Kinetochore Recruitment of CENP-F Illustrates How Paralog Divergence

Home , Kinetochore

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1 Kinetochore recruitment of CENP-F illustrates how paralog

2 divergence shapes kinetochore composition and function

4 Giuseppe Ciossani (1,*), Katharina Overlack (1,*), Arsen Petrovic (1), Pim Huis in ‘t 5 Veld (1), Carolin Körner (1), Sabine Wohlgemuth (1), Stefano Maffini (1) & Andrea 6 Musacchio (1,2,#)

8 (1) Department of Mechanistic Cell Biology, Max Planck Institute of Molecular 9 Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany

10 (2) Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, 11 Universitätsstraße, 45141 Essen, Germany

13 # Correspondence: [email protected]

14 * These authors contributed equally to this work

16 Keywords: kinetochore, centromere, chromosome, mitosis, cell cycle, CENP-E, CENP- 17 F, Bub1, BubR1, protein electroporation, spindle assembly checkpoint, mitotic 18 checkpoint, paralogs, duplication

20 Short title: Mechanism of kinetochore recruitment of CENP-F

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22 The metazoan proteins CENP-E and CENP-F are components of a fibrous layer 23 of mitotic kinetochores named the corona. Several features suggest that CENP-E 24 and CENP-F are paralogs: they are very large (approximately 2700 and 3200 25 residues, respectively), rich in predicted coiled-coil structure, C-terminally 26 prenylated, and endowed with microtubule-binding sites at their termini. In 27 addition, CENP-E contains an ATP-hydrolyzing motor domain that promotes 28 microtubule plus-end directed motion. Here, we show that CENP-E and CENP- 29 F are recruited to mitotic kinetochores independently of the Rod-Zwilch-ZW10 30 (RZZ) complex, the main corona constituent. We identify selective interactions of 31 CENP-E and CENP-F respectively with BubR1 and Bub1, paralogous proteins 32 involved in mitotic checkpoint control and chromosome alignment. While BubR1 33 is dispensable for kinetochore localization of CENP-E, Bub1 is stringently 34 required for CENP-F localization. Through biochemical reconstitution, we 35 demonstrate that the CENP-E:BubR1 and CENP-F:Bub1 interactions are direct 36 and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F 37 and a kinase domain in BubR1 or Bub1. Our findings are consistent with the 38 existence of ‘pseudo-symmetric’, paralogous Bub1:CENP-F and BubR1:CENP-E 39 axes, supporting evolutionary relatedness of CENP-E and CENP-F.

41 Introduction

42 The segregation of chromosomes from a mother cell to its daughters during cell division 43 relies on the function of specialized protein complexes, the kinetochores, as bridges 44 linking chromosomes to spindle microtubules (Musacchio and Desai, 2017). 45 Kinetochores are built on specialized chromosome loci known as centromeres, whose 46 hallmark is the enrichment of the histone H3 variant centromeric protein A (CENP-A, 47 also known as CenH3) (Earnshaw, 2015). CENP-A seeds kinetochore assembly by 48 recruiting CENP-C, CENP-N, and their associated protein subunits in the constitutive 49 centromere associated network (CCAN) (Cheeseman and Desai, 2008). These 50 centromere proximal ‘inner kinetochore’ subunits, in turn, recruit the centromere distal 51 ‘outer kinetochore’ subunits of the KMN complex (Knl1 complex, Mis12 complex, 52 Ndc80 complex), which promote ‘end-on’ microtubule binding and control the spindle 53 assembly checkpoint (SAC) (Musacchio and Desai, 2017).

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54 Early in mitosis, prior to end-on microtubule attachment, an additional fibrous structure, 55 the kinetochore corona, assembles as the outermost layer of the kinetochore (Figure 1A) 56 (Jokelainen, 1967; Magidson et al., 2015; McEwen et al., 1993; Rieder, 1982). The 57 corona’s main constituent is a trimeric protein complex named RZZ [from the name of 58 the fruit fly genes Rough Deal (ROD), Zwilch, and Zeste White 10 (ZW10)]. The ROD 59 subunit is structurally related to proteins that oligomerize near biological membranes to 60 promote vesicular trafficking, including Clathrin (Civril et al., 2010; Mosalaganti et al., 61 2017), leading to hypothesize that corona assembly results from RZZ polymerization 62 (Mosalaganti et al., 2017). The interaction of the RZZ complex with an adaptor subunit 63 named Spindly, in turn, further recruits the microtubule minus-end directed motor 64 cytoplasmic Dynein and its binding partner Dynactin to kinetochores, as well as the 65 Mad1:Mad2 complex, which is crucially required for SAC signaling (Barisic et al., 2010; 66 Basto et al., 2004; Buffin et al., 2005; Caldas et al., 2015; Chan et al., 2009; 67 Cheerambathur et al., 2013; Gama et al., 2017; Gassmann et al., 2008; Gassmann et al., 68 2010; Griffis et al., 2007; Howell et al., 2001; Kops et al., 2005; Matson and Stukenberg, 69 2014; Mische et al., 2008; Silio et al., 2015; Sivaram et al., 2009; Starr et al., 1998; Varma 70 et al., 2008; Williams et al., 1996; Wojcik et al., 2001; Yamamoto et al., 2008; Zhang et al., 71 2015).

72 Corona assembly leads to a broad expansion of the microtubule-binding interface of 73 kinetochores that may promote initial microtubule capture, congression towards the 74 metaphase plate, and SAC signaling (Basto et al., 2000; Buffin et al., 2005; Hoffman et al., 75 2001; Kops et al., 2005; Magidson et al., 2011; Magidson et al., 2015; Wynne and 76 Funabiki, 2015). Differently from the mature end-on attachments, initial attachments of 77 kinetochores engage the microtubule lattice and are therefore defined as lateral or side- 78 on. CENP-E, a kinesin-7 family member, plays a crucial role at this stage. Its inhibition 79 or depletion lead to severe and persistent chromosome alignment defects, with numerous 80 chromosomes failing to congress towards the spindle equator and stationing near the 81 spindle poles, causing chronic activation of the SAC (Kapoor et al., 2006; Kuhn and 82 Dumont, 2017; Magidson et al., 2011; Magidson et al., 2015; Putkey et al., 2002; Schaar et 83 al., 1997; Wood et al., 1997; Yao et al., 2000; Yen et al., 1991). Human CENP-E consists 84 of 2701 residues (Figure 1B) (Yen et al., 1992). Besides the globular N-terminal motor 85 domain, the rest of the CENP-E sequence forms a flexible and highly elongated (~230 86 nm) coiled-coil (Kim et al., 2008). The kinetochore-targeting domain of CENP-E

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87 encompasses residues 2126-2476 and is followed by a microtubule-binding region 88 (Figure 1B) (Chan et al., 1998; Liao et al., 1994). The distribution of CENP-E in an outer 89 kinetochore crescent shape similar to that of the RZZ supports the notion that CENP-E 90 is part of the kinetochore corona, but its persistence at kinetochores after disappearance 91 of the corona suggests a corona-independent localization mechanism (Cooke et al., 1997; 92 Hoffman et al., 2001; Magidson et al., 2015; Wynne and Funabiki, 2015; Yao et al., 1997; 93 Yen et al., 1992).

94 CENP-F (also known as Mitosin, 3210 residues in humans) is also a kinetochore corona 95 constituent during early mitosis that persists at kinetochores after corona shedding 96 (Casiano et al., 1993; Hussein and Taylor, 2002; Liao et al., 1995; Rattner et al., 1993; 97 Zhu, 1999; Zhu et al., 1995a; Zhu et al., 1995b). Like CENP-E, CENP-F is also highly 98 enriched in predicted coiled-coil domains (Figure 1B), but lacks an N-terminal motor 99 domain. Rather, it contains two highly basic microtubule-binding domains in the N- 100 terminal 385 residues and in the C-terminal 187 residues (Feng et al., 2006; Musinipally et 101 al., 2013; Volkov et al., 2015). Similarly to CENP-E, the kinetochore recruitment domain 102 of CENP-F is positioned in proximity of the C-terminus [encompassing residues 2581- 103 3210, the minimal domain tested for this function to date (Hussein and Taylor, 2002; 104 Zhu, 1999; Zhu et al., 1995a)]. The apparent similarity of CENP-E and CENP-F extends 105 to the fact that they are both post-translationally modified with a farnesyl prenol lipid 106 chain (isoprenoid) on canonical motifs positioned in their C-termini (Ashar et al., 2000). 107 These modifications contribute to kinetochore recruitment of CENP-E and CENP-F, 108 albeit to extents that differ in various reports (Holland et al., 2015; Hussein and Taylor, 109 2002; Moudgil et al., 2015; Schafer-Hales et al., 2007).

110 Previous studies identified CENP-F and BubR1 as binding partners of CENP-E (Chan 111 et al., 1998; Mao et al., 2003; Yao et al., 2000). BubR1 is a crucial constituent of the SAC, 112 a molecular network required to prevent premature mitotic exit (anaphase) in cells 113 retaining unattached or improperly attached kinetochores (Musacchio, 2015). BubR1 is a 114 subunit of the mitotic checkpoint complex (MCC), the SAC effector (Sudakin et al., 115 2001). Its structure is a constellation of domains and interaction motifs required to 116 mediate binding to other SAC proteins, and terminates in a kinase domain (Musacchio, 117 2015). It has been proposed that CENP-E stimulates BubR1 activity, and that 118 microtubule capture silences it (Mao et al., 2003; Mao et al., 2005). Later studies, 119 however, identified BubR1 as an inactive pseudokinase (Breit et al., 2015; Suijkerbuijk et

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120 al., 2012), and therefore the significance of CENP-E microtubule binding for the role of 121 BubR1 in the SAC remains unclear. Depletion or inactivation of CENP-E, however, is 122 compatible with a robust mitotic arrest (Schaar et al., 1997; Yen et al., 1991).

123 A yeast 2-hybrid (Y2H) interaction of CENP-F and Bub1 has also been reported but 124 never validated experimentally (Chan et al., 1998). Bub1, a paralog of BubR1, retained 125 genuine kinase activity in humans and it plays a function at the interface of mitotic 126 checkpoint signaling and kinetochore microtubule attachment (Raaijmakers et al., 2018; 127 Suijkerbuijk et al., 2012). Suggesting that the interaction of Bub1 and CENP-F is 128 functionally important, previous studies identified Bub1 as being essential for 129 kinetochore recruitment of CENP-F (Johnson et al., 2004; Klebig et al., 2009; Liu et al., 130 2006; Raaijmakers et al., 2018).

131 In our previous studies, we characterized in molecular detail how sequence divergence 132 impacted the protein interaction potential of the human Bub1 and BubR1 paralogs 133 (Overlack et al., 2017; Overlack et al., 2015). We described a molecular mechanism that 134 explains how Bub1, through an interaction with a phospho-aminoacid adaptor named 135 Bub3, can interact with kinetochores and promote the recruitment of BubR1 via a 136 pseudo-dimeric interface (Overlack et al., 2017; Overlack et al., 2015; Primorac et al., 137 2013). In view of these previous studies, here we have dissected the molecular basis of 138 the interactions of BubR1 and Bub1 with CENP-E and CENP-F. We provide strong 139 evidence for the sub-functionalization of these paralogous protein pairs.

140

141 Results and Discussion

142 Independent kinetochore localization of CENP-E, CENP-F, and the RZZ

143 Using specific antibodies (see Methods), we assessed the timing and specificity of 144 kinetochore localization of CENP-E, CENP-F, Zwilch, and Mad1. CENP-E showed 145 perinuclear localization until prometaphase, when it first appeared at kinetochores. It 146 persisted there until metaphase, and was then found at the spindle midzone after 147 anaphase onset (Figure 1C). This localization, which corresponds to previous 148 descriptions (Yen et al., 1991; Yen et al., 1992), is reminiscent of that of chromosome 149 passenger proteins (Earnshaw and Bernat, 1991). CENP-F, on the other hand, localized 150 to kinetochores already in prophase, where it was also temporarily visible at the nuclear 151 envelope, and persisted there until anaphase, with progressive weakening and dispersion

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152 (Figure 1D), as noted previously (Baffet et al., 2015; Bolhy et al., 2011; Hu et al., 2013; 153 Liao et al., 1995; Rattner et al., 1993). Also Zwilch (a subunit of the RZZ complex) and 154 Mad1 were already visible at kinetochores in prophase, but they became invisible at these 155 structures upon achievement of metaphase (Figure 1S1A-B), in agreement with the 156 notion that the corona becomes dissolved upon microtubule attachment (see 157 Introduction).

158 Thus, both CENP-E and CENP-F continue to localize to kinetochores well beyond the 159 timing of removal of the RZZ complex and Mad1, suggesting that they can be retained at 160 kinetochores independent of the corona. To test this directly, we identified conditions 161 for optimal depletion of Zwilch, CENP-E, or CENP-F by RNA interference (RNAi) 162 (Figure 2S1A-J). Depletion of Zwilch resulted in depletion of Mad1 from kinetochores 163 (Figure 2S1H-J), but left the kinetochore levels of CENP-E essentially untouched (Figure 164 2A). This observation is in agreement with previous studies showing that Mad2, whose 165 kinetochore localization requires Mad1 (Martin-Lluesma et al., 2002; Sharp-Baker and 166 Chen, 2001), is also depleted from kinetochores upon depletion of other RZZ subunits 167 (Caldas et al., 2015; Gassmann et al., 2008; Raaijmakers et al., 2018). The observation that 168 CENP-E retains kinetochore localization under conditions in which Mad1 appears to 169 become depleted seems inconsistent with a recent report proposing that Mad1 is 170 required for kinetochore localization of CENP-E (Akera et al., 2015), but agrees with 171 previous reports that failed to detect consequences on CENP-E localization upon 172 depletion of Mad1 (Martin-Lluesma et al., 2002; Sharp-Baker and Chen, 2001).

