Progress in the structural and functional characterization of kinetochores

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ScienceDirect Progress in the structural and functional characterization of kinetochores Marion E Pesenti1, John R Weir1 and Andrea Musacchio1,2 Kinetochores are macromolecular complexes built on a specialized chromatin domain called the centromere. Kinetochores provide a site of attachment for spindle microtubules during mitosis. They also control a cell cycle checkpoint, the spindle assembly checkpoint, which coordinates mitotic exit with the completion of chromosome alignment on the mitotic spindle. Correct kinetochore operation is therefore indispensable for accurate chromosome segregation. With multiple copies of at least 30 structural core subunits and a myriad of regulatory subunits, kinetochores are among the largest known macromolecular machines. Biochemical reconstitution and structural analysis, together with functional studies, are bringing to light the organizational principles of these complex and fascinating structures. We summarize recent work and identify a few challenges for future work. Addresses 1 Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany 2 Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universita¨tsstraße, 45141 Essen, Germany Corresponding author: Musacchio, Andrea ([email protected])

Current Opinion in Structural Biology 2016, 37:152–163 This review comes from a themed issue on Macromolecular machines and assemblies Edited by David Barford and Karl-Peter Hopfner

of kinetochores to stabilize bi-oriented sister chromatids selectively over incorrect configurations of attachment [3,4]. This ability builds on a feedback mechanism that ultimately modulates the binding affinity of kinetochores for microtubules through changes in kinetochore structure and chemistry (e.g. in phosphorylation status), but many of the molecular details of this process remain obscure [3,4]. Biochemical reconstitution of kinetochore function in vitro with samples of sufficient complexity and stability will likely become instrumental to build a molecular understanding of these phenomena, and indeed three distinct and highly complementary approaches pursued in recent years have yielded promising results and illustrated possible ways forward. These include 1) the biochemical reconstitution of parts of the kinetochore with purified recombinant components, often in conjunction with cross-linking and mass spectrometry [5,6]; 2) the reconstitution of kinetochores on synthetic chromatin in cell-free egg extracts of Xenopus laevis [7,8], and 3) the purification of endogenous, native kinetochores from S. cerevisiae and their investigation in reconstituted systems [9,10,11]. Because of their impact on understanding the connectivity of the kinetochore and in creating premises for its structural characterization, this review focuses mainly, but not exclusively, on results from the first approach.

The centromere

Introduction

The centromere is a specialized segment of chromatin that promotes the assembly of the kinetochore [12–14]. On chromosomes of most species, the centromere occupies a discrete site (monocentric chromosomes), but in certain species it extends to the entire length of the chromosome (e.g. the holocentric chromosomes of C. elegans). While their name seems to imply positioning in the midpoint of chromosomes (metacentric chromosomes), centromeres can also be positioned close to the ends (acrocentric or telocentric chromosomes).

In the cell division cycle of eukaryotes, chromosomes in a mother cell are replicated during S-phase to generate sister chromatids that are then parted to the daughter cells during mitosis. Bi-orientation of each sister chromatid pair, i.e. the attachment of sister chromatids to opposite poles of the mitotic spindle, is necessary for accurate chromosome segregation (Figure 1a). Bi-orientation is a gradual process, which usually starts with kinetochore attachment to the microtubule lattice (lateral attachment), and later develops into a stable end-on attachment with the microtubule’s plus end [1,2]. Accurate sister chromatid segregation results from the remarkable ability

Besides positional variability, also the underlying DNA sequence of centromeres differs considerably in different species. The ‘point’ centromeres of Saccharomyces cerevisiae and other yeasts extend over 125 base pairs (bps) of DNA and include three conserved DNA elements whose mutation impairs centromere function [12–14]. Point centromeres likely evolved from ‘regional’ centromeres that are common in most model organisms, from Schyzosaccharomyces pombe to humans [15]. Regional centromeres usually extend from tens of thousand up to million bps of DNA and are often enriched in repetitive DNA

http://dx.doi.org/10.1016/j.sbi.2016.03.003 0959-440/# 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

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Atomic force microscopy of membrane pore formation Pesenti, Weir and Musacchio 153

Figure 1

(a)

(b) Kinetochore Cohesin microtubules Chromosomes Kinetochore Kinetochore Kinetochore Centrosome microtubules

