Chromosoma (2014) 123:447–457 DOI 10.1007/s00412-014-0472-y

MINI-REVIEW

“Uno, nessuno e centomila”: the different faces of the budding yeast kinetochore Francesca Malvezzi & Stefan Westermann

Received: 3 March 2014 / Revised: 10 June 2014 / Accepted: 10 June 2014 / Published online: 26 June 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract “One, no one and one hundred thousand” is a masterpiece of Italian literature, written by Luigi Pirandello. The central theme is that in each individual there are multiple personalities, since one’s perception of one’s self differs from the view of others. As a consequence, a unique identity does not exist, but rather one hundred thousand. This concept can be very well applied to the kinetochore, one of the largest macromolecular complexes conserved in eukaryotes. The kinetochore is essential during cell division and fulfills different sophisticated functions, including linking chromosomes to spindle microtubules and delaying anaphase onset in case of incorrect bi-orientation. In order to perform these tasks, the kinetochore shapes its structure by recruiting different subunits, such as the components of the spindle assembly checkpoint (SAC) or the monopolin complex during meiosis. It also modifies its internal organization by rearranging intramolecular connections and acquiring a distinct identity at different time points of cell division. In this review, we describe recent insights into the changes in composition and configuration of the kinetochore in mitosis and meiosis, focusing on the kinetochore of Saccharomyces cerevisiae. Keywords Kinetochore . Nucleosome . Microtubules . Spindle checkpoint

Introduction One of the largest protein assemblies in eukaryotes is the kinetochore (KT), an extremely specialized structure located at the centromere of each chromosome. Even the simple F. Malvezzi : S. Westermann (*) Research Institute of Molecular Pathology (IMP), Dr. Bohr Gasse 7, 1030 Vienna, Austria e-mail: [email protected]

kinetochore of Saccharomyces cerevisiae has a size in excess of 5 MDa and is composed of more than 300 proteins. These are organized into a conserved architecture characterized by two different plates: the outer kinetochore, called KMN (based on its components KNL1-Zwint, Mis12 complex, and Ndc80 complex), which in budding yeast connects to a single microtubule (MT), and the inner kinetochore, named constitutive centromere-associated network (CCAN), which recognizes the 125-bp yeast centromere and forms a platform for the MT-binding complexes [reviewed in Biggins (2013)]. During cell division, the kinetochore has to fulfill different functions, both mechanical, such as providing a robust connection between chromatin and dynamic microtubules; and chemical, such as sensing mistakes in the microtubule attachment and communicating them to the cell cycle machinery. Given the sophisticated tasks it has to perform, it is not surprising that the kinetochore is anything but a static entity. It rather recruits specific subunits at different time points of cell division, thus changing its composition. It also adopts a variety of internal configurations by rearranging the intra- and intermolecular geometries in order to respond to different external stimuli. Therefore, the kinetochore can not only be seen as one macromolecular complex, but also as multiple different entities. This highly dynamic nature, which contrasts with other macromolecular complexes, such as the ribosome, the nuclear pore complex or fatty acid synthase, has made it difficult to obtain intact, native kinetochores for structural analysis. In this review, we describe recent insights into the dynamic changes of kinetochore composition and configuration in mitosis and meiosis. In particular, we focus on the budding yeast kinetochore and its architecture relative to microtubule attachment, its reorganization in anaphase and on the distinct geometry that the kinetochore acquires in meiosis in order to co-segregate sister chromatids.

448

Microtubules as key determinants of kinetochore composition and architecture The primary function of kinetochores is to establish a connection between chromosomes and microtubules. The presence or absence of microtubules, as well as the different ways in which kinetochores and microtubules can become engaged, causes critical changes in composition and organization. A peculiarity of the closed mitosis of budding yeast is that the MT-organizing center, named spindle pole body, is embedded into the nuclear envelope throughout the cell cycle (Robinow and Marak 1966). Moreover, the outer kinetochore is detected at the centromeric DNA by immunofluorescence microscopy at all stages (Wigge et al. 1998; Janke et al. 2001). The simultaneous presence of microtubules in the nucleus and MT-binding proteins at the kinetochore leads to the constitutive interaction of chromosomes with the spindle pole except during synthesis (S)-phase, when kinetochores are transiently disassembled or released from centromeres to allow DNA replication (Winey and O’Toole 2001; Tanaka et al. 2005; Kitamura et al. 2007). After reassembly, the kinetochore of one sister chromatid is laterally captured by microtubules, slides toward the pole and then turns into an end-on attachment (Tanaka et al. 2005). Subsequently, the other unoccupied kinetochore has to interact with microtubules coming from the opposite spindle pole in order to acquire biorientation before anaphase onset. This gradual process, characterized by an error correction mechanism that prevents a premature stabilization of misattached kinetochores, has been recently proposed also in budding yeast in both mitosis and meiosis, in contrast to the common belief that in this model organism a correct KT-MT attachment is achieved early in cell division (Meyer et al. 2013; Marco et al. 2013). Relative to microtubules, kinetochores can therefore exist in three main conformational states: unoccupied, laterally attached to the microtubule surface and bound end-on to the microtubule plus tip. The kinetochore composition in these different states changes as does its overall architecture (Fig. 1). Two main features specify the unbound state. The first is the lack of tension that is translated into a relaxation of the entire assembly as observed in vertebrates by comparing kinetochores in metaphase and after treatment with MTaltering drugs such as nocodazole and taxol (Wan et al. 2009; Uchida et al. 2009; Maresca and Salmon 2009; Suzuki et al. 2011). A similar shape modification is also observed in mono-oriented chromosomes, analyzing kinetochores attached to microtubules relative to their unattached sisters (Uchida et al. 2009). From the immuno-electron microscopy (EM) and superresolution microscopy analysis, it appears that the redistribution mainly affects the inner kinetochore, stretched by pulling microtubules (Wan et al. 2009; Suzuki et al. 2011). Since the change in the shape of the inner