173 Conversely, depletion of CENP-E or CENP-F in HeLa cells, or even their co-depletion 174 (Figure 2S2A), did not influence the kinetochore localization of Zwilch or Mad1 (Figure 175 2B-F and Figure 2S3A-D), as observed previously (Martin-Lluesma et al., 2002; Yang et 176 al., 2005). We note, however, that depletion of CENP-E in DLD-1 cells was reported to 177 have deleterious effects on Mad2 localization, while Mad1 or RZZ subunits were not 178 tested (Johnson et al., 2004). Based on these results, we conclude that kinetochore 179 localization of CENP-E and CENP-F does not require the kinetochore corona, nor does 180 it influence corona assembly. We also observed that CENP-E and CENP-F were not 181 reciprocally affected by their depletion (Figure 2G-H), indicating that they localize (at 182 least largely) independently to kinetochores, as previously suggested (Yao et al., 2000).

183 In most cells analyzed, depletion of CENP-F resulted in apparently normal metaphase 184 alignment, with only a slight increase in the fraction of cells presenting metaphase

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185 alignment defects (Figure 2S3B and Figure 2S4). In agreement with the effects of CENP- 186 F depletion being mild, duration of mitosis (caused by spindle assembly checkpoint 187 activation) was only marginally increased in cells depleted of CENP-F (Figure 2S4D). 188 Similarly mild effects from depleting CENP-F were observed previously (Bomont et al., 189 2005; Feng et al., 2006; Holt et al., 2005; Raaijmakers et al., 2018; Yang et al., 2005). On 190 the other hand, depletion of CENP-E (with or without additional depletion of CENP-F) 191 led to conspicuous chromosome alignment problems (Figure 2S3C-D), as reported 192 previously (Kapoor et al., 2006; Kuhn and Dumont, 2017; Magidson et al., 2011; 193 Magidson et al., 2015; Putkey et al., 2002; Schaar et al., 1997; Wood et al., 1997; Yao et 194 al., 2000; Yen et al., 1991).

195

196 CENP-E binds to the BubR1 pseudokinase domain

197 Previous studies identified a kinetochore-binding region in residues 2126-2476 of CENP- 198 E (Chan et al., 1998). By expression in insect cells, we generated a recombinant version 199 of a larger fragment of CENP-E (residues 2070-C) encompassing this region fused to 200 eGFP (eGFP-CENP-E2070-C) and purified it to homogeneity. After electroporation in 201 mitotic cells arrested by addition of the microtubule-depolymerizing drug nocodazole, 202 cells were fixed to assess the localization of eGFP-CENP-E2070-C. eGFP-CENP-E2070-C 203 localized robustly to mitotic kinetochores (Figure 3A), adopting the typical crescent-like 204 shape previously attributed to the corona (Hoffman et al., 2001; Magidson et al., 2015). 205 An equivalent mutant construct in which Cys2697 had been mutated to alanine to 206 prevent farnesylation also localized to kinetochores, even if at generally lower levels and 207 without showing a crescent-like distribution, suggesting that farnesylation is not strictly 208 required for kinetochore localization of CENP-E but that it might contribute to an 209 unknown aspect of corona assembly.

210 In previous yeast 2-hybrid (Y2H) analyses, a CENP-E segment encompassing residues 211 1958-2662 was found to interact with residues 410-1050 of BubR1 (Chan et al., 1998; 212 Yao et al., 2000). Even if shorter by more than 100 residues at the N-terminal end, 213 eGFP-CENP-E2070-C interacted directly with the dimeric BubR1:Bub3 complex in size 214 exclusion chromatography (SEC) analyses (Figure 3B), as evidenced by the shift in 215 elution volume of both proteins when combined at 16 µM and 4 µM concentration, 216 respectively. Similar observations were made when we mixed CENP-E2070-C with the

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217 BubR1 pseudokinase domain (KinD, residues 705–1050) (Figure 3C). eGFP-CENP- 218 E2070-C, however, did not interact with the paralogous Bub1:Bub3 dimer (Figure 3D). In 219 analytical centrifugation (AUC) sedimentation velocity experiments in which we 220 monitored the sedimentation of eGFP-CENP-E, addition of unlabeled BubR1:Bub3 at 221 3-fold higher concentration caused a complete shift of eGFP-CENP-E to a species with 222 higher sedimentation coefficient (S), indicative of complex formation (Figure 3E). The 223 high frictional ratio of this sample (an indication that the CENP-E structure is very 224 elongated, a consequence of its large coiled-coil content) prevented a quantitative 225 estimate of molecular mass. The analysis, however, strongly suggest that eGFP-CENP- 226 E2070-C adopts the highly elongated conformation of coiled-coils, as shown previously for 227 recombinant full length CENP-E from Xenopus laevis (Kim et al., 2008). Thus, a minimal 228 segment of CENP-E capable of kinetochore localization interacts directly with the 229 BubR1:Bub3 complex, and the BubR1 pseudokinase domain is sufficient for this 230 interaction, at least at the relatively high concentration of the SEC assay. In agreement 231 with our own previous studies (Breit et al., 2015), BubR1 did not show any catalytic 232 activity, nor did it become active in presence of eGFP-CENP-E2070-C (unpublished data).

233 In a previous study in egg extracts of Xenopus laevis, depletion of BubR1 was shown to 234 prevent kinetochore localization of CENP-E, an effect that could be rescued by re- 235 addition of wild type BubR1, but not of a deletion mutant lacking the kinase domain 236 (Mao et al., 2003). CENP-E kinetochore levels were also reduced in DLD-1 cells upon 237 depletion of BubR1 by RNAi (Johnson et al., 2004).

238 We therefore asked if BubR1 was also important for CENP-E recruitment in HeLa cells. 239 Furthermore, in view of evidence that Bub1 is required for kinetochore recruitment of 240 BubR1 (Klebig et al., 2009; Liu et al., 2006; Logarinho et al., 2008; Overlack et al., 2015), 241 we also monitored localization of CENP-E upon depletion of Bub1. Contrarily to the 242 previous observations in frogs and DLD-1 cells, but in agreement with other studies in 243 HeLa cells (Akera et al., 2015; Lampson and Kapoor, 2005; Liu et al., 2006), RNAi-based 244 depletion of Bub1 or BubR1 did not result in obvious adverse effects on the kinetochore 245 localization of CENP-E, even after co-depletion of Zwilch (Figure 3F-G and Figure 246 2S2B-E). These observations suggest that CENP-E, at least in HeLa cells, becomes 247 recruited through a different pathway that does not involve Bub1 and BubR1. After 248 application of highly specific small-molecule inhibitors, we found CENP-E kinetochore 249 localization to depend on the kinase activity of Aurora B and (to a lower extent) of

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250 Mps1, but not of Bub1 or of Plk1 (Figure 3S1A-D). The dependence of CENP-E on 251 Aurora B kinase activity for kinetochore localization, together with the central spindle 252 co-localization at anaphase of CENP-E with the chromosome passenger complex (CPC, 253 the catalytic subunit of which is Aurora B), leads to speculate that these proteins interact, 254 a hypothesis that will need to be formally tested in the future.

255

256 CENP-F binds to the Bub1 kinase domain

257 Next, we asked how CENP-F becomes recruited to kinetochores. CENP-F recruitment 258 was strictly dependent on the kinase activity of Aurora B, partly dependent on that of 259 Mps1 and Plk1, and not dependent on that of Bub1 (Figure 4A and Figures 4S1). This 260 pattern of kinetochore localization is reminiscent of that of Bub1, which has been 261 previously shown to be important for CENP-F kinetochore recruitment (Johnson et al., 262 2004; Klebig et al., 2009; Liu et al., 2006; Raaijmakers et al., 2018). In agreement with 263 these previous studies, RNAi-based depletion of Bub1 resulted in complete ablation of 264 CENP-F from kinetochores (Figure 4C, see panel I for quantification).

265 By expression in insect cells, we generated a recombinant fragment of CENP-F 266 encompassing its previously identified kinetochore-binding domain (residues 2688-C) 267 (Hussein and Taylor, 2002; Zhu, 1999; Zhu et al., 1995a) fused to an N-terminal 268 mCherry tag. In SEC experiments, mCherry-CENP-F2688-C bound Bub1:Bub3 directly, as 269 indicated by its altered elution volume in presence of the CENP-F construct (Figure 4D; 270 note that mCherry-CENP-F2688-C is highly elongated, as shown below, and therefore its 271 hydrodynamic radius, which determines elution volume in SEC experiments, is unlikely 272 to change as a result of an interaction with Bub1:Bub3). On the other hand, mCherry- 273 CENP-F2688-C failed to interact with Bub11-409:Bub3, where the Bub1 deletion mutant 274 Bub11-409 lacks a central region of Bub1 and its kinase domain (Figure 4E). Indeed, 275 mCherry-CENP-F2688-C bound the Bub1 kinase domain (Bub1KinD, residues 725–1085; 276 Figure 4F). Conversely, mCherry-CENP-F2688-C did not interact with the BubR1:Bub3 277 complex (Figure 4G). Thus, the kinetochore-targeting domain of CENP-F interacts 278 directly with the Bub1:Bub3 complex, and the kinase domain appears to be necessary 279 and partly sufficient for this interaction.

280 In agreement with these in vitro findings, we observed robust kinetochore localization of 281 endogenous CENP-F in HeLa cells previously depleted of Bub1 by RNAi and expressing

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282 an RNAi-resistant GFP-Bub1 transgene, while CENP-F kinetochore localization 283 appeared entirely compromised in Bub1-depleted cells expressing GFP-Bub11-788, which 284 lacks exclusively the Bub1 kinase domain (Figure 4H-I). Collectively, our observations 285 indicate that the kinase domain of Bub1 is sufficient for a direct interaction with CENP- 286 F in vitro, and necessary for kinetochore recruitment of CENP-F in HeLa cells. A very 287 recent study identified a similar requirement for the kinase domain of Bub1 in 288 kinetochore recruitment of CENP-F in HAP1 cells (Raaijmakers et al., 2018).

289

290 CENP-F dimerization is important for Bub1 binding

291 The mCherry-CENP-F2688-C construct that interacted with Bub1:Bub3 in SEC 292 experiments also localized to mitotic kinetochores when electroporated in HeLa cells 293 (Figure 5A). A farnesylation mutant of this construct on which Cys3207 had been 294 mutated to alanine retained kinetochore localization, although not as robustly as the wild 295 type counterpart (Figure 5A). This result suggests that farnesylation is not strictly 296 required for kinetochore recruitment of CENP-F, as already observed with CENP-E 297 (Figure 3A). Collectively, our results with electroporated farnesylation mutants of CENP- 298 E and CENP-F are in agreement with results obtained with farnesyl transferase 299 inhibitors, in which only partial repression of kinetochore recruitment of CENP-E and 300 CENP-F was observed (Holland et al., 2015).

301 By rotary shadowing electron microscopy (EM), which is particularly suited to the study 302 of elongated coiled-coil proteins, mCherry-CENP-F2688-C had the appearance of a highly 303 elongated (~40 nm) rod. Most likely, the latter corresponds to a predicted coiled-coil 304 comprised between residues 2688 and ~3000, flanked on one side by two globular 305 domains, most likely corresponding to mCherry, and on the other side by disordered 306 fragments corresponding to the last ~200 residues and containing the C-terminal 307 microtubule-binding domains (Figure 5B) (Feng et al., 2006; Musinipally et al., 2013; 308 Volkov et al., 2015). Collectively, these observations suggest that CENP-F2688-C contains a 309 parallel dimeric coiled-coil, like the one previously identified in CENP-E (Kim et al., 310 2008).

311 On the basis of previous studies implicating Cys2864 in kinetochore recruitment of mouse 312 CENP-F (Zhu, 1999), we generated a mutant version of human mCherry-CENP-F2688-C 313 in which the equivalent residue, Cys2961, was mutated to serine (our residue numbering is

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314 in accordance with the 3210-residue human CENP-F sequence in Uniprot). While wild 315 type mCherry-CENP-F2688-C interacted with Bub1:Bub3 in SEC experiments, as already 316 shown, the interaction was at least partially impaired when the mCherry-CENP-F2688- 317 C/C2961S mutant was analyzed, confirming the role of Cys2961 in kinetochore localization 318 and implicating this residue in the interaction with Bub1 (Figure 5C-D). Furthermore, 319 mCherry-CENP-F2688-C did not interact with GFP-CENP-E2070-C in SEC experiments, as 320 predicted based on previous work identifying the CENP-E-binding region of CENP-F 321 within a segment (residues 1804-2104) that precedes and is not included in CENP-F2688-C 322 (Figure 5E) (Chan et al., 1998; Yao et al., 2000).

323 The effects of the Cys2961 mutation made us ask if we could identify a minimal Bub1- 324 binding domain of CENP-F. For this, we further trimmed CENP-F. CENP-F2866-2990, 325 which is entirely encompassed within the predicted coiled-coil of CENP-F, appeared 326 dimeric by sedimentation velocity AUC and retained the ability to bind to the Bub1 327 kinase domain in a SEC experiment (Figure 6A-B). Similar results were obtained with an 328 even shorter CENP-F fragment, CENP-F2922-2990 (Figure 6C-D). CENP-F2950-2990, on the 329 other hand, appeared monomeric in AUC runs and was unable to interact with the Bub1 330 kinase domain (Figure 6E-F). These observations do not allow us to resolve whether 331 impaired binding to the Bub1 kinase domain CENP-F2950-2990 is due to loss of 332 dimerization or to trimming of residues directly involved in the interaction, but identify 333 CENP-F2922-2990 as a minimal Bub1-binding fragment of CENP-F. In agreement with 334 these observations, mCherry-CENP-F2866-2990 and mCherry-CENP-F2922-2990 labeled 335 kinetochores after electroporation in HeLa cells, albeit weakly in comparison to 336 mCherry-CENP-F2866-C, whereas mCherry-CENP-F2950-2990 did not localize to 337 kinetochores (Figure 6G).

338

339 Conclusions

340 Previous studies on human Bub1 and BubR1, including our own work, demonstrated 341 that these paralogs sub-functionalized in various ways, including 1) the selective 342 inactivation of the kinase domain in BubR1; 2) the development of phospho-aminoacid 343 recognition modules that contribute to the ability of Bub3 to recognize distinct 344 substrates; and 3) the interaction with distinct binding partners that subtends to distinct 345 functions in chromosome alignment and mitotic checkpoint signaling (Figure 7)

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346 (Overlack et al., 2017; Overlack et al., 2015; Primorac et al., 2013; Suijkerbuijk et al., 347 2012; van Hooff et al., 2017).