Metaphase Bi-oriented sister chromatids

H3 NCP PDB ID 1KX5

(c) Outer Inner KT KT

H2A H2B H4 H3 or CENP-A

Inner Outer KT KT

Centromere

H3

H3

H3 CA

Cohesin

CA H3

H3

Sister 1

H3

H3

KMN

CCAN

H3

CCAN

KT MT

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CENP-A NCP PDB ID 3AN2

KT MT

N C

Sister 2

65-100 nm CATD

CA

H3

CENP-A and H3 nucleosomes

KT MT

Kinetochore microtubule

CCAN

KMN

Constitutive centromereassociated network

KMN network Current Opinion in Structural Biology

Centromeres and kinetochores. (a) Schematic depiction of the mitotic spindle and chromosomes at metaphase Microtubules (brown) originating from the spindle poles capture chromosomes at kinetochores (yellow dots). In the enlargement, two sister chromatids are held together by the Cohesin complex. The sisters are bi-oriented, because their kinetochores are connected to opposite spindle poles. (b) Cartoon models of canonical H3-containing nucleosome core particles (NCP) and of CENP-A containing NCP (top and bottom, respectively). The position of the N-termini and C-termini and the CATD of CENP-A are indicated. (c) Schematic representation of centromeres and kinetochores. KT = kinetochore, MT = microtubule, CA = CENP-A nucleosome, H3 = H3 nucleosome, KMN = Knl1 complex, Mis12 complex, Ndc80 complex, CCAN = constitutive centromere-associated network.

sequences, such as the 171 bp human a-satellite. The identity of regional centromeres, however, may not be specified by the underlying DNA sequence. Rather, centromeres are maintained ‘epigenetically’, as the establishment of stably inherited, functional ‘neo-centromeres’ on non-repetitive, non-centromeric sequence domains exemplifies [16,17]. With the remarkable exception of the unconventional kinetochore of kinetoplastids [18], eukaryotic kinetochores (including those built both on point and on regional centromeres) contain at least a subset of the same protein orthologs, pointing to the existence of a common structural design. Understanding how this design is tailored to fit a variety of centromere organizations is an www.sciencedirect.com

interesting challenge. The hallmark of the inner kinetochore (the innermost part of the kinetochore that interfaces with the centromeric chromatin) is the histone H3 variant centromeric protein A (CENP-A, also called CenH3) [19,20]. Like H3, CENP-A can be reconstituted in a tight tetramer with H4, which additionally binds two H2A:H2B dimers to form nucleosome core particles analogous to those formed by canonical H3 [21] (Figure 1b). Likely, this is also the organization of CENP-A nucleosomes in vivo [12,13,22]. After its incorporation into chromatin, CENP-A is very tightly bound [23–25]. There might be as little as a single CENP-A nucleosome at point centromeres, and as much as a few hundreds at regional centromeres, interspersed in clusters within canonical H3 nucleosomes [12,13,26–29]. Current Opinion in Structural Biology 2016, 37:152–163

154 Macromolecular machines and assemblies

The constitutive centromere associated network (CCAN) The role of CENP-A as the kinetochore’s founding stone appears to require interactions with several additional inner kinetochore proteins collectively known as CCAN (constitutive centromere associated network, a name that emphasizes the continuous presence of most of these proteins at centromeres during the cell cycle), or Ctf19 complex in Saccharomyces cerevisiae [12,13] (Figure 1c). The primary function of CCAN is to generate a highly selective binding interface for CENP-A that allows the establishment of the outer kinetochore, the microtubulebinding interface. CCAN also participates in microtubule binding and in the deposition of new CENP-A. Several CCAN sub-complexes have significant autonomous stability and can be reconstituted in the absence of other subunits. These include the CENP-N:CENP-L sub-complex (CENP-NL), the CENP-H, CENP-I, CENP-K, CENP-M sub-complex (CENP-HIKM), the CENP-T:CENP-W sub-complex (CENP-TW, which might additional bind to two additional subunits named CENP-S and CENP-X, CENP-SX), and the CENPO:CENP-P:CENP-Q:CENP-R:CENP-U sub-complex (CENP-OPQRU, which can be further dissected in sub-complexes) (Figure 2a). Recent structure determinations of CENP-M (whose structure resembles that of Rasfamily small GTPases, but lacks required signatures of active GTPases, thus configuring a ‘pseudo small GTPase’), of the CENP-TWSX complex (containing histone fold domains), of a segment of the CENP-LN complex (discussed below), of the Ctf19CENPP :Mcm21CENP-O complex, and of the C-terminal dimerization domain of Mif2CENP-C by X-ray crystallography, and of the CENP-HIKM complex by negative stain electron microscopy (EM) shed light on the organization of CCAN subunits [30,31,32,33,34] (Figure 2a). Based on secondary and tertiary structure predictions, CENP-C, the largest CCAN subunit [35], is mostly intrinsically disordered. CENP-C has been suggested to act as an ‘instruction plan’ (a ‘blueprint’) for kinetochore assembly [36], in which the succession of docking sites for other kinetochore subunits along the primary structure spatially correlates with their organization along the outer-inner kinetochore axis (Figure 2b). Specifically, the N-terminal region of CENP-C interacts directly with the Mis12 complex in the outer kinetochore [37,38,39,40,41,42] (see below). Immediately downstream in the CENP-C sequence are binding sites for the CCAN sub-complexes CENP-HIKM and CENP-LN [36,39,43,44], and further downstream are two sequencerelated motifs (known as the ‘central motif’ or ‘central region’ and the ‘CENP-C motif’) that promote CENP-A binding [39,45,46]. Thus, the CENP-C sequence is designed to bridge the CENP-A nucleosome with the outer kinetochore, while also contributing to organize the Current Opinion in Structural Biology 2016, 37:152–163