Chromosoma (2014) 123:447–457

plate is uniform, it is likely that this event occurs also in the single microtubule-binding module of the yeast kinetochore. The second feature is the presence of a specific protein network called the spindle assembly checkpoint (SAC) and composed of Bub3, Mad1, Mad2, Mad3 and the kinases Bub1 and Mps1. The SAC is essential for sensing unattached chromosomes and delaying their segregation by inhibition of the anaphase-promoting complex (APC). Binding of SAC components to kinetochores is hierarchical. The upstream components of the pathway, Mps1, Bub1, and Bub3, are localized at chromosome clusters from S-phase to metaphase (Castillo et al. 2002; Gillett et al. 2004; Jelluma et al. 2010). These proteins are not exclusively located at unbound kinetochores, but they are also present in the “laterally attached” configuration, suggesting the recognition of a particular kinetochore architecture rather than the presence or absence of microtubules (Gillett et al. 2004; Shimogawa et al. 2010). On the other hand, Mad1 and Mad2 are detected only at unbound kinetochores and, therefore, they are commonly used as markers for KT-MT dissociation and SAC activation. Mad3 does not localize to kinetochores in budding yeast (Gillett et al. 2004). It binds instead to both Mad2 and Bub3 in solution, inhibiting the APC coactivator Cdc20 (Hardwick et al. 2000; Chao et al. 2012). Therefore, Mad3 does not act as sensor for unattached kinetochores, but as an effector of the signal that delays mitotic progression. Tremendous progress has been recently achieved to uncover the structural details of the Bub1 and Bub3 interaction with kinetochore components (Fig. 1a). Bub3 recognizes conserved MELT (M [E/D] [I/L] [S/T]) repeats of the outer protein Spc105, which are phosphorylated by Mps1 (London et al. 2012; Yamagishi et al. 2012; Primorac et al. 2013). The interaction is required for Bub1 localization; on the other hand, the presence of a fragment of Bub1 bound to Bub3 remarkably increases the Bub3 affinity for MELTp motifs. In humans, Bub1 additionally interacts with an N-terminal KI region (KI[D/N]XXXF[L/I]XXLK, where X is any residue) of Knl1 (Spc105 of metazoan) (Krenn et al. 2013). However, the KI motifs are not conserved in Spc105 and, therefore, in budding yeast Bub1 exclusively relies on Bub3 for its kinetochore recruitment. Recent work has also shed light into the mode of Mad1 and Mad2 binding. Mad1 and Mad2 form a heterotetramer (2 Mad1:2 Mad2) that, in the absence of microtubules, interacts with Bub1 and Bub3 at the kinetochore (Brady and Hardwick 2000; Sironi et al. 2002). The interaction is mediated by Mps1-phosphorylated residues in the central portion of Bub1 (aa 368-309) (London and Biggins 2014) and a Mad1 motif located in the coiled-coil region immediately upstream of the highly conserved C-terminal domain. Interestingly, the crystal structure of this domain in human Mad1 reveals a fold called RWD found in several other kinetochore components: the inner subunits Ctf19 and Mcm21, the Spc24-Spc25 heterodimer of the Ndc80 complex,

Chromosoma (2014) 123:447–457

449

a Unattached configuration

Cdc20

Signal? Ndc80 N

Mad2

Kar3

Mad1 Mps1

Stu2 KM CC

Mad1

?

Ndc80 complex

Mad2

Mps1

Nuf2

Bub1 Bub3

Bub3

N

Bub1

RWD domain

N

AN

RWD domain

Spc24 Spc25 Spc105 CEN DNA

b

End-on attachment

c

Lateral attachment

-

MT rescue

+

Kar3 Stu2 Mps1

Dam1 complex

+

Bub1 Bub3

Fig. 1 Representation of the different configurations of the kinetochore relative to microtubule binding. a The unoccupied kinetochore is shown in light gray, with the subunits of the spindle assembly checkpoint (SAC) highlighted in red. The kinesin Kar3 and the microtubule associatedprotein Stu2 are colored in light red because they are present at this configuration, but they require microtubules for their function. The details of kinetochore recruitment of SAC subunits are visualized in the box as a zoom in. Mps1 is recruited to the kinetochore in a still unclear fashion likely involving the N-terminus of Ndc80, which is also phosphorylated by the kinase. Mps1 phosphorylation of the Spc105 MELT repeats drives the recruitment of Bub3-Bub1. An unknown signal induces Bub1

phosphorylation by Mps1, which is then recognized by Mad1-Mad2, leading to the downstream events of the cascade that inhibit the APC co-activator Cdc20. The RWD domain of Spc24-Spc25 and Mad1 are highlighted. b At laterally-attached kinetochores, Kar3 is required for poleward movement while Stu2 translocates to the plus end of microtubules where it increases the frequency of microtubule rescues. The SAC components present at this configuration are in light red because they do not signal to the rest of the checkpoint cascade. The microtubule is colored in green. c Sixteen copies of the Dam1 complex are considered to encircle the plus-end tip of microtubules attached to kinetochores, thus forming a ring that connects to the kinetochore via the Ndc80 complex

the Csm1 homodimer of Monopolin, described below, and the C-terminus of Knl1, recently published (Wei et al. 2006; Corbett et al. 2010; Kim et al. 2012; Schmitzberger and Harrison 2012; Petrovic et al. 2014). In the latter three cases, the domain is used for kinetochore binding (in particular to the Mtw1 complex) and thus could resemble a recurrent interaction module in the kinetochore architecture (Malvezzi et al. 2013; Sarkar et al. 2013; Petrovic et al. 2014). In contrast to humans, budding yeast Mad1 (and thus Mad2) recruitment seems to rely exclusively on the interface between the Mad1

RWD domain and the Mps1-phosphorylated region of Bub1. In fact, the RWD domain is sufficient for Mad1 kinetochore targeting (Kastenmayer et al. 2005) and a Bub1-3A phosphodeficient mutant impairs Mad1 co-purification from both kinetochore particles and recombinantly purified proteins (Kastenmayer et al. 2005; London and Biggins 2014). However, the presence of Mad2 is essential for Mad1 recruitment to the centromere and it cannot be excluded that Mad2 establishes additional contacts with other kinetochore components (Gillett et al. 2004; London and Biggins 2014).

450

Despite these recent discoveries, we still do not fully understand the upstream events in the checkpoint cascade. One open question is how Mps1 is recruited to kinetochores. Biochemical studies have identified the N-terminus of Ndc80 (aa 1-257) as an Mps1 receptor, based on the observation that they interact in vitro (Kemmler et al. 2009). Moreover, Ndc80, in particular its unstructured N-terminal tail, is phosphorylated by Mps1 and mimicking phosphorylation leads to a checkpoint arrest that is not caused by an impaired KT-MT connection (Kemmler et al. 2009). Since the N-terminus of Ndc80 is involved in MT binding (Wei et al. 2007; Ciferri et al. 2008), it is tempting to think that particular MT-KT conformations leave the N-terminus exposed for binding to Mps1 and subsequently being phosphorylated. However, there is still no clear evidence that Ndc80 is the Mps1 recruiter to the kinetochore in vivo and further studies will be required to clarify this crucial aspect. Another key feature to be determined is how the absence of microtubules drives Bub1 phosphorylation by Mps1 with subsequent Mad1-Mad2 recruitment. Understanding the exact localization of Mps1 at the kinetochore and the change in the architecture of the MT-binding subunits upon microtubule attachment will help to elucidate this point. The two additional kinetochore configurations, lateral and end-on attachments, are specified by the presence of bound microtubules. Upon microtubule attachment, not only the inner kinetochore stretches as previously discussed, but the outer components undergo a structural rearrangement, as observed in vertebrates (Dong et al. 2007). The microtubule-associated protein Stu2 and the minusend-directed kinesin motor Kar3 are key features of laterally attached kinetochores (Fig. 1b) (Tanaka et al. 2005, 2007). Similar to Mps1, Bub1, and Bub3, these proteins are not only found in this state, but they are also detected at kinetochores that have not been yet captured by the spindle (Tytell and Sorger 2006; Tanaka et al. 2007). However, they exert their function in the presence of microtubules. Stu2 has been proposed to increase the frequency of MT rescues by translocating from the kinetochore to the microtubule plus end (Tanaka et al. 2005), a function that could be important for reducing the probability of a detachment event once the spindle has captured a kinetochore. Kar3, on the other hand, is necessary for poleward movement of laterally attached kinetochores. In Kar3-deleted cells or those harboring a motordead Kar3 mutant, chromatids are still captured, but they can be transported toward the pole only in the end-on configuration and they often remain at the same position, resulting in increased bi-orientation defects (Tanaka et al. 2005; Liu et al. 2011). Despite the evident importance of Stu2 and Kar3 for initial KT-MT interactions, nothing is known about their kinetochore receptors, although they are likely to be components of the microtubule-binding interface, such as Ndc80 and Spc105

