bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license.

1 The Ndc80 complex targets Bod1 to human mitotic 2 3 4

5 Katharina Schleicher*, Sara ten Have, Iain M. Porter†, and Jason R. Swedlow†

6

7

8 Centre for Regulation and Expression, College of Life Sciences, University of Dundee,

9 Dundee, United Kingdom, DD1 5EH

10

11 Author for correspondence: Jason R. Swedlow [email protected]

12 † These authors contributed equally to this study.

13 *Present address: Department of Physiology, Anatomy and Genetics, University of Oxford,

14 Sherrington Road, Oxford, OX1 3PT, United Kingdom

15 16 17 18 19 20 21 22 23 24 25 Keywords: 26 segregation, , PP2A-B56, PP2A inhibitor, Ndc80 complex, Bod1 bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license.

27 Abstract 28 Regulation of phosphatase activity by endogenous protein inhibitors is an

29 important mechanism to control protein phosphorylation in cells. We recently identified

30 Biorientation defective 1 (Bod1) as a small protein inhibitor of PP2A-B56. This phosphatase

31 controls the amount of phosphorylation of mitotic kinetochore and thus the

32 establishment of load-bearing chromosome-spindle attachments in time for accurate separation

33 of sister chromatids in . The PP2A-B56 regulatory protein Bod1 directly localises to

34 mitotic kinetochores and is required for correct segregation of mitotic . In this

35 report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression.

36 Kinetochore localisation of Bod1 increases from nuclear envelope breakdown until metaphase.

37 Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1’s binding and inhibition

38 of PP2A-B56, peaks in prometaphase, when PP2A-B56 localisation to kinetochores is highest.

39 Kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-

40 B56. Additionally, Bod1 depletion leads to a defect in the phosphorylation kinetics of Ndc80

41 at serine 55 (S55) within its N-terminus. Therefore, Ndc80 recruits a phosphatase inhibitor to

42 kinetochores which directly feeds forward to regulate Ndc80 phosphorylation and the stability

43 of the attachments of microtubules to kinetochores.

44 bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

45 1. Introduction 46 To preserve genome integrity, the two sister chromatids of each mitotic chromosome

47 must be distributed equally between daughter cells. Movement of sister chromatids to opposite

48 poles of a dividing cell requires attachment to spindle microtubules of opposing orientation.

49 Errors in the attachment process can lead to chromosome missegregation and aberrant

50 chromosome numbers. Such aneuploid karyotypes are the major cause of spontaneous

51 miscarriages in humans [1] and often observed in cancer genomes [2]. Spindle microtubule

52 attachment occurs at a multi-complex protein interface between mitotic chromosomes and the

53 spindle apparatus called the kinetochore [3]. The kinetochore consists of approximately 30 core

54 structural proteins that are arranged into several functional subcomplexes. As well as providing

55 a structural link between chromosomes and spindle microtubules, structural kinetochore

56 proteins also act as a signalling platform by recruiting checkpoint proteins, kinases, and

57 phosphatases. Early kinase activity destabilises kinetochore-microtubule interactions [4,5] to

58 prevent attachment errors [6]. Conversely, phosphatase activity is needed at later stages of the

59 attachment process to stabilise kinetochore-microtubule interactions that have passed the

60 quality-control and thus protect load-bearing attachments [7]. There is a conserved, dynamic

61 system comprised of at least eight kinases and two phosphatases that control microtubule-

62 kinetochore attachments [3,8].

63 The detailed role and function of protein phosphatases and their interplay at the

64 kinetochore is only beginning to be elucidated [9–11], but both are of great interest as they are

65 absolutely required to ensure faithful chromosome segregation. Protein phosphatase 1 (PP1)

66 dephosphorylates microtubule-binding kinetochore proteins to ultimately stabilise attachments

67 [12,13]. However, recruitment of PP1 to kinetochores requires the initial activity of protein

68 phosphatase 2A (PP2A) [14], highlighting the importance of coordinated timely activation of

69 these kinetochore components. PP2A is a heterotrimeric , composed of a scaffolding bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

70 (A) subunit, catalytic (C) subunit, and a regulatory (B) subunit [15]. It is targeted to the

71 kinetochore by the B56 family of B subunits [11,16,17]. The highest mitotic occupancy of

72 PP2A-B56 at kinetochores is reached in prometaphase and can be maximised by increasing the

73 number of unattached kinetochores with nocodazole [11]. Under the same conditions, PP1

74 localisation to kinetochores is low [12], suggesting that PP2A accumulation at unattached

75 kinetochores alone is not sufficient to recruit PP1 and that additional molecular signals are

76 required to activate PP2A-mediated PP1 recruitment in metaphase. .

77 We have recently identified a small kinetochore protein, Biorientation defective 1

78 (Bod1), that can specifically inhibit PP2A-B56 [9,18]. Bod1 is required for cognitive function

79 in humans and Drosophila models [19] and is thought to contribute to the early stages of

80 radiation-induced genomic instability [20]. Depletion of Bod1 from HeLa cells leads to

81 premature loss of phosphorylation on several kinetochore proteins, including MCAK and

82 CENPU, due to unregulated activity of PP2A-B56. It also causes an increase in aberrant

83 chromosome attachments and defective chromosome segregation. Bod1, together with CIP2A

84 [21], FAM122A [22], I1PP2A/ANP32A [23], I2PP2A/SET [24], TIP [25], and Arpp-19/Ensa

85 [26,27], forms part of a growing family of PP2A inhibitors that have important roles in

86 supporting cell division. However, little is known about the localisation of these PP2A

87 regulators or how they modulate the activity of PP2A towards different substrates.

88 We have studied the temporal recruitment and phospho-regulation of Bod1 at mitotic

89 kinetochores. Bod1 is recruited to kinetochores by the outer kinetochore protein Ndc80

90 (Nuclear division cycle protein 80, also known as highly expressed in cancer protein Hec1).