intermediate CCAN layer, likely explaining its role in the stabilization of mitotic kinetochores [28]. In certain organisms, including D. melanogaster and C. elegans, CENP-C is the only identified CCAN subunit [41,42,47,48], emphasizing this bridging function. Why certain CENP-C orthologs, including human CENP-C, contain two related CENP-A binding motifs is currently unclear [45], but this feature is not fully conserved in evolution.

Interactions of CCAN subunits with the CENPA nucleosome Three regions unique to CENP-A, including the Nterminal and C-terminal tails and a region comprising loop 1 and helix a2 (known as CENP-A centromeretargeting domain, CATD) functionally differentiate CENP-A from H3 by allowing it to interact with CCAN subunits, form a functional kinetochore, and propagate centromeric chromatin indefinitely (Figure 1b) [7,8,45,49–56]. Post-translational modifications altering the function of CENP-A have also been described [57,58], with mono-methylation of histone H4 in the CENP-A nucleosome being prominent and functionally involved in inner kinetochore assembly [59,60]. At least in vertebrates, kinetochore recruitment of CENP-C may depend on other CCAN subunits during interphase but not mitosis, at which point CENP-C can be recruited to CENP-A in the absence of other CCAN subunits, plausibly because (unknown) post-translational modifications enhance the interaction’s affinity [30,36,40,61–63]. Conversely, CENP-C depletion invariably results in the eviction of all other CCAN subunits from kinetochores, in both mitosis and interphase [30,36,64,65,66]. Although CENP-TW had been previously proposed to form a CENP-C independent axis of outer kinetochore assembly downstream of CENP-A [65,67,68], CENP-TW recruitment is in fact dependent on CENP-C (and other CCAN subunits, see below) [30,44,56,64,69,70,71]. High-affinity CENP-C binding requires both the CATD and the C-terminal tail of CENP-A [8,45,56,64,69]. Details of the interaction of CENP-C with the CENP-A C-terminal tail emerged from nuclear magnetic resonance (NMR) and crystallographic studies of complexes of CENP-C’s ‘central’ and ‘CENP-C’ motifs with an engineered chimeric nucleosome consisting of H3 with an engrafted CENP-A C-terminal tail [45] (Figure 2c). Positively charged residues in the N-terminal region of the CENP-C motif dock on an acidic patch of histone H2A and H2B (Figure 2d) previously shown to interact with other chromatin binding proteins, including LANA, RCC1, and the Sir3 BAH domain [72–74]. An aromatic Tyr-Trp dipeptide in the C-terminal region of the CENP-C motif, on the other hand, packs against the hydrophobic C-terminal region of CENP-A [45]. www.sciencedirect.com

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Figure 2

Iml3CENP-L:Chl4CENP-N PDB ID 4JE3

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Spc24:Spc25 KMN binding? binding N

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CENP-TWSX complex PDB ID 3VH5