Chromosoma (2014) 123:447–457

complexes (Wong et al. 2007). Biochemical studies are therefore needed in order to shed light on this crucial aspect. The conversion of a MT-KT attachment from lateral to endon requires the Dam1 complex, which is loaded onto the kinetochore only at this stage and is absent in the other two configurations (Fig. 1b, right panel) (Li et al. 2002; Tanaka et al. 2007). The Dam1 complex is a heterodecamer that can assemble into a higher order ring structure composed of 16 complexes which encircle the microtubule tip (Cheeseman et al. 2001; Westermann et al. 2005, 2006; Miranda et al. 2005). Although the presence of a ring at the KT-MT interface has also been observed in purified kinetochore particles (Gonen et al. 2012) and could suggest an elegant way of coupling the force of depolymerizing mirotubules with chromatid movement, there is still only weak direct evidence of the existence of a similar structure in vivo (McIntosh et al. 2013). In order to clarify the Dam1 oligomeric state, high-resolution structures of the Dam1 ring are needed to design specific point mutations and test the requirement of the ring for chromosome segregation. Biophysical and biochemical studies suggest that the Dam1 complex is able to track the plus ends of microtubules, sustain high pulling forces and reduce microtubule shrinkage upon tension (Asbury et al. 2006; Franck et al. 2007; Lampert et al. 2010; Tien et al. 2010; Volkov et al. 2013), characteristic features that ensure a stable KT-MT attachment. These properties are transmitted to the rest of the kinetochore via a microtubule-dependent interaction with the Ndc80 complex. The Dam1-Ndc80 association is at least partly mediated by a fungal-specific extension of the Ndc80 subunit near the microtubule-interacting calponin-homology (CH) domain (Lampert et al. 2013) and it increases the MT affinity of the Ndc80 complex in vitro. This suggests that the Dam1 ring could act not only as a force coupler, but also as an integral component of the MT-KT interface. The synergistic action of the Dam1 and the Ndc80 complexes is critical for correcting monopolar attachments by the mitotic kinase Ipl1. In a still unclear mechanism, absence of tension leads to phosphorylation of both Ndc80 and Dam1 subunits, which reduces the interaction of the kinetochore with the plus end and thus causes detachment from the spindle followed by SAC recruitment (Gestaut et al. 2008; Lampert et al. 2010; Tien et al. 2010; Sarangapani et al. 2013).

Kinetochore architecture at the anaphase onset A key execution point during cell division is the irreversible commitment to segregate sister chromatids at anaphase onset. At this crucial time, the kinetochore architecture appears to undergo dramatic changes: superresolution microscopy techniques analyzing pairwise distances between fluorophores have allowed to observe an overall reduction of the kinetochore length in anaphase from 70 to 45 nm (Joglekar et al.

Chromosoma (2014) 123:447–457

2009; Haase et al. 2013). The change in distribution mainly affects the KMN network, with the Ndc80 complex reducing its head-to-tail distance and, together with the Mtw1 complex, localizing closer to the Cse4 (CENP-A) nucleosome. A main question arises from this observation: what causes this conformational change? Several factors could be involved in kinetochore remodeling when chromatids are pulled to opposite spindle poles. First, an abrupt change of forces exerted on kinetochore subunits: from a situation of balanced tension, operated on one side by the microtubule plus end and on the other side by the cohesin complex that keeps the chromatid linked to its homologue, to an unbalanced situation, where the centromere experiences a net positive pulling force from the microtubule counteracted only by friction due to the viscous nucleoplasm. In the past years, several attempts of calculating forces sensed by the kinetochore have been performed [reviewed in Rago and Cheeseman (2013)], but technical difficulties and a limited knowledge of all the factors implicated have not yet allowed a clear answer. It is possible, however, that the variation of tension at the kinetochore would induce intramolecular changes in some subunits, in particular in those that are elongated and contain a certain degree of flexibility, such as the Ndc80 complex and the inner subunits Mif2 and Cnn1, leading to a partial remodeling of the kinetochore architecture (Wang et al. 2008; Westermann and Schleiffer 2013). A second factor implicated in a structural change of the anaphase kinetochore may be a reduction in the number of a few subunits (Joglekar et al. 2006). In some cases, as for the inner kinetochore complex CBF3 and the Dam1 complex, this could depend on their additional localization at the midzone of the mitotic spindle, where they seem to be involved in spindle stability and cell separation (Bouck and Bloom 2005; Buvelot et al. 2003). The chromosomal passenger complex (CPC), composed of the Aurora kinase Ipl1 and the proteins Bir1, Sli15, and Nbl1, is another component of the kinetochore that shows a dynamic localization during mitosis (Buvelot et al. 2003). In early mitosis, the CPC is detected at the centromere where it performs the essential function of destabilizing incorrect KT-MT attachments by phosphorylating Ndc80 and Dam1 subunits, as described in the previous paragraph. At this stage, the CBF3 complex or the Bub1 phosphorylation of histone H2A mediate the centromeric recruitment, while the localization at the midzone is inhibited by Cdk1 phosphorylation of both Ipl1 and Sli15 [reviewed in Carmena et al. (2012)]. Given Ipl1’s presence at the centromere, it has been hypothesized that a bipolar MT-attachment would stretch kinetochores and separate the outer substrates from the kinase, thus stabilizing attachments under tension (Liu et al. 2009). However, a recent demonstration of correct chromosome bioorientation in presence of a premature targeting of the CPC to the spindle challenges the idea of an “Ipl1 activity gradient” and suggests the existence of additional mechanisms for Ipl1

451

substrate recognition (Campbell and Desai 2013). At anaphase onset, when Cdk1 activity drops, the CPC is dephosphorylated by Cdc14 and the population present at the kinetochore translocates to the midzone thanks to the interaction with the plus-end tracking protein Bim1 (Zimniak et al. 2012). Here, Ipl1 phosphorylates different substrates in order to regulate spindle disassembly and delays abscission in case of incorrect sister chromatid separation (Norden et al. 2006; Buvelot et al. 2003; Woodruff et al. 2010). Finally, kinetochore remodeling could be a consequence of the reorganization of intermolecular connections in anaphase. During mitosis several kinetochore components experience extensive modifications through phosphorylation by kinases whose activity is high at mitotic entry and starts decreasing at anaphase (Funabiki and Wynne 2013). Phosphorylation is a suitable modification in the cell cycle because it is easily reversed by the action of phosphatases and therefore allows transient changes in protein conformations or protein-protein interactions [reviewed in Hunter (2012)]. The functional importance of the majority of kinetochore phosphorylations is still unknown, but it is likely that in addition to the regulation of the KT-MT interface from S-phase to metaphase, they also play a role in modulating intra-kinetochore connections. An example is given by the recruitment of the Ndc80 complex via Cnn1, a histone-fold containing protein of the inner kinetochore. Cnn1 is phosphorylated in early mitosis by several mitotic kinases, including Mps1, which targets a critical residue in the Ndc80-binding interface, thus impairing the interaction (Bock et al. 2012; Malvezzi et al. 2013). At this state, the fundamental Ndc80 recruiter within the kinetochore structure is the Mtw1 complex, mainly through the C-terminus of the Dsn1 subunit. A tempting speculation arises from the observation that also the C-terminal region of Dsn1 is an Mps1 target in vitro (Malvezzi and Westermann, unpublished observation). Phosphorylation in early mitosis may therefore regulate both interactions, by increasing the Ndc80-Mtw1 affinity on one hand and decreasing the Ndc80-Cnn1 association on the other. At anaphase onset, with a reduction in the kinase/phosphatase activity balance, Cnn1 competes with Dsn1 for Ndc80 binding and the Cnn1-Ndc80 connection is established (Schleiffer et al. 2012; Bock et al. 2012). It is possible that the change in binding partner for the Ndc80 complex could partially explain the spatial reorganization of the KMN observed by Joglekar et al. (2009) and Haase et al. (2013) (Fig. 2). In fact, vertebrate CENP-T (Cnn1) and the Mis12 complex (Mtw1 complex) are linked to centromeric chromatin at different positions: CENP-T seems to be part of a nucleosome-like structure close to H3-containing chromatin (Nishino et al. 2012; Takeuchi et al. 2013; Dornblut et al. 2014), while the Mis12 complex is linked to the CENP-A nucleosome through CENP-C (Mif2 in yeast) (Screpanti et al. 2011; Przewloka et al. 2011; Kato et al. 2013), a connection that we found to be conserved in fungi (unpublished data). In