91 Furthermore, Bod1 can protect phosphorylation of a key site in the N-terminal tail of Ndc80

92 that is required for microtubule attachment. These data refine our understanding of how PP2A

93 activity at the kinetochore is regulated and identify another target of the Bod1 phosphatase

94 inhibitor pathway. bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

95 2. Results

96 Bod1 localises to kinetochores throughout mitosis and is maximally 97 phosphorylated in prometaphase 98 To dissect the temporal regulation of Bod1 recruitment to kinetochores, we raised

99 peptide antibodies for immunofluorescence profiling in HeLa cells (figure 1a; electronic

100 supplementary material, figure S1). We quantified Bod1 kinetochore intensities within a 4 pixel

101 radius of anti-centromere antibody (ACA) staining as a reference (figure 1c). Bod1 is first

102 detected on kinetochores at nuclear envelope breakdown and reaches its maximum at

103 metaphase.

104 We showed previously that inhibition of PP2A-B56 by Bod1 is greatly enhanced by its

105 phosphorylation at T95 [9]. We therefore raised a phospho-specific antibody against this site

106 (figure 1b; electronic supplementary material, figure S1). Quantification of pT95 Bod1 at

107 kinetochores revealed that this post-translational modification peaks in prometaphase, before

108 maximal recruitment of the total protein (figure 1d). PP2A-B56 levels at kinetochores are

109 highest in prometaphase when attachments are weak [11]. The phosphorylation of Bod1 at

110 T95 therefore coincides with the recruitment of PP2A-B56, consistent with a role in inhibiting

111 PP2A-B56 activity and enabling correction of attachment errors in early mitosis.

112 Bod1 recruitment to kinetochores is independent of PP2A-B56 and Knl1 113 To test whether Bod1 and PP2A-B56 are co-recruited to kinetochores, we depleted

114 PP2A-B56 from HeLa cells using a pool of B56 isoform-specific siRNAs [11] and quantified

115 total Bod1 protein at the kinetochores (figure 2a-e). Surprisingly, there was no significant

116 change in Bod1 recruitment to kinetochores upon B56 depletion. Since it is difficult to achieve

117 complete knockdown of B56 isoforms via siRNA (figure 2e), we then depleted the outer

118 kinetochore protein Knl1, which acts as a scaffolding platform that recruits PP2A-B56 to

119 kinetochores via BubR1 [16,17,28]. As with B56 depletion, siRNA-mediated knockdown of

120 Knl1 did not affect Bod1 recruitment to kinetochores (figure 2f-h). We therefore conclude that bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

121 Bod1 is recruited to kinetochores independently of its target, PP2A-B56, and via a different

122 interaction platform.

123 The mitotic interactome of Bod1 contains many outer kinetochore 124 proteins including Ndc80 125 To discover candidate proteins that might target Bod1 to kinetochores, we combined

126 affinity purification of Bod1 with label-free quantitative mass spectrometry (MS) (figure 3a;

127 electronic supplementary material, figure S2). In mitotic lysates from HeLa cells expressing

128 Bod1-GFP, we identified and could quantify 3512 proteins. Of these, 42 were significantly

129 enriched in affinity purifications from Bod1-GFP expressing cells compared to cells expressing

130 GFP alone as a control (n = 4 biological replicates; electronic supplementary material, table

131 S1). Analysis of (GO) term annotation identified 95 centromere- and

132 kinetochore-associated proteins in the Bod1-GFP affinity purifications (electronic

133 supplementary material, table S2). Of these, Bod1 itself, Nuclear division cycle protein 80

134 (Ndc80; also known as Highly expressed in cancer protein (Hec1)), and Dynein intermediate

135 chain 1 (DYNC1I1) were significantly enriched in Bod1-GFP affinity purifications compared

136 to controls (figure 3b; electronic supplementary material, figure S3a). The most reproducible

137 kinetochore interactor was Ndc80; it was found in all four biological replicates of the

138 experiment. Furthermore, of all kinetochore proteins detected, Ndc80 exhibited the highest fold

139 change in Bod1-GFP affinity purifications compared to controls. Intensity analysis of the

140 centromeric region in HeLa cells, co-stained with Bod1 and Ndc80 antibodies, revealed that

141 the immunofluorescence signal of the two proteins overlaps at the outer kinetochore (figure

142 3c). The mitotic Bod1 interactome also contained components of the SET1B methyltransferase

143 complex with significant enrichment of ASH2L. This is consistent with previous results in

144 asynchronous HeLa cells [29]. bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

145 Bod1 directly interacts with the Ndc80 complex 146 To confirm the Bod1-Ndc80 interaction detected by MS analysis, we performed pull

147 down assays with purified Bod1-GST on Sepharose beads to validate Ndc80 as a bona fide

148 Bod1 interactor. Ndc80 localises to kinetochores as part of the heterotetrameric Ndc80

149 complex, consisting of Ndc80, Nuf2, Spc24, and Spc25 [30,31] (figure 4a). Bod1-GST coated

150 beads pulled out Ndc80, Nuf2 and Spc24 from mitotic HeLa cell lysates (figure 4b) (Spc25

151 was not tested). In order to determine whether this was a direct interaction with the complex,

152 we tethered recombinant Ndc80 Bonsai, a truncated form of the complex containing a GST-

153 Nuf2-Spc24 fusion and an Ndc80-Spc25 fusion that can be expressed in bacteria [32], to beads

154 and incubated them with recombinantly expressed Bod1-MBP or MBP alone (figure 4c,d).

155 Bod1-MBP interacted strongly with purified recombinant Ndc80 Bonsai complexes (figure

156 4e), supporting the proteomics and immunofluorescence data. Together these results

157 demonstrate Bod1 is part of the outer kinetochore and directly interacts with Ndc80.