Mcm21CENP-O:Ctf19CENP-P PDB ID 3ZXU

Mif2CENP-C Cupin PDB ID 2VPV

Hs CENP-C N Mis12 binding

C

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Inner KT

Central motif CENP-C motif

Hs 516-TVTKSRRISRRPSDWWVVKSEE-537 Hs 736-NVRRTKRTRLKPLEYWRGERIDY-758 (d)

PDB ID 4X23

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Cupin CENP-C (CENP-C motif (CENP-A dimerization) binding)

Central CENP-HIKLMN region (CENP-A binding binding)

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H3CENPA-C H2A H2B H4

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R Current Opinion in Structural Biology

The constitutive centromere associated network (CCAN). (a) Composition of the CCAN and gallery of currently available crystal and EM structures [30,31,33,34,121]. The EM map of the CENP-HIKM complex was fitted with a model of the CENP-M:CENP-I sub-complex, as described in reference [30]. (b) Schematic representation of human

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156 Macromolecular machines and assemblies

Although the C-terminal tail is relatively poorly conserved within CENP-A orthologs, it is more hydrophobic than H3’s, pointing to hydrophobicity as a key specificity determinant for CENP-C recognition [45]. CENP-C binding may contribute to a direct stabilization of the CENP-A nucleosome [23]. Besides contributing to the recruitment of CENP-C, the CATD domain of CENP-A has been also implicated in CENP-LN binding [54] (Figure 2c). The N-terminal region of CENP-N (missing in the crystal structure shown in Figure 2a) mediates the interaction [54], whose structural basis remains unknown. Iml3, the CENP-L ortholog of S. cerevisiae, is structurally related to a bacterial recombination-associated protein, RdgC [31,75], and binds the C-terminal domain of Chl4CENP-N [31] (Figure 2a). Interestingly, CENP-LN appears to have been retained in a handful of insect lineages with holocentric chromosomes, in which both CENP-A and CENP-C cannot be identified [48]. The CENP-A N-terminal tail has been recently shown to be important for CENP-T recruitment downstream of CENP-C, but it is unclear whether this is through a direct interaction [55,56]. The CENP-HIKM complex, which binds CENP-C and requires it for kinetochore localization (Figure 2b), also binds CENP-TW directly and is required for its localization [30,36]. CENP-HIKM is therefore another candidate for an interaction with the CENP-A N-terminal tail (Figure 2c). The latter has also been implicated in binding CENP-B [50,51,76], a conserved centromeric DNA binding protein that recognizes a 17-bp sequence called the CENP-B box, which in humans is found in a subset of the a-satellite repeats [77]. CENP-B interacts directly with the CENP-A:H4 complex as well as with CENP-C, and its depletion causes a reduction of the kinetochore levels of CENPC and a partial impairment of new CENP-A deposition, thus identifying CENP-B as being functionally important [51,76,78]. The importance of CENP-B, however, remains debated because neo-centromeres can be established in the absence of CENP-B; CENP-B knockout mice are viable; CENP-B is absent from the natural human Y chromosome; and CENP-B boxes cannot be identified in African Green monkey, despite the presence of CENP-B and alpha satellite DNA [12,13,79,80]. In summary, the CENP-A nucleosome is selected as a binding target by an array of inner kinetochore proteins in what may be a large macromolecular assembly. Importantly, all CCAN proteins reported to interact with

CENP-A, including CENP-B, CENP-C, CENP-LN, and CENP-TW, interact with each other directly or via other CCAN subunits, and may bind concomitantly to CENP-A [30,31,36,43,44,51,64,78], suggesting that CCAN subunits have the potential to form a stable, cooperative complex around the CENP-A nucleosome. Cooperativity may be crucial to understand the exquisite selectivity of the interaction of CCAN with CENP-A. Both CENP-C and CENP-LN also recognize canonical H3 nucleosomes, albeit with reduced affinity in comparison to CENP-A [45,54]. Combining binding sites for CENP-A in a single complex likely increases the specificity and selectivity of the interaction, a prediction that requires formal proof. CCAN subunits, including CENP-C and the CENPHIKM complex, have been involved in the deposition of new CENP-A [8,53,69,81–85], a mechanism at the heart of the epigenetic inheritance of centromeres. In metazoans, CENP-A loading takes place after mitotic exit and continues until the early G1 phase of the cell cycle [25,86]. It restores CENP-A levels after their two-fold dilution during DNA replication, when the CENP-A complement of the parent chromosome is portioned equally to the resulting sister chromatids [25,27]. Incorporation of new CENP-A normally takes place exclusively within the existing CENP-A domain. The CENP-A domain might therefore act as a ‘template’ for a reaction in which histone H3.3, believed to fill vacancies in centromeres created by 2-fold dilution of CENP-A during DNA replication, is replaced with CENP-A [8,87]. The reconstitution of this reaction in a cell free extract promises to be invaluable for dissecting its requirements [8]. A detailed description of this process goes beyond the scope of this review, but readers are referred to recent comprehensive reviews of this topic [12,13,88].