452 Fig. 2 Schematic representation of the Ndc80 recruitment pathways in anaphase. The alternative nucleosomecontaining Cnn1 may be located in the centromeric regions flanking the Cse4 nucleosome, as observed in vertebrates. Upon dephosphorylation, the Nterminus of Cnn1 binds to the Ndc80 complex, competing with the C-terminus of Dsn1. This rearrangement could lead to a reorganization of the outer kinetochore architecture. The concominant existence of the two pathways is still unclear

Chromosoma (2014) 123:447–457

Dam1 complex

-

Ndc80 complex

+ Dsn1 C-terminus Mtw1 complex

C

D Mif2 N

Cse4 nucleosome H3 nucleosome

Cnn1 N-tail Cnn1 alternative nucleosome

Kinetochore in anaphase

the point centromere of budding yeast, where the single Cse4 nucleosome is flanked by H3-containing chromatin, the establishment of the Cnn1-Ndc80 connection would likely alter the KMN geometry. However, a more detailed analysis of the centromere localization of Cnn1 in budding yeast will clarify this point in the future. Since a deletion of Cnn1 does not show any clear mitotic defects in an otherwise wild-type strain (Bock et al. 2012; Schleiffer et al. 2012), a still open question is as follows: why does a critical MT-binding component, such as the Ndc80 complex, establish a new nonessential interaction in late mitosis? One could advance at least four hypotheses: first, the new link may optimize the orientation of the outer kinetochore relative to microtubule, in order to sustain the driving force of depolymerization. Second, it allows other connections to be established, for example through the C-terminus of Dsn1. Third, it may simplify the MT-chromosome linkage by reducing the number of molecular interfaces between complexes. In

the Cnn1 pathway in fact there is only one bond between the microtubule and the DNA, the Ndc80-Cnn1 interaction, which leads to a simpler and more robust linkage. Indeed, Cnn1 is one of the few kinetochore proteins tested which is able to segregate mini-chromosomes when artificially recruited, in contrast to Mif2 (Kiermaier et al. 2009; Schleiffer et al. 2012). Finally, because of the simplicity of the Cnn1 pathway, a very speculative idea is that it could be a vestigial feature of the first mechanism used for segregating DNA, a specialized histone N-terminal tail.

Kinetochore architecture in meiosis Meiosis is a specialized cell division in which a single DNA replication event is followed by two sequential rounds of chromosome segregation, meiosis I and meiosis II, resulting in a 50 % reduction of the overall genetic material. While

Chromosoma (2014) 123:447–457

meiosis II is similar to a mitotic division in which sister chromatids are segregated to opposite spindle poles, meiosis I separates the replicated chromatids from their homologues. There are three principal events that make this specialized mode of segregation possible: first, crossovers between homologues during prophase I allow them to jointly align; second, a meiosis-specific cohesin complex is retained at the centromere throughout the first division, keeping sister chromatids together; third, kinetochores of sister chromatids acquire the capacity to bind to a single microtubule (Winey et al. 2005; Lee and Amon 2001). In order for this last feature to occur, kinetochores have to drastically change their geometry compared to the mitotic architecture. A key player in this process is the yeastspecific complex monopolin, composed of the subunits Csm1, Lrs4, Mam1 and the kinase Hrr25 (Tóth et al. 2000; Petronczki et al. 2006; Rabitsch et al. 2003). The complex forms during entry into meiosis, when Mam1 and Hrr25 are expressed and Csm1 and Lrs4 are released from the nucleolus (Rabitsch et al. 2003), where they are involved in rDNA silencing and maintenance (Huang et al. 2006). Monopolin is recruited to kinetochores in a manner that requires the phosphorylation of Lrs4 for a stable binding (Matos et al. 2008) and the absence of attached microtubules, which is accomplished by two mechanisms. On one hand, Ipl1 is involved in detaching chromatids and inhibiting the formation of the spindle by binding to the nascent microtubules at the poles; on the other hand, the kinetochore association of critical MT-binding components, such as Ndc80 and the Dam1C subunit Hsk3, is prevented and their expression is downregulated (Miller et al. 2012; Kim et al. 2013; Meyer et al. 2013). The inhibition of KT-MT interactions in prophase I is critical for kinetochore recruitment of monopolin even if the manner in which microtubules would impair the interaction is elusive: KT-MT attachment could occlude binding sites for monopolin either directly or by inducing a conformational change of the kinetochore architecture. Another component required for the assembly of a functional monopolin at the kinetochore is the condensin complex, which seems to create a higher order structure of chromatin permissive for Mam1 recruitment (Brito et al. 2010b). However, the relationship between monopolin and condensin is still enigmatic and could differ between yeast species, as shown by work in Schizosaccharomyces pombe and Candida albicans (Tada et al. 2011; Burrack et al. 2013). Despite the recent progress on the structure and function of monopolin, its mode of action at the kinetochore is not completely understood. A first hypothesis is based on the structural features of the complex (Fig. 3a) (Corbett et al. 2010; Corbett and Harrison 2012). The scaffold is composed by four Csm1 and two Lrs4 molecules that are organized as follows: Csm1 assembles into a homodimer with an RWD domain at the C-terminus and a coiled-coil N-terminus that