158 Ndc80 is essential for Bod1 kinetochore recruitment 159 To test if the Ndc80 complex was necessary for Bod1 kinetochore recruitment in cells,

160 we depleted Ndc80 from HeLa cells using siRNA. Ndc80 depletion also reduced the

161 immunofluorescence signal of its direct binding partner Nuf2 (figure 5). In contrast, we

162 observed only a minor reduction in Knl1 signal, indicating that Ndc80 siRNA did not

163 destabilise the entire outer kinetochore. Crucially, Ndc80 depletion resulted in significant loss

164 of Bod1 from kinetochores, concomitant with an increase in localisation of B56. The increase

165 in B56 localisation recapitulates our previous observations that siRNA depletion of Bod1

166 elevates B56 levels at kinetochores [9] and suggests that the localisation of Bod1 to

167 kinetochores might limit PP2A-B56 accumulation at these sites. bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

168 Bod1 depletion results in loss of Ndc80 phosphorylation at its N-terminal 169 tail 170 siRNA-mediated depletion of Bod1 leads to mitotic arrest as cells are unable to

171 maintain chromosome alignment in metaphase until onset [18]. This biorientation

172 phenotype is accompanied by an increase in syntelic kinetochore-microtubule attachments, an

173 attachment conformation in which a pair of sister kinetochores connects to spindle

174 microtubules emanating from the same pole. This form of attachment can lead to erroneous

175 mitosis, , and cell death [33] and therefore needs to be corrected before cells

176 progress through mitosis. Correction of syntelic attachments is enabled by phosphorylation of

177 outer kinetochore proteins [4,5,7,34], among them the N-terminus of Ndc80. Upon

178 phosphorylation, the affinity of these proteins to microtubules is reduced and attachments are

179 destabilised [5]. To evaluate if Ndc80 phosphorylation is dependent on Bod1 levels, we probed

180 Ndc80 phosphorylation at its N-terminal serine 55 (S55) in Bod1 depleted cells using a

181 phospho-specific antibody against this site. First, we compared control metaphase cells with

182 Bod1 depleted cells that exhibited a clear biorientation phenotype. Bod1 depleted cells with

183 biorientation phenotypes exhibited a 65% decrease in Ndc80 phosphorylation at S55 (figure

184 6a,b). However, Bod1 depletion and development of the bioriention phenotype also led to a

185 small but significant reduction (~30%) in the intensity of total Ndc80 protein at the kinetochore.

186 We therefore aimed to understand the effects of Bod1 depletion on Ndc80 phosphorylation at

187 S55 in cells just entering mitosis, before development of the mature chromosome misalignment

188 phenotype.

189 To selectively sample cells between nuclear envelope breakdown and metaphase, we

190 first synchronised cells at the G2/M transition with RO-3306, a Cdk1 inhibitor [35]. The cells

191 were then released into mitosis. Their medium was supplemented with the proteasome inhibitor

192 MG132 to limit mitotic progression to , prometaphase, and metaphase (figure 6c).

193 Thirty minutes after RO-3306 release we assessed both Ndc80 pS55 kinetochore intensities bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

194 and mitotic progression. At the early stages of mitosis, when error correction takes place, Bod1-

195 depleted cells had significantly lower Ndc80 pS55 levels than control cells (figure 6c). Bod1-

196 depleted cells also showed a delay in mitotic progression, even in these early stages of mitosis,

197 which is consistent with previous findings [18].

198 Our results suggest a model where Bod1 holds PP2A-B56 activity in check during

199 prometaphase. This ensures that phosphorylation of Ndc80 and possibly other kinetochore

200 substrates can occur, allowing turnover of attachments (figure 6d). Once amphitelic

201 kinetochore-microtubules attachment are achieved, we observe rapid dephosphorylation of

202 Bod1 at T95. We have previously shown that loss of Bod1 phosphorylation prevents its

203 inhibition of PP2A-B56 [9]. Active PP2A-B56 can then dephosphorylate the RVSF and SILK

204 motifs on KNL1 [36], resulting in recruitment of PP1 [12] and stabilization of kinetochore-

205 microtubule attachments by PP1-mediated dephosphorylation of Ndc80 [13].

206 3. Discussion 207 Using a combination of proteomics, in vitro recombinant interaction studies, and in vivo

208 siRNA mediated localisation studies, we have demonstrated that the PP2A-B56 regulator Bod1

209 is recruited to kinetochores by Ndc80. Using quantitative immunofluorescence, we have also

210 demonstrated that Bod1 is required to prevent premature dephosphorylation of Ndc80 during

211 early mitosis. Together with MCAK S92 [15] and CENP-U/PBIP1 T78 [16], Ndc80 S55 adds

212 to the list of phospho-epitopes at kinetochores lost upon Bod1 depletion. Bod1 therefore

213 controls the phosphorylation of distinct groups of kinetochore proteins, all of which are

214 implicated in the establishment of proper amphitelic kinetochore – microtubule attachments.

215 Bod1 is present at kinetochores throughout mitosis, from nuclear envelope breakdown

216 until the end of anaphase [18]. Here, quantitative analysis has demonstrated that Bod1

217 localisation to kinetochores peaks during metaphase. Phosphorylation of Bod1 at T95 is bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

218 essential for its inhibitory function against PP2A-B56 [9]. In contrast to the total population of

219 Bod1, the pool of phopsho-T95 Bod1 peaked at kinetochores during prometaphase. This timing

220 potentiates the inhibitory activity towards PP2A-B56 when phosphatase localisation to

221 kinetochores is greatest and its activity needs to be properly regulated to allow correction of

222 erroneous attachments [11].

223 While PP2A-B56 is recruited to kinetochores in a Knl1-dependent manner [16,17], we

224 show here that Bod1 relies on the Ndc80 complex for kinetochore binding instead. Inter-

225 complex interactions within the KMN network are an emerging theme in attachment

226 regulation: The mitotic checkpoint kinase Mps1 is recruited to kinetochores through Ndc80

227 [14,37,38], but it phosphorylates MELT motifs on Knl1 [39–41]. Whether dynamic structural

228 changes of the outer kinetochore allow interaction between components of the different KMN

229 subcomplexes or medium affinity binding surfaces allow protein turn-over and localised

230 gradients of diffusible proteins remains to be determined.

231 4. Materials and methods

232 Cell lines and cell culture 233 HeLa S3 cells were maintained in EMEM (Lonza) and supplemented with 10% fetal calf 234 serum, 2mM L-glutamine, 100U/ml penicillin and 100mg/ml streptomycin. A cell line stably 235 expressing Bod1-GFP was generated using HeLa cells harbouring a single Flp recombination 236 target (FRT) site in their genome (a kind gift from Patrick Meraldi [42]) and maintained in the 237 media described above with an additional 200 mg/ml hygromycin. Cells lines were maintained 238 at 37°C with 5% CO2 in a humidified incubator. For transfections, cells were seeded in 6-well 239 dishes and transfected with 300ng plasmid DNA per well using Effectene transfection reagent 240 (Qiagen) or with 33nM siRNA oligo duplexes or medium GC control siRNA (Invitrogen) using 241 lipofectamine 2000 (Invitrogen). Cells were split onto coverslips the next day. 242 Immunofluorescence staining of the cells and immunoblot analysis were performed 48h after 243 siRNA transfection.