348 In this study, we report an additional aspect of this sub-functionalization, and show that 349 the kinase domains of BubR1 and Bub1 interact respectively with C-terminal regions of 350 CENP-E and CENP-F that encompass the kinetochore-targeting domains of these 351 proteins. Both interactions are direct and were reproduced with recombinant proteins. 352 Neither interaction appears to be crucially required for downstream signaling events. In 353 the BubR1 case, it appears well established that deletion of the pseudokinase domain in 354 human cells is compatible with its functions in the SAC as a subunit of the MCC, the 355 SAC effector (Musacchio, 2015). We, and others, have also shown that depletion of 356 BubR1 does not affect CENP-E kinetochore localization in HeLa cells [this study and 357 (Akera et al., 2015; Lampson and Kapoor, 2005)]. In Xenopus egg extracts, however, the 358 kinase domain of BubR1 has been implicated in CENP-E recruitment and this 359 interaction has been shown to be important for SAC silencing (Mao et al., 2003). Given 360 the very complex evolutionary history of the Bub1 and BubR1 paralogs or of the 361 singleton from which they originate, these apparent differences may genuinely reflect 362 different evolutionary paths in these organisms (Suijkerbuijk et al., 2012; van Hooff et al., 363 2017). A question for future work that this study raises regards the detailed mechanism 364 of kinetochore recruitment of CENP-E in human cells, which remains unknown.

365 The role of Bub1 in CENP-F kinetochore recruitment was already established in 366 previous work (Berto et al., 2018; Johnson et al., 2004; Liu et al., 2006; Raaijmakers et al., 367 2018), and a very recent study implicated the Bub1 kinase domain in CENP-F 368 kinetochore recruitment (Raaijmakers et al., 2018). Here, we have extended this previous 369 study by showing that the interaction of the Bub1 kinase domain and CENP-F is direct, 370 and by identifying a minimal CENP-F domain involved in this interaction and capable of 371 kinetochore localization. Our identification of a minimal kinetochore-targeting domain 372 of HsCENP-F within residues 2922-2990 agrees with a previous study that made use of 373 CENP-F deletion mutants (Zhu, 1999). It also agrees with another study, currently in 374 press, that identified distinct binding domains in CENP-F for nuclear envelope and for 375 kinetochore localization (Berto et al., 2018). Specifically, residues 2655 to 2860 of mouse 376 CENP-F (corresponding to residues 2866-3072 of HsCENP-F) were sufficient for 377 kinetochore recruitment (Berto et al., 2018). Within this fragment, a specialized N- 378 terminal sub-domain (residues 2655-2723, corresponding to HsCENP-F residues 2866-

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379 2933) bound Nup133, a subunit of the nuclear pore complex Nup107-Nup160, and 380 mediated CENP-F recruitment to the nuclear envelope shortly before mitosis, but was 381 not required for kinetochore recruitment. A specialized C-terminal sub-domain (residues 382 2724-2860, corresponding to HsCENP-F residues 2934-3072), on the other hand, was 383 required for kinetochore recruitment (Berto et al., 2018). As previous studies identified 384 CENP-E as binding partner of CENP-F (Chan et al., 1998; Mao et al., 2003; Yao et al., 385 2000), CENP-E may reinforce binding of CENP-F to kinetochores, as shown by Berto 386 and colleagues (Berto et al., 2018). However, there is now sufficient evidence to conclude 387 that this interaction is clearly not sufficient to promote stable binding of CENP-F to 388 kinetochores in absence of Bub1.

389 Our observation that CENP-F depletion results in very mild chromosome alignment 390 defects, in line with other reports (see e.g. (Raaijmakers et al., 2018)), is surprising. 391 CENP-F has been implicated in Dynein recruitment and regulation through a pathway 392 involving Nde1, Ndel1, and Lis1, the product of the lissencephaly type 1 gene (Baffet et 393 al., 2015; Bolhy et al., 2011; Hu et al., 2013; Simoes et al., 2018; Vergnolle and Taylor, 394 2007). However, CENP-F is not sufficient for stable kinetochore recruitment of Dynein, 395 as it does not seem to be able to complement the very strong reduction or loss of 396 kinetochore Dynein in cells depleted of the RZZ complex or Spindly [see for instance 397 (Barisic et al., 2010; Chan et al., 2009; Gassmann et al., 2010)]. The latter appears 398 therefore to be the dominant factor in Dynein recruitment to kinetochores. It is 399 plausible, however, that the consequences of CENP-F depletion are exacerbated by 400 concomitant depletion of RZZ (Simoes et al., 2018).

401 As already discussed in the Introduction, various common features of CENP-E and 402 CENP-F support the speculation that they are distantly related paralogs. Their ability to 403 interact with the kinase domains of BubR1 and Bub1, themselves paralogs, lends strong 404 further credit to this hypothesis. Apparent lack of strong functional consequences from 405 disrupting these interactions may indicate that they may have become vestigial in some 406 species. It is also possible, however, that these interactions play more important roles 407 during development or in specific cell types. The dissection described here will allow 408 testing this hypothesis in future work.

409

410 Acknowledgments

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411 We thank all members of the Musacchio laboratory for helpful discussions and 412 comments. A.M. gratefully acknowledges the Max Planck Society, the European 413 Research Council (ERC) Advanced Investigator Grant RECEPIANCE (proposal 414 number nº 669686), and the European Union’s Horizon 2020 research and innovation 415 programme under the Marie Sklodowska-Curie grant agreement No 675737. We are very 416 grateful to Dr. Valerie Doye and her collaborators for sharing with us unpublished 417 results.

418

419 Material & Methods

420 Plasmids The codon optimized cDNAs of Homo sapiens CENP-E (Q02224) and CENP- 421 F (P49454) were synthetized at GeneWiz. CENP-E and CENP-F constructs were 422 subcloned respectively in pLIB-eGFP and pLIB-mCherry or pET-mCherry, modified 423 versions of the pLIB (Weissmann et al., 2016) and pET-28 vectors for expression of

424 proteins with N-terminal PreScission-cleavable His6-eGFP or His6-mCherry tag. Site- 425 directed mutagenesis was performed by PCR (Sawano and Miyawaki, 2000). All 426 constructs were sequence verified. The vectors for the co-expression of full length Bub1 427 and BubR1 proteins with Bub3, as well as that for the Bub1 & BubR1 constructs were 428 described previously (Breit et al., 2015; Overlack et al., 2015).

429 Protein expression and purification Expression and purification of eGFP-CENP- 430 E2070-C and mCherry-CENP-F2866-C wild-type and mutants was carried out in insect cells 431 using a pBig system (Weissmann et al., 2016). Baculoviruses were generated in Sf9 cells 432 and use to infect Tnao38 cells for 48-96 hours at 27°C. Cells were collected by 433 centrifugation, washed in PBS and then frozen at -80ºC. CENP-E expressing cell pellets 434 were resuspended in lysis buffer (50 mM Sodium Phosphate buffer pH 8.0, 500 mM 435 NaCl, 5 % (w/v) glycerol and 0.5 mM TCEP) supplemented with protease inhibitor 436 cocktail, lysed by sonication and cleared by centrifugation at 100.000 g at 4°C. The 437 supernatant was filtered and loaded on a 5 ml HisTrap FF column (GE Healthcare) 438 equilibrated in lysis buffer. After washing with lysis buffer, the protein was eluted with a 439 linear gradient of 0-250 mM imidazole in 10 column volumes. The fractions of interest 440 were pooled, concentrated with a 50 kDa cut-off Amicon concentrator (Millipore) and 441 loaded onto a Superose 6 Increase 10/300 (GE Healthcare) equilibrated in SEC buffer 442 (50 mM Hepes pH 8.0, 200 mM NaCl, 5% (w/v) glycerol and 0.5 mM TCEP). CENP-E

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443 containing fractions were concentrated, flash-frozen in liquid nitrogen and stored at - 444 80°C. The purification protocol for the mCherry-CENP-F2866-C constructs is identical to 445 that of eGFP-CENP-E2070-C, but the lysis and the SEC buffers were at pH 7.0.

446 The constructs mCherry-CENP-F2866-2990, mCherry-CENP-F2922-2990 and mCherry-CENP- 2950-2990 447 F were expressed in E. coli BL21 (DE3) RP plus cells grown at 37ºC to O.D.600 = 2 448 and then induced with 0.25 mM IPTG for 16 h at 25ºC. Cell were collected by 449 centrifugation, washed in PBS and then frozen at -80ºC. Cell pellets were resuspended in 450 lysis buffer (50 mM Sodium Phosphate buffer pH 7.5, 500 mM NaCl, 5 % (w/v) glycerol 451 and 2 mM β-mercaptoethanol) supplemented with protease inhibitor cocktail, lysed by 452 sonication and cleared by centrifugation at 70.000 g at 4°C. The supernatant was filtered 453 and loaded on a 5 ml HisTrap FF column (GE Healthcare) equilibrated in lysis buffer. 454 After washing with lysis buffer, the protein was eluted with a linear gradient of 0-500 455 mM imidazole in 10 column volumes. The fractions of interest were pooled, 456 concentrated with a 10 kDa cut-off Amicon concentrator (Millipore) and loaded onto a 457 HiLoad Superdex 75 16/60 (GE Healthcare) equilibrated in SEC buffer (50 mM Sodium 458 Phosphate buffer pH 7.0, 200 mM NaCl, 5% (w/v) glycerol and 1 mM TCEP).

459 Expression and purification of Bub1 and BubR1 constructs, as well as of Bub1:Bub3 and 460 BubR1:Bub3 complexes was carried out as described (Breit et al., 2015; Overlack et al., 461 2015).

462 Analytical SEC analysis 4 µM Bub1 and BubR1 protein constructs or Bub1:Bub3 and 463 BubR1:Bub3 complexes were mixed with 16 µM CENP-E and CENP-F proteins 464 respectively, in 30 µl final volume. Analytical size exclusion chromatography was carried 465 out at 4°C on a Superose 6 5/150 or Superdex 75 5/150 in a buffer containing 50 mM 466 HEPES pH 8.0, 100 mM NaCl, 5% (w/v) glycerol and 0.5 mM TCEP at a flow rate of 467 0.12 ml/min on an ÄKTA micro system. Elution of proteins was monitored at 280 nm, 468 488 nm (eGFP-tag) and 587 nm (mCherry-tag). 50 µl fractions were collected and 469 analysed by SDS-PAGE and Coomassie blue staining.

470 Analytical Ultracentrifugation Sedimentation velocity AUC was performed at 42,000 471 rpm at 20°C in a Beckman XL-A ultracentrifuge. Protein samples were loaded into 472 standard double-sector centerpieces. The cells were scanned every minute and 500 scans 473 were recorded for every sample. 6 µM mCherry-CENP-F2866-2990, mCherry-CENP-F2922- 474 2990 and mCherry-CENP-F2950-2990 were scanned at 587 nm. 7 µM eGFP-CENP-E2070-C

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475 alone or mixed with 21 µM BubR1:Bub3 were instead scanned at 488 nm. Data were 476 analyzed using the program SEDFIT (Brown and Schuck, 2006) with the model of 477 continuous c(s) distribution. The partial specific volumes of the proteins, buffer density, 478 and buffer viscosity were estimated using the program SEDNTERP. Data figures were 479 generated using the program GUSSI (Brautigam, 2015).

480 Protein Electroporation For eGFP-CENP-E protein electroporation, HeLa cells were 481 arrested in G2 with a 9 µM RO-3306 treatment for 15 hours (Millipore) and then 482 released into mitosis for 3 hours in presence of 3.3 µM nocodazole. Mitotic cells were 483 then collected by mitotic shake-off, washed with PBS and counted. Approximatively 484 3x106 cells were then electroporated (Neon Transfection System Kit, Thermo Fisher) 485 with 10 µM eGFP-CENP-E. Following electroporation, cells were allowed to recover in 486 media with 3.3 µM nocodazole for 4 hours and then fixed and prepared for 487 immunofluorescence analysis. For mCherry-CENP-F protein electroporation, HeLa cells 488 were treated for 16 hours with 0.33 µM Nocodazole (Sigma) to synchronise cells in 489 mitosis. Mitotic cells were then collected by mitotic shake-off, washed with PBS and 490 counted. Approximatively 3x106 cells were then electroporated with 5 µM mCherry- 491 CENP-F. Following electroporation, cells were allowed to recover in media with 3.3 µM 492 nocodazole for 4 hours and then fixed and prepared for immunofluorescence analysis.

493 Low-angle metal shadowing and electron microscopy mCherry-CENP-F2688-C 494 fractions from the elution peak of an analytical size-exclusion chromatography column 495 were diluted 1:1 with spraying buffer (200 mM ammonium acetate and 60% glycerol) and 496 air-sprayed as described (Baschong and Aebi, 2006; Huis in 't Veld et al., 2016) onto 497 freshly cleaved mica pieces of approximately 2x3 mm (V1 quality, Plano GmbH). 498 Specimens were mounted and dried in a MED020 high-vacuum metal coater (Bal-tec). A 499 Platinum layer of approximately 1 nm and a 7 nm Carbon support layer were evaporated 500 subsequently onto the rotating specimen at angles of 6-7° and 45° respectively. Pt/C 501 replicas were released from the mica on water, captured by freshly glow-discharged 400- 502 mesh Pd/Cu grids (Plano GmbH), and visualized using a LaB6 equipped JEM-1400 503 transmission electron microscope (JEOL) operated at 120 kV. Images were recorded at a 504 nominal magnification of 60,000x on a 4k x 4k CCD camera F416 (TVIPS), resulting in 505 0.18 nm per pixel.

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506 Mammalian Plasmids Plasmids were derived from the pCDNA5/FRT/TO-EGFP- 507 IRES, a previously modified version (Krenn et al., 2012) of the pCDNA5/FRT/TO 508 vector (Invitrogen). To create N-terminally-tagged EGFP-Bub1 truncation constructs, 509 the Bub1 sequence was obtained by PCR amplification from the previously generated 510 pCDNA5/FRT/TO-EGFP-Bub1-IRES vector (Krenn et al., 2012) and subcloned in 511 frame with the GFP-tag. All Bub1 constructs were RNAi resistant (Kiyomitsu et al., 512 2007). pCDNA5/FRT/TO-based plasmids were used for generation of stable cell lines. 513 All plasmids were verified by sequencing.