CCAN and the outer kinetochore The ‘outer kinetochore’ is the microtubule-binding interface, and its core is the KMN network, a 10-subunit super-complex of the Knl1, Mis12, and Ndc80 complexes [89,90] (Figure 3a). With its 55 nm long axis, the 4subunit Ndc80 complex is a highly elongated, coiled-coil rich ‘antenna’ which binds the Mis12 complex at the centromere-proximal end and captures microtubules at the centromere-distal end [91,92,93,94,95,96,97,98]. A closely packed arrangement of two calponin-homology (CH) domains on the Ndc80 and Nuf2 subunits promotes microtubule binding [93,96,98,99] (Figure 3b). The latter is further dynamically modulated through Aurora

(Figure 2 Legend Continued) CENP-C (943 residues) with its main functional regions. The colored boxes in the background represent the outer and inner kinetochore, as already shown in Figure 1c. The sequence of the related ‘Central motif’ and ‘CENP-C motif’ are shown. (c) Cartoon model of the complex of the CENP-C motif with a chimeric nucleosome incorporating the C-terminal tail of CENP-A [45]. Putative CCAN partners binding to the N-termini or C-termini and to the CATD of the closely related CENP-A nucleosomes are shown. (d) Enlargement of the boxed area in (c) showing the meandering CENP-C motif and its main interactions at the surface of the chimeric nucleosome. Current Opinion in Structural Biology 2016, 37:152–163

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Figure 3

Microtubule

(a)

Nuf2 (CH domain)

Ndc80 (CH domain)

P P P

Coiled-coil Spc24 (RWD domain)

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Knl1 C-term domain (RWD) PDB ID 4NF9

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Zwint

Nsl1

Knl1 Knl1 C-terminal domain (RWD domain)

10 nm

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Ndc80 (CH domain)

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Spc25 (RWD domain)

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Ndc80-CBonsai PDB ID 2VE7

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NDC80 complex

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β−Tubulin Ndc80Bonsai bound to microtubules PDB ID 3IZ0

Spc24 (RWD domain) Spc24:Spc25 complex with CENP-T N-terminal region PDB ID 3VZA Current Opinion in Structural Biology

The KMN network and its interactions with microtubules and CCAN. (a) Schematic view of the KMN network with the main interactions discussed in the text. The inset on the upper left shows a negative-stain electron micrograph of a reconstituted sample containing the Mis12 and Ndc80 complexes (reproduced from reference [37]). Scale bar = 50 nm. Next to it is a model interpreting the electron micrograph based on known features of the Ndc80 and Mis12 complexes. In EM micrographs, the Ndc80 complex is often seen to bend at a fixed position along the length of the complex, which is believed to correspond to a 30-residue insertion in the Ndc80 subunit [92,93,135]. The complex likely straightens when bound to microtubules, as shown in the larger schematic representation. (b) Molecular model of the interaction of the Ndc80Bonsai with microtubules. The model is based on fitting of high-resolution structures of Ndc80Bonsai and microtubules in a cryo-EM map at medium resolution [96,98]. (c) A single particle electron microscopy (EM) 3D reconstruction of the KMN network obtained from negatively stained images, reproduced from reference [108]. The reconstructions were obtained from a sample containing Ndc80Bonsai, a simplified version of the Ndc80 complex that retains the kinetochore targeting and microtubule binding

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158 Macromolecular machines and assemblies