453

interacts with another Csm1 homodimer in the presence of two Lrs4 molecules. The so formed assembly has a characteristic “V-shaped structure” with the globular domains at the two peripheries of the complex at a distance of 10 nm from each other. The meiosis-specific subunits Mam1 and Hrr25 bind to each globular domain via Mam1. Since each RWD domain of Csm1 can directly interact with the N-terminus of Dsn1 (Sarkar et al. 2013), it was suggested that monopolin may physically crosslink the two sister kinetochores, thus co-orienting them toward the same microtubule (Fig. 3b, c) (Corbett and Harrison 2012). Another possibility is that monopolin could block one sister kinetochore from microtubule binding through crosslinking Dsn1 subunits of the same MT-binding module. However, in this last case, it is not clear how monopolin would inactivate only one of the two kinetochores, allowing a MT-KT interaction to occur. It is interesting to note that a part of monopolin, in particular the V-scaffold composed of Csm1 and Lrs4, is released from the nucleolus also during mitosis and is present at the kinetochore specifically in anaphase, where it may increase chromosome segregation fidelity by an unknown mechanism (Fig. 3b) (Brito et al. 2010a). The kinetochore recruitment of this minimal complex could differ from the meiotic counterpart. It has been observed in vitro that Csm1-Lrs4 not only binds to Dsn1, but also to Mif2 through the same conserved surface present twice in each RWD domain (Corbett et al. 2010). However, it is not known whether Csm1-Lrs4 can simultaneously bind to Dsn1 and Mif2 or if the interaction is competitive. The addition of the meiosis-specific subunit Mam1 occludes one of the two binding sites and the resulting monopolin complex shows binding only to Dsn1 with a reduced stoichiometry, so that each domain interacts with only one kinetochore receptor (Corbett and Harrison 2012). Therefore, the geometry of the KTmonopolin association in the mitotic anaphase could drastically differ from the meiosis I arrangement, at least with regards to the binding stoichiometry, reflecting the different role of monopolin in the two cell divisions. It is intriguing to observe that the same subunit of the Mtw1 complex, Dsn1, seems to be involved in both the intramolecular rearrangements of the kinetochore in anaphase: on one hand, the competition with Cnn1 for Ndc80 interaction and, on the other hand, the binding of the minimal monopolin complex. It would be interesting to test whether these two rearrangements are interdependent and whether the monopolin-Dsn1 interaction is phospho-regulated similarly to the Cnn1-Ndc80 connection. A structural analysis of the binding mode of monopolin to the kinetochore both in meiosis I and mitotic anaphase is required in order to clarify monopolin function and the effects of its recruitment on the overall kinetochore architecture.

454

Chromosoma (2014) 123:447–457

a

b

Meiosis

Anaphase in mitosis

Lrs4 V-scaffold (Minimal monopolin)

Csm1

N

Hrr25 Mam1

Csm1 RWD domain

N

Mtw1 complex

10 nm

M Nn

Conserved binding site

putative monopolin Mtw1C binding site binding site ?

Ns

N

D

Dsn1 Mif2 homodimer

c

monopolin mode of action as:

Crosslinker of sister kinetochores

Inhibitor of a sister kinetochore

-

Inactivated kinetochore

+ monopolin Sister kinetochores

Sister chromatids

Fig. 3 Role of the monopolin complex at the kinetochore. a Schematic representation of the monopolin complex, showing the characteristic Vshaped structure. The meiosis-specific subunits Hrr25 and Mam1 bind to one of the two conserved sites present in the RWD domain of Csm1, encircled with a dotted square. The unoccupied binding site available for kinetochore recruitment is highlighted in red. b Possible recruitment modes of monopolin to the kinetochore. In meiosis, the subunit Dsn1 of the Mtw1 complex binds to the Csm1 conserved site using the N-terminus. Therefore, two Mtw1 complexes can interact with monopolin, coming from the same or two different kinetochores (see c). During anaphase, in mitosis, only the V-scaffold is recruited to the kinetochore. This

minimal monopolin has four available sites for binding to the Mtw1 complex. In addition to Dsn1, the minimal monopolin has been proposed to interact with Mif2, which in turn harbors a binding site for the Mtw1 complex, as does the vertebrate homolog CENP-C. c Possible models of the monopolin mode of action in co-orienting sister kinetochores during meiosis: monopolin could cross-link the sister kinetochores, thus forcing the formation of a single microtubule-binding site (left panel) or blocking microtubule binding at only one of the two sister kinetochores (right panel). The chromosomes configuration in meiosis is represented as a cartoon. The homologous chromosomes are shown in red and gray, while the sister chromatids are in darker and lighter color

Closing remarks

The conformational change of the kinetochore in anaphase is still obscure and it is likely that at this particular time point of mitosis, the dephosphorylation of kinetochore subunits drives a dramatic rearrangement of multiple intermolecular connections. Our knowledge of protein interactions between CCAN components or at the CCAN and KMN interface is still very limited. Mapping these connections by reconstituting subassemblies of the kinetochore in vitro and identifying post-translational modifications at these binding sites will allow the prediction of conformational changes at the kinetochore during the cell cycle.

Over the past years, the field has started to realize that the kinetochore is a highly dynamic structure, which is able to modify its architecture by recruiting multiple subunits and changing its internal geometry in order to fulfill different tasks. A current challenge and a high priority for the future is to analyze in atomic details how the additional subunits are recruited and what kind of conformational changes they induce in the overall architecture of the complex. Moreover, the complete set of subunits that dynamically bind to the kinetochore has not been yet indentified and in the future, the exact composition of the kinetochore at different time points of the cell cycle has to be determined. At present, this could be achieved thanks to the recent technical advancements in quantitative mass spectrometry.

Acknowledgments The authors wish to thank members of the Westermann lab for discussion. Research in the Westermann lab has received funding from the European Research Council under the European Community’s Seventh Framework Program (FP7/2007-2013)/ERC

Chromosoma (2014) 123:447–457 grant agreement no. [203499] and by the Austrian Science Fund FWF (SFB F34-B03).

References Asbury CL, Gestaut DR, Powers AF, Franck AD, Davis TN (2006) The Dam1 kinetochore complex harnesses microtubule dynamics to produce force and movement. Proc Natl Acad Sci U S A 103(26): 9873–9878. doi:10.1073/pnas.0602249103 Biggins S (2013) The composition, functions, and regulation of the budding yeast kinetochore. Genetics 194(4):817–846. doi:10.1534/ genetics.112.145276 Bock LJ, Pagliuca C, Kobayashi N, Grove RA, Oku Y, Shrestha K, Alfieri C et al (2012) Cnn1 inhibits the interactions between the KMN complexes of the yeast kinetochore. Nat Cell Biol 14(6):614– 624. doi:10.1038/ncb2495 Bouck DC, Bloom KS (2005) The kinetochore protein ndc10p is required for spindle stability and cytokinesis in yeast. Proc Natl Acad Sci U S A 102(15):5408–5413. doi:10.1073/pnas.0405925102 Brady DM, Hardwick KG (2000) Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function. Curr Biol CB 10(11):675–678 Brito IL, Monje-Casas F, Amon A (2010a) The Lrs4-Csm1 monopolin complex associates with kinetochores during anaphase and is required for accurate chromosome segregation. Cell Cycle (Georgetown, Tex) 9(17):3611–3618 Brito IL, Yu H-G, Amon A (2010b) Condensins promote coorientation of sister chromatids during meiosis I in budding yeast. Genetics 185(1):55–64. doi:10.1534/genetics.110.115139 Burrack LS, Applen Clancey SE, Chacón JM, Gardner MK, Berman J (2013) Monopolin recruits condensin to organize centromere DNA and repetitive DNA sequences. Mol Biol Cell 24(18):2807–2819. doi:10.1091/mbc.E13-05-0229 Buvelot S, Tatsutani SY, Vermaak D, Biggins S (2003) The budding yeast Ipl1/Aurora protein kinase regulates mitotic spindle disassembly. J Cell Biol 160(3):329–339. doi:10.1083/jcb.200209018 Campbell CS, Desai A (2013) Tension sensing by Aurora B kinase is independent of survivin-based centromere localization. Nature 497(7447):118–121. doi:10.1038/nature12057 Carmena M, Wheelock M, Funabiki H, Earnshaw WC (2012) The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nat Rev Mol Cell Biol 13(12):789–803. doi: 10.1038/nrm3474 Castillo AR, Meehl JB, Morgan G, Schutz-Geschwender A, Winey M (2002) The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p. J Cell Biol 156(3):453–465. doi:10.1083/jcb.200111025 Chao WCH, Kulkarni K, Zhang Z, Kong EH, Barford D (2012) “Structure of the mitotic checkpoint complex.” Nature: 1–7. doi: 10.1038/nature10896 Cheeseman IM, Brew C, Wolyniak M, Desai A, Anderson S, Muster N, Yates JR, Huffaker TC, Drubin DG, Barnes G (2001) Implication of a novel multiprotein Dam1p complex in outer kinetochore function. J Cell Biol 155(7):1137–1145. doi:10.1083/jcb.200109063 Ciferri C, Pasqualato S, Screpanti E, Varetti G, Santaguida S, Dos Reis G, Maiolica A et al (2008) Implications for kinetochore-microtubule attachment from the structure of an engineered Ndc80 Complex. Cell 133(3):427–439. doi:10.1016/j.cell.2008.03.020 Corbett KD, Harrison SC (2012) Molecular architecture of the yeast monopolin complex. Cell Rep 1(6):583–589. doi:10.1016/j.celrep. 2012.05.012 Corbett KD, Calvin K, Yip L-SE, Walz T, Amon A, Harrison SC (2010) The monopolin complex crosslinks kinetochore components to