514 Cell culture and transfection HeLa cells were grown in DMEM (PAN Biotech) 515 supplemented with 10 % FBS (Clontech), penicillin and streptomycin (GIBCO) and 516 2 mM L-glutamine (PAN Biotech). Flp-In T-REx HeLa cells used to generate stable 517 doxycycline-inducible cell lines were a gift from S.S. Taylor (University of Manchester, 518 Manchester, England, UK). Flp-In T-REx host cell lines were maintained in DMEM 519 with 10 % tetracycline-free FBS (Clontech) supplemented with 50 µg/ml Zeocin 520 (Invitrogen). Flp-In T-REx HeLa expression cell lines were generated as previously 521 described (Krenn et al., 2012). Briefly, Flp-In T-Rex HeLa host cells were cotransfected 522 with a ratio of 9:1 (w/w) pOG44:pcDNA5/FRT/TO expression plasmid using X- 523 tremeGene transfection agent (Roche). 48 h after transfection, Flp-In T-Rex HeLa 524 expression cell lines were put under selection for two weeks in DMEM with 10 % 525 tetracycline-free FBS (Invitrogen) supplemented with 250 µg/ml Hygromycin (Roche) 526 and 5 µg/ml Blasticidin (ICN Chemicals). The resulting foci were pooled and tested for 527 expression. Gene expression was induced by addition of 0.5 µg/ml doxycycline (Sigma) 528 for 24 h. 529 siBUB1 (Dharmacon, 5’-GGUUGCCAACACAAGUUCU-3’) or siBUBR1 (Dharmacon, 530 5’-CGGGCAUUUGAAUAUGAAA-3’) duplexes were transfected with Lipofectamine 531 2000 (Invitrogen) at 50 nM for 24 h. siCENP-E (Dharmacon, 5’- 532 AAGGCUACAAUGGUACUAUAU-3’) and siCENP-F (Dharmacon, 5'- 533 CAAAGACCGGUGUUACCAAG-3' and 5'-AAGAGAAGACCCCAAGUCAUC-3') 534 duplexes were transfected at 60 nM with LipofectamineRNAiMAX (Invitrogen) for 24 h. 535 siZwilch (SMART pool from Dharmacon, #L-019377-00-0005) duplexes were 536 transfected with LipofectamineRNAiMAX at 120 nM for 72 h.

537 Immunoblotting To generate mitotic populations for immunoblotting experiments, 538 cells were treated with 330 nM nocodazole for 16 h. Mitotic cells were then harvested by

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539 shake off and lysed in lysis buffer [150 mM KCl, 75 mM Hepes, pH 7.5, 1.5 mM EGTA,

540 1.5 mM MgCl2, 10 % glycerol, and 0.5 % Triton-X 100 supplemented with protease 541 inhibitor cocktail (Serva) and PhosSTOP phosphatase inhibitors (Roche)]. Cleared cell 542 lysates were resuspended in sample buffer, boiled and analyzed by SDS-PAGE using 3- 543 8 % gradient gels (NuPAGE® Tris-Acetate Gels, Life technologies) and Western 544 blotting. The following antibodies were used: anti-CENP-E (rabbit, ab133583, 1:500), 545 anti-CENP-F (rabbit, Novus NB500-101, 1:500) and anti-Tubulin (mouse, Sigma T9026, 546 1:10000). Secondary antibodies were anti–mouse (Amersham) or anti–rabbit (Amersham) 547 affinity-purified with horseradish peroxidase conjugate (working dilution 1:10000). After 548 incubation with ECL Western blotting system (GE Healthcare), images were acquired 549 with the ChemiDocTM MP Imaging System (BioRad) in 16-bit TIFF format. Images were 550 cropped and converted to 8-bit using Image J software (NIH). Brightness and contrast 551 were adjusted using Photoshop CS5 (Adobe).

552 Live cell imaging Cells were plated on a 24-well µ-Plate (Ibidi®). The medium was

553 changed to CO2 Independent Medium (Gibco®) 6 h before filming. DNA was stained 554 by addition of the SiR-Hoechst-647 Dye (Spirochrome) to the medium 1 h before 555 imaging. Cells were imaged every 5 to 10 min in a heated chamber (37 °C) on a 3i 556 Marianas™ system (Intelligent Imaging Innovations Inc.) equipped with Axio Observer 557 Z1 microscope (Zeiss), Plan-Apochromat 40x/1.4NA oil objective, M27 with DIC III 558 Prism (Zeiss), Orca Flash 4.0 sCMOS Camera (Hamamatsu) and controlled by Slidebook 559 Software 6.0 (Intelligent Imaging Innovations Inc).

560 Immunofluorescence HeLa cells and Flp-In T-REx HeLa cells were grown on 561 coverslips precoated with poly-D-Lysine (Millipore, 15 µg/ml) and poly-L-Lysine 562 (Sigma), respectively. Asynchronously growing cells or cells that were arrested in 563 prometaphase by the addition of nocodazole (Sigma-Aldrich) were fixed using 4 % 564 paraformaldehyde. Cells were stained for Bub1 (mouse, ab54893, 1:400), BubR1 (rabbit, 565 Bethyl A300-386A-1, 1:1000), Tubulin (mouse, DM1a Sigma, 1:500), CENP-E (mouse, 566 ab5093, 1:200), CENP-F (rabbit, Novus NB500-101, 1:300), Zwilch (rabbit, in-house 567 made, SI520, 1:900), Mad1 labelled with AlexaFluor-488 (mouse, in-house made, Clone 568 BB3-8, 1:200), pT232-AurB (rabbit, Rockland #660-401-667, 1:2000), Plk1 (mouse, 569 ab17057, 1:300), pS10H3 (mouse, ab14955, 1:3000), pT121 H2A (rabbit, active motif 570 #39391, 1:2000) and CREST/anti-centromere antibodies (Antibodies, Inc., 1:100), 571 diluted in 2 % BSA-PBS for 1.5 h.

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572 For testing the effect of various kinase inhibitors on CENP-E and CENP-F kinetochore 573 localization, the protocol was adapted in the following way: Cells were pre-permeabilized 574 with 0.5 % triton-X-100 solution in PHEM (Pipes, Hepes, EGTA, MgCl2) buffer for 2 575 min before fixation with 4% PFA-PHEM for 15 min. After blocking the cells with 3 % 576 BSA-PHEM buffer supplemented with 0.1 % triton-X-100, they were incubated at room 577 temperature for 1-2 h with primary antibodies diluted in blocking buffer. Washing steps 578 were performed in PHEM-T buffer.

579 Goat anti–human (Invitrogen), goat anti–mouse (Jackson ImmunoResearch 580 Laboratories, Inc.) and goat anti-rabbit (Jackson ImmunoResearch Laboratories, Inc.) 581 fluorescently labeled antibodies were used as secondary antibodies. DNA was stained 582 with 0.5 µg/ml DAPI (Serva) and coverslips were mounted with Mowiol mounting 583 media (Calbiochem). Cells were imaged at room temperature using a spinning disk 584 confocal device on the 3i Marianas™ system equipped with an Axio Observer Z1 585 microscope (Zeiss), a CSU-X1 confocal scanner unit (Yokogawa Electric Corporation), 586 Plan-Apochromat 63x or 100x/1.4NA Oil Objectives (Zeiss) and Orca Flash 4.0 sCMOS 587 Camera (Hamamatsu). Images were acquired as z-sections at 0.27 µm. Images were 588 converted into maximal intensity projections, exported and converted into 8-bit. 589 Quantification of kinetochore signals was performed on unmodified 16-bit z-series 590 images using Imaris 7.3.4 32-bit software (Bitplane). After background subtraction, all 591 signals were normalized to CREST. At least 117 kinetochores were analyzed per 592 condition. Measurements were exported in Excel (Microsoft) and graphed with 593 GraphPad Prism 6.0 (GraphPad Software).

594 Cell synchronization To test the effect of various kinase activities on CENP-E and 595 CENP-F kinetochore localization, cells were synchronized using a double thymidine 596 arrest. Cells were released from the first 18 h thymidine (2 mM; Sigma–Aldrich) block by 597 washing them with fresh pre-warmed media several times. After releasing them for the 598 next 9 h, cells were exposed to thymidine (2 mM) a second time for 15 h. Afterwards, 599 cells were released into S-phase for 4 h and then nocodazole (330 nM) was added to the 600 media for the next 3-4 h to enrich for the mitotic cell population. Kinase activity 601 inhibitors, BI 2536 (500 nM; Calbiochem), Hesperadin (500 nM; Calbiochem), Reversine 602 (500 nM; Calbiochem) or BAY-320 (10 µM; kindly received from Dr. Gerhard 603 Siemeister, Bayer GmbH, Berlin) were added in the presence of the proteasome 604 inhibitor, MG132 (10 µM; Calbiochem) to the cells for 90 min before fixing these cells

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605 for immunofluorescence.

606 Chromosome alignment For analysis of the effect of CENP-F depletion on 607 chromosome alignment, cells were fixed after RNAi either asynchronously or after an 608 additional treatment with 10 µM MG-132 for 2 h. Cells were stained for CENP-F, 609 Tubulin and CREST. DNA was labeled with DAPI. The number of metaphase cells with 610 aligned chromosomes and with misaligned chromosomes was scored for each condition. 611 At least 595 cells (without synchronization) or 92 cells (with synchronization) were 612 analyzed per condition.

613

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614 Uncategorized References 615 Akera, T., Goto, Y., Sato, M., Yamamoto, M., and Watanabe, Y. (2015). Mad1 promotes 616 chromosome congression by anchoring a kinesin motor to the kinetochore. Nature cell 617 biology 17, 1124-1133.

618 Ashar, H.R., James, L., Gray, K., Carr, D., Black, S., Armstrong, L., Bishop, W.R., and 619 Kirschmeier, P. (2000). Farnesyl transferase inhibitors block the farnesylation of CENP- 620 E and CENP-F and alter the association of CENP-E with the microtubules. The Journal 621 of biological chemistry 275, 30451-30457.

622 Baffet, A.D., Hu, D.J., and Vallee, R.B. (2015). Cdk1 Activates Pre-mitotic Nuclear 623 Envelope Dynein Recruitment and Apical Nuclear Migration in Neural Stem Cells. 624 Developmental cell 33, 703-716.

625 Barisic, M., Sohm, B., Mikolcevic, P., Wandke, C., Rauch, V., Ringer, T., Hess, M., Bonn, 626 G., and Geley, S. (2010). Spindly/CCDC99 is required for efficient chromosome 627 congression and mitotic checkpoint regulation. Molecular biology of the cell 21, 1968- 628 1981.

629 Baschong, W., and Aebi, U. (2006). Glycerol Spraying/Low Angle Rotary Metal 630 Shadowing, Vol 3 (Elsevier Science).

631 Basto, R., Gomes, R., and Karess, R.E. (2000). Rough deal and Zw10 are required for the 632 metaphase checkpoint in Drosophila. Nature cell biology 2, 939-943.

633 Basto, R., Scaerou, F., Mische, S., Wojcik, E., Lefebvre, C., Gomes, R., Hays, T., and 634 Karess, R. (2004). In vivo dynamics of the rough deal checkpoint protein during 635 Drosophila mitosis. Current biology : CB 14, 56-61.

636 Berto, A., Yu, J., Morchoisne-Bohly, S., Bertipaglia, C., Vallee, R., Dumont, J., 637 Ochsenbein, F., Guerois, R., and Doye, V. (2018). Disentangling the molecular 638 determinants for Cenp-F localiation to nuclear pores and kinetochores. EMBO reports in 639 press.

640 Bolhy, S., Bouhlel, I., Dultz, E., Nayak, T., Zuccolo, M., Gatti, X., Vallee, R., Ellenberg, 641 J., and Doye, V. (2011). A Nup133-dependent NPC-anchored network tethers 642 centrosomes to the nuclear envelope in prophase. The Journal of cell biology 192, 855- 643 871.

644 Bomont, P., Maddox, P., Shah, J.V., Desai, A.B., and Cleveland, D.W. (2005). Unstable 645 microtubule capture at kinetochores depleted of the centromere-associated protein 646 CENP-F. The EMBO journal 24, 3927-3939.

647 Brautigam, C.A. (2015). Calculations and Publication-Quality Illustrations for Analytical 648 Ultracentrifugation Data. Methods Enzymol 562, 109-133.

649 Breit, C., Bange, T., Petrovic, A., Weir, J.R., Muller, F., Vogt, D., and Musacchio, A. 650 (2015). Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic 651 Checkpoint Kinase Bub1. PLoS One 10, e0144673.

21 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

652 Brown, P.H., and Schuck, P. (2006). Macromolecular size-and-shape distributions by 653 sedimentation velocity analytical ultracentrifugation. Biophys J 90, 4651-4661.

654 Buffin, E., Lefebvre, C., Huang, J., Gagou, M.E., and Karess, R.E. (2005). Recruitment 655 of Mad2 to the kinetochore requires the Rod/Zw10 complex. Current biology : CB 15, 656 856-861.

657 Caldas, G.V., Lynch, T.R., Anderson, R., Afreen, S., Varma, D., and DeLuca, J.G. (2015). 658 The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 659 kinetochore localization in human cells. Open biology 5.

660 Casiano, C.A., Landberg, G., Ochs, R.L., and Tan, E.M. (1993). Autoantibodies to a 661 novel cell cycle-regulated protein that accumulates in the nuclear matrix during S phase 662 and is localized in the kinetochores and spindle midzone during mitosis. Journal of cell 663 science 106 ( Pt 4), 1045-1056.

664 Chan, G.K., Schaar, B.T., and Yen, T.J. (1998). Characterization of the kinetochore 665 binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F 666 and hBUBR1. The Journal of cell biology 143, 49-63.

667 Chan, Y.W., Fava, L.L., Uldschmid, A., Schmitz, M.H., Gerlich, D.W., Nigg, E.A., and 668 Santamaria, A. (2009). Mitotic control of kinetochore-associated dynein and spindle 669 orientation by human Spindly. The Journal of cell biology 185, 859-874.