B kinase-dependent phosphorylation of a positively charged and intrinsically disordered fragment that precedes the CH domain at the N-terminus of Ndc80 [93,94,96,100–106] (Figure 3a). The 4-subunit Mis12 complex has so far escaped high resolution structural analysis, but negative stain EM revealed an elongated, approximately cylindrical structure with a long axis of 22 nm [6,107–110] (Figure 3c). The Mis12 complex takes roots in the inner kinetochore via a direct interaction with CENP-C (already discussed above, see Figure 2b), and, at least in S. cerevisiae, with additional Ctf19CCAN subunits [37,38,110]. The interactions with the Ndc80 complex and with the 2-subunit Knl1 complex take place on adjacent binding sites at the opposite end of the Mis12 complex. They involve short linear motifs of the Nsl1 and Dsn1 subunits of the Mis12 complex, which engage RWD (RING finger, WD repeat, DEAD-like helicases) domains located in the Spc24 and Spc25 subunits of the Ndc80 complex and in the Cterminal region of the Knl1 subunit of the Knl1 complex [6,32,93,97,108,111–113] (Figure 3a). Interestingly, RWD domains were also identified in the Ctf19CENPP :Mcm21CENP-O complex [33], in the SAC protein Mad1 [114], as well as in the Csm1 subunit of the Monopolin complex of S. cerevisiae, required to fuse sister kinetochores during meiosis I [115,116]. Thus, RWD domains, which in the brand observed at kinetochores are always preceded by coiled-coil segments of variable length, are one of the most represented building blocks of the kinetochore [33,108]. The CENP-TW complex also has an established role in outer kinetochore assembly [32,53,65,68,70,71,113, 117,118,119]. Histone fold domains (HFDs) allow CENP-T and CENP-W dimerization, and have been shown to promote binding to CENP-HIKM and DNA [30,32,120]. In complex with two additional HFD proteins, CENP-S and CENP-X, CENP-TW has been hypothesized to form a nucleosome-like structure (Figure 2a) capable of inducing supercoiling on DNA and of protecting approximately 100 bp of DNA from partial nuclease digestion [121,122]. Both properties do not necessarily imply the establishment of a nucleosome-like structure, and recent studies suggest that CENP-TW binds DNA in inter-nucleosomal regions [122,123]. While depletion or deletion of CENP-TW has dramatic effects on outer kinetochore organization (see next paragraphs), depletion of CENPSX has not [63], making it unlikely that CENP-TW and CENP-SX are obligate partners in outer kinetochore assembly.

Outer kinetochore assembly requires an extended Nterminal tail of CENP-T. Two related, short linear motifs in the first 100 residues of this tail (Figure 2a) have the potential to interact with the RWD domains of the Spc24 and Spc25 subunits of the Ndc80 complex in a manner that, at least in some species, is facilitated by phosphorylation [32,65,68,71,113,117] (visualized in the crystal structure shown in Figure 3d, reference [32]; this interaction is identified in Box 1 in Figure 4a). Whether the two related motifs of CENP-T can bind concomitantly to Spc24:Spc25 is currently unclear [68]. Importantly, Mis12 and CENP-T bind competitively to the RWD domains of the Ndc80 complex, and are therefore mutually exclusive binders [32,113]. Nevertheless, both complexes may be saturated with Ndc80 at kinetochores, as depletion of either strongly reduces Ndc80 kinetochore levels [65,68,71,118].

A single kinetochore module? The second part of the CENP-T tail (residues 100–450 in humans) has been implicated in kinetochore recruitment of the Mis12 complex, or possibly of an entire KMN network [53,68,71,118]. This pattern is highly reminiscent of CENP-C (Figure 4a, Box 2 and Box 3), but it is unclear whether Mis12 interact directly with CENP-T. Copy number measurements suggest that CENP-C and CENP-T may form distinct complexes with the KMN network [71], but also this remains unproven. Overall, these interactions predict that 3 (and up to 4) Ndc80 complexes may associate with each of the two CENP-A molecules in the CENP-A nucleosomes (1 through each CENP-C and at least 2, and possibly up to 3 through each CENP-T), thus increasing the relative stoichiometry of the Mis12 and Ndc80 complexes (and of their binding partners) over CCAN components (Figure 4a). Reconstitution experiments hold great promise to shed light on this model of outer kinetochore organization. In conclusion of this section, it is important to note that the KMN network is not only the main microtubule receptor at the kinetochore, but it also acts as a recruitment platform for numerous other proteins, including spindle assembly checkpoint (SAC) components and additional regulators of kinetochore-microtubule attachment, including the Dam1 complex, the SKA complex, the Kinastrin/SKAP:Astrin complex, MCAK, CENP-E, Kif18a, and Dynein. Due to space limitations, we are unable to discuss the roles of these additional proteins and of the SAC in this review, and refer to recent reviews on this topic [3,124].

