An essential part of cell proliferation in eukaryotes is the accurate segregation of sister chromatids to opposite poles of the cell during mitosis. Previous studies have shown that error in this process – mis-segregation of chromosomes – generates aneuploidy cells which are linked to human diseases like cancers and congenital disorders (1, 2). Forces generated by microtubules (MTs) upon linked kinetochores (KTs) is the major factor of chromosome segregation during anaphase (3). Hence, KTs has to be correctly attached/captured by spindle MTs on both ends and aligned along the metaphase plate before the chromosomes are segregated (4). This essay will describe four KT-MT interactions during the pro-metaphase and metaphase stages of the cell cycle: capture, transport, error correction and bi-orientation (summarized in Figure 1). Most of the interactions mentioned are based on the model system of budding yeast Saccharomyces cerevisiae (S. cerevisiae) due to its simple machinery (a KT interacts with a MT as compared to higher organisms where a KT interacts with multiple MTs) (5,6), the size of its centromere (only 130bp) (7) and the large information available about it. At the same time, components of the KT are conserved in other organisms (8).
The first contact between MT and KT is needed so that the subsequent mechanisms can take place (Fig 1). Notably, this initial contact between KT and MT occurs during pro-metaphase (breakdown of nuclear envelope) in vertebrate cells while in budding yeast, it takes place during S phase (complete KT assembly) (9-13). ‘Open’ mitosis occurs in the former allowing interaction between KTs and MTs extending from MT-organizing centres (MTOCs) only after nuclear breakdown (14). Yeast cells undergo ‘closed’ mitosis and the KTs are attached to MTs from MTOCs – known as spindle-pole bodies (SPBs) – for most of the cell cycle (15-16).
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Upon centromere DNA replication, KTs are disassembled and transient detachment of the centromere from MTs take place (17). MTs then quickly recapture and reassemble KTs localized around SPBs during the S phase (16). This is further supported by the high occurrence of turnover of Cse4 protein at the S phase as compared to the other phases in the cell cycle (18). Initial KT-MT interactions are discovered to be conserved from yeast to vertebrate cells (9, 11, 12, 19-20); KTs attach to the lateral surface of a MT (lattice) before being transported along the MT. The advantage of having a larger surface area for capture provided by MT lattice as compared to the tips is a highly possible reason for this conservation (21).
Carazo-Salas and his colleagues demonstrated inXenopus laevisegg extracts that ‘MTs extend from centrosomes preferentially in the direction of chromosomes’ (22). The presence of RanGTP concentration gradient and its linked proteins surrounding chromosomes drive a MT assembly bias and thus, improves the efficiency of the search-and-capture process (23). Ran – a small Ras-like GTPase – switches between GTP (active) and GDP bound forms through interactions with other proteins like RCC1 and RanGAP (24). In S. cerevisiae though, MTs extend in all directions due to two possible factors; its small nuclear size and having a closed mitotic nature which would hinder the formation of a RanGTP gradient (12, 25). Besides +TIPs, such as Bim1, Bik1 and Stu2, RanGTP also plays a role in MT rescue/extension (26, 27).
MTs generated from KT were discovered in yeast, Drosophila melanogaster, and vertebrate cells (28-30). The interaction between KT-derived MTs with spindle pole-MTs ‘guides’ KT loading onto the lattice of the latter. The presence of KT-derived MTs speed up the interactions between KT and MT as evidenced by their increase when KT remains unattached (30). In Drosophila cells, the minus (distal) ends of KT-derived MTs reach spindle poles and becomes part of the spindle whereas the plus (distal) ends in budding yeast disappear once KT-derived MTs are attached to a spindle-pole MT (28,30,31).
Several complexes are involved in both the capture mechanism and transport along MT such as CBF3, Ndc80, Mtw1 and Ctf19 (12), of which Ndc80 will be further discussed in the following section.
Once KTs are loaded onto MT (lateral attachment), they are transported (sliding) towards the minus-end of the spindle pole (SP) along the MT (9, 32) (Fig 1). KT transport is important for bi-orientation as it brings KTs to close proximity with the mitotic spindle. ATP-driven motor proteins at SP ends such as dynein (vertebrate cells) and kinesin (budding yeast) super-families promote this sliding process (12, 33-35). Kar3 (member of kinesin super-family) and dynein are involved in KT sliding towards the pole and pulling respectively (35, 36). It is suggested that other regulators (not yet identified) act antagonistically with Kar3 as KT transport still takes place in cells with deleted Kar3 genes. Lateral attachment is useful for the initial KT capture as it provides a larger surface area for contact with KTs as compared to end-on attachment which uses the MT tips.
While the KT is sliding along the MT lattice, the MT continuously extend and shrink at their plus ends. However, the shrinking end (distal to SP) regularly catches up to KT causing it to be tethered onto the plus end of the MT (end-on attachment) with subsequent pulling towards the pole as the MT shrinks (35). As the rate of MT shrinkage is much faster than the poleward sliding of KT, Stu2 provides MT rescue which prevents the KT from falling off the lateral MT (35). It is suggested that the Dam1 complex (budding yeast) and Bub1 (metazoan cells) are involved in the conversion of lateral to end-on attachment (35, 70).