455 regulate chromosome-microtubule attachments. Cell 142(4):556– 567. doi:10.1016/j.cell.2010.07.017 Dong Y, Vanden Beldt KJ, Meng X, Khodjakov A, Mcewen BF (2007) The outer plate in vertebrate kinetochores is a flexible network with multiple microtubule interactions. Nat Cell Biol 9(5):516–522. doi:10.1038/ncb1576 Dornblut C, Quinn N, Monajambashi S, Prendergast L, van Vuuren C, Munch S, Deng W et al (2014) A CENP-S/X complex assembles at the centromere in S and G2 phases of the human cell cycle. Open Biol 4(2):130229. doi:10.1091/mbc.E03-11-0827 Franck AD, Powers AF, Gestaut DR, Gonen T, Davis TN, Asbury CL (2007) Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis. Nat Cell Biol 9(7):832–837. doi:10.1038/ncb1609 Funabiki H, Wynne DJ (2013) Making an effective switch at the kinetochore by phosphorylation and dephosphorylation. Chromosoma 122(3):135–158. doi:10.1007/s00412-013-0401-5 Gestaut DR, Graczyk B, Cooper J, Widlund PO, Zelter A, Wordeman L, Asbury CL, Davis TN (2008) Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation. Nat Cell Biol 10(4):407–414. doi:10.1038/ ncb1702 Gillett ES, Espelin CW, Sorger PK (2004) Spindle checkpoint proteins and chromosome-microtubule attachment in budding yeast. J Cell Biol 164(4):535–546. doi:10.1083/jcb.200308100 Gonen S, Akiyoshi B, Iadanza MG, Shi D, Duggan N, Biggins S, Gonen T (2012) The structure of purified kinetochores reveals multiple microtubule-attachment sites. Nat Struct Mol Biol 19(9):925–929. doi:10.1038/nsmb.2358 Haase J, Mishra PK, Stephens A, Haggerty R, Quammen C, Taylor RM II,Yeh E, Basrai MA, Bloom K (2013) “A 3D map of the yeast kinetochore reveals the presence of core and accessory centromerespecific histone.” Curr Biol Elsevier Ltd: 1–6. doi:10.1016/j.cub. 2013.07.083 Hardwick KG, Johnston RC, Smith DL, Murray AW (2000) MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p. J Cell Biol 148(5):871–882 Huang J, Brito IL, Villén J, Gygi SP, Amon A, Moazed D (2006) Inhibition of homologous recombination by a cohesin-associated clamp complex recruited to the rDNA recombination enhancer. Genes Dev 20(20):2887–2901. doi:10.1101/gad.1472706 Hunter T (2012) Why nature chose phosphate to modify proteins. Philos Trans R Soc LondB Biol Sci 367(1602):2513–2516. doi:10.1098/ rstb.2012.0013 Janke C, Ortiz J, Lechner J, Shevchenko A, Shevchenko A, Magiera MM, Schramm C, Schiebel E (2001) The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control. EMBO J 20(4):777–791. doi:10.1093/emboj/20.4.777 Jelluma N, Dansen TB, Sliedrecht T, Kwiatkowski NP, Kops GJPL (2010) Release of Mps1 from kinetochores is crucial for timely anaphase onset. J Cell Biol 191(2):281–290. doi:10.1083/jcb. 201003038 Joglekar AP, Bouck DC, Molk JN, Bloom KS, Salmon ED (2006) Molecular architecture of a kinetochore-microtubule attachment site. Nat Cell Biol 8(6):581–585. doi:10.1038/ncb1414 Joglekar AP, Bloom K, Salmon ED (2009) In vivo protein architecture of the eukaryotic kinetochore with nanometer scale accuracy. Curr Biol 19(8):694–699. doi:10.1016/j.cub.2009.02.056 Kastenmayer JP, Lee MS, Hong AL, Spencer FA, Basrai MA (2005) The C-terminal half of saccharomyces cerevisiae Mad1p mediates spindle checkpoint function, chromosome transmission fidelity and CEN association. Genetics 170(2):509–517. doi:10.1534/genetics. 105.041426 Kato H, Jiang J, Zhou B-R, Rozendaal M, Feng H, Ghirlando R, Sam Xiao T, Straight AF, Bai Y (2013) A conserved mechanism for

