Kinetochore recruitment of CENP-F illustrates how paralog ...3 54 Early in mitosis, prior to end-on...

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Kinetochore recruitment of CENP-F illustrates how paralog 1

divergence shapes kinetochore composition and function 2

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Giuseppe Ciossani (1,*), Katharina Overlack (1,*), Arsen Petrovic (1), Pim Huis in ‘t 4

Veld (1), Carolin Körner (1), Sabine Wohlgemuth (1), Stefano Maffini (1) & Andrea 5

Musacchio (1,2,#) 6

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(1) Department of Mechanistic Cell Biology, Max Planck Institute of Molecular 8

Physiology, Otto-Hahn-Straße 11, 44227 Dortmund, Germany 9

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

Universitätsstraße, 45141 Essen, Germany 11

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# Correspondence: [email protected] 13

* These authors contributed equally to this work 14

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Keywords: kinetochore, centromere, chromosome, mitosis, cell cycle, CENP-E, CENP-16

F, Bub1, BubR1, protein electroporation, spindle assembly checkpoint, mitotic 17

checkpoint, paralogs, duplication 18

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

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.CC-BY-NC-ND 4.0 International licenseavailable under anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (which wasthis version posted March 4, 2018. ; https://doi.org/10.1101/276204doi: bioRxiv preprint

https://doi.org/10.1101/276204

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The metazoan proteins CENP-E and CENP-F are components of a fibrous layer 22

of mitotic kinetochores named the corona. Several features suggest that CENP-E 23

and CENP-F are paralogs: they are very large (approximately 2700 and 3200 24

residues, respectively), rich in predicted coiled-coil structure, C-terminally 25

prenylated, and endowed with microtubule-binding sites at their termini. In 26

addition, CENP-E contains an ATP-hydrolyzing motor domain that promotes 27

microtubule plus-end directed motion. Here, we show that CENP-E and CENP-28

F are recruited to mitotic kinetochores independently of the Rod-Zwilch-ZW10 29

(RZZ) complex, the main corona constituent. We identify selective interactions of 30

CENP-E and CENP-F respectively with BubR1 and Bub1, paralogous proteins 31

involved in mitotic checkpoint control and chromosome alignment. While BubR1 32

is dispensable for kinetochore localization of CENP-E, Bub1 is stringently 33

required for CENP-F localization. Through biochemical reconstitution, we 34

demonstrate that the CENP-E:BubR1 and CENP-F:Bub1 interactions are direct 35

and require similar determinants, a dimeric coiled-coil in CENP-E or CENP-F 36

and a kinase domain in BubR1 or Bub1. Our findings are consistent with the 37

existence of ‘pseudo-symmetric’, paralogous Bub1:CENP-F and BubR1:CENP-E 38

axes, supporting evolutionary relatedness of CENP-E and CENP-F. 39

40

Introduction 41

The segregation of chromosomes from a mother cell to its daughters during cell division 42

relies on the function of specialized protein complexes, the kinetochores, as bridges 43

linking chromosomes to spindle microtubules (Musacchio and Desai, 2017). 44

Kinetochores are built on specialized chromosome loci known as centromeres, whose 45

hallmark is the enrichment of the histone H3 variant centromeric protein A (CENP-A, 46

also known as CenH3) (Earnshaw, 2015). CENP-A seeds kinetochore assembly by 47

recruiting CENP-C, CENP-N, and their associated protein subunits in the constitutive 48

centromere associated network (CCAN) (Cheeseman and Desai, 2008). These 49

centromere proximal ‘inner kinetochore’ subunits, in turn, recruit the centromere distal 50

‘outer kinetochore’ subunits of the KMN complex (Knl1 complex, Mis12 complex, 51

Ndc80 complex), which promote ‘end-on’ microtubule binding and control the spindle 52

assembly checkpoint (SAC) (Musacchio and Desai, 2017). 53



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Early in mitosis, prior to end-on microtubule attachment, an additional fibrous structure, 54

the kinetochore corona, assembles as the outermost layer of the kinetochore (Figure 1A) 55

(Jokelainen, 1967; Magidson et al., 2015; McEwen et al., 1993; Rieder, 1982). The 56

corona’s main constituent is a trimeric protein complex named RZZ [from the name of 57

the fruit fly genes Rough Deal (ROD), Zwilch, and Zeste White 10 (ZW10)]. The ROD 58

subunit is structurally related to proteins that oligomerize near biological membranes to 59

promote vesicular trafficking, including Clathrin (Civril et al., 2010; Mosalaganti et al., 60

2017), leading to hypothesize that corona assembly results from RZZ polymerization 61

(Mosalaganti et al., 2017). The interaction of the RZZ complex with an adaptor subunit 62

named Spindly, in turn, further recruits the microtubule minus-end directed motor 63

cytoplasmic Dynein and its binding partner Dynactin to kinetochores, as well as the 64

Mad1:Mad2 complex, which is crucially required for SAC signaling (Barisic et al., 2010; 65

Basto et al., 2004; Buffin et al., 2005; Caldas et al., 2015; Chan et al., 2009; 66

Cheerambathur et al., 2013; Gama et al., 2017; Gassmann et al., 2008; Gassmann et al., 67

2010; Griffis et al., 2007; Howell et al., 2001; Kops et al., 2005; Matson and Stukenberg, 68

2014; Mische et al., 2008; Silio et al., 2015; Sivaram et al., 2009; Starr et al., 1998; Varma 69

et al., 2008; Williams et al., 1996; Wojcik et al., 2001; Yamamoto et al., 2008; Zhang et al., 70

2015). 71

Corona assembly leads to a broad expansion of the microtubule-binding interface of 72

kinetochores that may promote initial microtubule capture, congression towards the 73

metaphase plate, and SAC signaling (Basto et al., 2000; Buffin et al., 2005; Hoffman et al., 74

2001; Kops et al., 2005; Magidson et al., 2011; Magidson et al., 2015; Wynne and 75

Funabiki, 2015). Differently from the mature end-on attachments, initial attachments of 76

kinetochores engage the microtubule lattice and are therefore defined as lateral or side-77

on. CENP-E, a kinesin-7 family member, plays a crucial role at this stage. Its inhibition 78

or depletion lead to severe and persistent chromosome alignment defects, with numerous 79

chromosomes failing to congress towards the spindle equator and stationing near the 80

spindle poles, causing chronic activation of the SAC (Kapoor et al., 2006; Kuhn and 81

Dumont, 2017; Magidson et al., 2011; Magidson et al., 2015; Putkey et al., 2002; Schaar et 82

al., 1997; Wood et al., 1997; Yao et al., 2000; Yen et al., 1991). Human CENP-E consists 83

of 2701 residues (Figure 1B) (Yen et al., 1992). Besides the globular N-terminal motor 84

domain, the rest of the CENP-E sequence forms a flexible and highly elongated (~230 85

nm) coiled-coil (Kim et al., 2008). The kinetochore-targeting domain of CENP-E 86



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encompasses residues 2126-2476 and is followed by a microtubule-binding region 87

(Figure 1B) (Chan et al., 1998; Liao et al., 1994). The distribution of CENP-E in an outer 88

kinetochore crescent shape similar to that of the RZZ supports the notion that CENP-E 89

is part of the kinetochore corona, but its persistence at kinetochores after disappearance 90

of the corona suggests a corona-independent localization mechanism (Cooke et al., 1997; 91

Hoffman et al., 2001; Magidson et al., 2015; Wynne and Funabiki, 2015; Yao et al., 1997; 92

Yen et al., 1992). 93

CENP-F (also known as Mitosin, 3210 residues in humans) is also a kinetochore corona 94

constituent during early mitosis that persists at kinetochores after corona shedding 95

(Casiano et al., 1993; Hussein and Taylor, 2002; Liao et al., 1995; Rattner et al., 1993; 96

