The structure of the kinetochore

Kinetochores are multi-protein complexes that assemble in prophase at the centromeres of chromosomes and thereby physically link centromeric DNA to the plus-tips of spindle microtubules (Cheeseman et al., 2006; DeLuca et al., 2006). Their key functions are the end-on attachment of microtubules to generate or transduce forces that are required for chromosome movement and to generate spindle assembly checkpoint signals to prevent missegregation (DeLuca et al., 2005). Based on transmission electron microscopy stud-ies and immunofluorescence analysis the kinetochore structure can be divided into the inner kinetochore (constitutive centromere associated network, CCAN) and outer kineto-chore (KNL1/Mis12/Ndc80-network, KMN network) (Brinkley & Stubblefield, 1966; Cheese-manet al., 2006; DeLucaet al., 2005; Suzukiet al., 2011; Wanet al., 2009). In the absence of microtubule attachment the outer kinetochores are decorated by a fibrous corona con-taining proteins for microtubule attachments as well as components of the spindle assembly checkpoint (Cookeet al., 1997; Rieder & Salmon, 1998; Williamset al., 1996).

Centromeres are chromosomal regions that are specified by the presence of nucleosomes that contain the Histone H3 variant CENP-A (centromeric protein A) (Palmer et al., 1991;

Sullivan et al., 1994). The assembly of the vertebrate kinetochore at the centromeric region is mediated by two parallel pathways (Figure 3.4): CENP-C interacts directly with

3 Introduction 13

CENP-A nucleosomes (Carroll et al., 2010; Kato et al., 2013) and binds the Nnf1 sub-unit of the Mis12-complex (Gascoigne et al., 2011; Petrovic et al., 2010; Screpanti et al., 2011). The Nsl1 subunit of Mis12-complex in turn interacts with Spc24/Spc25 heads of the Ndc80-complex and KNL1. Thereby, the Mis12-complex connects the inner and outer kinetochore (Petrovic et al., 2010, 2014) and positions them correctly to stimulate their microtubule-binding activities (Welburnet al., 2010). The second pathway is mediated by CENP-T in complex with CENP-W/-X/-S, which forms a nucleosome-like structure and binds and wraps centromeric DNA (Horiet al., 2008; Nishinoet al., 2012). The N-terminal region of CENP-T interacts with Spc24/Spc25 (Gascoigneet al., 2011; Nishinoet al., 2013) and the interaction of Spc24/Spc25 with the Mis12-complex or CENP-T is mutually exclusive (Nishino et al., 2013).

Whereas CENP-A, CENP-C and CENP-T (CCAN proteins) localize at the centromere during the cell cycle, the Mis12-complex and KNL1 localizes to centromeres during S-phase and the Ndc80-complex is recruited in late G2 (Gascoigne & Cheeseman, 2013). Kinetochore as-sembly is regulated during the cell cycle by cytoplasmic localization of the Ndc80-complex during interphase. Upon nuclear envelope breakdown the Ndc80-complex can assemble on the centromere (Gascoigne & Cheeseman, 2013) and in addition the Cdk1 mediated phos-phorylation of CENP-T enhances its binding affinity to the Ndc80-complex (Gascoigneet al., 2011; Nishinoet al., 2013).

Mis12

Figure 3.4. Assembly of the outer kinetochore.

The constitutive centromere associated network (CCAN) assembles on nucleosomes composed of A. CENP-C and CENP-CENP-T interact with the Mis12-complex and Ndc80-complex, respectively. For simplicity only the CENP-CENP proteins interacting with components of the KMN are depicted. The Mis12-complex binds to KNL1 and the Ndc80-complex, which both mediate microtubule attachment. The microtubule-binding of KNL1 is not shown. In addition KNL1 recruits Bub3/Bub1 and Bub3/BubR1 as well as Zwint-1 for checkpoint signalling. Modified from (Corbett &

Desai, 2014).

The KMN network is the core microtubule-attachment site and forms the bridge between the centromere associated proteins and the plus-tips of microtubules (Figure 3.4). It is com-posed of KNL1, the Mis12-complex (Nnf1, Mis12, Dsn1 and Nsl1) and the Ndc80-complex (Ndc80/Hec1, Nuf2, Spc24 and Spc25) (Cheesemanet al., 2006; Petrovicet al., 2010).

14 3 Introduction

The Ndc80-complex is essential for kinetochore-microtubule interactions as it represents the linker that has both microtubule and kinetochore binding sites (Cheesemanet al., 2006;

DeLucaet al., 2006). This complex is composed of four proteins: Ndc80/Hec1, Nuf2, Spc24 and Spc25. Hec1/Nuf2 and Spc24/Spc25 form heterodimers with globular heads at each end and are separated by a large coiled coil region. The C-terminal coiled coils of Hec1/Nuf2 interact with the N-terminal coiled coils of Spc25/Spc24 (Ciferri et al., 2005; Wei et al., 2005). The outer head (Hec1/Nuf2) harbours two calponin homology (CH) domains to bind directly to microtubules (Ciferri et al., 2008; Weiet al., 2005). The affinity to microtubules is increased by an unstructured, highly positively charged N-terminal tail of Hec1. The tail mediates microtubule-binding by electrostatic interactions (Ciferriet al., 2008; Guimaraes et al., 2008; Miller et al., 2008) and forms intermolecular contacts to facilitate oligomer-ization of Ndc80-complexes to allow cooperative association with the microtubule lattice (Alushin et al., 2010; Ciferri et al., 2008). It is proposed that the Hec1/Nuf2 heads form initial, low affinity contacts with microtubules and that the N-terminal tail promotes stable end-on attachment. Phosphorylation of the N-terminal tail by Aurora-B reduces the micro-tubule-binding of Hec1in-vitro (Cheeseman et al., 2006; Ciferri et al., 2008) and leads to destabilization of kinetochore-microtubule attachments (DeLuca et al., 2011; Guimaraes et al., 2008; Milleret al., 2008).

