Kinetochore structure and assembly

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Organization of the kinetochore in monocentric chromosome has been revealed by electron microscopy (Brinkley and Stubblefiend, 1966; Maiato et al., 2006; McEwen et al., 2007; Dong et al., 2007). The kinetochore appears as a layered structure, in which the inner layer, the inner kinetochore, is adjacent to the centromeric chromatin and the outer layer, the outer kinetochore, contacts microtubules (Fig.5.). Poleward surface of the outer plate has been shown to contain fibrillar material, called the fibrous corona (Ris and Witt, 1981; McEwen et al., 2007). Immunoelectron microscopy enabled to localize a subset of proteins belonging to different kinetochore regions (McEwen et al., 2007).

The laminar ultrastructure is correlated with a hierarchical assembly of the kinetochore constituents, beginning with the centromeric chromatin and finishing at the outer kinetochore (Fig.5.; Liu et al., 2006; Przewloka et al., 2007; Cheeseman and Desai, 2008). Some proteins of the outer kinetochore and the fibrous corona do not follow the hierarchy of assembly. Their localization at the kinetochore is often transient and dependent on kinetochore-microtubule attachment or entry into the next stage of division. The majority of the kinetochore components assemble only during division, when the interaction of chromosomes with microtubules is to be established. Kinetochore structure disassembles upon exit from division.

The functional kinetochore comprises of over 90 proteins (Cheeseman and Desai, 2008). Most of them are conserved among species (Westermann et al, 2007; Cheeseman and Desai, 2008; Welburn and Cheeseman, 2008). Dysfunction of many kinetochore constituents compromises correct attachment of microtubules to chromosomes and leads to incorrect chromosome positing within the spindle in mitosis (Kops et al., 2010). Nevertheless, the exact molecular functions and the pathway of recruitment of individual kinetochore proteins are yet to be determined.

Purification of Cenp-A nucleosomes from human and chicken cells enabled to identify a subset of centromeric proteins, which make up a base of the kinetochore (Foltz et al., 2006; Okada et al., 2006). These proteins form two complexes: Cenp-A-nucleosome-associated complex (NAC) and Cenp-A-nucleosome distal complex (CAD). As both complexes are constitutively associated with centromeric chromatin throughout the cell cycle, they are together referred to as CCAN, the constitutive centromere-associated network. CCAN components are often considered as proteins belonging to the inner kinetochore. CCAN includes Cenp-C, Cenp-H, Cenp-I, Cenp-K-U). All CCAN proteins lay downstream of Cenp-A in the hierarchy of assembly (Cheeseman and Desai, 2008). However, CenpH/I/K and Cenp-M/N have been suggested to function in tethering and/or stabilisation of new Cenp-A on chromatin (Okada et al., 2006). Cenp-C and Cenp-H/I/K are also involved in localization of outer kinetochore components in vertebrate cells (Okada et al., 2006; Liu et al., 2006). CCAN proteins have not been identified beyond vertebrates except for Cenp-C, which has also been shown to be required for assembly of outer kinetochore proteins in C. elegans, Drosophila and in yeast (Cheeseman et al., 2004; Przewloka et al., 2011; Tanaka et. al., 2009).

Recruitment of distal kinetochore proteins is cell cycle regulated and takes place in late G2 phase or at specific time of division (Cheeseman and Desai, 2008). The mechanism that dictates the assembly of the kinetochore during division is, however, not well known.

The core kinetochore comprises of 3 main protein complexes: Mis12/MIND/Mtw1 (referred to as Mis12 complex), Ndc80/HEC1 (referred to as Ndc80 complex) and Spc105/KNL1/Blinkin, collectively called KMN (KNL1/Mis12/Ndc80) network (Fig.5.; Cheeseman and Desai, 2008). Mis12 complex belongs to the inner kinetochore and some fraction of it seems to be present at kinetochores during interphase in human cells (Kline et al., 2006). Mis12 complex is composed of Mis12, Nsl1R, Nnf1R and Dsn1/KNL-3 proteins (Cheeseman and Desai, 2008). Dsn1 has not been identified in Drosophila (Przewloka et al., 2009). In human cells, the subunits of the complex are mutually dependent for their localization (Kline et al., 2006). This dependency is, however, not entirely conserved between organisms (Przewloka et al., 2007). Mis12 complex has been proposed to bind to CCAN/Cenp-C to act as a docking site for outer kinetochore proteins (Scerpanti et al., 2011). Mis12 complex is required for assembly of constituents of Ndc80 complex and Spc105 (Kline et al., 2006; Cheesman et al., 2004; Przewloka et al., 2007; Schittenhelm et al., 2009).

Spc105 interacts with many kinetochore proteins and thus it has been thought of as a geometric scaffold supporting connection between the components of the inner and outer kinetochore. Another model based on study in Drosphila depicts Spc105 as a protein important for recruitment of outer kinetochore proteins rather than constitute the geometric linker of kinetochore subcomplexes (Schittenhelm et al., 2009). In C.elegans Spc105 homologue, KNL1, directly interacts with microtubules (Cheeseman et al., 2006). In the hierarchy, Spc105/KNL1 is upstream of Ndc80 complex in Drosophila and C. elegans (Przewloka et al., 2007; Cheeseman et al., 2004). In human, localization of Ndc80 does not depend on Spc105 (Cheeseman et al., 2008). In Drosophila and C.elegans, Spc105 is also required for recruitment of components of Mis12 complex to kinetochores (Schittenhelm et al., 2009; Cheeseman et al., 2004).

Ndc80 complex is recruited exclusively during mitosis (McCleland et al., 2003; Cheeseman and Desai, 2008). In yeast and humans, Ndc80 complex consists of Ndc80/Hec1, Nuf2, Spc24 and Spc25 subunits. The components of Ndc80 complex form two heterodimers, in which Spc24/Spc25 heterodimer interacts with Ndc80/Nuf2 heterodimer via coiled-coil domains (Ciferri et al., 2005; Wei et al., 2005). Together, they form a rod-like structure, which bridges Mis12 complex of the inner kinetochore with outer kinetochore plate. While Spc24 and Spc25 heterodimer directed towards Mis12 complex, Ndc80/Hec1 and Nuf2 heterodimer faces the spindle (Ciferri ert al., 2008; DeLuca et al., 2006). In Drosophila, Spc24 homolog has not been identified (Przewloka et al., 2007). Nuf2 and Ndc80 interact tightly and form the kinetochore-microtubule interface (Alushin et al., 2010). Ndc80 was shown to directly bind microtubules (Cheesman et al., 2006; Wilson-Kubalek, 2008; Alushin et al., 2010).

The core kinetochore complexes provide a platform for assembly of other kinetochore proteins, including components of the fibrous corona, many of which associate transiently with the kinetochore (Maiato el al., 2004). Among recruited proteins are microtubule motors such as dynein and Cenp-E, and microtubule regulators like EB1, components of the spindle assembly checkpoint and proteins of the nuclear pore complex (see 1.1.3.3. and 1.1.3.4.; Starr, 1998; Cooke et al., 1997; Karess et al., 2004; Obuse et al., 2004; Kops et al., 2005; Zuccolo et al., 2007; Kiyomitsu et al., 2007).