670 Cheerambathur, D.K., Gassmann, R., Cook, B., Oegema, K., and Desai, A. (2013). 671 Crosstalk between microtubule attachment complexes ensures accurate chromosome 672 segregation. Science 342, 1239-1242.

673 Cheeseman, I.M., and Desai, A. (2008). Molecular architecture of the kinetochore- 674 microtubule interface. Nature reviews Molecular cell biology 9, 33-46.

675 Civril, F., Wehenkel, A., Giorgi, F.M., Santaguida, S., Di Fonzo, A., Grigorean, G., 676 Ciccarelli, F.D., and Musacchio, A. (2010). Structural analysis of the RZZ complex 677 reveals common ancestry with multisubunit vesicle tethering machinery. Structure 18, 678 616-626.

679 Cooke, C.A., Schaar, B., Yen, T.J., and Earnshaw, W.C. (1997). Localization of CENP-E 680 in the fibrous corona and outer plate of mammalian kinetochores from prometaphase 681 through anaphase. Chromosoma 106, 446-455.

682 Delorenzi, M., and Speed, T. (2002). An HMM model for coiled-coil domains and a 683 comparison with PSSM-based predictions. Bioinformatics 18, 617-625.

684 Earnshaw, W.C. (2015). Discovering centromere proteins: from cold white hands to the 685 A, B, C of CENPs. Nature reviews Molecular cell biology 16, 443-449.

686 Earnshaw, W.C., and Bernat, R.L. (1991). Chromosomal passengers: toward an 687 integrated view of mitosis. Chromosoma 100, 139-146.

688 Feng, J., Huang, H., and Yen, T.J. (2006). CENP-F is a novel microtubule-binding 689 protein that is essential for kinetochore attachments and affects the duration of the 690 mitotic checkpoint delay. Chromosoma 115, 320-329.

22 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

691 Gama, J.B., Pereira, C., Simoes, P.A., Celestino, R., Reis, R.M., Barbosa, D.J., Pires, H.R., 692 Carvalho, C., Amorim, J., Carvalho, A.X., et al. (2017). Molecular mechanism of dynein 693 recruitment to kinetochores by the Rod-Zw10-Zwilch complex and Spindly. The Journal 694 of cell biology 216, 943-960.

695 Gassmann, R., Essex, A., Hu, J.S., Maddox, P.S., Motegi, F., Sugimoto, A., O'Rourke, 696 S.M., Bowerman, B., McLeod, I., Yates, J.R., 3rd, et al. (2008). A new mechanism 697 controlling kinetochore-microtubule interactions revealed by comparison of two dynein- 698 targeting components: SPDL-1 and the Rod/Zwilch/Zw10 complex. Genes & 699 development 22, 2385-2399.

700 Gassmann, R., Holland, A.J., Varma, D., Wan, X., Civril, F., Cleveland, D.W., Oegema, 701 K., Salmon, E.D., and Desai, A. (2010). Removal of Spindly from microtubule-attached 702 kinetochores controls spindle checkpoint silencing in human cells. Genes & development 703 24, 957-971.

704 Griffis, E.R., Stuurman, N., and Vale, R.D. (2007). Spindly, a novel protein essential for 705 silencing the spindle assembly checkpoint, recruits dynein to the kinetochore. The 706 Journal of cell biology 177, 1005-1015.

707 Gruber, M., Soding, J., and Lupas, A.N. (2005). REPPER--repeats and their periodicities 708 in fibrous proteins. Nucleic Acids Res 33, W239-243.

709 Hoffman, D.B., Pearson, C.G., Yen, T.J., Howell, B.J., and Salmon, E.D. (2001). 710 Microtubule-dependent changes in assembly of microtubule motor proteins and mitotic 711 spindle checkpoint proteins at PtK1 kinetochores. Molecular biology of the cell 12, 1995- 712 2009.

713 Holland, A.J., Reis, R.M., Niessen, S., Pereira, C., Andres, D.A., Spielmann, H.P., 714 Cleveland, D.W., Desai, A., and Gassmann, R. (2015). Preventing farnesylation of the 715 dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase 716 inhibitors. Molecular biology of the cell 26, 1845-1856.

717 Holt, S.V., Vergnolle, M.A., Hussein, D., Wozniak, M.J., Allan, V.J., and Taylor, S.S. 718 (2005). Silencing Cenp-F weakens centromeric cohesion, prevents chromosome 719 alignment and activates the spindle checkpoint. Journal of cell science 118, 4889-4900.

720 Howell, B.J., McEwen, B.F., Canman, J.C., Hoffman, D.B., Farrar, E.M., Rieder, C.L., 721 and Salmon, E.D. (2001). Cytoplasmic dynein/dynactin drives kinetochore protein 722 transport to the spindle poles and has a role in mitotic spindle checkpoint inactivation. 723 The Journal of cell biology 155, 1159-1172.

724 Hu, D.J., Baffet, A.D., Nayak, T., Akhmanova, A., Doye, V., and Vallee, R.B. (2013). 725 Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry 726 in brain progenitor cells. Cell 154, 1300-1313.

727 Huis in 't Veld, P.J., Jeganathan, S., Petrovic, A., John, J., Singh, P., Weissmann, F., 728 Bange, T., and Musacchio, A. (2016). Molecular basis of outer kinetochore assembly on 729 CENP-T. Elife pii: e21007.

23 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

730 Hussein, D., and Taylor, S.S. (2002). Farnesylation of Cenp-F is required for G2/M 731 progression and degradation after mitosis. Journal of cell science 115, 3403-3414.

732 Johnson, V.L., Scott, M.I., Holt, S.V., Hussein, D., and Taylor, S.S. (2004). Bub1 is 733 required for kinetochore localization of BubR1, Cenp-E, Cenp-F and Mad2, and 734 chromosome congression. Journal of cell science 117, 1577-1589.

735 Jokelainen, P.T. (1967). The ultrastructure and spatial organization of the metaphase 736 kinetochore in mitotic rat cells. J Ultrastruct Res 19, 19-44.

737 Kapoor, T.M., Lampson, M.A., Hergert, P., Cameron, L., Cimini, D., Salmon, E.D., 738 McEwen, B.F., and Khodjakov, A. (2006). Chromosomes can congress to the metaphase 739 plate before biorientation. Science 311, 388-391.

740 Kim, Y., Heuser, J.E., Waterman, C.M., and Cleveland, D.W. (2008). CENP-E combines 741 a slow, processive motor and a flexible coiled coil to produce an essential motile 742 kinetochore tether. The Journal of cell biology 181, 411-419.

743 Kiyomitsu, T., Obuse, C., and Yanagida, M. (2007). Human Blinkin/AF15q14 is required 744 for chromosome alignment and the mitotic checkpoint through direct interaction with 745 Bub1 and BubR1. Developmental cell 13, 663-676.

746 Klebig, C., Korinth, D., and Meraldi, P. (2009). Bub1 regulates chromosome segregation 747 in a kinetochore-independent manner. The Journal of cell biology 185, 841-858.

748 Kops, G.J., Kim, Y., Weaver, B.A., Mao, Y., McLeod, I., Yates, J.R., 3rd, Tagaya, M., and 749 Cleveland, D.W. (2005). ZW10 links mitotic checkpoint signaling to the structural 750 kinetochore. The Journal of cell biology 169, 49-60.

751 Krenn, V., Wehenkel, A., Li, X., Santaguida, S., and Musacchio, A. (2012). Structural 752 analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 753 interaction. The Journal of cell biology 196, 451-467.

754 Kuhn, J., and Dumont, S. (2017). Spindle assembly checkpoint satisfaction occurs via 755 end-on but not lateral attachments under tension. The Journal of cell biology 216, 1533- 756 1542.

757 Lampson, M.A., and Kapoor, T.M. (2005). The human mitotic checkpoint protein 758 BubR1 regulates chromosome-spindle attachments. Nature cell biology 7, 93-98.

759 Liao, H., Li, G., and Yen, T.J. (1994). Mitotic regulation of microtubule cross-linking 760 activity of CENP-E kinetochore protein. Science 265, 394-398.

761 Liao, H., Winkfein, R.J., Mack, G., Rattner, J.B., and Yen, T.J. (1995). CENP-F is a 762 protein of the nuclear matrix that assembles onto kinetochores at late G2 and is rapidly 763 degraded after mitosis. The Journal of cell biology 130, 507-518.

764 Liu, S.T., Rattner, J.B., Jablonski, S.A., and Yen, T.J. (2006). Mapping the assembly 765 pathways that specify formation of the trilaminar kinetochore plates in human cells. The 766 Journal of cell biology 175, 41-53.

24 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

767 Logarinho, E., Resende, T., Torres, C., and Bousbaa, H. (2008). The human spindle 768 assembly checkpoint protein Bub3 is required for the establishment of efficient 769 kinetochore-microtubule attachments. Molecular biology of the cell 19, 1798-1813.

770 Lupas, A., Van Dyke, M., and Stock, J. (1991). Predicting coiled coils from protein 771 sequences. Science 252, 1162-1164.

772 Magidson, V., O'Connell, C.B., Loncarek, J., Paul, R., Mogilner, A., and Khodjakov, A. 773 (2011). The spatial arrangement of chromosomes during prometaphase facilitates spindle 774 assembly. Cell 146, 555-567.

775 Magidson, V., Paul, R., Yang, N., Ault, J.G., O'Connell, C.B., Tikhonenko, I., McEwen, 776 B.F., Mogilner, A., and Khodjakov, A. (2015). Adaptive changes in the kinetochore 777 architecture facilitate proper spindle assembly. Nature cell biology 17, 1134-1144.

778 Mao, Y., Abrieu, A., and Cleveland, D.W. (2003). Activating and silencing the mitotic 779 checkpoint through CENP-E-dependent activation/inactivation of BubR1. Cell 114, 87- 780 98.

781 Mao, Y., Desai, A., and Cleveland, D.W. (2005). Microtubule capture by CENP-E 782 silences BubR1-dependent mitotic checkpoint signaling. The Journal of cell biology 170, 783 873-880.

784 Martin-Lluesma, S., Stucke, V.M., and Nigg, E.A. (2002). Role of Hec1 in spindle 785 checkpoint signaling and kinetochore recruitment of Mad1/Mad2. Science 297, 2267- 786 2270.

787 Matson, D.R., and Stukenberg, P.T. (2014). CENP-I and Aurora B act as a molecular 788 switch that ties RZZ/Mad1 recruitment to kinetochore attachment status. The Journal of 789 cell biology 205, 541-554.

790 McEwen, B.F., Arena, J.T., Frank, J., and Rieder, C.L. (1993). Structure of the colcemid- 791 treated PtK1 kinetochore outer plate as determined by high voltage electron microscopic 792 tomography. The Journal of cell biology 120, 301-312.

793 Mische, S., He, Y., Ma, L., Li, M., Serr, M., and Hays, T.S. (2008). Dynein light 794 intermediate chain: an essential subunit that contributes to spindle checkpoint 795 inactivation. Molecular biology of the cell 19, 4918-4929.

796 Mosalaganti, S., Keller, J., Altenfeld, A., Winzker, M., Rombaut, P., Saur, M., Petrovic, 797 A., Wehenkel, A., Wohlgemuth, S., Muller, F., et al. (2017). Structure of the RZZ 798 complex and molecular basis of its interaction with Spindly. The Journal of cell biology 799 216, 961-981.

800 Moudgil, D.K., Westcott, N., Famulski, J.K., Patel, K., Macdonald, D., Hang, H., and 801 Chan, G.K. (2015). A novel role of farnesylation in targeting a mitotic checkpoint 802 protein, human Spindly, to kinetochores. The Journal of cell biology 208, 881-896.

803 Musacchio, A. (2015). The Molecular Biology of Spindle Assembly Checkpoint Signaling 804 Dynamics. Current biology : CB 25, R1002-1018.

25 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

805 Musacchio, A., and Desai, A. (2017). A Molecular View of Kinetochore Assembly and 806 Function. Biology (Basel) 6.

807 Musinipally, V., Howes, S., Alushin, G.M., and Nogales, E. (2013). The microtubule 808 binding properties of CENP-E's C-terminus and CENP-F. J Mol Biol 425, 4427-4441.

809 Overlack, K., Bange, T., Weissmann, F., Faesen, A.C., Maffini, S., Primorac, I., Muller, 810 F., Peters, J.M., and Musacchio, A. (2017). BubR1 Promotes Bub3-Dependent APC/C 811 Inhibition during Spindle Assembly Checkpoint Signaling. Current biology : CB 27, 812 2915-2927 e2917.

813 Overlack, K., Primorac, I., Vleugel, M., Krenn, V., Maffini, S., Hoffmann, I., Kops, G.J., 814 and Musacchio, A. (2015). A molecular basis for the differential roles of Bub1 and 815 BubR1 in the spindle assembly checkpoint. Elife 4, e05269.

816 Primorac, I., Weir, J.R., Chiroli, E., Gross, F., Hoffmann, I., van Gerwen, S., Ciliberto, 817 A., and Musacchio, A. (2013). Bub3 reads phosphorylated MELT repeats to promote 818 spindle assembly checkpoint signaling. Elife 2, e01030.

819 Putkey, F.R., Cramer, T., Morphew, M.K., Silk, A.D., Johnson, R.S., McIntosh, J.R., and 820 Cleveland, D.W. (2002). Unstable kinetochore-microtubule capture and chromosomal 821 instability following deletion of CENP-E. Developmental cell 3, 351-365.

822 Raaijmakers, J.A., van Heesbeen, R., Blomen, V.A., Janssen, L.M.E., van Diemen, F., 823 Brummelkamp, T.R., and Medema, R.H. (2018). BUB1 Is Essential for the Viability of 824 Human Cells in which the Spindle Assembly Checkpoint Is Compromised. Cell Rep 22, 825 1424-1438.

826 Rattner, J.B., Rao, A., Fritzler, M.J., Valencia, D.W., and Yen, T.J. (1993). CENP-F is a 827 .ca 400 kDa kinetochore protein that exhibits a cell-cycle dependent localization. Cell 828 Motil Cytoskeleton 26, 214-226.

829 Rieder, C.L. (1982). The formation, structure, and composition of the mammalian 830 kinetochore and kinetochore fiber. Int Rev Cytol 79, 1-58.