(Figure 3 Legend Continued) domains but is deprived of most of the coiled-coil region of Ndc80 [93]. The full length Ndc80 complex is approximately 55 nm in length. The C-terminal region of Knl1, whose crystal structure showed it consists of tandem RWD domains [108], was also included in the reconstruction. (d) Crystal structure of the complex of one of the two CENP-T motifs and the Spc24:Spc25 RWD globular region, required for kinetochore targeting [32]. Current Opinion in Structural Biology 2016, 37:152–163

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Atomic force microscopy of membrane pore formation Pesenti, Weir and Musacchio 159

Figure 4

(a)

Mis12 N

Box 1 Box 2 Box 3

?

CENP-TW CENP-A CENP-H CENP-I CENP-K CENP-M

x2 CENP-L CENP-N

CENP-C Point

(b) H3 CA

H3

H3

H3

Cohesin H3

H3

H3

CA H3

Outer Inner KT KT

Regional H3 CA

H3

H3

H3

Cohesin

CA

H3

H3

H3

H3

H3

H3

H3

H3

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Current Opinion in Structural Biology

Deposition of CENP-A and modular organization of kinetochores. (a) Summary of interactions between CCAN and KMN subunits discussed in the text. The interaction between CENP-T and the Mis12 complex shown in Box 2 is hypothetical and therefore accompanied by a question mark. CENP-C dimerizes via its C-terminal domain. The significance of CENP-C dimerization, and how it reflects on the stoichiometries shown in the figure is unclear. (b) Kinetochores in point and regional centromeres might be built using the kinetochore module shown in a.

[46,125,126] (Figure 1c). There is evidence that pulling by bound microtubules stretches the kinetochore (intrakinetochore stretch) along the direction of the interkinetochore axis, which in turn has been proposed to regulate SAC function [46,127–130]. It is currently unclear whether intra-kinetochore stretch is caused by a single, discrete change in the structure of the kinetochore, or rather by multiple small changes, or even by simple re-orientations of kinetochore parts along the axis of measurement. Investigating the structural basis of this mechanism is important and likely to shed light on fundamental principles of kinetochore function. Importantly, besides the Ndc80 complex, also two subunits of the CCAN sub-complex CENP-OPQRU have been identified for carrying microtubule-binding sites [131–133]. It is possible, but unproven, that this binding site coincides with the recently described active force generator acting at kinetochores during poleward movement [130]. The interactions discussed in the previous paragraphs describe a continuous link between the CENP-A nucleosome and the KMN network in the outer kinetochore, with a connectivity that is largely conserved in S. cerevisiae and humans, the model systems where biochemical reconstitution of kinetochores is most advanced. These interactions suggest the existence of a ‘unit module’ of kinetochore structure, likely built around a single CENPA nucleosome (Figure 4a). Because there likely is a single CENP-A nucleosome in S. cerevisiae [134], a single copy of this unit may exist in this organism. Regional kinetochores, on the other hand, may be built by convolution of the unit module with the multiple CENP-A nucleosomes present in these structures (Figure 4b). In conjunction with the considerable progress of reconstitution approaches, discussed in this review, affinity purification has recently led to isolate the S. cerevisiae kinetochore, yielding samples that were used for force measurements with microtubules and for low-resolution structural analyses by electron microscopy [9,10,11]. This approach is greatly promising and likely to shed new crucial insights in the future, especially if coupled with improvements in sample homogeneity. Analogous but more challenging approaches might be attempted with regional kinetochores. We conclude that structural and mechanistic studies have the potential to discover fundamental principles of kinetochore function in the near future.

Conflict of interest Application of an ingenious pseudo-super resolution method to measure average label separation along the inter-kinetochore axis revealed that the ‘depth’ of the kinetochore, from the outermost Ndc80 complex to the innermost CENP-A nucleosome, is 65–100 nm, with the Ndc80 complex spanning roughly 50% or more of it www.sciencedirect.com

The authors declare no competing financial interests.

Acknowledgements A.M. gratefully acknowledges funding by the European Research Council (ERC) Advanced Investigator award RECEPIANCE (GA number 669686) and the DFG’s Collaborative Research Centre (CRC) 1093. Current Opinion in Structural Biology 2016, 37:152–163

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Current Opinion in Structural Biology 2016, 37:152–163