456 centromeric nucleosome recognition by centromere protein CENPC. Science (New York, NY) 340(6136):1110–1113. doi:10.1126/ science.1235532 Kemmler S, Stach M, Knapp M, Ortiz J, Pfannstiel J, Ruppert T, Lechner J (2009) Mimicking Ndc80 phosphorylation triggers spindle assembly checkpoint signalling. EMBO J 28(8):1099–1110. doi:10.1038/ emboj.2009.62 Kiermaier E, Woehrer S, Peng Y, Mechtler K, Westermann S (2009) A Dam1-based artificial kinetochore is sufficient to promote chromosome segregation in budding yeast. Nat Cell Biol 11(9):1109–1115. doi:10.1038/ncb1924 Kim S, Sun H, Tomchick DR, Yu H, Luo X (2012) Structure of human Mad1 C-terminal domain reveals its involvement in kinetochore targeting. Proc Natl Acad Sci U S A 109(17):6549–6554. doi:10. 1073/pnas.1118210109 Kim S, Meyer R, Chuong H, Dawson DS (2013) Dual mechanisms prevent premature chromosome segregation during meiosis. Genes Dev 27(19):2139–2146. doi:10.1101/gad.227454.113 Kitamura E, Tanaka K, Kitamura Y, Tanaka TU (2007) Kinetochore microtubule interaction during S phase in Saccharomyces Cerevisiae. Genes Dev 21(24):3319–3330. doi:10.1101/gad.449407 Krenn V, Overlack K, Primorac I, van Gerwen S, Musacchio A (2013) KI motifs of human Knl1 enhance assembly of comprehensive spindle checkpoint complexes around MELT repeats. Curr Biol CB. doi:10. 1016/j.cub.2013.11.046 Lampert F, Hornung P, Westermann S (2010) The Dam1 complex confers microtubule plus end-tracking activity to the Ndc80 kinetochore complex. J Cell Biol 189(4):641–649. doi:10. 1083/jcb.200912021 Lampert F, Mieck C, Alushin GM, Nogales E, Westermann S (2013) Molecular requirements for the formation of a kinetochoremicrotubule interface by Dam1 and Ndc80 complexes. J Cell Biol 200(1):21–30. doi:10.1083/jcb.201210091 Lee B, Amon A (2001) Meiosis: how to create a specialized cell cycle. Curr Opin Cell Biol 13(6):770–777 Li Y, Bachant J, Alcasabas AA, Wang Y, Qin J, Elledge SJ (2002) The mitotic spindle is required for loading of the DASH complex onto the kinetochore. Genes Dev 16(2):183–197. doi:10.1101/gad. 959402 Liu D, Vader G, Vromans MJM, Lampson MA, Lens SMA (2009) Sensing chromosome bi-orientation by spatial separation of Aurora B kinase from kinetochore substrates. Science (New York, NY) 323(5919):1350–1353. doi:10.1126/science.1167000 Liu H, Jin F, Liang F, Tian X, Wang Y (2011) The Cik1/Kar3 motor complex is required for the proper kinetochore-microtubule interaction after stressful DNA replication. Genetics 187(2):397–407. doi: 10.1534/genetics.110.125468 London N, Biggins S (2014) Mad1 kinetochore recruitment by Mps1mediated phosphorylation of Bub1 signals the spindle checkpoint. Genes Dev. doi:10.1101/gad.233700.113 London N, Ceto S, Ranish JA, Biggins S (2012) Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores. Curr Biol CB 22(10):900–906. doi:10.1016/j.cub.2012.03. 052 Malvezzi F, Litos G, Schleiffer A, Heuck A, Mechtler K, Clausen T, Westermann S (2013) A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors. EMBO J 32(3):409–423. doi:10.1038/emboj.2012.356 Marco E, Dorn JF, Hsu P, Jaqaman K, Sorger PK, Danuser G (2013) “S. cerevisiae chromosomes biorient via gradual resolution of syntely between S phase and anaphase.” Cell 154 (5). Elsevier Inc.: 1127– 39. doi:10.1016/j.cell.2013.08.008 Maresca TJ, Salmon ED (2009) Intrakinetochore stretch is associated with changes in kinetochore phosphorylation and spindle assembly checkpoint activity. J Cell Biol 184(3):373–381. doi:10.1083/jcb. 200808130

Chromosoma (2014) 123:447–457 Matos J, Lipp JJ, Bogdanova A, Guillot S, Okaz E, Junqueira M, Shevchenko A, Zachariae W (2008) Dbf4-dependent CDC7 kinase links DNA replication to the segregation of homologous chromosomes in meiosis I. Cell 135(4):662–678. doi:10.1016/j.cell.2008. 10.026 McIntosh JR, O'Toole E, Zhudenkov K, Morphew M, Schwartz C, Ataullakhanov FI, Grishchuk EL (2013) Conserved and divergent features of kinetochores and spindle microtubule ends from five species. J Cell Biol 200(4):459–474. doi:10.1083/jcb.201209154 Meyer RE, Kim S, Obeso D, Straight PD, Winey M, Dawson DS (2013) Mps1 and Ipl1/Aurora B act sequentially to correctly orient chromosomes on the meiotic spindle of budding yeast. Science (New York, NY) 339(6123):1071–1074. doi:10.1126/science.1232518 Miller MP, Unal E, Brar GA, Amon A (2012) Meiosis I chromosome segregation is established through regulation of microtubulekinetochore interactions. eLife 1:e00117. doi:10.7554/eLife.00117 Miranda JJL, De Wulf P, Sorger PK, Harrison SC (2005) The yeast DASH complex forms closed rings on microtubules. Nat Struct Mol Biol 12(2):138–143. doi:10.1038/nsmb896 Nishino T, Takeuchi K, Gascoigne KE, Suzuki A, Hori T, Oyama T, Morikawa K, Cheeseman IM, Fukagawa T (2012) CENP-T-W-S-X forms a unique centromeric chromatin structure with a histone-like fold. Cell 148(3):487–501. doi:10.1016/j.cell.2011.11.061 Norden C, Mendoza M, Dobbelaere J, Kotwaliwale CV, Biggins S, Barral Y (2006) The no cut pathway links completion of cytokinesis to spindle midzone function to prevent chromosome breakage. Cell 125(1):85–98. doi:10.1016/j.cell.2006.01.045 Petronczki M, Matos J, Mori S, Gregan J, Bogdanova A, Schwickart M, Mechtler K, Shirahige K, Zachariae W, Nasmyth K (2006) Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1. Cell 126(6):1049–1064. doi:10.1016/j.cell.2006.07. 029 Petrovic A, Mosalaganti S, Keller J, Mattiuzzo M, Overlack K, Krenn V, De Antoni A et al (2014) “Modular assembly of RWD domains on the Mis12 complex underlies outer kinetochore organization.” Mol Cell 53 (4). Elsevier Inc.: 591–605. doi:10.1016/j.molcel.2014.01. 019 Primorac I, Weir JR, Chiroli E, Gross F, Hoffmann I, van Gerwen S, Ciliberto A, Musacchio A (2013) Bub3 reads phosphorylated Melt repeats to promote spindle assembly checkpoint signaling. eLife 2: e01030. doi:10.7554/eLife.01030 Przewloka MR, Venkei Z, Bolanos-Garcia VM, Debski J, Dadlez M, Glover DM (2011) CENP-C is a structural platform for kinetochore assembly. Curr Biol CB 21(5):399–405. doi:10.1016/j.cub.2011.02. 005 Rabitsch KP, Petronczki M, Javerzat JP, Genier S, Chwalla B, Schleiffer A, Tanaka TU, Nasmyth K (2003) Kinetochore recruitment of two nucleolar proteins is required for homolog segregation in meiosis I. Dev Cell 4(4):535–548 Rago F, Cheeseman IM (2013) Review series: the functions and consequences of force at kinetochores. J Cell Biol 200(5):557–565. doi: 10.1083/jcb.201211113 Robinow CF, Marak J (1966) A fiber apparatus in the nucleus of the yeast cell. J Cell Biol 29(1):129–151 Sarangapani KK, Akiyoshi B, Duggan NM, Biggins S, Asbury CL (2013) Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms. Proc Natl Acad Sci U S A 110(18):7282–7287. doi:10.1073/ pnas.1220700110 Sarkar S, Rajesh T, Shenoy JZ, Dalgaard LN, Hoffmann E, Millar JBA, Arumugam P (2013) Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I. PLoS Genet 9(7):e1003610. doi:10.1371/ journal.pgen.1003610 Schleiffer A, Maier M, Litos G, Lampert F, Hornung P, Mechtler K, Westermann S (2012) CENP-T proteins are conserved centromere