831 Sawano, A., and Miyawaki, A. (2000). Directed evolution of green fluorescent protein by 832 a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic 833 Acids Res 28, E78.

834 Schaar, B.T., Chan, G.K., Maddox, P., Salmon, E.D., and Yen, T.J. (1997). CENP-E 835 function at kinetochores is essential for chromosome alignment. The Journal of cell 836 biology 139, 1373-1382.

837 Schafer-Hales, K., Iaconelli, J., Snyder, J.P., Prussia, A., Nettles, J.H., El-Naggar, A., 838 Khuri, F.R., Giannakakou, P., and Marcus, A.I. (2007). Farnesyl transferase inhibitors 839 impair chromosomal maintenance in cell lines and human tumors by compromising 840 CENP-E and CENP-F function. Mol Cancer Ther 6, 1317-1328.

841 Sharp-Baker, H., and Chen, R.H. (2001). Spindle checkpoint protein Bub1 is required for 842 kinetochore localization of Mad1, Mad2, Bub3, and CENP-E, independently of its kinase 843 activity. The Journal of cell biology 153, 1239-1250.

26 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

844 Silio, V., McAinsh, A.D., and Millar, J.B. (2015). KNL1-Bubs and RZZ Provide Two 845 Separable Pathways for Checkpoint Activation at Human Kinetochores. Developmental 846 cell 35, 600-613.

847 Simoes, P.A., Celestino, R., Carvalho, A.X., and Gassmann, R. (2018). NudE regulates 848 dynein at kinetochores but is dispensable for other dynein functions in the C. elegans 849 early embryo. Journal of cell science 131.

850 Sivaram, M.V., Wadzinski, T.L., Redick, S.D., Manna, T., and Doxsey, S.J. (2009). 851 Dynein light intermediate chain 1 is required for progress through the spindle assembly 852 checkpoint. The EMBO journal 28, 902-914.

853 Starr, D.A., Williams, B.C., Hays, T.S., and Goldberg, M.L. (1998). ZW10 helps recruit 854 dynactin and dynein to the kinetochore. The Journal of cell biology 142, 763-774.

855 Sudakin, V., Chan, G.K., and Yen, T.J. (2001). Checkpoint inhibition of the APC/C in 856 HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. The 857 Journal of cell biology 154, 925-936.

858 Suijkerbuijk, S.J., van Dam, T.J., Karagoz, G.E., von Castelmur, E., Hubner, N.C., 859 Duarte, A.M., Vleugel, M., Perrakis, A., Rudiger, S.G., Snel, B., et al. (2012). The 860 vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase. 861 Developmental cell 22, 1321-1329.

862 van Hooff, J.J., Tromer, E., van Wijk, L.M., Snel, B., and Kops, G.J. (2017). Evolutionary 863 dynamics of the kinetochore network in eukaryotes as revealed by comparative 864 genomics. EMBO reports 18, 1559-1571.

865 Varma, D., Monzo, P., Stehman, S.A., and Vallee, R.B. (2008). Direct role of dynein 866 motor in stable kinetochore-microtubule attachment, orientation, and alignment. The 867 Journal of cell biology 182, 1045-1054.

868 Vergnolle, M.A., and Taylor, S.S. (2007). Cenp-F links kinetochores to 869 Ndel1/Nde1/Lis1/dynein microtubule motor complexes. Current biology : CB 17, 1173- 870 1179.

871 Volkov, V.A., Grissom, P.M., Arzhanik, V.K., Zaytsev, A.V., Renganathan, K., McClure- 872 Begley, T., Old, W.M., Ahn, N., and McIntosh, J.R. (2015). Centromere protein F 873 includes two sites that couple efficiently to depolymerizing microtubules. The Journal of 874 cell biology 209, 813-828.

875 Weissmann, F., Petzold, G., VanderLinden, R., Huis In 't Veld, P.J., Brown, N.G., 876 Lampert, F., Westermann, S., Stark, H., Schulman, B.A., and Peters, J.M. (2016). biGBac 877 enables rapid gene assembly for the expression of large multisubunit protein complexes. 878 Proceedings of the National Academy of Sciences of the United States of America 113, 879 E2564-2569.

880 Williams, B.C., Gatti, M., and Goldberg, M.L. (1996). Bipolar spindle attachments affect 881 redistributions of ZW10, a Drosophila centromere/kinetochore component required for 882 accurate chromosome segregation. The Journal of cell biology 134, 1127-1140.

27 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

883 Wojcik, E., Basto, R., Serr, M., Scaerou, F., Karess, R., and Hays, T. (2001). Kinetochore 884 dynein: its dynamics and role in the transport of the Rough deal checkpoint protein. 885 Nature cell biology 3, 1001-1007.

886 Wood, K.W., Sakowicz, R., Goldstein, L.S., and Cleveland, D.W. (1997). CENP-E is a 887 plus end-directed kinetochore motor required for metaphase chromosome alignment. 888 Cell 91, 357-366.

889 Wynne, D.J., and Funabiki, H. (2015). Kinetochore function is controlled by a phospho- 890 dependent coexpansion of inner and outer components. The Journal of cell biology 210, 891 899-916.

892 Yamamoto, T.G., Watanabe, S., Essex, A., and Kitagawa, R. (2008). SPDL-1 functions as 893 a kinetochore receptor for MDF-1 in Caenorhabditis elegans. The Journal of cell biology 894 183, 187-194.

895 Yang, Z., Guo, J., Chen, Q., Ding, C., Du, J., and Zhu, X. (2005). Silencing mitosin 896 induces misaligned chromosomes, premature chromosome decondensation before 897 anaphase onset, and mitotic cell death. Mol Cell Biol 25, 4062-4074.

898 Yao, X., Abrieu, A., Zheng, Y., Sullivan, K.F., and Cleveland, D.W. (2000). CENP-E 899 forms a link between attachment of spindle microtubules to kinetochores and the mitotic 900 checkpoint. Nature cell biology 2, 484-491.

901 Yao, X., Anderson, K.L., and Cleveland, D.W. (1997). The microtubule-dependent 902 motor centromere-associated protein E (CENP-E) is an integral component of 903 kinetochore corona fibers that link centromeres to spindle microtubules. The Journal of 904 cell biology 139, 435-447.

905 Yen, T.J., Compton, D.A., Wise, D., Zinkowski, R.P., Brinkley, B.R., Earnshaw, W.C., 906 and Cleveland, D.W. (1991). CENP-E, a novel human centromere-associated protein 907 required for progression from metaphase to anaphase. The EMBO journal 10, 1245- 908 1254.

909 Yen, T.J., Li, G., Schaar, B.T., Szilak, I., and Cleveland, D.W. (1992). CENP-E is a 910 putative kinetochore motor that accumulates just before mitosis. Nature 359, 536-539.

911 Zhang, G., Lischetti, T., Hayward, D.G., and Nilsson, J. (2015). Distinct domains in 912 Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint. Nature 913 communications 6, 7162.

914 Zhu, X. (1999). Structural requirements and dynamics of mitosin-kinetochore interaction 915 in M phase. Mol Cell Biol 19, 1016-1024.

916 Zhu, X., Chang, K.H., He, D., Mancini, M.A., Brinkley, W.R., and Lee, W.H. (1995a). 917 The C terminus of mitosin is essential for its nuclear localization, 918 centromere/kinetochore targeting, and dimerization. The Journal of biological chemistry 919 270, 19545-19550.

920 Zhu, X., Mancini, M.A., Chang, K.H., Liu, C.Y., Chen, C.F., Shan, B., Jones, D., Yang- 921 Feng, T.L., and Lee, W.H. (1995b). Characterization of a novel 350-kilodalton nuclear

28 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

922 phosphoprotein that is specifically involved in mitotic-phase progression. Mol Cell Biol 923 15, 5017-5029.

924

925

29 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

926 Figure Legends

927 Figure 1. The corona proteins CENP-E and CENP-F localize at kinetochores 928 with distinct timing

929 A) Schematic representation of the corona structure and function. MT, microtubules; IP, 930 inner plate of kinetochore; OP, outer plate of kinetochore. B) Schematic organization of 931 the CENP-E and CENP-F full-length proteins. Coiled coil regions were predicted with 932 coils, pcoils and marcoils (Delorenzi and Speed, 2002; Gruber et al., 2005; Lupas et al., 933 1991) using default parameters (ncoils and paircoils: windows size 21). To combine all 934 three coiled coil prediction algorithms, we applied a scoring system in which we assigned 935 for each residue two points for a high significance (P-value >=0.9) and one point for low 936 significance (P-value >=0.8). Two additional points were granted for an identical register 937 position in coiled coil if predicted by all three programs, resulting in a maximum score of 938 8. C, D) Representative images of fixed Hela cells treated for fluorescence staining with 939 the indicated antibodies. The panel illustrates the localization of CENP-E (C) and 940 CENP-F (D) in the different phases of the cell cycle. Scale bar: 10 µm.

941

942 Figure 1S1. Zwilch and Mad1 localization through the cell cycle

943 A, B) Representative images of Hela cells showing Zwilch (A) and Mad1 (B) localization 944 in the different phases of the cell cycle. Scale bar: 10 µm.

945

946 Figure 2. Kinetochore localization of RZZ and Mad1 are independent of CENP-E 947 and CENP-F

948 A-H) Representative images and quantification of proteins kinetochore levels in Hela 949 cells mock treated or depleted of Zwilch (A), CENP-E (B, E, H), CENP-F (C, F, G) or 950 co-depleted of CENP-E and CENP-F (D). Scale bar: 10 µm. Zwilch depletion does not 951 affect the localization of CENP-E (A). CENP-E depletion does not affect the 952 localization of Zwilch (B), Mad1 (E) and CENP-F (H). Similarly, CENP-F depletion 953 does not interfere with the recruitment of Zwilch (C), Mad1 (F) and CENP-E (G). Co- 954 depletion of CENP-E and CENP-F has no effects on localization of Zwilch (D). The 955 graphs show mean intensity of one (B, C, E), two (D, F) or three (A, G, H) experiments; 956 the error bars indicate SEM and the mean values for non-depleted cells are set to 1.

30 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

957

958 Figure 2S1. Efficient siRNA depletion of CENP-E, CENP-F and Zwilch

959 A) Western blot showing CENP-E protein levels in mock treated or CENP-E depleted 960 cells. B) Representative images of HeLa cells, either mock-treated or depleted of CENP- 961 E, showing that CENP-E can be efficiently depleted. Scale bar: 10 µm. C) Quantification 962 of CENP-E kinetochore levels in cells treated as in panel (B). The graph shows mean 963 intensity of two independent experiments; the error bars indicate SEM and the mean 964 value for non-depleted cells is set to 1. D) Western blot showing CENP-F protein levels 965 in mock treated or CENP-F depleted cells. E) Representative images of HeLa cells mock 966 treated or depleted of CENP-F showing successful CENP-F depletion. Scale bar: 10 µm. 967 F) Quantification of CENP-F kinetochore levels in cells treated as in panel (E). The 968 graph shows mean intensity of three independent experiments; the error bars indicate 969 SEM and the mean value for non-depleted cells is set to 1. G) Quantification of Zwilch 970 kinetochore levels in cells treated as in Figure 2A (upper panel) and in (H). The graphs 971 show mean intensity of three (CENP-E experiment) or two (Mad1 experiment) 972 independent experiments; the error bars indicate SEM and the mean value for non- 973 depleted cells is set to 1. H) Representative images of HeLa cells mock treated or 974 depleted of Zwilch showing that Zwilch depletion leads to reduced Mad1 levels. Scale 975 bar: 10 µm. I) Quantification of Mad1 kinetochore levels in cells treated as in (H). The 976 graph shows mean intensity of two independent experiments; the error bars indicate 977 SEM and the mean value for non-depleted cells is set to 1. J) Correlation of Mad1 and 978 Zwilch levels in 38 cells, 19 of which were mock treated, while the other 19 were RNAi 979 depleted of Zwilch. Cells are from two independent experiments. AU, arbitrary units.

980

981 Figure 2S2. Zwilch and BubR1 co-depletion does not affect CENP-E kinetochore 982 recruitment

983 A) Upper panel - Representative images of HeLa cells either mock treated or co-depleted 984 of CENP-E and CENP-F showing effective co-depletion of the proteins. Scale bar: 10 985 µm. Lower panel - Quantification of co-depletion efficiency. The graph shows mean 986 intensity of two independent experiments; the error bars indicate SEM and the mean 987 values for non-depleted cells are set to 1. B) Representative images of HeLa cells mock 988 treated or co-depleted of Zwilch and BubR1 showing efficient depletion of both the

31 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

989 proteins. Scale bar: 10 µm. C) Representative images of HeLa cells mock treated or co- 990 depleted of Zwilch and BubR1 showing that CENP-E localization is not affected. Scale 991 bar: 10 µm. D) Representative images of HeLa cells mock treated or co-depleted of 992 Zwilch and Bub1 showing efficient depletion of both proteins. Two cells with different 993 Zwilch levels and the same low Bub1 levels are shown for the RNAi condition. Scale bar: 994 10 µm. E) Representative images of Hela cells mock treated or co-depleted of Zwilch 995 and Bub1, showing that CENP-E is not lost from KTs upon Zwilch and Bub1 co- 996 depletion. Two cells with different depletion efficiency for Zwilch are shown for the 997 RNAi condition. Scale bar: 10 µm.

998

999 Figure 2S3. Kinetochore localization of RZZ (Zwilch) is not affected by CENP-E 1000 or CENP-F depletion also in presence of microtubules

1001 A-C) Representative images of a prometaphase and metaphase HeLa cells either mock 1002 treated (A) or individually depleted of CENP-F (B), co-depleted of both CENP-E and 1003 CENP-F (C), individually depleted of CENP-E (D) in absence of nocodazole. Neither 1004 CENP-E nor CENP-F are required for kinetochore localization of Zwilch even in the 1005 presence of spindle microtubules. Scale bar: 10 µm.