Zhu, 1999; Zhu et al., 1995a; Zhu et al., 1995b). Like CENP-E, CENP-F is also highly 97

enriched in predicted coiled-coil domains (Figure 1B), but lacks an N-terminal motor 98

domain. Rather, it contains two highly basic microtubule-binding domains in the N-99

terminal 385 residues and in the C-terminal 187 residues (Feng et al., 2006; Musinipally et 100

al., 2013; Volkov et al., 2015). Similarly to CENP-E, the kinetochore recruitment domain 101

of CENP-F is positioned in proximity of the C-terminus [encompassing residues 2581-102

3210, the minimal domain tested for this function to date (Hussein and Taylor, 2002; 103

Zhu, 1999; Zhu et al., 1995a)]. The apparent similarity of CENP-E and CENP-F extends 104

to the fact that they are both post-translationally modified with a farnesyl prenol lipid 105

chain (isoprenoid) on canonical motifs positioned in their C-termini (Ashar et al., 2000). 106

These modifications contribute to kinetochore recruitment of CENP-E and CENP-F, 107

albeit to extents that differ in various reports (Holland et al., 2015; Hussein and Taylor, 108

2002; Moudgil et al., 2015; Schafer-Hales et al., 2007). 109

Previous studies identified CENP-F and BubR1 as binding partners of CENP-E (Chan 110

et al., 1998; Mao et al., 2003; Yao et al., 2000). BubR1 is a crucial constituent of the SAC, 111

a molecular network required to prevent premature mitotic exit (anaphase) in cells 112

retaining unattached or improperly attached kinetochores (Musacchio, 2015). BubR1 is a 113

subunit of the mitotic checkpoint complex (MCC), the SAC effector (Sudakin et al., 114

2001). Its structure is a constellation of domains and interaction motifs required to 115

mediate binding to other SAC proteins, and terminates in a kinase domain (Musacchio, 116

2015). It has been proposed that CENP-E stimulates BubR1 activity, and that 117

microtubule capture silences it (Mao et al., 2003; Mao et al., 2005). Later studies, 118

however, identified BubR1 as an inactive pseudokinase (Breit et al., 2015; Suijkerbuijk et 119



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al., 2012), and therefore the significance of CENP-E microtubule binding for the role of 120

BubR1 in the SAC remains unclear. Depletion or inactivation of CENP-E, however, is 121

compatible with a robust mitotic arrest (Schaar et al., 1997; Yen et al., 1991). 122

A yeast 2-hybrid (Y2H) interaction of CENP-F and Bub1 has also been reported but 123

never validated experimentally (Chan et al., 1998). Bub1, a paralog of BubR1, retained 124

genuine kinase activity in humans and it plays a function at the interface of mitotic 125

checkpoint signaling and kinetochore microtubule attachment (Raaijmakers et al., 2018; 126

Suijkerbuijk et al., 2012). Suggesting that the interaction of Bub1 and CENP-F is 127

functionally important, previous studies identified Bub1 as being essential for 128

kinetochore recruitment of CENP-F (Johnson et al., 2004; Klebig et al., 2009; Liu et al., 129

2006; Raaijmakers et al., 2018). 130

In our previous studies, we characterized in molecular detail how sequence divergence 131

impacted the protein interaction potential of the human Bub1 and BubR1 paralogs 132

(Overlack et al., 2017; Overlack et al., 2015). We described a molecular mechanism that 133

explains how Bub1, through an interaction with a phospho-aminoacid adaptor named 134

Bub3, can interact with kinetochores and promote the recruitment of BubR1 via a 135

pseudo-dimeric interface (Overlack et al., 2017; Overlack et al., 2015; Primorac et al., 136

2013). In view of these previous studies, here we have dissected the molecular basis of 137

the interactions of BubR1 and Bub1 with CENP-E and CENP-F. We provide strong 138

evidence for the sub-functionalization of these paralogous protein pairs. 139

140

Results and Discussion 141

Independent kinetochore local izat ion o f CENP-E, CENP-F, and the RZZ 142

Using specific antibodies (see Methods), we assessed the timing and specificity of 143

kinetochore localization of CENP-E, CENP-F, Zwilch, and Mad1. CENP-E showed 144

perinuclear localization until prometaphase, when it first appeared at kinetochores. It 145

persisted there until metaphase, and was then found at the spindle midzone after 146

anaphase onset (Figure 1C). This localization, which corresponds to previous 147

descriptions (Yen et al., 1991; Yen et al., 1992), is reminiscent of that of chromosome 148

passenger proteins (Earnshaw and Bernat, 1991). CENP-F, on the other hand, localized 149

to kinetochores already in prophase, where it was also temporarily visible at the nuclear 150

envelope, and persisted there until anaphase, with progressive weakening and dispersion 151



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(Figure 1D), as noted previously (Baffet et al., 2015; Bolhy et al., 2011; Hu et al., 2013; 152

Liao et al., 1995; Rattner et al., 1993). Also Zwilch (a subunit of the RZZ complex) and 153

Mad1 were already visible at kinetochores in prophase, but they became invisible at these 154

structures upon achievement of metaphase (Figure 1S1A-B), in agreement with the 155

notion that the corona becomes dissolved upon microtubule attachment (see 156

Introduction). 157

Thus, both CENP-E and CENP-F continue to localize to kinetochores well beyond the 158

timing of removal of the RZZ complex and Mad1, suggesting that they can be retained at 159

kinetochores independent of the corona. To test this directly, we identified conditions 160

for optimal depletion of Zwilch, CENP-E, or CENP-F by RNA interference (RNAi) 161

(Figure 2S1A-J). Depletion of Zwilch resulted in depletion of Mad1 from kinetochores 162

(Figure 2S1H-J), but left the kinetochore levels of CENP-E essentially untouched (Figure 163

2A). This observation is in agreement with previous studies showing that Mad2, whose 164

kinetochore localization requires Mad1 (Martin-Lluesma et al., 2002; Sharp-Baker and 165

Chen, 2001), is also depleted from kinetochores upon depletion of other RZZ subunits 166

(Caldas et al., 2015; Gassmann et al., 2008; Raaijmakers et al., 2018). The observation that 167

CENP-E retains kinetochore localization under conditions in which Mad1 appears to 168

become depleted seems inconsistent with a recent report proposing that Mad1 is 169

required for kinetochore localization of CENP-E (Akera et al., 2015), but agrees with 170

previous reports that failed to detect consequences on CENP-E localization upon 171

depletion of Mad1 (Martin-Lluesma et al., 2002; Sharp-Baker and Chen, 2001). 172

Conversely, depletion of CENP-E or CENP-F in HeLa cells, or even their co-depletion 173

(Figure 2S2A), did not influence the kinetochore localization of Zwilch or Mad1 (Figure 174

2B-F and Figure 2S3A-D), as observed previously (Martin-Lluesma et al., 2002; Yang et 175

al., 2005). We note, however, that depletion of CENP-E in DLD-1 cells was reported to 176

have deleterious effects on Mad2 localization, while Mad1 or RZZ subunits were not 177

tested (Johnson et al., 2004). Based on these results, we conclude that kinetochore 178

localization of CENP-E and CENP-F does not require the kinetochore corona, nor does 179

it influence corona assembly. We also observed that CENP-E and CENP-F were not 180

reciprocally affected by their depletion (Figure 2G-H), indicating that they localize (at 181

least largely) independently to kinetochores, as previously suggested (Yao et al., 2000). 182

In most cells analyzed, depletion of CENP-F resulted in apparently normal metaphase 183

alignment, with only a slight increase in the fraction of cells presenting metaphase 184



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alignment defects (Figure 2S3B and Figure 2S4). In agreement with the effects of CENP-185