The higher stability of end-on attachment compared to lateral attachment makes it a better choice for maintaining KT-MT interactions. Notably, in budding yeast, KTs never ‘fall off’ from MTs when they are attached end-on to but they could detach from the lateral surface of MTs while they are transported to the pole (12, 67). Dam1 complex is also involved in the conversion of free energy from depolymerising MTs to generate a pulling force along the MT, hence the end-on pulling of KTs towards the pole (71). The Kar3 driving force for KT sliding however, relies on ATP hydrolysis which is more energy consuming.
Interface of KT-MT attachment
KTs are large and highly conserved proteins complexes with several components identified for their roles in the KT-MT interaction from recent papers (37). Two complexes, Ndc80 and Dam1 will be further discussed in this essay.
The Ndc80 complex is made up of four components; Ndc80 (Hec1 in mammals), Nuf2, Spc24 and Spc25 forming a hetero-tetrameric rod structure with two globular domains at both ends (Ndc80-Nuf2 and Spc24-Spc25 respectively) (38, 39). Ndc80-Nuf2 is orientated towards the MTs while Spc24 and Spc25 faces the inner KT.
In vitro studies carried out by Cheeseman et al.and Wei et al. showed that the Ndc80-Nuf2 domain acts as a link between KT and the MT lattice which was further validated by in vivo experiments carried out in S. cerevisiae (39, 40). Furthermore, calponin-homology (CH) domain which was observed in protein EB1 (a MT-associated protein) was also found in Ndc80-Nuf2 (41). The N-terminus (a basic region of 80-100 residues) extending outwards from the CH domain into the MT seems to have functions in both inter-complex and KT-MT interaction with the latter regulated through phosphorylation by Aurora B kinase (42, 43).
The other globular domain of Spc24-Spc25 bridges the Ndc80 and Mis12 complexes. The KMN network in worms consisting of KNL1 and complexes Ndc80 and Mis12 provides a stronger attractive force towards MT as compared to just the Ndc80 complex alone (40). It is suggested that the presence of KNL1 provides extra interface for lateral attachment of KT.
Dam1 Complex in Yeast
The Dam1/DASH complex is made up of 10 proteins and discovered in both budding and fission yeast (37, 44). The complex does not have roles in KT capture or poleward transport along the MT lattice. Instead, it plays an integral role for bi-orientation. When lateral attachment is converted to end-on attachment, the Dam1 complex is able to detect the shrinking plus end of MT and hence, attaches itself onto the KT (35). The assembly of Dam1 complexes into a ring structure surrounding the MT assists the transport of KT towards the spindle pole by end-on pulling (45). Notably, the attachment of Dam1 complex onto KT is Ndc80 complex-dependent. End-on attachment is affected when the relationship between the two complexes are interrupted whereas no effect was observed in lateral attachment (21). This proves that the two complexes work in concert to provide stable end-on attachment.
Error correction & Bi-orientation
Upon successful transport of KT towards the spindle pole, the next step for accurate segregation is sister kinetochore bi-orientation; each sister chromatid containing a KT each should be attached to a MT (in yeast) or a number of MTs (vertebrate cells) from opposite poles (Fig 1). Thus, it is very important for the cell to be able to differentiate between right and wrong MT attachments as well as being able to correct the latter (46).
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Monotelic attachment or mono-orientation is the attachment between a single KT and a single MT. When its sister KT is attached to another MT at the opposite pole, it is therefore, a bi-orientation or amphitelic attachment. Wrong MT attachments could happen: synthelic attachment where sister KTs are attached to the same pole or merotelic attachment where a KT is attached to two poles.
Tanaka stated that ‘any connection between kinetochores, which can provide tension upon bi-orientation, is sufficient to facilitate bi-orientation probably by stabilizing kinetochore-to-pole microtubule connections in mitosis in a similar way to meiosis I’ (64) (Fig 1). Therefore, although back-to-back geometry of sister KTs (not discussed in this paper) is a mechanism which avoids wrong KT attachments and helps promote bi-orientation, it is not necessary as long as tension is induced across sister KTs (13). For example, in synthelic attachments where tension is absent between sister KTs, the KT-MT attachment would be removed and replaced with a new one and bi-orientation can be established.
Cohesin, a protein complex made up four conserved subunits (Scc1, Scc3, Smc1 and Smc3) in yeast and vertebrate cells is mostly localized at centromeres (65, 66). It is important in generating tension (counter-acting forces) by providing a physical connection between the MT and centromeric chromatins (67). The tension indicates a stable KT attachment with spindle pole MTs and segregation can then takes place once cohesin subunit Scc1p is cleaved by separin (68).
Aurora B/Ipl1 kinase
Aurora B kinase (Ipl1 in budding yeast) has been shown to be an important player in the error correction mechanism for the avoidance of the stabilization of inaccurate attachments (Fig 2). When Aurora B was inhibited, a build-up of syntelic attachments were observed and when the inhibitor was washed-out, the activated kinase was able to correct the wrong attachments (47). A similar study carried out using Ipl1 in S. cerevisiae produced similar results suggesting that the kinase enables turnover of the KT-MT association to eliminate wrong attachments (do not produce tension across sister KTs) (48).