244 Generation of peptide antibodies 245 Sheep were immunized with the immunogenic Bod1 peptide NH2-CRNGLRQ[pS]VVQS- 246 COOH and serum containing the polyclonal antibody was collected in three batches. The third 247 batch, obtained 91 days after the initial immunisation and 7 days after the third antigen booster 248 injection, was used for antibody purification. 5mg NH2-CRNGLRQSVVQS-COOH peptide 249 were added to 5ml activated Affigel-10 (Biorad) in 0.1M Na phosphate buffer pH7.8. Coupling 250 of was allowed to take place over night. Then, residual iodoacetate groups were blocked with bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

251 0.2% β-mercaptoethanol and non-covalently bound peptide was removed by consequtive 252 washes with 0.1M NaHCO3, 1M Na2CO3, water, 0.2M glycine-HCl pH2.0, 150mM NaCl, 253 and TBS. The resin was stored in 0.1% NaN3 in TBS. For antibody purification, 4ml serum 254 were diluted 1:1 with TBS and passed through a 0.2µm filter. The diluted serum was run over 255 the column ten times. The column was then washed with TBS, 0.5M NaCl, 20mM Tris-HCl 256 pH7.4, 0.2% Triton-X in TBS, and TBS. A low pH elution was performed with 0.15M NaCl, 257 0.2M glycine-HCl pH2.0 collecting 1ml fractions with each tube containing 0.1ml 2MTris-HCl 258 pH8.5. After re-equilibrating the pH of the column by washing with TBS, a second, guadinuim 259 hydrochloride elution was performed with 6M GuanidinHCl in TBS. Samples of all fractions 260 were spotted onto nitrocellulose membranes and protein content was visualised with Ponceau 261 S (Sigma). All fractions that contained antibody proteins were pooled and dialysed into TBS 262 over night. Antibodies were stored in 0.1% sodium azide in TBS at 4°C. 263

264 Immunofluorescence and microscopy 265 Cells were seeded on coverslips (thickness 1.5) 24h before fixation. Cells were pre- 266 permeabilised with ice-cold cytoskeleton (CSK) buffer (100mM NaCl, 300mM sucrose, 3mM 267 MgCl2, 10mM PIPES (pH6.8)) containing 0.1% Triton X-100 for 3min at 4°C before fixation 268 with 37% paraformaldehyde in PBS at room temperature. Samples were re-hydrated with TBS- 269 T before blocking with 1% normal donkey serum in AbDil (0.25% v/v Tween-20, 2% w/v 270 BSA, 0.1% w/v NaN3 in TBS). Primary antibodies were added, diluted in AbDil, for 1h. Cells 271 were carefully washed with TBS-T and secondary antibodies (1:500 in AbDil, Jackson 272 ImmunoResearch) were added for 30min. Cells were washed again and 4',6-Diamidino-2- 273 phenylindole (DAPI, Sigma) was added at 1µg/ml in TBS for 10min. Coverslips were washed 274 with TBS and mounted onto microscope slides by inverting them into mounting medium (0.5 275 % p-phenylenediamine (Free Base; Sigma) in 20 mM Tris, pH 8.8, 90 % glycerol). Primary 276 antibodies included polyclonal sheep Bod1 antibodies (0.5µg/ml), mouse anti-B56α (1:100, 277 BD Biosciences), mouse anti-Ndc80 (1:500, Abcam [9G3]), mouse anti-Nuf2 (1:300, Abcam), 278 rabbit anti-Dsn1 (1:300, GeneTex), rabbit anti-CASC5 [Knl1] (1:1000, Abcam), rabbit anti- 279 Mlf1 [CENPU] (1:200, Rockland), rabbit anti-Ndc80 (phospho-Ser55) antibody (1:300, 280 GeneTex), rat anti-tubulin (1:500, AbD Serotec), human anti-centromere autoantisera [ACA] 281 (1:1000, a kind gift from Sara Marshall, Ninewells Hospital, Dundee). Secondary antibodies 282 (Jackson ImmunoResearch) were used at 1:150. Three-dimensional deconvolution image data 283 sets were acquired on a DeltaVision imaging system (Applied Precision) equipped with an 284 Olympus 1-UB836 microscope, CCD camera (CoolSNAP_HQ/ICX285), and 100x/1.4 NA 285 Plan-Apochromat oil immersion objectives (Olympus). Z stacks were collected 0.2µm apart 286 and deconvolved using softWoRx (Applied Precision). 287

288 Image analysis 289 Image data were imported into OMERO and quantification of kinetochore intensities was 290 performed using OMERO.mtools [43]. Kinetochores were identified from deconvolved image 291 stacks covering the whole cell by segmenting them based on anti-centromere antibody (ACA) 292 staining using Otsu thresholding. The minimum object size was set to 50 pixels and the 293 perimeter of the automatically generated mask was expanded by 4 pixels to include the outer 294 kinetochore in the analysis. The fluorescence signal within this mask was measured and 295 background staining was quantified in a 2 pixel annulus with a 1 pixel gap to the perimeter of 296 each segmented mask. Fluorescence intensity at the kinetochore was then calculated as the 297 summed fluorescence intensity within the mask subtracting the product of its size and the bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

298 average background intensity in the 2 pixel annulus. Only positive values were taken into 299 consideration for statistical analysis. 300 All images were stored in OMERO, and figures were generated using OMERO.figure. 301