F depletion being mild, duration of mitosis (caused by spindle assembly checkpoint 186

activation) was only marginally increased in cells depleted of CENP-F (Figure 2S4D). 187

Similarly mild effects from depleting CENP-F were observed previously (Bomont et al., 188

2005; Feng et al., 2006; Holt et al., 2005; Raaijmakers et al., 2018; Yang et al., 2005). On 189

the other hand, depletion of CENP-E (with or without additional depletion of CENP-F) 190

led to conspicuous chromosome alignment problems (Figure 2S3C-D), as reported 191

previously (Kapoor et al., 2006; Kuhn and Dumont, 2017; Magidson et al., 2011; 192

Magidson et al., 2015; Putkey et al., 2002; Schaar et al., 1997; Wood et al., 1997; Yao et 193

al., 2000; Yen et al., 1991). 194

195

CENP-E binds to the BubR1 pseudokinase domain 196

Previous studies identified a kinetochore-binding region in residues 2126-2476 of CENP-197

E (Chan et al., 1998). By expression in insect cells, we generated a recombinant version 198

of a larger fragment of CENP-E (residues 2070-C) encompassing this region fused to 199

eGFP (eGFP-CENP-E2070-C) and purified it to homogeneity. After electroporation in 200

mitotic cells arrested by addition of the microtubule-depolymerizing drug nocodazole, 201

cells were fixed to assess the localization of eGFP-CENP-E2070-C. eGFP-CENP-E2070-C 202

localized robustly to mitotic kinetochores (Figure 3A), adopting the typical crescent-like 203

shape previously attributed to the corona (Hoffman et al., 2001; Magidson et al., 2015). 204

An equivalent mutant construct in which Cys2697 had been mutated to alanine to 205

prevent farnesylation also localized to kinetochores, even if at generally lower levels and 206

without showing a crescent-like distribution, suggesting that farnesylation is not strictly 207

required for kinetochore localization of CENP-E but that it might contribute to an 208

unknown aspect of corona assembly. 209

In previous yeast 2-hybrid (Y2H) analyses, a CENP-E segment encompassing residues 210

1958-2662 was found to interact with residues 410-1050 of BubR1 (Chan et al., 1998; 211

Yao et al., 2000). Even if shorter by more than 100 residues at the N-terminal end, 212

eGFP-CENP-E2070-C interacted directly with the dimeric BubR1:Bub3 complex in size 213

exclusion chromatography (SEC) analyses (Figure 3B), as evidenced by the shift in 214

elution volume of both proteins when combined at 16 µM and 4 µM concentration, 215

respectively. Similar observations were made when we mixed CENP-E2070-C with the 216



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BubR1 pseudokinase domain (KinD, residues 705–1050) (Figure 3C). eGFP-CENP-217

E2070-C, however, did not interact with the paralogous Bub1:Bub3 dimer (Figure 3D). In 218

analytical centrifugation (AUC) sedimentation velocity experiments in which we 219

monitored the sedimentation of eGFP-CENP-E, addition of unlabeled BubR1:Bub3 at 220

3-fold higher concentration caused a complete shift of eGFP-CENP-E to a species with 221

higher sedimentation coefficient (S), indicative of complex formation (Figure 3E). The 222

high frictional ratio of this sample (an indication that the CENP-E structure is very 223

elongated, a consequence of its large coiled-coil content) prevented a quantitative 224

estimate of molecular mass. The analysis, however, strongly suggest that eGFP-CENP-225

E2070-C adopts the highly elongated conformation of coiled-coils, as shown previously for 226

recombinant full length CENP-E from Xenopus laevis (Kim et al., 2008). Thus, a minimal 227

segment of CENP-E capable of kinetochore localization interacts directly with the 228

BubR1:Bub3 complex, and the BubR1 pseudokinase domain is sufficient for this 229

interaction, at least at the relatively high concentration of the SEC assay. In agreement 230

with our own previous studies (Breit et al., 2015), BubR1 did not show any catalytic 231

activity, nor did it become active in presence of eGFP-CENP-E2070-C (unpublished data). 232

In a previous study in egg extracts of Xenopus laevis, depletion of BubR1 was shown to 233

prevent kinetochore localization of CENP-E, an effect that could be rescued by re-234

addition of wild type BubR1, but not of a deletion mutant lacking the kinase domain 235

(Mao et al., 2003). CENP-E kinetochore levels were also reduced in DLD-1 cells upon 236

depletion of BubR1 by RNAi (Johnson et al., 2004). 237

We therefore asked if BubR1 was also important for CENP-E recruitment in HeLa cells. 238

Furthermore, in view of evidence that Bub1 is required for kinetochore recruitment of 239

BubR1 (Klebig et al., 2009; Liu et al., 2006; Logarinho et al., 2008; Overlack et al., 2015), 240

we also monitored localization of CENP-E upon depletion of Bub1. Contrarily to the 241

previous observations in frogs and DLD-1 cells, but in agreement with other studies in 242

HeLa cells (Akera et al., 2015; Lampson and Kapoor, 2005; Liu et al., 2006), RNAi-based 243

depletion of Bub1 or BubR1 did not result in obvious adverse effects on the kinetochore 244

localization of CENP-E, even after co-depletion of Zwilch (Figure 3F-G and Figure 245

2S2B-E). These observations suggest that CENP-E, at least in HeLa cells, becomes 246

recruited through a different pathway that does not involve Bub1 and BubR1. After 247

application of highly specific small-molecule inhibitors, we found CENP-E kinetochore 248

localization to depend on the kinase activity of Aurora B and (to a lower extent) of 249



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Mps1, but not of Bub1 or of Plk1 (Figure 3S1A-D). The dependence of CENP-E on 250

Aurora B kinase activity for kinetochore localization, together with the central spindle 251

co-localization at anaphase of CENP-E with the chromosome passenger complex (CPC, 252

the catalytic subunit of which is Aurora B), leads to speculate that these proteins interact, 253

a hypothesis that will need to be formally tested in the future. 254

255

CENP-F binds to the Bub1 kinase domain 256

Next, we asked how CENP-F becomes recruited to kinetochores. CENP-F recruitment 257

was strictly dependent on the kinase activity of Aurora B, partly dependent on that of 258

Mps1 and Plk1, and not dependent on that of Bub1 (Figure 4A and Figures 4S1). This 259

pattern of kinetochore localization is reminiscent of that of Bub1, which has been 260

previously shown to be important for CENP-F kinetochore recruitment (Johnson et al., 261

2004; Klebig et al., 2009; Liu et al., 2006; Raaijmakers et al., 2018). In agreement with 262

these previous studies, RNAi-based depletion of Bub1 resulted in complete ablation of 263

CENP-F from kinetochores (Figure 4C, see panel I for quantification). 264

By expression in insect cells, we generated a recombinant fragment of CENP-F 265

encompassing its previously identified kinetochore-binding domain (residues 2688-C) 266

(Hussein and Taylor, 2002; Zhu, 1999; Zhu et al., 1995a) fused to an N-terminal 267

mCherry tag. In SEC experiments, mCherry-CENP-F2688-C bound Bub1:Bub3 directly, as 268

indicated by its altered elution volume in presence of the CENP-F construct (Figure 4D; 269

note that mCherry-CENP-F2688-C is highly elongated, as shown below, and therefore its 270

hydrodynamic radius, which determines elution volume in SEC experiments, is unlikely 271

to change as a result of an interaction with Bub1:Bub3). On the other hand, mCherry-272

CENP-F2688-C failed to interact with Bub11-409:Bub3, where the Bub1 deletion mutant 273

Bub11-409 lacks a central region of Bub1 and its kinase domain (Figure 4E). Indeed, 274

mCherry-CENP-F2688-C bound the Bub1 kinase domain (Bub1KinD, residues 725–1085; 275