Chromosoma (2014) 123:447–457 receptors of the Ndc80 complex. Nat Cell Biol 14(6):604–613. doi: 10.1038/ncb2493 Schmitzberger F, Harrison SC (2012) RWD domain: a recurring module in kinetochore architecture shown by a Ctf19-Mcm21 complex structure. EMBO Rep 13(3):216–222. doi:10.1038/embor.2012.1 Screpanti E, De Antoni A, Gregory M, Alushin AP, Melis T, Nogales E, Musacchio A (2011) Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore. Curr Biol CB 21(5):391– 398. doi:10.1016/j.cub.2010.12.039 Shimogawa MM, Wargacki MM, Muller EG, Davis TN (2010) Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint. Cell Cycle (Georgetown, Tex) 9(17): 3619–3628 Sironi L, Mapelli M, Knapp S, De Antoni A, Jeang K-T, Musacchio A (2002) Crystal structure of the tetrameric mad1-Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint. EMBO J 21(10):2496–2506. doi:10.1093/emboj/21.10. 2496 Suzuki A, Hori T, Nishino T, Usukura J, Miyagi A, Morikawa K, Fukagawa T (2011) Spindle microtubules generate tensiondependent changes in the distribution of inner kinetochore proteins. J Cell Biol 193(1):125–140. doi:10.1083/jcb.201012050 Tada K, Susumu H, Sakuno T, Watanabe Y (2011) Condensin association with histone H2A shapes mitotic chromosomes. Nature 474(7352): 477–483. doi:10.1038/nature10179 Takeuchi K, Nishino T, Mayanagi K, Horikoshi N, Osakabe A, Tachiwana H, Hori T, Kurumizaka H, Fukagawa T (2013) The centromeric nucleosome-like CENP-T-W-S-X complex induces positive supercoils into DNA. Nucleic Acids Res. doi:10.1093/nar/ gkt1124 Tanaka K, Mukae N, Dewar H, van Breugel M, James EK, Prescott AR, Antony C, Tanaka TU (2005) Molecular mechanisms of kinetochore capture by spindle microtubules. Nature 434(7036):987–994. doi: 10.1038/nature03483 Tanaka K, Kitamura E, Kitamura Y, Tanaka TU (2007) Molecular mechanisms of microtubule-dependent kinetochore transport toward spindle poles. J Cell Biol 178(2):269–281. doi:10.1083/jcb. 200702141 Tien JF, Umbreit NT, Gestaut DR, Franck AD, Cooper J, Wordeman L, Gonen T, Asbury CL, Davis TN (2010) Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by Aurora B. J Cell Biol 189(4):713–723. doi:10. 1083/jcb.200910142 Tóth A, Rabitsch KP, Gálová M, Schleiffer A, Buonomo SB, Nasmyth K (2000) Functional genomics identifies monopolin: a kinetochore protein required for segregation of homologs during meiosis I. Cell 103(7):1155–1168 Tytell JD, Sorger PK (2006) Analysis of kinesin motor function at budding yeast kinetochores. J Cell Biol 172(6):861–874. doi:10. 1083/jcb.200509101 Uchida KSK, Takagaki K, Kumada K, Hirayama Y, Noda T, Hirota T (2009) Kinetochore stretching inactivates the spindle assembly checkpoint. J Cell Biol 184(3):383–390. doi:10.1083/jcb. 200811028 Volkov VA, Zaytsev AV, Gudimchuk N, Grissom PM, Gintsburg AL, Ataullakhanov FI, McIntosh JR, Grishchuk EL (2013) Long tethers

457 provide high-force coupling of the Dam1 ring to shortening microtubules. Proc Natl Acad Sci U S A 110(19):7708–7713. doi:10. 1073/pnas.1305821110 Wan X, O'quinn RP, Pierce HL, Joglekar AP, Gall WE, Deluca JG, Carroll CW et al (2009) Protein architecture of the human kinetochore microtubule attachment site. Cell 137(4):672–684. doi:10. 1016/j.cell.2009.03.035 Wang H-W, Long S, Ciferri C, Westermann S, Drubin D, Barnes G, Nogales E (2008) Architecture and flexibility of the yeast Ndc80 kinetochore complex. J Mol Biol 383(4):894–903. doi:10.1016/j. jmb.2008.08.077 Wei RR, Schnell JR, Larsen NA, Sorger PK, Chou JJ, Harrison SC (2006) Structure of a central component of the yeast kinetochore: the Spc24p/Spc25p globular domain. Structure (London, England : 1993) 14(6):1003–1009. doi:10.1016/j.str.2006.04.007 Wei RR, Al-Bassam J, Harrison SC (2007) The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment. Nat Struct Mol Biol 14(1):54–59. doi:10.1038/nsmb1186 Westermann S, Schleiffer A (2013) Family matters: structural and functional conservation of centromere-associated proteins from yeast to humans. Trends Cell Biol 23(6):260–269. doi:10.1016/j.tcb.2013. 01.010 Westermann S, Avila-Sakar A, Wang H-W, Niederstrasser H, Wong J, Drubin DG, Nogales E, Barnes G (2005) Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex. Mol Cell 17(2):277–290. doi:10.1016/j.molcel.2004. 12.019 Westermann S, Wang H-W, Avila-Sakar A, Drubin DG, Nogales E, Barnes G (2006) The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends. Nature 440(7083):565–569. doi:10.1038/nature04409 Wigge PA, Jensen ON, Holmes S, Souès S, Mann M, Kilmartin JV (1998) Analysis of the saccharomyces spindle pole by matrixassisted laser desorption/ionization (MALDI) mass spectrometry. J Cell Biol 141(4):967–977 Winey M, O'Toole ET (2001) The spindle cycle in budding yeast. Nat Publ Group 3(1):E23–E27. doi:10.1038/35050663 Winey M, Morgan GP, Straight PD, Giddings TH, Mastronarde DN (2005) Three-dimensional ultrastructure of saccharomyces cerevisiae meiotic spindles. Mol Biol Cell 16(3):1178–1188. doi: 10.1091/mbc.E04-09-0765 Wong J, Nakajima Y, Westermann S, Shang C, Kang J s, Goodner C, Houshmand P et al (2007) A protein interaction map of the mitotic spindle. Mol Biol Cell 18(10):3800–3809. doi:10.1091/mbc.E0706-0536 Woodruff JB, Drubin DG, Barnes G (2010) Mitotic spindle disassembly occurs via distinct subprocesses driven by the anaphase-promoting complex, Aurora B kinase, and kinesin-8. J Cell Biol 191(4):795– 808. doi:10.1083/jcb.201006028 Yamagishi Y, Yang C-H, Tanno Y, Watanabe Y (2012) MPS1/Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components. Nat Cell Biol 14(7):746–752. doi:10.1038/ncb2515 Zimniak T, Fitz V, Zhou H, Lampert F, Opravil S, Mechtler K, StoltBergner P, Westermann S (2012) Spatiotemporal regulation of Ipl1/Aurora activity by direct Cdk1 phosphorylation. Curr Biol CB 22(9):787–793. doi:10.1016/j.cub.2012.03.007