During the pro-metaphase and metaphase, Aurora B/Ipl1 kinase is concentrated around the centromeres. Turnover of the KT-MT interaction happens when the kinase phosphorylates KT compartments causing the dissociation of the interaction. Amongst the substrates for this protein include Dam1, Ndc80 and KNL1 complexes. Phosphorylation of Dam1 and Ndc80 occurs at the C- and N-terminus respectively (43). Association with Ndc80 complex is also affected in the former while the affinity of the latter for MT and inter-complex interactions are decreased. When sister chromatids bi-orient and tension is generated across KTs, Dam1 and Ndc80 becomes de-phosphorylated (49). Thus, the KT-MT turnover is halted to maintain the bi-orientated state (48).
The Chromosomal Passenger Complex (CPC)
The regulation of Aurora B includes the kinase activity of the other chromosomal passengers in CPC in mammalian cells; INCENP and Survivin (50). Hence, the complex is very crucial for bi-orientation. Besides that, the function and localization of CPC varies at the different mitotic phases in mitosis (51). TD-60 is suggested to be the fourth CPC subunit based on its localization, but it still remains to be seen if it is part of the complex (52, 53).
Compromising the chromosomal passengers causes mitotic error such as synthelic attachment and defects in bi-orientation (48, 54). This defects are observed in vertebrates, yeast and worms indicating that CPC is conserved (55).
Other regulators: Bub1, Haspin, Mps1
Two mechanisms regulate CPC by targeting the centromeres. Bub1 protein phosphorylates histone H2A, a process that recruits Borealin/Bir1 (56). The other mechanism is the phosphorylation of histone H3 by protein Haspin causing the binding of Survivin/Bir1 (57). These two pathways are partly antagonistic to each other but in the presence of both, Aurora B is highly localized in the inner centromeric regions (58). Hence, although mutations in Bub1 or Haspin causes error in sister KT bi-orientation, the defect of both mechanisms would cause more damage.
Protein kinase Mps1 regulates KT-MT turnover especially in sister KTs which do not have tension applied across them (59). In a study by Jelluma et al., ‘Mps1 in mammalian cells regulate Aurora B kinase activity by phosphorylating Borealin/Dasra B that forms a complex with Aurora B (60). However, in S. cerevisiae, Borealin/Dasra B is not conserved indicating a possibility that Mps1 and Aurora B could work in parallel pathways (59). Other functions of Mps1 include chromosome segregation, the spindle assembly checkpoint (SAC) and duplication of MTOCs (61, 62).
Spindle-assembly checkpoint (SAC)
SAC ensures that KTs are properly captured and bi-orientated before chromosome separation takes place. SAC differs from error correction; it provides the means for error correction to happen and therefore, it does not promote bi-orientation on its own (21). At the same time, Aurora B, Mps1 and Bub1 are also part of the SAC mechanism(63). These proteins produce unattached KTs through error correction, which in turn promotes SACtes SAC (48). However, the relationship between SAC and error correction is not clearly understood as phosphorylating KT and SAC components by Aurora B and Mps1 protein kinases could partially signal SAC, independent of the KT-MT attachment turnover (21).
Bi-orientation of sister KTs is necessary for segregation which ensures the accurate passing-down of genetic information. This paper states the numerous studies which have shown the few but essential KT-MT interactions required for bi-orientation. At the same time, a number of questions have yet to be answered, such as the relationship between the complexes for conversion of lateral to end-on attachment (21). Even more importantly, it is understood that these mechanisms are conserved from yeast to vertebrates. With novel advances in biochemistry, genetics and proteomics, it would be of necessity and great benefit to identify their roles in eukaryotes. In light of the recent finding of the open complex of the cohesin ring (69), it shows that this field still has much to be discovered.
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Figure 1: Step-wise overview of kinetochore–microtubule (KT-MT) interactions during pro-metaphase (steps 1–3), metaphase (step 4) and anaphase A (step 5 – not discussed in this paper) (3).
- KTs are captured by the lateral surface of a spindle-pole MT once nuclear envelope breaks down in metazoan cells (9, 11) or when KT assembly is complete in budding yeast (17).
- Captured KTs are transported along the MT’s lateral surface towards the spindle pole (sliding) (9, 11) before tethered at the end (end-coupled pulling) and transported on a shrinking MT (35).
- Sister KTs attach to MTs from opposite poles. If both KTs attach to MTs from the same pole, turnover/re-orientation takes place until sister KTs are bi-oriented (32).
- Tension is applied during bi-orientation (32). Unlike budding yeast where a KT interacts with only a MT, in metazoan cells, tension on chromosomes can increase the number of KT derived MTs (68).
- When bi-orientation is established for all KTs, cohesion between sister chromatids are removed and separates to opposite poles (66). End-on attachment remains as MTs depolymerize towards the spindle pole (3).
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