at the sister kinetochores plays a key role in this modulation as the enzymatic activities at the KT are activated differently depending on the amount of tensions imparted to the sister kinetochores, which determine the closeness between the enzymes and the substrates. One key regulatory kinase in this process is Aurora B, a conserved serine/threonine kinase [132]. Aurora B (Ipl1 in budding yeast) is highly enriched at the inner centromere in the form of chromosomal passenger complex (CPC), which also consists of three other components, Borealin, INCEP and Survivin [133]. The functionally active form of Aurora B has also been found to be associated with the spindle MTs [105,134,135]. A primary function of Aurora B is to destabilize incorrect KT-MT attachments or improper KT-MT interactions, so that cells get a fresh chance to re-establish the correct attachments. Essentially, it imparts a negative effect on the MT-binding activity of the KT by phosphorylating several key proteins at the KT-MT interface. Major targets of Aurora B at the outer KT are KMN network complex proteins. Phosphorylation of Ndc80 and KNL1 inhibit their MT-binding abilities, presumably by creating negative charges that prevent their interactions with the negatively charged MT surface [27,30,40,42,43,136]. Phosphorylation of the Mis12 complex component Dsn1 also affects KT-MT attachment by modulating the MT-binding conformations of Ndc80 and KNL1 [137]. Aurora B-mediated phosphorylation of KMN substrates correlates linearly with the KT-MT attachment errors. When a proper bi-orientation of the spindles and the correct KT-MT attachment are established, the sister KTs are stretched towards the spindle poles, keeping Aurora B farthest from the KMN network. In contrast, when there is a wrong KT-MT attachment or the bi-orientation is not achieved, not enough tension is generated to stretch the sister KTs and, therefore, centromeric Aurora B is moved closer to the KMN network, resulting in their phosphorylation and destabilization of the attachment. This allows a further chance to re-establish the correct attachment [136,138]. To re-establish the correct attachment and bi-orientation, a counteracting mechanism is involved to ensure dephosphorylation of the KMN proteins and favor KT-MT re-attachment. Although many more details are yet to be known in this pathway, it has been shown that KNL1 phosphorylation by Aurora B negatively regulates binding of KNL1 to protein phosphatase 1 (PP1), which dephosphorylates outer kinetochore proteins including KMN [139]. Phosphorylation and dephosphorylation at multiple sites of KMN located at different distances from the inner centromere play crucial roles in fine-tuning KT-MT attachment and ensuring fidelity of the attachment.

Aurora B also targets several other key proteins at the KT-MT interface, e.g. the Dam1 complex and the Ska complex proteins. Dam1 phosphorylation by Aurora B (Ip11) in yeast has been shown to play a role in the establishment of bi-orientation, presumably by destabilizing Dam1 interaction with Ndc80 [59,140,141]. In human cells, Aurora B phosphorylates Ska1 and Ska3, which negatively regulates interaction between the Ska complex and the KMN network [142]. However, it is yet to be fully understood how Dam1 or Ska phosphorylation affects KT-MT attachment stability. Interestingly, a recent study has shown that Ska complex promotes Aurora B activity and such an action is required to limit Ska association with the MTs and KT thereby maintaining k-MT stability and dynamics optimum [143]. Another earlier study showed that Aurora B activity is enhanced in the presence of EB1 on the spindle MTs [105]. Together, these findings indicate a possibility that EB1–Ska interaction could play a role in fine-tuning Aurora B activity at the KT-MT interface. There are also other spindle-associated substrates of Aurora B that directly regulate MT assembly–disassembly dynamics. The kinesin-13 family protein MCAK (Kif2c), which is a microtubule depolymerizer, is phosphorylated by Aurora B, and the MT depolymerization at the kinetochore is negatively regulated by this phosphorylation [144,145]. There is another line of thought that selective correction of KT-MT mal-attachment may not always be necessary, but the stochastic KT-MT attachment and detachment caused by MT dynamics can facilitate bi-orientation. In that aspect, Aurora B has been shown to control MT dynamics by regulating Kif2b and MCAK localization at the plus ends [146].

Beside Aurora B, there are also other kinases that regulate KT-MT attachment. Polo-like kinase 1 (Plk1) phosphorylates several KT components and stabilizes KT-MT attachment [147]. Plk1 and Aurora B activity also appear to be regulated parallelly. Another kinase, Nek2A, stabilizes KT-MT attachment by phosphorylating Ndc80 [148]. Furthermore, it is now understood that not just phosphorylation, but the dynamic balance between phosphorylation and dephosphorylation of the substrates, is crucial for KT-MT attachment and bi-orientation. Two phosphatases, PP1 and PP2, are mainly known to play crucial roles in this pathway. While PP2 counteracts with Aurora B and Plk1-mediated substrate phosphorylation on the unattached KT, thereby stabilizing initial KT-MT interaction [149], PP1 is involved in facilitating bi-orientation by removing Aurora B phosphorylation of its substrates involved in KT-MT attachment stability [139,150].