1006

1007 Figure 2S4. Mitotic phenotype of CENP-F depletion

1008 A) Examples of the scored categories, aligned metaphase and metaphases with 1009 misalignments in mock treated or CENP-F depleted HeLa cells. Scale bar: 10 µm. B) 1010 Percentages of cells in the depicted categories in unsynchronized cells from one 1011 experiment. C) Percentages of cells in the depicted categories in cells treated with 10 µM 1012 MG for 2h before fixation from one experiment. D) Mean duration of mitosis of HeLa 1013 cells in presence or absence of endogenous CENP-F. Cell morphology was used to 1014 measure entry into and exit from mitosis by time-lapse microscopy (n>76 per condition 1015 per experiment) from two independent experiments. Error bars indicate SEM.

1016

1017 Figure 3. CENP-E interacts with the BubR1 pseudo-kinase domain, but BubR1 is 1018 not required for its kinetochore localization

1019 A) Representative images of mitotic Hela cells electroporated with eGFP, eGFP-CENP-

32 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1020 E2070-C WT or eGFP-CENP-E2070-C C2997A mutant (preventing CENP-E farnesylation). 1021 Scale bar: 5 µm. Both the WT and the un-farnesylated mutant CENP-E constructs 1022 localize at kinetochores. B-D) Elution profiles and SDS-PAGE analysis of SEC 1023 experiments of eGFP-CENP-E2070-C with BubR1:Bub3 complex (B), BubR1 pseudo- 1024 kinase domain (KinD) construct (C) and Bub1:Bub3 complex (D). A shift in elution 1025 volume is observed for the BubR1:Bub3 complex and the BubR1 pseudo-kinase domain 1026 construct, indicative of complex formation. The interaction of CENP-E with BubR1 is 1027 specific, as no shift is observed with the Bub1:Bub3 complex. E) Sedimentation velocity 1028 AUC profiles of eGFP-CENP-E2070-C alone and in complex with BubR1:Bub3. AU,

1029 arbitrary units; MWtheo, predicted molecular weight assuming stoichiometry of 1. A 1030 reliable estimation of the molecular mass of the proteins in the samples was unsuccessful, 1031 likely because of the very elongated and flexible structure of both CENP-E and BubR1. 1032 F) Representative images of stable Flp-In T-REx cells mock treated or depleted of 1033 endogenous Bub1, BubR1, or both, showing that CENP-E kinetochore localization is 1034 unaffected under any of the conditions. Scale bar: 10 µm. G) Quantification of CENP-E 1035 kinetochore levels in cells treated as in (F). The graph shows mean intensity of two 1036 independent experiments, the error bars indicate SEM. The mean value for non-depleted 1037 cells expressing GFP was set to 1.

1038

1039 Figure 3S1. CENP-E kinetochore localization sensitivity to kinases inhibition

1040 A-D) Representative images and quantification of CENP-E kinetochore levels in mitotic 1041 Hela cells treated with the indicated concentrations of the indicated kinase inhibitors. 1042 Scale bar: 10 µm. CENP-E localization is sensitive to Aurora B (A), Mps1 (B) and, to a 1043 lesser extent, Plk1 (D) but not to Bub1 (C) inhibition. Reduction in P-T232 Aurora B 1044 (Aurora B activation segment) staining was used as a positive control for Aurora B 1045 inhibition (A), while reduction in Bub1 localization was used as positive control for Mps1 1046 inhibition (B). Bub1 inhibition was confirmed by reduction in P-T121 H2A staining (C). 1047 The graphs show mean intensity of one (C, I), two (A) or four (B) experiments. The 1048 error bars indicate SEM and the mean values for DMSO-treated cells are set to 1.

1049

1050 Figure 4. CENP-F interaction with the Bub1 kinase domain is necessary for its 1051 kinetochore localization

33 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1052 A) Representative images of mitotic HeLa cells treated with 500 nM Hesperadin, 1053 showing that CENP-F kinetochore localization is strictly dependent on Aurora B kinase 1054 activity. Reduction in P-S10-H3 staining was used as a control for the Aurora B 1055 inhibition. Scale bar: 10 µm. B) Quantification of CENP-F kinetochore levels in cells 1056 treated as in A. The graph shows mean intensity of three experiments. The error bars 1057 indicate SEM and the mean values for DMSO-treated cells are set to 1. C) 1058 Representative images of GFP-expressing stable HeLa Flp-In T-REx cell lines mock 1059 treated or depleted of Bub1, showing that CENP-F kinetochore recruitment depends on 1060 the presence of Bub1 at kinetochores. D-G) Elution profiles and SDS-PAGE analysis of 1061 SEC experiments of mCherry-CENP-F2688-C with the Bub1FL:Bub3 complex; FL, full 1062 length (D), the Bub11-409:Bub3 complex (E), the Bub1 kinase domain (KinD) (F), and the 1063 BubR1:Bub3 complex (G). A shift in the elution volume is only observed for the Bub1 1064 constructs containing the C-terminal kinase domain (D, F) The interaction of CENP-F 1065 with Bub1 is specific, as no shift is observed for the BubR1:Bub3 complex (G). H) 1066 Representative images of stable HeLa Flp-In T-REx cell lines depleted of endogenous 1067 Bub1 and expressing GFP-Bub1 full length or lacking the kinase domain (Bub11-788). 1068 CENP-F kinetochore recruitment depends on the Bub1 kinase domain, as Bub11-788 does 1069 not rescue CENP-F localization, while full length Bub1 does. Scale bar: 10 µm. I) 1070 Quantification of CENP-F kinetochore levels in cells of panels C and H. The graph 1071 shows mean intensity of three independent experiments, the error bars indicate SEM. 1072 The mean value for non-depleted cells expressing GFP is set to 1.

1073

1074 Figure 4S1. Sensitivity to kinases inhibition of CENP-F kinetochore localization

1075 A-E) Representative images and quantification of CENP-F kinetochore levels in mitotic 1076 Hela cells treated with the indicated type and concentrations of kinase inhibitors. Scale 1077 bar: 10 µm. CENP-F localization is sensitive to Mps1 (A) and partially to Plk1 (D) 1078 inhibition. Despite a requirement for the Bub1 kinase domain for CENP-F kinetochore 1079 recruitment, Bub1 catalytic activity is dispensable (B, C). Reduction in Bub1 localization 1080 was used as a control for Mps1 inhibition (A) and reduction in P-T121-H2A staining was 1081 used as a control for Bub1 inhibition (E). Loss of Plk1 localization was used as control 1082 for Plk1 inhibition (D). The graphs show mean intensity of one (B, C, E) or two (A, D) 1083 experiments. The error bars indicate SEM and the mean values for DMSO-treated cells 1084 are set to 1. F) Representative images of HeLa cells mock treated or depleted of CENP-

34 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1085 F showing that CENP-F depletion does not affect the kinase activity of Bub1, as no 1086 changes are detected for the P-T121-H2A signal. Scale bar: 10 µm.

1087

1088 Figure 5. Requirements for CENP-F kinetochore localization

1089 A) Representative images of mitotic HeLa cells electroporated with mCherry, mCherry- 1090 CENP-F2688-C WT or mCherry-CENP-F2688-C C3207A (farnesylation) mutant. Scale bar: 5 1091 µm. As for CENP-E, both the WT and the un-farnesylated mutant CENP-F constructs 1092 localize at kinetochore. B) mCherry-CENP-F2688-C sample was visualized by electron 1093 microscopy after glycerol spraying and low-angle platinum shadowing (right panel). The 1094 elongated shape of the observed particles is consistent with the secondary structure 1095 expected for the mCherry-tag coiled-coil construct (right panel). C-D) SEC elution 1096 profiles and SDS-PAGE analysis of binding experiments with 16 µM each of mCherry- 1097 CENP-F2688-C WT (C) or C2961S mutant (D) and 4 µM Bub1FL:Bub3 complex. The shift 1098 in elution volume of Bub1:Bub3 is observed with both wild type and mutant CENP-F, 1099 but it significantly less pronounced for the CENP-F mutant, suggesting that the C2961S 1100 mutation reduces the affinity of CENP-F for the Bub1 kinase domain without 1101 completely abolishing it. E) SEC elution profile and SDS-PAGE analysis of a binding 1102 experiment with 16 µM each of mCherry-CENP-F2688-C and eGFP-CENP-E2070-C. No 1103 shift is observed, indicating that the tested constructs do not interact.

1104

1105 Figure 6. Identification of a minimal CENP-F construct for binding to Bub1

1106 A) Sedimentation velocity AUC results of the indicated mCherry-CENP-F constructs.

1107 MWobs, observed molecular weight; MWtheo, the predicted molecular weight of the 1108 monomer; Frict. ratio denotes the frictional ratio. AU, arbitrary units. mCherry-CENP- 1109 F2866-2990 forms a dimer. B) Elution profiles and SDS-PAGE analysis of SEC experiments 1110 of the Bub1 kinase domain (KinD) with mCherry-CENP-F2866-2990. The shift in elution 1111 volume of Bub1KinD indicated binding. The red asterisk indicated a breakdown product of 1112 mCherry that is produced during boiling in sample buffer. C) As in (A) but with the 1113 CENP-F2922-2990 construct, which is also dimeric. D) As in (B) but with the CENP-F2922-2990 1114 construct. Also in this case, an interaction with the kinase domain of CENP-F is clearly 1115 discernible. E) As in (A) but with the CENP-F2950-2990 construct, which is monomeric. F) 1116 As in (B) but with the CENP-F2950-2990 construct. In this case, no interaction with the

35 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1117 kinase domain of CENP-F is discernible. G) Representative images of mitotic HeLa cells 1118 electroporated with the indicated constructs. mCherry-CENP-F2688-C (positive control), 1119 mCherry-CENP-F2866-2990, and mCherry-CENP-F2922-2990 localized to kinetochores, 1120 whereas mCherry (negative control) and mCherry-CENP-F2950-2990 did not. Scale bar: 5 1121 µm.

1122

1123 Figure 7 Schematic of the interactions of Bub1 and BubR1 paralogs

1124 The schematic summarizes the interactions occurring at kinetochores between CENP-E, 1125 CENP-F, Bub1, and BubR1. Bub1 is recruited to the kinetochore subunit Knl1 (see 1126 Introduction) after phosphorylation by the SAC kinase Mps1. There, Bub1 recruits 1127 BubR1 through a pseudo-dimeric interaction (Musacchio, 2015). CENP-F kinetochore 1128 localization strictly depends on Bub1, while CENP-E recruitment requires a wider and 1129 still uncharacterized network of interactions, indicated by a question mark. RZZ and 1130 Mad1 recruitment, as well as the corona expansion, appear to be independent from 1131 CENP-E and CENP-F, and are not shown. An interaction of CENP-E and CENP-F has 1132 also been identified (grey arrow), but is not sufficient for CENP-F localization in absence 1133 of Bub1.

1134

36 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 A B Coiled Coil 0 KT-MT Corona Attached 1 CENP-E 2701 attachment CENP-E kinetochore Kinesin CENP-F + MT Domain KT localization binding KT-MT CENP-F Spindle 2070 2701 attachment Dynein MT SAC Dynactin Coiled 1 inactivation RZZ & Coil 0 Spindly CENP-F SAC MAD1 1 3210 MT CENP-E KT MT activation MAD2 IP Unattached binding binding localization binding OP kinetochore 2688 3210 C D Inter- Pro- Prometa- Meta- Ana- Telo- early Inter- Pro- Prometa- Meta- Ana- Telo- early phase phase phase phase phase phase G1 phase phase phase phase phase phase G1 N RS EPFTubulin CENP-F CREST DNA N RS EPETubulin CENP-E CREST DNA

Ciossani, Overlack et al. Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A DNA CREST Zwilch Tubulin B DNA CREST Mad1 Tubulin

Inter- Inter- phase phase

Pro- Pro- phase phase

Prometa- Prometa- phase phase

Meta- Meta- phase phase

Ana- Ana- phase phase

Telo- Telo- phase phase

early early G1 G1

Ciossani, Overlack et al. Figure 1S1 E A CENP-E RNAi

DNA CREST Mad1 CENP-E Zwilch RNAi DNA CREST CENP-E Zwilch bioRxiv preprint

MAD1 intensity CENP-E intensity not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade CENP-E RNAi - normalized to CREST Zwilch RNAi normalized to CREST + - 0.0 0.5 1.0 0.0 0.5 1.0 + - + - doi: + https://doi.org/10.1101/276204 B F DNA CREST Mad1 CENP-F CENP-E RNAi DNA CREST Zwilch CENP-E CENP-F RNAi

Zwilch intensity available undera MAD1 intensity

Ciossani, Overlack etal.Figure 2 normalized to CREST CENP-E RNAi 0.0 0.5 1.0 CENP-F RNAi normalized to CREST - - 0.0 0.5 1.0 + - ; + - this versionpostedMarch4,2018. CC-BY-NC-ND 4.0Internationallicense + + G C DNA CREST CENP-E CENP-F CENP-F RNAi DNA CREST Zwilch CENP-F CENP-F RNAi Zwilch intensity CENP-E intensity

normalized to CREST CENP-F RNAi - CENP-F RNAi 0.0 0.5 1.0 normalized to CREST + - 0.0 0.5 1.0 The copyrightholderforthispreprint(whichwas + - + - + . D + CENP-FRNAi H CENP-E RNAi DNA CREST Zwilch CENP-E CENP-E +CENP-FRNAi CENP-E RNAi DNA CREST CENP-F CENP-E Zwilch intensity CENP-F intensity normalized to CREST 0.0 0.5 1.0 - CENP-E RNAi normalized to CREST + - 0.0 0.5 1.0 + - + - + bioRxiv preprint CENP-E (316kDa) CENP-F (368kDa) not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade Tubulin (50kDa) Tubulin (50kDa) F D C A CENP-E RNAi CENP-F RNAi doi: CENP-F intensity CENP-E intensity normalized to CREST normalized to CREST https://doi.org/10.1101/276204 0.0 0.5 1.0 0.0 0.5 1.0 100 % 100 % mock 50 % 50 % mock + - + -

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5 % CENP-E RNAi RNAi 100 % 100 % -50 -260 (kDa) -50 MW -260 (kDa) MW ; this versionpostedMarch4,2018. CC-BY-NC-ND 4.0Internationallicense E B DNA CREST CENP-F DNA CREST CENP-E CENP-F RNAi - - CENP-E RNAi Ciossani, Overlack etal.Figure2S1 The copyrightholderforthispreprint(whichwas + + . G I Zwilch RNAi Zwilch RNAi Mad1 intensity Zwilch intensity Zwilch intensity

Mad1 inZwilchRNAi normalized to CREST Zwilch normalized to CREST normalized to CREST RNAi 0.0 0.5 1.0 0.0 0.5 1.0 Zwilch RNAiefficiency Zwilch RNAiefficiency 0.0 0.5 1.0 (Cenp-E exp) (Mad1 exp) + - + - + -

Mad1 intensity (AU) J 0.00 0.01 0.02 0.03 0.04 H . . . 0.3 0.2 0.1 0.0 DNA CREST Zwilch Mad1 R y=0.08298*x+0.006827 2 : 0.8345 - Zwilch RNAi Zwilch intensity(AU) + bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A CENP-E + CENP-F RNAi B Zwilch + BubR1 RNAi C Zwilch + BubR1 RNAi

- + - + - + DNA CREST Zwilch BubR1

DNA CREST CENP-E CENP-F DNA CREST Zwilch CENP-E

D Zwilch + Bub1 RNAi E Zwilch + Bub1 RNAi 1.0 - + + - + + en sity CR ES T int to to F

ed 0.5 li z enp -E /- C no rm a 0.0 CENP-E RNAi - + - + + CENP-F RNAi CENP-E CENP-F DNA CREST Zwilch Bub1 DNA CREST Zwilch CENP-E

Ciossani, Overlack et al. Figure 2S2 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. A DNA CREST Zwilch Tubulin

Prometa- phase mock

Meta- phase

B Prometa- phase

CENP-F RNAi Meta- phase

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Prometa- phase

CENP-E RNAi Meta- phase

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percentage of cells B 100 20 40 60 80 0 DNA CREST CENP-F Tubulin Metaphase

aligned Metaphase aligned NO TREATMENT available undera

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Duration of mitosis (h) F RNAi 0.0 0.5 1.0 1.5 C

percentage of cells Metaphase aligned 100 20 40 60 80 0 +

Metaphase aligned The copyrightholderforthispreprint(whichwas 10 uMMG-132for2h Metaphase with misalignment .