302 Affinity purification and immunoblotting 303 For affinity purification, HeLa S3 cells were arrested in 5µM STLC for 18h. After gentle 304 mitotic shake-off, cells were resuspended in lysis buffer (20mM Tris acetate pH 7.5, 1mM 305 EGTA, 1mM EDTA, 10mM Na-β-glycerophosphate, 5mM Na-pyrophosphate, 1mM Na- 306 orthovanadate, 50mM NaF, 1µM microcystin, 0.27M sucrose, 10µg/ml leupeptin, 10µg/ml 307 pepstatin, 10µg/ml aprotinin), containing 0.01% and 0.05% Triton X-100 for stable and 308 transient transfections respectively, and disrupted with four rounds of freeze fracturing. After 309 depleting insoluble proteins by centrifugation (4°C, 10.000rpm, 5min), affinity purification 310 was performed using GFP-Binder (Chromotek) for 90min at 4°C and constant agitation. 311 Purified samples were washed and resolved by SDS-PAGE. Immunoblotting was performed 312 using standard procedures, and secondary antibody was detected using either Clarity Western 313 ECL Substrate (BioRad) and X-Ray films (Kodak) or the Odyssey Clx infrared detection 314 system (LI-COR). Primary antibodies included mouse anti-B56α (1:500, Abcam), mouse anti- 315 B56δ (1:500, Abcam), mouse anti-Ndc80 (1:1000, Abcam [9G3]), mouse anti-Nuf2 (1:1000, 316 Abcam), rabbit anti-Spc24 (1:1000, Abcam [EPR11548(B)]), mouse anti-MBP (1:20,000, 317 NEB), goat anti-GST (1:5000, Abcam), mouse anti-Vinculin (1:10,000, Abcam [SPM227]), 318 mouse anti-GFP (1:1000, Roche), rabbit anti-Bod1 (1:500, Abcam), polyclonal sheep anti- 319 Bod1 (2ug/ml). Secondary antibodies were sheep anti-mouse IgG, HRP-linked (1:10,000, GE 320 Healthcare), goat anti-rabbit IgG, HRP-linked (1:5000, Cell Signalling), donkey anti-goat IgG, 321 HRP-linked (1:20,000, Promega), donkey anti-sheep HRP (1:20,000, Sigma), IRDye 680LT 322 donkey anti-mouse IgG (H+L) (1:20,000, LI-COR), IRDye 800CW donkey anti-goat IgG 323 (H+L) (1:20,000, LI-COR). LI-COR images were quantified using Image Studio software V2.0 324 (LI-COR), with signal intensity normalized to input protein levels. 325

326 Mass spectrometry 327 Eight 15cm plates of stably Bod1-GFP or GFP transfected cells or two 15cm plates of 328 transiently Bod1-GFP or GFP transfected cells were arrested in mitosis and affinity purification 329 using GFP-Binder (Chromotek) was performed as described above. Proteins were eluted with 330 2xSDS buffer and the full eluate was run on a 4-12% SDS-PAGE. Bands were visualised using 331 Coomassie Brilliant Blue and lanes were cut into 4 gel pieces. Gel pieces were subsequently 332 de-stained with ammonium bicarbonate and acetonitrile as an organic solvent and dried 333 completely in a vacuum centrifuge. 10mM DTT was used to reduce cystine disulphide bonds 334 and the resulting thiol groups were irreversibly alkylated to S-carboxyamidomethylcysteine 335 with 55mM iodoacetamide. Excess iodoacetamide was removed and gel pieces were dried in a 336 vacuum centrifuge before enzymatic digest of the proteins. In-gel digest was performed with 337 20ng/µl trypsin in 50mM ammonium bicarbonate at 37°C o/n. Tryptic peptides were extracted 338 from the gel by repeated addition of 0.1%TFA/acetonitrile extraction solution and sonication. 339 Peptide samples were cleaned for mass spectrometry using a C18-Ziptip protocol. Mass 340 spectrometry was performed on an LTQ Orbitrap Velos Pro instrument (Thermo Fisher 341 Scientific). Mass spectrometry raw data were processed in the MaxQuant software package V 342 1.3.0.5 utilising the Uniprot Human database (09/08/2012) [44]. Parameters applied include: 343 minimum peptide length = 7, Protein FDR = 0.01, Site FDR = 0.01. Peptides with variable 344 modifications (N-terminal acetylation of the protein, oxMet, and pyroGlu) and fixed 345 modifications (S-carboxyamidomethylcysteine) were accounted for in the analysis. Shotgun bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

346 proteomics data analysis, including statistical analysis and GO term analysis, was performed 347 using the Perseus software package V1.5.5.3 [45]. Statistical test performed was an unpaired 348 Student’s t-test with a threshold p-value of 0.05. 349

350 Protein expression and purification 351 Ndc80 Bonsai was expressed and purified as described previously [32]. For production of 352 Bod1-MBP and MBP, 5ml LB medium containing the appropriate selection marker were 353 inoculated with transformed BL21 E. coli. After 18h at 37°C, starter cultures were transferred 354 into 2l conical flasks containing 500ml LB medium with the selection antibiotic. Cultures were 355 grown in shaking incubators at 37°C up to OD600 = 0.4. After adding 100mM benzylalcohol 356 for 30min at 37°C, recombinant protein production was induced by addition of 0.1mM 357 Isopropyl β-D-1-thiogalactopyranoside (IPTG).Protein expression was allowed for 18h at 358 18°C.Bacteria were harvested by ultracentrifugation (5250xg, 4°C, 30min, slow 359 deceleration)and lysed by resuspending them in PBS containing a protease inhibitor cocktail 360 (Roche) and adding 1mg/ml lysozyme. Cells were incubated at 4°C under constant agitation 361 for 30min after which Triton X-100 was added to a final concentration of 1%. The suspension 362 was sonicated for 30s on ice and left to incubate another 30min at 4°C. The lysate was sonicated 363 twice more and insoluble debris was pelleted by ultracentrifugation (26.000xg, 4°C, 1h). For 364 protein purification, 1ml amylose resin (NEB) was pre-equilibrated with binding buffer (50mM 365 Tris-HCl pH 7.5, 100mM NaCl, 1mM DTT) and the soluble fraction of the protein lysate was 366 added after passing through a 0.2μm filter. Binding was allowed for 2h at 4°C under constant 367 agitation. The recombinant protein bound to beads was washed with binding buffer. To elute 368 the protein, 500μl binding buffer containing 20mM maltose were added and samples were 369 incubated for 90min at 4°C under agitation. The supernatant was transferred into a Slide-A- 370 Lyzer dialysis cassette (Pierce) and dialysed into interaction buffer (20mM Tris–HCl, 20mM 371 NaCl, 10% glycerol, 1mM EGTA, 1mM DTT) over night. The concentration of dialysed 372 protein was determined using a Bradford colorimetric assay. If protein concentrations were 373 below 0.2mg/ml, protein solutions were concentrated using Vivaspin columns (GE Healthcare) 374 at 4750rpm, 4°C. Proteins were aliquoted and stored at -80°C. 375