Structure of EB1–Ska oligomer: MT-binding ring model

Ultrastructural analyses by atomic force microscopy and negatively stained EM have shown that the EB1–Ska complex can decorate MTs with extended structures that appear as rings that encircle the MT lattice (Figure 3). These ring structures appear at regular intervals and wrap around the MTs nearly orthogonally to the MT axis. The inner sides (lumens) of the ring-like structures are so closely associated with the MT lattice that they can be considered more as MT-binding structures rather than movable rings like the Dam1 rings [88]. Ska complex alone, however, does not form similar structures; rather it forms hair-like fibrils, termed ‘meshwork’, on the MTs [82,85,88]. Interestingly, EB1 when complexed with Ska1 alone can also form ring-like MT-binding structures like EB1–Ska, although the EB1–Ska rings are more regular with larger thickness and more precise repeatability on the MTs than the EB1–Ska1 rings. This indicates additional roles of Ska2 and Ska3 in modulating the structures. EB1 alone does not form any such ordered structures, rather it forms discrete globular patches on the MT surface. It has been postulated that those patches can act as nucleating centers to recruit Ska proteins to the MT surface and transform the fibril-like Ska structures to ring-like MT-binding structures. The increased MT-stabilization and formation of MT-wrapped ring structures by the EB1–Ska complex suggest that the inter-protofilament association during MT depolymerization could be stabilized more efficiently by these extended structures.

EB1–Ska ring-like MT-binding structures vs Dam1 rings

The yeast Dam1–DASH complex stabilizes MTs and promotes MT polymerization [72]. Similar to Dam1, EB1–Ska1/Ska complex stimulates MT polymerization, stabilizes MTs against depolymerization, and induces MT bundling [88]. Like the Dam1 oligomers, structures formed by either EB1–Ska1 or EB1–Ska complex appear to encircle the MT lattice and organize orthogonally to the MT axis. Although it is tempting to suggest from these observations that EB1–Ska structures could play functionally analogous roles in vertebrates to Dam1 rings in yeast, attention needs to be paid to some distinctive differences between the EB1–Ska rings and the Dam1 rings. The Dam1 rings are wider in diameter (∼50 nm) and they do not seem to wrap so closely on the MT lattice like the EB1–Ska rings (diameter ∼40 nm) (Figure 3). Dam1 rings have been shown to be capable of sliding along the MTs in vitro [66,76]. Their ability to slide along the MTs has been proposed as a favorable mechanism to couple KT movements during MT depolymerization. In principle, the sliding can be favorable, as the Dam1 rings are much wider in diameter than MTs (∼25–30 nm). However, the Dam1 ring components also mediate interactions with MTs and stabilize MT lattice [72]. Therefore, a critical balance between the attachment and sliding could probably be important for efficient KT coupling by the Dam1 rings. EB1–Ska ring-like structures, on the contrary, wrap around the MTs so closely to the MT lattice that they appear as MT-bound structures. It is less feasible to conceive that these structures would move or slide along the MT lengths. A more favorable mechanism that would favor KT coupling to the depolymerizing MTs by these structures could be that the structures can disassemble and reassemble at or near the plus end following depolymerization (Figure 4). EB1 can provide a platform to anchor Ska complex to the plus end and move together with Ska in the form of the MT-wrapped structures away from the depolymerizing end. This can, in principle, be possible for both the depolymerization- and polymerization-driven KT movements, since both Ska1 and EB1 can recognize curved as well as straight MT protofilaments [82,83,99]. It would be interesting to investigate whether the intact EB1–Ska rings move along the MTs like the Dam1 rings and to identify regulators that are involved in the movement.

Models showing the possible mechanisms of KT-MT attachment in human cells vs yeast.

Figure 4.
Models showing the possible mechanisms of KT-MT attachment in human cells vs yeast.

In humans, the EB1–Ska complex oligomerizes into ring-like microtubule-binding structures on the MT lattice. The Ska complex (Ska1, Ska2, Ska3) alone forms hair-like extensions on the microtubules and EB1 forms globular patches on the microtubules. EB1–Ska complex forms ring-like microtubule-binding structures that appear to wrap around the microtubule lattice nearly orthogonally to the microtubule axis. The ring-like structures provide a larger platform that facilitates Ndc80 attachment to the dynamic K-fibers. In this model, it is proposed that the ring structures can couple kinetochore attachment to the dynamic microtubule plus ends either by moving along the microtubule lengths via a dynamic assembly–disassembly–reassembly process following the polymerization–depolymerization dynamics of the microtubules, or by translocating along the microtubule lengths in the form of microtubule-bound structures. In yeast, the Dam1 complex consisting of ten proteins, oligomerizes into ring structures on the microtubules and stabilizes MT-KT attachments by facilitating Ndc80 attachment to the K-fibers. The wider diameter of the Dam1 rings also enables tracking on the microtubule lattice to further facilitate microtubule depolymerization-driven processive movement of the kinetochore. Organization of the Dam1 components was adapted from [151].