Figure 4F). Conversely, mCherry-CENP-F2688-C did not interact with the BubR1:Bub3 276

complex (Figure 4G). Thus, the kinetochore-targeting domain of CENP-F interacts 277

directly with the Bub1:Bub3 complex, and the kinase domain appears to be necessary 278

and partly sufficient for this interaction. 279

In agreement with these in vitro findings, we observed robust kinetochore localization of 280

endogenous CENP-F in HeLa cells previously depleted of Bub1 by RNAi and expressing 281



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an RNAi-resistant GFP-Bub1 transgene, while CENP-F kinetochore localization 282

appeared entirely compromised in Bub1-depleted cells expressing GFP-Bub11-788, which 283

lacks exclusively the Bub1 kinase domain (Figure 4H-I). Collectively, our observations 284

indicate that the kinase domain of Bub1 is sufficient for a direct interaction with CENP-285

F in vitro, and necessary for kinetochore recruitment of CENP-F in HeLa cells. A very 286

recent study identified a similar requirement for the kinase domain of Bub1 in 287

kinetochore recruitment of CENP-F in HAP1 cells (Raaijmakers et al., 2018). 288

289

CENP-F dimerizat ion is important for Bub1 binding 290

The mCherry-CENP-F2688-C construct that interacted with Bub1:Bub3 in SEC 291

experiments also localized to mitotic kinetochores when electroporated in HeLa cells 292

(Figure 5A). A farnesylation mutant of this construct on which Cys3207 had been 293

mutated to alanine retained kinetochore localization, although not as robustly as the wild 294

type counterpart (Figure 5A). This result suggests that farnesylation is not strictly 295

required for kinetochore recruitment of CENP-F, as already observed with CENP-E 296

(Figure 3A). Collectively, our results with electroporated farnesylation mutants of CENP-297

E and CENP-F are in agreement with results obtained with farnesyl transferase 298

inhibitors, in which only partial repression of kinetochore recruitment of CENP-E and 299

CENP-F was observed (Holland et al., 2015). 300

By rotary shadowing electron microscopy (EM), which is particularly suited to the study 301

of elongated coiled-coil proteins, mCherry-CENP-F2688-C had the appearance of a highly 302

elongated (~40 nm) rod. Most likely, the latter corresponds to a predicted coiled-coil 303

comprised between residues 2688 and ~3000, flanked on one side by two globular 304

domains, most likely corresponding to mCherry, and on the other side by disordered 305

fragments corresponding to the last ~200 residues and containing the C-terminal 306

microtubule-binding domains (Figure 5B) (Feng et al., 2006; Musinipally et al., 2013; 307

Volkov et al., 2015). Collectively, these observations suggest that CENP-F2688-C contains a 308

parallel dimeric coiled-coil, like the one previously identified in CENP-E (Kim et al., 309

2008). 310

On the basis of previous studies implicating Cys2864 in kinetochore recruitment of mouse 311

CENP-F (Zhu, 1999), we generated a mutant version of human mCherry-CENP-F2688-C 312

in which the equivalent residue, Cys2961, was mutated to serine (our residue numbering is 313



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in accordance with the 3210-residue human CENP-F sequence in Uniprot). While wild 314

type mCherry-CENP-F2688-C interacted with Bub1:Bub3 in SEC experiments, as already 315

shown, the interaction was at least partially impaired when the mCherry-CENP-F2688-316C/C2961S mutant was analyzed, confirming the role of Cys2961 in kinetochore localization 317

and implicating this residue in the interaction with Bub1 (Figure 5C-D). Furthermore, 318

mCherry-CENP-F2688-C did not interact with GFP-CENP-E2070-C in SEC experiments, as 319

predicted based on previous work identifying the CENP-E-binding region of CENP-F 320

within a segment (residues 1804-2104) that precedes and is not included in CENP-F2688-C 321

(Figure 5E) (Chan et al., 1998; Yao et al., 2000). 322

The effects of the Cys2961 mutation made us ask if we could identify a minimal Bub1-323

binding domain of CENP-F. For this, we further trimmed CENP-F. CENP-F2866-2990, 324

which is entirely encompassed within the predicted coiled-coil of CENP-F, appeared 325

dimeric by sedimentation velocity AUC and retained the ability to bind to the Bub1 326

kinase domain in a SEC experiment (Figure 6A-B). Similar results were obtained with an 327

even shorter CENP-F fragment, CENP-F2922-2990 (Figure 6C-D). CENP-F2950-2990, on the 328

other hand, appeared monomeric in AUC runs and was unable to interact with the Bub1 329

kinase domain (Figure 6E-F). These observations do not allow us to resolve whether 330

impaired binding to the Bub1 kinase domain CENP-F2950-2990 is due to loss of 331

dimerization or to trimming of residues directly involved in the interaction, but identify 332

CENP-F2922-2990 as a minimal Bub1-binding fragment of CENP-F. In agreement with 333

these observations, mCherry-CENP-F2866-2990 and mCherry-CENP-F2922-2990 labeled 334

kinetochores after electroporation in HeLa cells, albeit weakly in comparison to 335

mCherry-CENP-F2866-C, whereas mCherry-CENP-F2950-2990 did not localize to 336

kinetochores (Figure 6G). 337

338

Conclusions 339

Previous studies on human Bub1 and BubR1, including our own work, demonstrated 340

that these paralogs sub-functionalized in various ways, including 1) the selective 341

inactivation of the kinase domain in BubR1; 2) the development of phospho-aminoacid 342

recognition modules that contribute to the ability of Bub3 to recognize distinct 343

substrates; and 3) the interaction with distinct binding partners that subtends to distinct 344

functions in chromosome alignment and mitotic checkpoint signaling (Figure 7) 345



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(Overlack et al., 2017; Overlack et al., 2015; Primorac et al., 2013; Suijkerbuijk et al., 346

2012; van Hooff et al., 2017). 347

In this study, we report an additional aspect of this sub-functionalization, and show that 348

the kinase domains of BubR1 and Bub1 interact respectively with C-terminal regions of 349

CENP-E and CENP-F that encompass the kinetochore-targeting domains of these 350

proteins. Both interactions are direct and were reproduced with recombinant proteins. 351

Neither interaction appears to be crucially required for downstream signaling events. In 352

the BubR1 case, it appears well established that deletion of the pseudokinase domain in 353

human cells is compatible with its functions in the SAC as a subunit of the MCC, the 354

SAC effector (Musacchio, 2015). We, and others, have also shown that depletion of 355

BubR1 does not affect CENP-E kinetochore localization in HeLa cells [this study and 356

(Akera et al., 2015; Lampson and Kapoor, 2005)]. In Xenopus egg extracts, however, the 357

kinase domain of BubR1 has been implicated in CENP-E recruitment and this 358

interaction has been shown to be important for SAC silencing (Mao et al., 2003). Given 359

the very complex evolutionary history of the Bub1 and BubR1 paralogs or of the 360

singleton from which they originate, these apparent differences may genuinely reflect 361

different evolutionary paths in these organisms (Suijkerbuijk et al., 2012; van Hooff et al., 362

2017). A question for future work that this study raises regards the detailed mechanism 363

of kinetochore recruitment of CENP-E in human cells, which remains unknown. 364

The role of Bub1 in CENP-F kinetochore recruitment was already established in 365

previous work (Berto et al., 2018; Johnson et al., 2004; Liu et al., 2006; Raaijmakers et al., 366

2018), and a very recent study implicated the Bub1 kinase domain in CENP-F 367

kinetochore recruitment (Raaijmakers et al., 2018). Here, we have extended this previous 368

study by showing that the interaction of the Bub1 kinase domain and CENP-F is direct, 369

and by identifying a minimal CENP-F domain involved in this interaction and capable of 370

kinetochore localization. Our identification of a minimal kinetochore-targeting domain 371

of HsCENP-F within residues 2922-2990 agrees with a previous study that made use of 372