376 Pull down experiments 377 150pmol Ndc80Bonsai, coupled to glutathione beads, were pre-incubated with 0.01% insulin 378 in interaction buffer (20mM Tris–HCl, 20mM NaCl, 10% glycerol, 1mM EGTA, 1mM DTT, 379 Complete protease inhibitors (Roche)) for 20min at 4°C. 1nmol MBP or Bod1-MBP was added 380 to the beads and binding was allowed to take place for 1h at 4°C. After washing with interaction 381 buffer, proteins were eluted with SDS loading buffer and all eluate was loaded for immunoblot 382 analysis. 25pmols MBP or Bod1-MBP were loaded as input controls. Band intensity was 383 determined using the ImageStudio software package. Total amount of protein in the pull down 384 was determined by using the input as a reference. 385

386 Statistical analysis 387 Statistical significance tests were performed using Sigma Plot 12.5 (Systat Software Inc.). For 388 pairwise comparison, datasets were tested for normal distribution and then analysed by 389 Student’s t test (for Gaussian distributions) or by Mann-Whitney U test (for Non-Gaussian 390 distributions). For group-wise comparison, datasets were compared by Kruskal-Wallis one- bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

391 way analysis of variance (ANOVA) on ranks, followed by pairwise multiple comparison 392 procedures (Dunn's Method). 393

394 siRNAs 395 Knl1 was depleted using 5′-GCAUGUAUCUCUUAAGGAA-3′ [36]. siRNA targeting Bod1 396 was 5’-GCCACAAAUAGAACGAGCAAUUCAU-3’ [18]. Ndc80 was depleted using an 397 siRNA with the sequence 5’-AAGTTCAAAAGCTGGATGATCTT-3’ [46]. All isoforms of 398 the B56 PP2A regulatory subunit were depleted using a pool of 5’- 399 GCUCAAAGAUGCCACUUCA-3’ (B56α/PPP2R5A), 5’-CGCAUGAUCUCAGUGAAUA- 400 3’ (B56β(PPP2R5B)), 5’-GGAUUUGCCUUACCACUAA-3’ (B56γ/PPP2R5C), 5′- 401 UCCAUGGACUGAUCUAUAA-3′ (B56δ/PPP2R5D), 5’-UUAAUGAACUGGUGGACUA- 402 3’ (B56ε/PPP2R5E), described in [11]. Stealth™ RNAi siRNA Negative Control, Med GC 403 (Invitrogen) was used for control transfections.

404 Data accessibility 405 The mass spectrometry proteomics data have been deposited to the ProteomeXchange

406 Consortium [47] via the PRIDE partner repository [48] with the dataset identifier PXD006322.

407 Acknowledgements 408 We are grateful to Jennifer G DeLuca for the gift of the Ndc80Bonsai expression construct and

409 Adrian T Saurin for the Knl1 siRNA. We thank the Dundee Centre for Advanced Scientific

410 Technologies and Fingerprints Proteomics in Dundee for their support and all the members of

411 our laboratory for helpful discussions.

412 Author contributions 413 KS, IMP, and JRS conceived the experimental strategy. KS performed and analysed the

414 experiments. StH performed and analysed the mass spectrometry with input from KS. KS, IMP

415 and JRS wrote the manuscript.

416 Competing interest 417 The authors declare no conflict of interest.

418 Funding 419 This work was supported by a Cancer Research UK PhD fellowship (C5314/A11784) to KS.

420 Imaging and image analysis was supported by two Wellcome Trust Strategic Awards bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

421 (097945/B/11/Z and 095931/Z/11/Z) and an MRC Next Generation Optical Microscopy Award

422 (MR/K015869/1).

423

424 Figure Legends 425

426 Figure 1. profiles of Bod1 kinetochore recruitment and phosphorylation.

427 HeLa cells were fixed in paraformaldehyde and stained with (a) a Bod1 pan-specific peptide

428 antibody raised in sheep or (b) a T95 phospho-specific antibody (both green). Mitotic cells

429 were imaged and the kinetochore was marked as the interface between centromeric region

430 (anti-centromere antibody, ACA, in blue) and spindle microtubules (tubulin, red). Top panel

431 shows a single z-section of each cell cycle stage. Bottom panels are magnifications of the same

432 cell (section indicated by white boxes). Scale bars are 1 µm. Quantification of (c) total Bod1

433 or (d) T95-specific phospho-Bod1fluorescence intensity at the kinetochore corresponding to

434 experiments shown in (a) and (b). Background-corrected summed intensity of centromeric

435 Bod1 staining was quantified. n = 10 cells per mitotic phase. Error bars represent standard

436 error. Asterisks indicate significance in multiple comparison after ANOVA on ranks. Int:

437 interphase, Pro: prophase, PM: prometaphase, Met: metaphase, Ana: anaphase, CK:

438 cytokinesis.