Figure 4.
Models showing the possible mechanisms of KT-MT attachment in human cells vs yeast.

In humans, the EB1–Ska complex oligomerizes into ring-like microtubule-binding structures on the MT lattice. The Ska complex (Ska1, Ska2, Ska3) alone forms hair-like extensions on the microtubules and EB1 forms globular patches on the microtubules. EB1–Ska complex forms ring-like microtubule-binding structures that appear to wrap around the microtubule lattice nearly orthogonally to the microtubule axis. The ring-like structures provide a larger platform that facilitates Ndc80 attachment to the dynamic K-fibers. In this model, it is proposed that the ring structures can couple kinetochore attachment to the dynamic microtubule plus ends either by moving along the microtubule lengths via a dynamic assembly–disassembly–reassembly process following the polymerization–depolymerization dynamics of the microtubules, or by translocating along the microtubule lengths in the form of microtubule-bound structures. In yeast, the Dam1 complex consisting of ten proteins, oligomerizes into ring structures on the microtubules and stabilizes MT-KT attachments by facilitating Ndc80 attachment to the K-fibers. The wider diameter of the Dam1 rings also enables tracking on the microtubule lattice to further facilitate microtubule depolymerization-driven processive movement of the kinetochore. Organization of the Dam1 components was adapted from [151].

Energetics in MT assembly dynamics and KT-MT interaction

Although a large number of microtubule and kinetochore proteins are involved in regulation of KT-MT attachment, the mechanochemical energy associated with the assembly–disassembly of MTs and MT interactions with these proteins is the actual underlying factor that drives the process. Dynamic instability of MTs involves stabilization or destabilization of the interactions of adjacent tubulin dimers in the MT lattice, and those interactions occur through discrete lateral and longitudinal non-covalent bonds. Earlier theoretical studies have estimated bond energies of these non-covalent interactions [152] and have derived relations of MT assembly–disassembly dynamics with the bond energetics [153]. Bond energy (ΔG) for a lateral interaction is in the range −3.2 to −5.7 kBT and that of the longitudinal interaction is in the range −6.8 to −9.5 kBT, where kB is the Boltzmann constant and T is the temperature. Considering the free energy for immobilizing a tubulin dimer in the MT lattice in addition to these bond energies, the energy associated with the conformational stress imparted to a tubulin-GDP dimer in the MT lattice has been estimated to be 2.1–2.5 kBT. It has also been found that the coupling between GTP-tubulin subunit addition and the rate of GTP-tubulin hydrolysis to GDP-tubulin is a critical determinant for favoring the assembly or the disassembly state of the MTs [8,153]. Interaction between α-subunit of one tubulin dimer and β-subunit of the other catalyses hydrolysis of GTP between the two subunits. The energy of GTP hydrolysis is stored as strain energy in the MT lattice. Simulation-based modeling has shown that even a small change in the lateral and longitudinal interactions between the tubulin dimers results in a robust effect on MT dynamics. In other words, small changes in bond energetics can cause a large difference in MT disassembly rates. From the nature of bond energetics, one can also predict the mechanisms of action of the proteins or regulators that bind to MTs and alter dynamic instability. For example, binding of XMAP215 has been shown to specifically increase the strength of the longitudinal bonds, supporting the fact that XMAP215 mostly binds to MTs along a protofilament and is less likely to bind across the protofilaments [153,154]. More such analyses will be needed to understand the binding mechanisms of other regulators including the Dam1 complex rings and the EB1–Ska ring structures.