CENP-F deletion mutants (Zhu, 1999). It also agrees with another study, currently in 373

press, that identified distinct binding domains in CENP-F for nuclear envelope and for 374

kinetochore localization (Berto et al., 2018). Specifically, residues 2655 to 2860 of mouse 375

CENP-F (corresponding to residues 2866-3072 of HsCENP-F) were sufficient for 376

kinetochore recruitment (Berto et al., 2018). Within this fragment, a specialized N-377

terminal sub-domain (residues 2655-2723, corresponding to HsCENP-F residues 2866-378



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2933) bound Nup133, a subunit of the nuclear pore complex Nup107-Nup160, and 379

mediated CENP-F recruitment to the nuclear envelope shortly before mitosis, but was 380

not required for kinetochore recruitment. A specialized C-terminal sub-domain (residues 381

2724-2860, corresponding to HsCENP-F residues 2934-3072), on the other hand, was 382

required for kinetochore recruitment (Berto et al., 2018). As previous studies identified 383

CENP-E as binding partner of CENP-F (Chan et al., 1998; Mao et al., 2003; Yao et al., 384

2000), CENP-E may reinforce binding of CENP-F to kinetochores, as shown by Berto 385

and colleagues (Berto et al., 2018). However, there is now sufficient evidence to conclude 386

that this interaction is clearly not sufficient to promote stable binding of CENP-F to 387

kinetochores in absence of Bub1. 388

Our observation that CENP-F depletion results in very mild chromosome alignment 389

defects, in line with other reports (see e.g. (Raaijmakers et al., 2018)), is surprising. 390

CENP-F has been implicated in Dynein recruitment and regulation through a pathway 391

involving Nde1, Ndel1, and Lis1, the product of the lissencephaly type 1 gene (Baffet et 392

al., 2015; Bolhy et al., 2011; Hu et al., 2013; Simoes et al., 2018; Vergnolle and Taylor, 393

2007). However, CENP-F is not sufficient for stable kinetochore recruitment of Dynein, 394

as it does not seem to be able to complement the very strong reduction or loss of 395

kinetochore Dynein in cells depleted of the RZZ complex or Spindly [see for instance 396

(Barisic et al., 2010; Chan et al., 2009; Gassmann et al., 2010)]. The latter appears 397

therefore to be the dominant factor in Dynein recruitment to kinetochores. It is 398

plausible, however, that the consequences of CENP-F depletion are exacerbated by 399

concomitant depletion of RZZ (Simoes et al., 2018). 400

As already discussed in the Introduction, various common features of CENP-E and 401

CENP-F support the speculation that they are distantly related paralogs. Their ability to 402

interact with the kinase domains of BubR1 and Bub1, themselves paralogs, lends strong 403

further credit to this hypothesis. Apparent lack of strong functional consequences from 404

disrupting these interactions may indicate that they may have become vestigial in some 405

species. It is also possible, however, that these interactions play more important roles 406

during development or in specific cell types. The dissection described here will allow 407

testing this hypothesis in future work. 408

409

Acknowledgments 410



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We thank all members of the Musacchio laboratory for helpful discussions and 411

comments. A.M. gratefully acknowledges the Max Planck Society, the European 412

Research Council (ERC) Advanced Investigator Grant RECEPIANCE (proposal 413

number nº 669686), and the European Union’s Horizon 2020 research and innovation 414

programme under the Marie Sklodowska-Curie grant agreement No 675737. We are very 415

grateful to Dr. Valerie Doye and her collaborators for sharing with us unpublished 416

results. 417

418

Material & Methods 419

Plasmids The codon optimized cDNAs of Homo sapiens CENP-E (Q02224) and CENP-420

F (P49454) were synthetized at GeneWiz. CENP-E and CENP-F constructs were 421

subcloned respectively in pLIB-eGFP and pLIB-mCherry or pET-mCherry, modified 422

versions of the pLIB (Weissmann et al., 2016) and pET-28 vectors for expression of 423

proteins with N-terminal PreScission-cleavable His6-eGFP or His6-mCherry tag. Site-424

directed mutagenesis was performed by PCR (Sawano and Miyawaki, 2000). All 425

constructs were sequence verified. The vectors for the co-expression of full length Bub1 426

and BubR1 proteins with Bub3, as well as that for the Bub1 & BubR1 constructs were 427

described previously (Breit et al., 2015; Overlack et al., 2015). 428

Protein expression and purification Expression and purification of eGFP-CENP-429

E2070-C and mCherry-CENP-F2866-C wild-type and mutants was carried out in insect cells 430

using a pBig system (Weissmann et al., 2016). Baculoviruses were generated in Sf9 cells 431

and use to infect Tnao38 cells for 48-96 hours at 27°C. Cells were collected by 432

centrifugation, washed in PBS and then frozen at -80ºC. CENP-E expressing cell pellets 433

were resuspended in lysis buffer (50 mM Sodium Phosphate buffer pH 8.0, 500 mM 434

NaCl, 5 % (w/v) glycerol and 0.5 mM TCEP) supplemented with protease inhibitor 435

cocktail, lysed by sonication and cleared by centrifugation at 100.000 g at 4°C. The 436

supernatant was filtered and loaded on a 5 ml HisTrap FF column (GE Healthcare) 437

equilibrated in lysis buffer. After washing with lysis buffer, the protein was eluted with a 438

linear gradient of 0-250 mM imidazole in 10 column volumes. The fractions of interest 439

were pooled, concentrated with a 50 kDa cut-off Amicon concentrator (Millipore) and 440

loaded onto a Superose 6 Increase 10/300 (GE Healthcare) equilibrated in SEC buffer 441

(50 mM Hepes pH 8.0, 200 mM NaCl, 5% (w/v) glycerol and 0.5 mM TCEP). CENP-E 442



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containing fractions were concentrated, flash-frozen in liquid nitrogen and stored at -443

80°C. The purification protocol for the mCherry-CENP-F2866-C constructs is identical to 444

that of eGFP-CENP-E2070-C, but the lysis and the SEC buffers were at pH 7.0. 445

The constructs mCherry-CENP-F2866-2990, mCherry-CENP-F2922-2990 and mCherry-CENP-446

F2950-2990 were expressed in E. coli BL21 (DE3) RP plus cells grown at 37ºC to O.D.600 = 2 447

and then induced with 0.25 mM IPTG for 16 h at 25ºC. Cell were collected by 448

centrifugation, washed in PBS and then frozen at -80ºC. Cell pellets were resuspended in 449

lysis buffer (50 mM Sodium Phosphate buffer pH 7.5, 500 mM NaCl, 5 % (w/v) glycerol 450

and 2 mM β-mercaptoethanol) supplemented with protease inhibitor cocktail, lysed by 451

sonication and cleared by centrifugation at 70.000 g at 4°C. The supernatant was filtered 452

and loaded on a 5 ml HisTrap FF column (GE Healthcare) equilibrated in lysis buffer. 453

After washing with lysis buffer, the protein was eluted with a linear gradient of 0-500 454

mM imidazole in 10 column volumes. The fractions of interest were pooled, 455

concentrated with a 10 kDa cut-off Amicon concentrator (Millipore) and loaded onto a 456

HiLoad Superdex 75 16/60 (GE Healthcare) equilibrated in SEC buffer (50 mM Sodium 457

Phosphate buffer pH 7.0, 200 mM NaCl, 5% (w/v) glycerol and 1 mM TCEP). 458

Expression and purification of Bub1 and BubR1 constructs, as well as of Bub1:Bub3 and 459

BubR1:Bub3 complexes was carried out as described (Breit et al., 2015; Overlack et al., 460