439 Figure 2. PP2A-B56 and Knl1 are dispensable for Bod1 recruitment to

440 kinetochores. HeLa cells were treated with either control siRNA or a smart pool siRNA

441 targeting all PP2A-B56 isoforms as described previously [11]. After 48h cells were fixed in

442 paraformaldehyde and stained with (a) the total Bod1 peptide antibody or (b) a PP2A-B56α

443 isoform-specific antibody. Metaphase control cells or B56siRNA-treated cells showing the

444 characteristic B56-depletion phenotype of metaphase chromosome alignment defects were

445 imaged. The last panels are magnifications of the same cell (section indicated by white boxes). bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

446 Scale bars are 1 µm. Total (c) Bod1 and (d) B56α intensities at kinetochores were quantified.

447 n = 10 cells per condition. Statistical test used was Student’s t‐test. Three asterisks indicate

448 highly significant difference. ns: no significant difference in fluorescence intensity. Error bars

449 represent standard error. (e) Immunoblot corresponding to the B56 depletion experiments in

450 (a-d) using antibodies against two of the five targeted B56 isoforms and vinculin as a loading

451 control. (f) HeLa cells were treated with either control or Knl1 siRNA for 48h, fixed in

452 paraformaldehyde, and co-stained with both total Bod1 peptide antibody and a Knl1-specific

453 antibody. Metaphase cells were imaged for both treatment conditions. The last panels are

454 magnifications of the same cell (section indicated by white boxes). Scale bars are 1 µm. Total

455 (g) Bod1 and (h) Knl1 intensities at kinetochores were quantified. n = 10 cells per condition.

456 Statistical test used was Student’s t‐test. Three asterisks indicate highly significant difference.

457 ns: no significant difference in fluorescence intensity. Error bars represent standard error.

458 Figure 3. Proteomics analysis of mitotic Bod1-GFP affinity purifications. (a) GFP

459 binder was used to affinity purify Bod1-GFP and associated proteins from mitotic HeLa cell

460 lysates as described previously [9]. (b) Volcano plot of the mitotic Bod1 interactome detected

461 by LFQ shotgun mass spectrometry. The fold change in Bod1-GFP samples vs control

462 (represented by log2 of their intensity ratios) is plotted against –log10 of the p-value obtained in

463 an unpaired Student’s t-test with a threshold p-value of 0.05. All kinetochore proteins detected

464 are highlighted in blue, kinetochore proteins above the significance threshold are labelled.

465 Components of the Set1b complex are highlighted in maroon and labelled. n = 4 biological

466 replicates. (c) HeLa cells were fixed in paraformaldehyde and stained for Ndc80 (red), Bod1

467 (green), and a marker of the centromeric region (ACA, blue). A pair of kinetochores is shown.

468 White line indicates the region chosen for a line profile (bottom panel) of fluorescence

469 intensities across the kinetochore pair. bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

470 Figure 4. The Ndc80 complex interacts with Bod1 in mitotic HeLa cell extracts

471 and in vitro. (a) Graphic representation of the full length Ndc80 complex. Regions of interest,

472 including the microtubule and centromere binding regions of the complex, are labelled. (b) Co-

473 precipitation of Ndc80 complex components Ndc80, Nuf2, and Spc24 from mitotic HeLa cell

474 extracts with purified Bod1-GST. (c) Representation of the recombinant truncated Ndc80

475 Bonsai complex (as described in [32]). Ndc80-Spc25 and GST-Nuf2-Spc24 are expressed as

476 fusion proteins. Both fusion constructs are co-expressed in E. coli from a dual-expression

477 vector. (d) 150pmol recombinant Ndc80 Bonsai, consisting of dimers of one Ndc80-Spc25

478 fusion protein with one GST-Nuf2-Spc24 fusion protein, was immobilised on Sepharose beads

479 and incubated with 1nmol Bod1-MBP or MBP. Binding was allowed for 1h. Proteins were

480 resolved by SDS-PAGE and immunoblotted using simultaneous detection of the MBP (red)

481 epitope tag on Bod1 and the GST (green) epitope tag on Nuf2-Spc24. (e) Amount of bound

482 protein in (d) was quantified relative to the input. Statistical test used was Student’s t‐test. Error

483 bars represent standard error. n = 9 separate experiments.

484 Figure 5. The Ndc80 complex is essential for Bod1 recruitment to mitotic

485 kinetochores. (a) HeLa cells were treated with either control siRNA or Ndc80 siRNA. After

486 48h cells were fixed in paraformaldehyde and stained with the indicated kinetochore proteins

487 (green). Kinetochores of chromosomes on the metaphase plate are shown. Localisation to the

488 kinetochore, i.e. the interface between centromeric region (anti-centromere antibody, ACA, in

489 blue) and spindle microtubules (tubulin, red) was compared. Scale bars are 1 µm. (b)

490 Quantification of the images in (a). n = 10 cells per condition. Error bars represent standard

491 error. Three asterisks indicate highly significant difference in Student’s t test.

492 Figure 6. Bod1 depletion interferes with phosphorylation of the Ndc80 N-terminal

493 tail. (a) HeLa cells were treated with control or Bod1 siRNA for 48h, fixed in

494 paraformaldehyde and stained with a phospho-specific antibody against a phosphoepitope bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

495 (pS55) in the Ndc80 N-terminal tail, or a total Ndc80 antibody. Metaphase cells or cells with

496 the characteristic Bod1 chromosome misalignment phenotype were imaged. Kinetochores of

497 chromosomes on the metaphase plate are shown. Scale bars are 1 µm. (b) pS55 and total Ndc80

498 intensities at kinetochores were quantified. n = 10 cells per condition. Statistical test used was

499 Student’s t‐test. Error bars represent standard error. Three asterisks indicate highly significant

500 difference. (c) Timeline and quantification of treatments to compare kinetics of Ndc80

501 phosphorylation in control and Bod1-depleted HeLa cells in early mitosis. 26h after

502 transfection, cells were synchronised at G2/M transition with the Cdk1 inhibitor RO-3306 for

503 16h. They were then released into culture medium containing the proteasome inhibitor MG132

504 to prevent mitotic exit and fixed 30min after their release from G1/M arrest. pS55 Ndc80

505 fluorescence intensities at kinetochores of mitotic cells were quantified. n = 50 mitotic cells

506 per condition. Three asterisks indicate high significance in pairwise multiple comparison after

507 ANOVA on ranks (p < 0.001). (d) A unified model integrating data presented in this paper with

508 previous findings on the temporal regulation of kinetochore phosphatases in mitosis. Bod1

509 phosphorylation in early mitosis can inhibit PP2A-B56 activity [9] and prevent premature

510 dephosphorylation of Ndc80 at its N-terminus. Dephosphorylation of Bod1 in later stages by

511 an unknown mechanism, coincides with a surge in phosphatase activity, recruitment of PP1,

512 and therefore enables PP1-mediated dephosphorylation of kinetochore epitopes in metaphase

513 [12–14,49–51]. bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license.