Summary

The attachment force required to physically move a chromosome is ∼700 pN and the force of attachment per k-MT has been experimentally estimated to be >10 pN [155]. The force associated with MT flux at the minus end is expected to further add up to this value. For an effective MT depolymerization-driven KT movement, such a large amount of force is to be generated by k-MT depolymerization. The strain energy, i.e. the energy of GTP hydrolysis, is stored in the MT lattice and it is available for performing work. However, this energy needs to converted into work that can generate the required force of attachment. Findings of indirect in vitro experiments suggested that Ndc80 binds to MTs with low affinity and it can sustain MT depolymerization-driven movement of KT-equivalent objects against opposing forces only up to 3 pN [56]. Subsequent studies identified additional MT-binding proteins, such as Dam1 complex (in yeast) and Ska complex (in metazoans), which can significantly enhance the affinity of Ndc80 complex to MTs. Moreover, Dam1 complex forms ring structures on the MT lattice. Unlike Ndc80, binding of the rings with the MTs is highly stable and the strong binding allows very little diffusion of the rings over MT lattice. Dam1 rings are able to track on the depolymerizing plus end and they can harness the strain energy of MT lattice generating ∼40−60 pN of force per MT [13,68], which satisfies the required force for KT attachment. In metazoans, Ska complex has been shown to stabilize KT-MT attachment. Ska can oligomerize into large globular structures and it can track the depolymerizing MT plus ends and stabilize the rate of MT disassembly in vitro in a manner similar to the Dam1 complex [82,83]. Our recent study has shown that Ska interacts with the MT plus end tip protein EB1 and their interaction is critical for KT-MT attachments. Moreover, EB1–Ska complex can form ring-like structures on the MT lattice in vitro [88], pointing towards the possibility of ring-mediated KT coupling in higher eukaryotes. More studies are needed to understand the involvement of Ska oligomers and the EB1–Ska ring structures in force generation at the KT. In this aspect, involvement of additional proteins that are known to regulate k-MT assembly and KT-MT interaction also needs to be explored (Figure 5).

Cartoon representing possible mechanisms of action of the factors that regulate k-MT assembly dynamics and MT attachment with the outer kinetochore.

Figure 5.
Cartoon representing possible mechanisms of action of the factors that regulate k-MT assembly dynamics and MT attachment with the outer kinetochore.

While the majority of the proteins, both individually and in the form of subcomplexes, stabilize microtubule assembly, some factors like MCAK destabilize microtubule assembly. In yeast, the multi-protein complex Dam1 forms rings on the MTs and stabilizes MT assembly, whereas in human cells, EB1–Ska complex ring-like MT-binding structures seems to play a similar role.

Figure 5.
Cartoon representing possible mechanisms of action of the factors that regulate k-MT assembly dynamics and MT attachment with the outer kinetochore.

While the majority of the proteins, both individually and in the form of subcomplexes, stabilize microtubule assembly, some factors like MCAK destabilize microtubule assembly. In yeast, the multi-protein complex Dam1 forms rings on the MTs and stabilizes MT assembly, whereas in human cells, EB1–Ska complex ring-like MT-binding structures seems to play a similar role.

Concluding remarks and future perspectives

Mechanisms of mitotic chromosome congression and segregation are likely to be conserved across eukaryotic organisms. The molecular players associated with the mitotic machinery and KT-MT interaction by and large bear significant homology. The data from S. cerevisiae and human cells and the supporting observations from the reconstituted protein systems suggest commonality in KT-MT attachment and KT coupling, which are driven by large oligomeric platforms. Although the platforms appear to have architectural similarity, they differ in other aspects, such as molecular components, modes of organization and mechanisms of action. The ring/ring-like structures formed by Dam1 components in yeast and EB1–Ska protein complexes of humans reinforce the possibility that formation of ring or ring-like molecular platforms on the MTs may be functionally relevant for harnessing the force of MT depolymerization to couple chromosome movement between species. However, several aspects are yet to be investigated in order to understand the functional involvement of these structures. In particular, for the EB1–Ska structures, one important aspect to elucidate is whether and how the EB1–Ska structures couple KTs to the depolymerizing MTs. Real time analysis with reconstituted MT and purified KT systems could provide useful insights into this aspect. Real-time analysis directly in cells by ultra-high-resolution microscopy, such as super-resolution microscopy, could reveal the relevance of these structures in vivo. Mechanisms underlying the organization of EB1 and Ska proteins to the ring-like assembly and their stoichiometry and dynamics, and furthermore, potential roles of other MT or KT proteins including +TIPs proteins in addition to EB1 in modulation of the structures need to be deciphered. Since EB1 is conserved from yeast to humans, another interesting possibility to explore is whether the S. cerevisiae EB1 ortholog, Bim1P, also imparts similar functions at the KT-MT interface in yeast and if so, how that is linked to Dam1 complex functions.

Abbreviations

     
  • CCAN

    constitutive centromere-associated network

  •  
  • CH

    calponin homology

  •  
  • CPC

    chromosomal passenger complex

  •  
  • EB

    end binding

  •  
  • EM

    electron microscopy

  •  
  • KT

    kinetochore

  •  
  • MT

    microtubule

  •  
  • k-MT

    kinetochore-attached microtubule

  •  
  • KT-MT

    complex between kinetochore and microtubule

  •  
  • Plk1

    Polo-like kinase 1

  •  
  • PP1

    protein phosphatase 1

  •  
  • Ska

    spindle and kinetochore associated

  •  
  • +TIP

    plus end tracking protein

Acknowledgments

We thank Professor S. Murty Srinivasula for critical reading of the manuscript and suggestions. Financial support from the Department of Biotechnology, Govt. of India is thankfully acknowledged. We apologize to people whose work could not be cited here due to space constraints.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contributed equally to this work.