2015). 461

Analytical SEC analysis 4 µM Bub1 and BubR1 protein constructs or Bub1:Bub3 and 462

BubR1:Bub3 complexes were mixed with 16 µM CENP-E and CENP-F proteins 463

respectively, in 30 µl final volume. Analytical size exclusion chromatography was carried 464

out at 4°C on a Superose 6 5/150 or Superdex 75 5/150 in a buffer containing 50 mM 465

HEPES pH 8.0, 100 mM NaCl, 5% (w/v) glycerol and 0.5 mM TCEP at a flow rate of 466

0.12 ml/min on an ÄKTA micro system. Elution of proteins was monitored at 280 nm, 467

488 nm (eGFP-tag) and 587 nm (mCherry-tag). 50 µl fractions were collected and 468

analysed by SDS-PAGE and Coomassie blue staining. 469

Analytical Ultracentrifugation Sedimentation velocity AUC was performed at 42,000 470

rpm at 20°C in a Beckman XL-A ultracentrifuge. Protein samples were loaded into 471

standard double-sector centerpieces. The cells were scanned every minute and 500 scans 472

were recorded for every sample. 6 µM mCherry-CENP-F2866-2990, mCherry-CENP-F2922-4732990 and mCherry-CENP-F2950-2990 were scanned at 587 nm. 7 µM eGFP-CENP-E2070-C 474



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alone or mixed with 21 µM BubR1:Bub3 were instead scanned at 488 nm. Data were 475

analyzed using the program SEDFIT (Brown and Schuck, 2006) with the model of 476

continuous c(s) distribution. The partial specific volumes of the proteins, buffer density, 477

and buffer viscosity were estimated using the program SEDNTERP. Data figures were 478

generated using the program GUSSI (Brautigam, 2015). 479

Protein Electroporation For eGFP-CENP-E protein electroporation, HeLa cells were 480

arrested in G2 with a 9 µM RO-3306 treatment for 15 hours (Millipore) and then 481

released into mitosis for 3 hours in presence of 3.3 µM nocodazole. Mitotic cells were 482

then collected by mitotic shake-off, washed with PBS and counted. Approximatively 483

3x106 cells were then electroporated (Neon Transfection System Kit, Thermo Fisher) 484

with 10 µM eGFP-CENP-E. Following electroporation, cells were allowed to recover in 485

media with 3.3 µM nocodazole for 4 hours and then fixed and prepared for 486

immunofluorescence analysis. For mCherry-CENP-F protein electroporation, HeLa cells 487

were treated for 16 hours with 0.33 µM Nocodazole (Sigma) to synchronise cells in 488

mitosis. Mitotic cells were then collected by mitotic shake-off, washed with PBS and 489

counted. Approximatively 3x106 cells were then electroporated with 5 µM mCherry-490

CENP-F. Following electroporation, cells were allowed to recover in media with 3.3 µM 491

nocodazole for 4 hours and then fixed and prepared for immunofluorescence analysis. 492

Low-angle metal shadowing and electron microscopy mCherry-CENP-F2688-C 493

fractions from the elution peak of an analytical size-exclusion chromatography column 494

were diluted 1:1 with spraying buffer (200 mM ammonium acetate and 60% glycerol) and 495

air-sprayed as described (Baschong and Aebi, 2006; Huis in 't Veld et al., 2016) onto 496

freshly cleaved mica pieces of approximately 2x3 mm (V1 quality, Plano GmbH). 497

Specimens were mounted and dried in a MED020 high-vacuum metal coater (Bal-tec). A 498

Platinum layer of approximately 1 nm and a 7 nm Carbon support layer were evaporated 499

subsequently onto the rotating specimen at angles of 6-7° and 45° respectively. Pt/C 500

replicas were released from the mica on water, captured by freshly glow-discharged 400-501

mesh Pd/Cu grids (Plano GmbH), and visualized using a LaB6 equipped JEM-1400 502

transmission electron microscope (JEOL) operated at 120 kV. Images were recorded at a 503

nominal magnification of 60,000x on a 4k x 4k CCD camera F416 (TVIPS), resulting in 504

0.18 nm per pixel. 505



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Mammalian Plasmids Plasmids were derived from the pCDNA5/FRT/TO-EGFP-506

IRES, a previously modified version (Krenn et al., 2012) of the pCDNA5/FRT/TO 507

vector (Invitrogen). To create N-terminally-tagged EGFP-Bub1 truncation constructs, 508

the Bub1 sequence was obtained by PCR amplification from the previously generated 509

pCDNA5/FRT/TO-EGFP-Bub1-IRES vector (Krenn et al., 2012) and subcloned in 510

frame with the GFP-tag. All Bub1 constructs were RNAi resistant (Kiyomitsu et al., 511

2007). pCDNA5/FRT/TO-based plasmids were used for generation of stable cell lines. 512

All plasmids were verified by sequencing. 513

Cell culture and transfection HeLa cells were grown in DMEM (PAN Biotech) 514

supplemented with 10 % FBS (Clontech), penicillin and streptomycin (GIBCO) and 515

2 mM L-glutamine (PAN Biotech). Flp-In T-REx HeLa cells used to generate stable 516

doxycycline-inducible cell lines were a gift from S.S. Taylor (University of Manchester, 517

Manchester, England, UK). Flp-In T-REx host cell lines were maintained in DMEM 518

with 10 % tetracycline-free FBS (Clontech) supplemented with 50 µg/ml Zeocin 519

(Invitrogen). Flp-In T-REx HeLa expression cell lines were generated as previously 520

described (Krenn et al., 2012). Briefly, Flp-In T-Rex HeLa host cells were cotransfected 521

with a ratio of 9:1 (w/w) pOG44:pcDNA5/FRT/TO expression plasmid using X-522

tremeGene transfection agent (Roche). 48 h after transfection, Flp-In T-Rex HeLa 523

expression cell lines were put under selection for two weeks in DMEM with 10 % 524

tetracycline-free FBS (Invitrogen) supplemented with 250 µg/ml Hygromycin (Roche) 525

and 5 µg/ml Blasticidin (ICN Chemicals). The resulting foci were pooled and tested for 526

expression. Gene expression was induced by addition of 0.5 µg/ml doxycycline (Sigma) 527

for 24 h. 528

siBUB1 (Dharmacon, 5’-GGUUGCCAACACAAGUUCU-3’) or siBUBR1 (Dharmacon, 529

5’-CGGGCAUUUGAAUAUGAAA-3’) duplexes were transfected with Lipofectamine 530

2000 (Invitrogen) at 50 nM for 24 h. siCENP-E (Dharmacon, 5’-531

AAGGCUACAAUGGUACUAUAU-3’) and siCENP-F (Dharmacon, 5'-532

CAAAGACCGGUGUUACCAAG-3' and 5'-AAGAGAAGACCCCAAGUCAUC-3') 533

duplexes were transfected at 60 nM with LipofectamineRNAiMAX (Invitrogen) for 24 h. 534

siZwilch (SMART pool from Dharmacon, #L-019377-00-0005) duplexes were 535

transfected with LipofectamineRNAiMAX at 120 nM for 72 h. 536

Immunoblotting To generate mitotic populations for immunoblotting experiments, 537

cells were treated with 330 nM nocodazole for 16 h. Mitotic cells were then harvested by 538



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

1.5 mM MgCl2, 10 % glycerol, and 0.5 % Triton-X 100 supplemented with protease 540

inhibitor cocktail (Serva) and PhosSTOP phosphatase inhibitors (Roche)]. Cleared cell 541

lysates were resuspended in sample buffer, boiled and analyzed by SDS-PAGE using 3-542