Metaphase with + misalignment C mock enp- F RNAi bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

eGFP A GFP CREST CREST E eGFP-CENP-E2070-C + BubR1:Bub3 1.0

2070-C Control eGFP-CENP-E 0.8 BubR1-Bub3 (1:1)

MWtheo= 182 kDa 0.6 2070-C

2070-C

WT eGFP-CENP-E

eGFP- 0.4 MWtheo = 103 kDa c(S) (AU/S) CENP-E (monomer) 0.2 2070-C 0.0 0 2 4 6 8 10 12 14 eGFP- C2697A sedimentation coefficient (S) CENP-E

B Superose 6 Increase 5/150 C Superose 6 Increase 5/150 D Superose 6 Increase 5/150 488 280 GFP 2070-C 488 280 GFP 2070-C 488 280 GFP 2070-C 40 CENP-E + CENP-E + 40 CENP-E + BR1-B3 40 BR1KinD B1FL-B3 GFPCENP-E2070-C GFPCENP-E2070-C GFPCENP-E2070-C 30 30 30 BR1-B3 BR1KinD B1FL-B3 20 20 20 Absorbance Absorbance

Absorbance 10 10 10 280/488 nm (mAU) 280/488 nm (mAU) 0 280/488 nm (mAU) 0 0 1.0 1.5 2.0 2.5 1.0 1.5 2.0 2.5 1.0 1.5 2.0 2.5 Elution volume (ml) Elution volume (ml) Elution volume (ml)

150 150 150 100 GFPCENP-E2070-C 100 GFPCENP-E2070-C 100 GFPCENP-E2070-C 75 75 75 50 50 50 37 37 37

25 25 25

150 BubR1 150 Bub1 100 150 100 75 100 75 75 50 50 50 37 Bub3 KinD 37 Bub3 37 BubR1

25 25 25 150 BubR1 150 Bub1 100 GFP 2070-C 100 GFPCENP-E2070-C 150 CENP-E 75 100 GFPCENP-E2070-C 75 75 50 50 50 Bub3 KinD Bub3 37 37 BubR1 37

25 25 25

F RNAi G mock Bub1 BubR1 Bub1+BubR1 1.0

0.5 CENP-E intensity

normalized to CREST 0.0 Bub1 - + RNAi

1.0

0.5 CENP-E intensity

normalized to CREST 0.0 DNA CREST CENP-E BubR1 - + RNAi

Ciossani, Overlack et al. Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A DNA CREST CENP-E P-T232 AurB

CENP-E in Hesp DMSO

1.0 CR ES T en sity o int E ed t

li z 0.5 500 nM ENP - Hesperadin C no rm a 0.0

DMSO 500 nM Hesp 500 nM Hesperadin

B DNA CREST CENP-E Bub1 CENP-E in Rev

1.0 CR ES T en sity o int DMSO E ed t 0.5 li z ENP - C

no rm a 0.0

500 nM 500 nM Reversine DMSO Reversine

CENP-E in P-T121-H2A in 10 µM BAY-320 C DNA CREST CENP-E P-T121 H2A 10 µM BAY-320 1.5 1.5 DMSO

CR ES T 1.0 1.0 en sity o int E ed t

li z 0.5 0.5 ENP - pH2A intensity C normalized to CREST no rm a 10 µM BAY-320 0.0 0.0

DMSO 10 µM DMSO 10 µM BAY-320 BAY-320

DNA CREST CENP-E CENP-E in BI-2536 D 1.5

1.0 CR ES T en sity DMSO o int E ed t

li z 0.5 ENP - C no rm a BI2563 500 nM 0.0 nM

DMSO 500 -2563 BI

Ciossani, Overlack et al. Figure 3S1 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A DNA CREST CENP-F P-S10-H3 B

1.0 DMSO

0.5

CENP-F intensity 0.0 normalized to CREST 500 nM Hesp Hesperadin DMSO (500 nM) C D Superose 6 Increase 5/150 E Superose 6 Increase 5/150 587 280 mCh 2688-C 40 mChCENP-F2688-C 40 587 280 CENP-F GFP FL + B11-409-B3 Bub1 + B1 -B3 - + mCh 2688-C mCh 2688-C RNAi 30 CENP-F 30 CENP-F FL 1-409 20 B1 -B3 20 B1 -B3 Absorbance 10 Absorbance 10 280/587 nm (mAU) 0 280/587 nm (mAU) 0 1.0 1.5 2.0 2.5 1.0 1.5 2.0 2.5 Elution volume (ml) Elution volume (ml)

150 150 100 mCh 2688-C 100 mChCENP-F2688-C 75 CENP-F 75 50 50 37 37

25 25

150 Bub1 150 100 100 75 75

50 50 1-409

DNA CREST CENP-F GFP Bub1 37 Bub3 37 Bub3

25 25

150 Bub1 150 H GFP-Bub1 100 mChCENP-F2688-C 100 mChCENP-F2688-C 75 75 Bub1 FL 1-788 50 50 Bub11-409 + + RNAi 37 Bub3 37 Bub3

25 25

F Superose 6 Increase 5/150 G Superose 6 Increase 5/150

587 280 mCh 2688-C 587 280 mCh 2688-C 40 CENP-F 40 CENP-F FL KinD + BR1 -B3 + B1 mCh 2688-C 30 mChCENP-F2688-C 30 CENP-F FL KinD BR1-B3 20 B1 20 Absorbance 10 10 Absorbance 280/587 nm (mAU)

280/587 nm (mAU) 0 0 1.0 1.5 2.0 2.5 1.0 1.5 2.0 2.5 Elution volume (ml) Elution volume (ml)

DNA CREST CENP-F GFP 150 150 100 mCh 2688-C 100 mChCENP-F2688-C 75 CENP-F 75 50 50 37 37

I 25 25 1.0 150 150 BubR1 100 100 75 75 50 50 0.5 KinD 37 Bub1 37 Bub3

25 25 Cenp-F intensity

normalized to CREST 150 0.0 150 - + + + 100 mChCENP-F2688-C BubR1 Bub1 RNAi 75 100 mChCENP-F2688-C FL 75 1-788 50 GFP GFP 50 37 Bub1KinD 37 Bub3 GFP-Bub1 25 GFP-Bub1 25

Ciossani, Overlack et al. Figure 4 A C B BAY-320 BAY-320 Reversine bioRxiv preprint

10 µM DMSO 3 µM DMSO 500 nM DMSO not certifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade N RS EPFBub1 CENP-F CREST DNA N RS CENP-F CREST DNA N RS CENP-F CREST DNA doi: https://doi.org/10.1101/276204 available undera ; this versionpostedMarch4,2018. CC-BY-NC-ND 4.0Internationallicense

CENP-F intensity CENP-F intensity normalized to CREST normalized to CREST 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 DMSO

DMSO CENP-F intensity 3 µM normalized to CREST BAY-320 0.0 0.5 1.0 Ciossani, Overlack etal.Figure4S1

10 µM DMSO BAY-320 The copyrightholderforthispreprint(whichwas 500 nM Rev . E D F BAY-320 BI 10 µM DMSO 500 nM DMSO

N RS P-T121-H2A CREST DNA N RS P-T121-H2A CREST DNA N RS EPFPlk1 CENP-F CREST DNA

+ -

CENP-F RNAi CENP-F P-H2A intensity normalized to CREST 0.0 0.5 1.0 1.5

DMSO CENP-F intensity normalized to CREST 0.0 0.5 1.0 1.5 10 µM BAY-320 DMSO

500 nM BI-2563 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A mCherry B mCherry CREST CREST mCherry-CENP-F2688-C control mCherry 2688-C WT mCherry CENP-F

2688-C 2688 3210 mCherry Kinetochore MT C3207A mCherry 50 nm

CENP-F localisation

C Superose 6 Increase 5/150 D Superose 6 Increase 5/150 E Superose 6 Increase 5/150

mCh 2688-C 587 280 mCh 2688-C/WT 60 587 280 mCh 2688-C/mChC2961S 488 280 CENP-F 60 CENP-F CENP-F 40 GFP 2070-C FL FL + CENP-E 50 + B1 -B3 50 + B1 -B3 30 40 mChCENP-F2688-C/WT 40 mChCENP-F2688-C/C2961S mChCENP-F2688-C 30 30 20 B1FL-B3 B1FL-B3 GFPCENP-E2070-C 20 20 Absorbance Absorbance Absorbance 10 10 10 280/587 nm (mAU) 280/587 nm (mAU)

0 0 280/488/587 nm (mAU) 0 1.0 1.5 2.0 2.5 1.0 1.5 2.0 2.5 1.0 1.5 2.0 2.5 Elution volume (ml) Elution volume (ml) Elution volume (ml)

150 150 150 100 GFP 2070-C mCh 2688-C 100 CENP-E 100 CENP-F mCh 2688-C/C2961S 75 75 CENP-F 75 50 50 50 37 37 37

25 25 25

150 Bub1 150 100 150 Bub1 100 mCh 2688-C 75 100 75 CENP-F 75 50 50 50 37 Bub3 37 Bub3 37 25 25 25

150 150 Bub1 150 Bub1 100 100 GFPCENP-E2070-C mCh 2688-C 100 mCh 2688-C/C2961S mCh 2688-C 75 CENP-F 75 CENP-F 75 CENP-F 50 50 50 Bub3 37 37 Bub3 37

25 25 25

Ciossani, Overlack et al. Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A mCherry-CENP-F2866-2990 C mCherry-CENP-F2922-2990 E mCherry-CENP-F2950-2990 1.6 2.0 1.4 1.5 1.2 MW = 43.5 kDa MW = 36.8 kDa MW = 33.6 kDa theo theo 1.5 theo 1.0 MW = 83.1 kDa MW = 67.2 kDa MW = 34.3 kDa obs 1.0 obs obs 0.8 Frict. ratio = 1.7 Frict. ratio = 1.6 1.0 Frict. ratio = 1.4 0.6 c(S) (AU/S) 0.4 c(S) (AU/S) 0.5 c(S) (AU/S) 0.5 0.2 0.0 0.0 0.0 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 0 2 4 6 8 10 12 14 sedimentation coefficient (S) sedimentation coefficient (S) sedimentation coefficient (S)

Superdex 75 Increase 5/150 B Superdex 75 Increase 5/150 D F Superdex 75 Increase 5/150 80 80 587 280 60 587 280 mCh 2866-2990 mCh 2922-2990 CENP-F CENP-F 587 280 mCh 2950-2990 70 KinD 70 + B1KinD 50 CENP-F + B1 + B1KinD 60 mCh 2866-2990 60 mChCENP-F2922-2990 CENP-F 40 mCh 9250-2990 50 50 CENP-F KinD B1KinD 40 B1 40 30 B1KinD 30 30 20 Absorbance Absorbance 20 20 Absorbance 10 280/587 nm (mAU) 280/587 nm (mAU) 10 10 280/587 nm (mAU) 0 0 0 0.8 1.0 1.2 1.4 1.6 1.8 0.8 1.0 1.2 1.4 1.6 1.8 1.0 1.2 1.4 1.6 1.8 2.0 Elution volume (ml) Elution volume (ml) Elution volume (ml) 100 100 100 75 75 75 50 50 50 Bub1KD Bub1KinD KinD 37 37 37 Bub1 25 25 25 20 20 20

100 100 100 75 75 75 50 50 50 mCh 2866-2990 CENP-F mCh 2922-2990 mCh 2950-2990 37 37 CENP-F 37 CENP-F 25 * 25 * 25 *

20 20 20 100 100 100 75 75 75 50 mChCENP-F2866-2990 50 50 KinD mCh 2922-2990 Bub1KD 37 Bub1 37 CENP-F mCh 2950-2990 * Bub1KinD 37 CENP-F 25 25 * 25 * 20 20 20

G Control CENP-F2688-C CENP-F2866-2990 CENP-F2922-2990 CENP-F2950-2990 mCherry CREST CREST mCherry

Ciossani, Overlack et al. Figure 6 bioRxiv preprint doi: https://doi.org/10.1101/276204; this version posted March 4, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Bub1 BubR1 kinase pseudo kinase

Bub3 Bub3 CENP-F CENP-E Knl1 P ?

TPR TPR

Mps1 Kinetochore

Ciossani, Overlack et al. Figure 7