Recently it was shown that the N-terminal tail harbours two phosphorylation clusters at the beginning (aa1-20) and the end of the tail (aa41-80), close to the CH domain of Hec1.

Aurora-B mediated phosphorylation in the first cluster alters mainly the tail-mediated oli-ogmerization. Whereas phosphorylation in the second cluster leads to a reduction in mi-crotubule-binding and oligomerization (Alushin et al., 2012). Phosphorylation results in a reduction in microtubule plus-tip stabilization which leads to the disassembly of the bound microtubules and this results finally in their detachment (Umbreit et al., 2012). As previ-ously proposed (Alushinet al., 2010) upon initial microtubule-binding the Hec1/Nuf2 heads are separated from Aurora-B phosphorylation due to increased intra-kinetochore stretch-ing (Maresca & Salmon, 2009). Upon de-phosphorylation of the N-terminus high-affinity Ndc80-complex oligomers are formed into a linear array enabling coupled binding along protofilaments (Alushinet al., 2010, 2012).

Aurora-B phosphorylates both clusters of the N-terminal tail in prometaphase. Upon attach-ment, phosphorylation sites in the first cluster remain at an intermediate level and phos-phorylation in the second cluster is absent (DeLucaet al., 2011). It is proposed that upon attachment the phosphorylation in the second cluster decreases resulting in microtubule binding and cooperative interactions by oligomerization, which prevents the accessibility

3 Introduction 15

of Aurora-B to these sites. Therefore the phosphorylation-status of Hec1 determines the stability of kinetochore-microtubule interactions. High phosphorylation level in prometa-phase promote labile interactions allowing detachment and reattachment. In metaprometa-phase phosphorylation is low resulting in the establishment of stable chromosome bi-orientation and silencing of the checkpoint (DeLucaet al., 2011).

Per se the Ndc80-complex has a weak microtubule-binding affinity, which is increased upon interaction with KNL1 and the Mis12-complex (Cheesemanet al., 2006). The Mis12-complex forms a rod-like structure consisting of the Nnf1, Mis12, Dsn1 and Nsl1 subunits (Klineet al., 2006; Petrovicet al., 2010). The Mis12-complex interacts with KNL1 via Nsl1 and Dsn1 (Kiy-omitsuet al., 2007). Dsn1 is regulated by Aurora-B phosphorylation, which influences the microtubule-binding affinity of the KMN network (Welburnet al., 2010). The Mis12-complex links the inner and outer kinetochore (Petrovic et al., 2010). KNL1 (kinetochre null, also called Blinkin, Bub-linking kinetochore protein) (Desaiet al., 2003; Kiyomitsu et al., 2007) contributes to microtubule binding via its N-terminal microtubule-binding region (Cheese-man et al., 2006; Welburn et al., 2010) and is required for checkpoint activation and si-lencing (Cheesemanet al., 2008; Kiyomitsuet al., 2007). How KNL1 plays a role in check-point activation and silencing is described in Section 3.4. Additionally, the N-terminus of KNL1 harbours PP1-binding motifs (Liuet al., 2010a). The C-terminus of KNL1 consists of a globular domain, which mediates direct interaction with the Nsl1 and Dsn1 subunit of the Mis12-complex (Kiyomitsuet al., 2007; Petrovicet al., 2010, 2014).

The Ndc80-complex is only able to bind to the straight microtubule lattice. But upon mi-crotubule de-polymerization tubulin adopts a bent conformation and the mimi-crotubule peels backward and shows a curved conformation (Mandelkow et al., 1991). To couple chro-mosome movement with microtubule de-polymerization additional factors must exist that maintain processive kinetochore-microtubule attachment under these conditions. In verte-brates this is mediated by the Ska (spindle and kinetochore associated)-complex, which is composed of three subunits (Ska1, Ska2 and Ska3), whereas each is present in two copies (Gaitanos et al., 2009; Hanisch et al., 2006). The Ska-complex binds directly to micro-tubules and is able to form oligomers (Schmidt et al., 2012). This enables the Ska-com-plex to bind to curved protofilaments (depolymerizing microtubules) and to diffuse along the the microtubule lattice (Schmidtet al., 2012; Welburnet al., 2009). The Ska-complex cooperates with the Ndc80-complex to couple chromosome movement to dynamic micro-tubule plus-tips (Gaitanos et al., 2009; Schmidt et al., 2012; Welburn et al., 2009) and is required to maintain stable end-on kinetochore-microtubule attachments (Gaitanos et al., 2009; Hanischet al., 2006; Welburnet al., 2009). Aurora-B mediated phosphorylation of the

16 3 Introduction

Ska-complex prevents its interaction with the subunits of the KMN network. Consequently phosphorylated Ska-complex is not recruited to kinetochores and stable kinetochore-micro-tubule attachments are not established (Chanet al., 2012; Schmidtet al., 2012).

3.2.2 Initial lateral attachment and conversion to stable end-on attachment