8 % gradient gels (NuPAGE® Tris-Acetate Gels, Life technologies) and Western 543

blotting. The following antibodies were used: anti-CENP-E (rabbit, ab133583, 1:500), 544

anti-CENP-F (rabbit, Novus NB500-101, 1:500) and anti-Tubulin (mouse, Sigma T9026, 545

1:10000). Secondary antibodies were anti–mouse (Amersham) or anti–rabbit (Amersham) 546

affinity-purified with horseradish peroxidase conjugate (working dilution 1:10000). After 547

incubation with ECL Western blotting system (GE Healthcare), images were acquired 548

with the ChemiDocTM MP Imaging System (BioRad) in 16-bit TIFF format. Images were 549

cropped and converted to 8-bit using Image J software (NIH). Brightness and contrast 550

were adjusted using Photoshop CS5 (Adobe). 551

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

changed to CO2 Independent Medium (Gibco®) 6 h before filming. DNA was stained 553

by addition of the SiR-Hoechst-647 Dye (Spirochrome) to the medium 1 h before 554

imaging. Cells were imaged every 5 to 10 min in a heated chamber (37 °C) on a 3i 555

Marianas™ system (Intelligent Imaging Innovations Inc.) equipped with Axio Observer 556

Z1 microscope (Zeiss), Plan-Apochromat 40x/1.4NA oil objective, M27 with DIC III 557

Prism (Zeiss), Orca Flash 4.0 sCMOS Camera (Hamamatsu) and controlled by Slidebook 558

Software 6.0 (Intelligent Imaging Innovations Inc). 559

Immunofluorescence HeLa cells and Flp-In T-REx HeLa cells were grown on 560

coverslips precoated with poly-D-Lysine (Millipore, 15 µg/ml) and poly-L-Lysine 561

(Sigma), respectively. Asynchronously growing cells or cells that were arrested in 562

prometaphase by the addition of nocodazole (Sigma-Aldrich) were fixed using 4 % 563

paraformaldehyde. Cells were stained for Bub1 (mouse, ab54893, 1:400), BubR1 (rabbit, 564

Bethyl A300-386A-1, 1:1000), Tubulin (mouse, DM1a Sigma, 1:500), CENP-E (mouse, 565

ab5093, 1:200), CENP-F (rabbit, Novus NB500-101, 1:300), Zwilch (rabbit, in-house 566

made, SI520, 1:900), Mad1 labelled with AlexaFluor-488 (mouse, in-house made, Clone 567

BB3-8, 1:200), pT232-AurB (rabbit, Rockland #660-401-667, 1:2000), Plk1 (mouse, 568

ab17057, 1:300), pS10H3 (mouse, ab14955, 1:3000), pT121 H2A (rabbit, active motif 569

#39391, 1:2000) and CREST/anti-centromere antibodies (Antibodies, Inc., 1:100), 570

diluted in 2 % BSA-PBS for 1.5 h. 571



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For testing the effect of various kinase inhibitors on CENP-E and CENP-F kinetochore 572

localization, the protocol was adapted in the following way: Cells were pre-permeabilized 573

with 0.5 % triton-X-100 solution in PHEM (Pipes, Hepes, EGTA, MgCl2) buffer for 2 574

min before fixation with 4% PFA-PHEM for 15 min. After blocking the cells with 3 % 575

BSA-PHEM buffer supplemented with 0.1 % triton-X-100, they were incubated at room 576

temperature for 1-2 h with primary antibodies diluted in blocking buffer. Washing steps 577

were performed in PHEM-T buffer. 578

Goat anti–human (Invitrogen), goat anti–mouse (Jackson ImmunoResearch 579

Laboratories, Inc.) and goat anti-rabbit (Jackson ImmunoResearch Laboratories, Inc.) 580

fluorescently labeled antibodies were used as secondary antibodies. DNA was stained 581

with 0.5 µg/ml DAPI (Serva) and coverslips were mounted with Mowiol mounting 582

media (Calbiochem). Cells were imaged at room temperature using a spinning disk 583

confocal device on the 3i Marianas™ system equipped with an Axio Observer Z1 584

microscope (Zeiss), a CSU-X1 confocal scanner unit (Yokogawa Electric Corporation), 585

Plan-Apochromat 63x or 100x/1.4NA Oil Objectives (Zeiss) and Orca Flash 4.0 sCMOS 586

Camera (Hamamatsu). Images were acquired as z-sections at 0.27 µm. Images were 587

converted into maximal intensity projections, exported and converted into 8-bit. 588

Quantification of kinetochore signals was performed on unmodified 16-bit z-series 589

images using Imaris 7.3.4 32-bit software (Bitplane). After background subtraction, all 590

signals were normalized to CREST. At least 117 kinetochores were analyzed per 591

condition. Measurements were exported in Excel (Microsoft) and graphed with 592

GraphPad Prism 6.0 (GraphPad Software). 593

Cell synchronization To test the effect of various kinase activities on CENP-E and 594

CENP-F kinetochore localization, cells were synchronized using a double thymidine 595

arrest. Cells were released from the first 18 h thymidine (2 mM; Sigma–Aldrich) block by 596

washing them with fresh pre-warmed media several times. After releasing them for the 597

next 9 h, cells were exposed to thymidine (2 mM) a second time for 15 h. Afterwards, 598

cells were released into S-phase for 4 h and then nocodazole (330 nM) was added to the 599

media for the next 3-4 h to enrich for the mitotic cell population. Kinase activity 600

inhibitors, BI 2536 (500 nM; Calbiochem), Hesperadin (500 nM; Calbiochem), Reversine 601

(500 nM; Calbiochem) or BAY-320 (10 µM; kindly received from Dr. Gerhard 602

Siemeister, Bayer GmbH, Berlin) were added in the presence of the proteasome 603

inhibitor, MG132 (10 µM; Calbiochem) to the cells for 90 min before fixing these cells 604



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for immunofluorescence. 605

Chromosome alignment For analysis of the effect of CENP-F depletion on 606

chromosome alignment, cells were fixed after RNAi either asynchronously or after an 607

additional treatment with 10 µM MG-132 for 2 h. Cells were stained for CENP-F, 608

Tubulin and CREST. DNA was labeled with DAPI. The number of metaphase cells with 609

aligned chromosomes and with misaligned chromosomes was scored for each condition. 610

At least 595 cells (without synchronization) or 92 cells (with synchronization) were 611

analyzed per condition. 612

613



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phosphoprotein that is specifically involved in mitotic-phase progression. Mol Cell Biol 92215, 5017-5029. 923

924

925



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Figure Legends 926

Figure 1. The corona proteins CENP-E and CENP-F localize at kinetochores 927

with distinct timing 928

A) Schematic representation of the corona structure and function. MT, microtubules; IP, 929

inner plate of kinetochore; OP, outer plate of kinetochore. B) Schematic organization of 930

the CENP-E and CENP-F full-length proteins. Coiled coil regions were predicted with 931

coils, pcoils and marcoils (Delorenzi and Speed, 2002; Gruber et al., 2005; Lupas et al., 932

1991) using default parameters (ncoils and paircoils: windows size 21). To combine all 933

three coiled coil prediction algorithms, we applied a scoring system in which we assigned 934

for each residue two points for a high significance (P-value >=0.9) and one point for low 935

significance (P-value >=0.8). Two additional points were granted for an identical register 936

position in coiled coil if predicted by all three programs, resulting in a maximum score of 937

8. C, D) Representative images of fixed Hela cells treated for fluorescence staining with 938

the indicated antibodies. The panel illustrates the localization of CENP-E (C) and 939