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673 bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license. Figure 1 (a) Interphase Prophase Prometaphase Metaphase Anaphase Cytokinesis Bod1

tubulin DNA ACA

1µm 5x Zoom

(b) Interphase Prophase Prometaphase Metaphase Anaphase Cytokinesis tubulin DNA pT95 Bod1 pT95

ACA 5x Zoom

(c) (d) Bod1 antibody pT95 Bod1 antibody 25000 20000 * 20000 * 15000 15000 10000 10000 Intensity [A.U.] Intensity [A.U.] 5000 5000 Summed Fluorescence Summed Fluorescence Summed Fluorescence Summed Fluorescence 0 0 Int Pro PM Met Ana CK Int Pro PM Met Ana CK bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license. Figure 2 (a) Bod1 Bod1 (b) 5x Zoom B56α B56α 5x Zoom ACA ACA tubulin DNA ACA ACA tubulin DNA CTRL siRNA CTRL siRNA B56 siRNA pool B56 siRNA pool (c) Bod1 (d) B56α (e) CTRL B56 ns (P = 0.182) (P = <0.001) siRNA siRNA 10000 14000 12000 B56α 8000 10000 6000 8000 4000 6000 *** B56δ 4000 2000 Intensity [A.U.]

Intensity [A.U.] 2000 0 0 vinculin Summed Fluorescence Summed Fluorescence Summed Fluorescence Summed Fluorescence CTRL B56 CTRL B56 siRNA siRNA siRNA siRNA Bod1 ns (P = 0.101) (f) (g) 100000 Bod1 Knl1 Bod1 Knl1 5x Zoom ACA ACA ACA DNA 80000 60000 40000 20000 Intensity [A.U.] 0

Summed Fluorescence Summed Fluorescence CTRL Knl1 siRNA siRNA CTRL siRNA (h) Knl1 (P = <0.001) 300000

200000

100000 Knl1 siRNA

Intensity [A.U.] *** 0

Summed Fluorescence Summed Fluorescence CTRL Knl1 siRNA siRNA bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license. Figure 3 (a) (b) 4.5

? 4 NDC80 BOD1 Bod1 GFP

GFP 3.5 binder 3

(c) 2.5 Loading... 2 ACA Ndc80 Bod1 -log10 (p-value) ASH2L 1.5 DYNC1I1 1.2 ACA p = 0.05 1 Ndc80

1 HCFC1 0.8 Bod1 RBBP5 0.6 SETD1B

0.4 0.5 Proteins with p < 0.05 WDR82 0.2 WDR5 Kinetochore proteins CXXC1 0 DPY30 SET1D methyltransferase complex 0 OGT

Relative Intensity Relative 0 10 20 30 40 other proteins distance [pixel] -6 -4 -2 0 2 4 6 8 10 12 14 16 fold change Bod1-GFP [log2 (Bod1 IP / ctrl IP)] bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license. Figure 4 (a) CH domain N-terminal (b) domain IP unbound HeLa GST Bod1- GST Bod1- input GST GST Hairpin 75kD- Ndc80 Nuf2 Ndc80 Nuf2 50kD- 25kD- Spc24 Loop Microtubule 50kD- Bod1-GST (GST antibody) Spc24 Spc25 Ndc80 complex GST (full length) 25kD- Centromere binding

(c) (d) input bound unbound (e) Ndc80 Bonsai MBP Bod MBP Bod MBP Bod binding 20 Nuf2 (P = 0.005) 15 ** Ndc80 75kD- Spc24 - Bod1-MBP 10 Spc25 GST-Nuf2-Spc24 50kD- - (Ndc80 bonsai) 5

- MBP 0 Ndc80 Bonsai [pmol] Bonsai complex

Protein bound to the Ndc80 bound to Protein MBP Bod1- MBP bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license. Figure 5 (a) Ndc80 ACA tubulin Nuf2 Knl1 B56α Bod1 CTRL siRNA Ndc80 siRNA (b) Ndc80 Nuf2 Knl1 B56a Bod1 (P = <0.001) (P = <0.001) 140000 (P = <0.001) (P = <0.001) (P = <0.001) 50000 200000 120000 30000 15000 100000 *** 40000 150000 *** 20000 80000 10000 30000 100000 60000 20000 *** 10000 *** 40000 5000 *** 50000

Intensity [A.U.] 10000 20000 0 0 0 0 0 Summed Fluorescence Summed Fluorescence CTRL Ndc80 CTRL Ndc80 CTRL Ndc80 CTRL Ndc80 CTRL Ndc80 siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA siRNA bioRxiv preprint doi: https://doi.org/10.1101/131664; this version posted April 27, 2017. 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 4.0 International license. Figure 6

(a) (c) Transfect Split cells on 10µM 10µM Fix & CTRL siRNA Bod1 siRNA with siRNA coverslips RO-3306 MG132 stain for IF

18h 8h 16h 30 min

tubulin 30min release from RO ACA pS55 Ndc80 600000 ***

400000 tubulin

Ndc80 200000 ACA pS55 Ndc80 Intensity

0 P PM M (b) P PM M 200000 pS55 Ndc80 CTRL siRNA Bod1 siRNA (P = <0.001) 150000 (d) Prometaphase Metaphase

100000 P

50000 *** P P P P P Intensity [A.U.] P P P P P 0 Bod1 Bod1 PP1 Summed Fluorescence Summed Fluorescence CTRL Bod1 P T95 P siRNA siRNA Bod1 PP2A T95 Nuf2 Nuf2 BubR1 Bub1 Ndc80 Ndc80 Bub3 Mad2 200000 Ndc80 Mad1 (P = <0.001) Spc24 Spc24

outer kinetochore outer Spc25 kinetochore outer Spc25 150000 Knl1 Knl1 100000 *** Mis12 C Mis12 C

50000

Intensity [A.U.] CCAN CCAN 0

Summed Fluorescence Summed Fluorescence CTRL Bod1 inner kinetochore inner kinetochore siRNA siRNA inner centromere inner centromere