CENP-F (D) in the different phases of the cell cycle. Scale bar: 10 µm. 940

941

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

A, B) Representative images of Hela cells showing Zwilch (A) and Mad1 (B) localization 943

in the different phases of the cell cycle. Scale bar: 10 µm. 944

945

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

and CENP-F 947

A-H) Representative images and quantification of proteins kinetochore levels in Hela 948

cells mock treated or depleted of Zwilch (A), CENP-E (B, E, H), CENP-F (C, F, G) or 949

co-depleted of CENP-E and CENP-F (D). Scale bar: 10 µm. Zwilch depletion does not 950

affect the localization of CENP-E (A). CENP-E depletion does not affect the 951

localization of Zwilch (B), Mad1 (E) and CENP-F (H). Similarly, CENP-F depletion 952

does not interfere with the recruitment of Zwilch (C), Mad1 (F) and CENP-E (G). Co-953

depletion of CENP-E and CENP-F has no effects on localization of Zwilch (D). The 954

graphs show mean intensity of one (B, C, E), two (D, F) or three (A, G, H) experiments; 955

the error bars indicate SEM and the mean values for non-depleted cells are set to 1. 956



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31

957

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

A) Western blot showing CENP-E protein levels in mock treated or CENP-E depleted 959

cells. B) Representative images of HeLa cells, either mock-treated or depleted of CENP-960

E, showing that CENP-E can be efficiently depleted. Scale bar: 10 µm. C) Quantification 961

of CENP-E kinetochore levels in cells treated as in panel (B). The graph shows mean 962

intensity of two independent experiments; the error bars indicate SEM and the mean 963

value for non-depleted cells is set to 1. D) Western blot showing CENP-F protein levels 964

in mock treated or CENP-F depleted cells. E) Representative images of HeLa cells mock 965

treated or depleted of CENP-F showing successful CENP-F depletion. Scale bar: 10 µm. 966

F) Quantification of CENP-F kinetochore levels in cells treated as in panel (E). The 967

graph shows mean intensity of three independent experiments; the error bars indicate 968

SEM and the mean value for non-depleted cells is set to 1. G) Quantification of Zwilch 969

kinetochore levels in cells treated as in Figure 2A (upper panel) and in (H). The graphs 970

show mean intensity of three (CENP-E experiment) or two (Mad1 experiment) 971

independent experiments; the error bars indicate SEM and the mean value for non-972

depleted cells is set to 1. H) Representative images of HeLa cells mock treated or 973

depleted of Zwilch showing that Zwilch depletion leads to reduced Mad1 levels. Scale 974

bar: 10 µm. I) Quantification of Mad1 kinetochore levels in cells treated as in (H). The 975

graph shows mean intensity of two independent experiments; the error bars indicate 976

SEM and the mean value for non-depleted cells is set to 1. J) Correlation of Mad1 and 977

Zwilch levels in 38 cells, 19 of which were mock treated, while the other 19 were RNAi 978

depleted of Zwilch. Cells are from two independent experiments. AU, arbitrary units. 979

980

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

recruitment 982

A) Upper panel - Representative images of HeLa cells either mock treated or co-depleted 983

of CENP-E and CENP-F showing effective co-depletion of the proteins. Scale bar: 10 984

µm. Lower panel - Quantification of co-depletion efficiency. The graph shows mean 985

intensity of two independent experiments; the error bars indicate SEM and the mean 986

values for non-depleted cells are set to 1. B) Representative images of HeLa cells mock 987

treated or co-depleted of Zwilch and BubR1 showing efficient depletion of both the 988



https://doi.org/10.1101/276204


32

proteins. Scale bar: 10 µm. C) Representative images of HeLa cells mock treated or co-989

depleted of Zwilch and BubR1 showing that CENP-E localization is not affected. Scale 990

bar: 10 µm. D) Representative images of HeLa cells mock treated or co-depleted of 991

Zwilch and Bub1 showing efficient depletion of both proteins. Two cells with different 992

Zwilch levels and the same low Bub1 levels are shown for the RNAi condition. Scale bar: 993

10 µm. E) Representative images of Hela cells mock treated or co-depleted of Zwilch 994

and Bub1, showing that CENP-E is not lost from KTs upon Zwilch and Bub1 co-995

depletion. Two cells with different depletion efficiency for Zwilch are shown for the 996

RNAi condition. Scale bar: 10 µm. 997

998

Figure 2S3. Kinetochore localization of RZZ (Zwilch) is not affected by CENP-E 999

or CENP-F depletion also in presence of microtubules 1000

A-C) Representative images of a prometaphase and metaphase HeLa cells either mock 1001

treated (A) or individually depleted of CENP-F (B), co-depleted of both CENP-E and 1002

CENP-F (C), individually depleted of CENP-E (D) in absence of nocodazole. Neither 1003

CENP-E nor CENP-F are required for kinetochore localization of Zwilch even in the 1004

presence of spindle microtubules. Scale bar: 10 µm. 1005

1006

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

A) Examples of the scored categories, aligned metaphase and metaphases with 1008

misalignments in mock treated or CENP-F depleted HeLa cells. Scale bar: 10 µm. B) 1009

Percentages of cells in the depicted categories in unsynchronized cells from one 1010

experiment. C) Percentages of cells in the depicted categories in cells treated with 10 µM 1011

MG for 2h before fixation from one experiment. D) Mean duration of mitosis of HeLa 1012

cells in presence or absence of endogenous CENP-F. Cell morphology was used to 1013

measure entry into and exit from mitosis by time-lapse microscopy (n>76 per condition 1014

per experiment) from two independent experiments. Error bars indicate SEM. 1015

1016

Figure 3. CENP-E interacts with the BubR1 pseudo-kinase domain, but BubR1 is 1017

not required for its kinetochore localization 1018

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



https://doi.org/10.1101/276204


33

E2070-C WT or eGFP-CENP-E2070-C C2997A mutant (preventing CENP-E farnesylation). 1020

Scale bar: 5 µm. Both the WT and the un-farnesylated mutant CENP-E constructs 1021

localize at kinetochores. B-D) Elution profiles and SDS-PAGE analysis of SEC 1022

experiments of eGFP-CENP-E2070-C with BubR1:Bub3 complex (B), BubR1 pseudo-1023

kinase domain (KinD) construct (C) and Bub1:Bub3 complex (D). A shift in elution 1024

volume is observed for the BubR1:Bub3 complex and the BubR1 pseudo-kinase domain 1025

construct, indicative of complex formation. The interaction of CENP-E with BubR1 is 1026

specific, as no shift is observed with the Bub1:Bub3 complex. E) Sedimentation velocity 1027

AUC profiles of eGFP-CENP-E2070-C alone and in complex with BubR1:Bub3. AU, 1028

arbitrary units; MWtheo, predicted molecular weight assuming stoichiometry of 1. A 1029

reliable estimation of the molecular mass of the proteins in the samples was unsuccessful, 1030

likely because of the very elongated and flexible structure of both CENP-E and BubR1. 1031

F) Representative images of stable Flp-In T-REx cells mock treated or depleted of 1032

endogenous Bub1, BubR1, or both, showing that CENP-E kinetochore localization is 1033

unaffected under any of the conditions. Scale bar: 10 µm. G) Quantification of CENP-E 1034

kinetochore levels in cells treated as in (F). The graph shows mean intensity of two 1035

independent experiments, the error bars indicate SEM. The mean value for non-depleted 1036

cells expressing GFP was set to 1. 1037

1038

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

A-D) Representative images and quantification of CENP-E kinetochore levels in mitotic 1040

Hela cells treated with the indicated concentrations of the indicated kinase inhibitors. 1041

Scale bar: 10 µm. CENP-E localization is sensitive to Aurora B (A), Mps1 (B) and, to a 1042

lesser extent, Plk1 (D) but not to Bub1 (C) inhibition. Reduction in P-T232 Aurora B 1043

(Aurora B activation segment) staining was used as a positive control for Aurora B 1044

inhibition (A), while reduction in Bub1 localization was used as positive control for Mps1 1045

inhibition (B). Bub1 inhibition was confirmed by reduction in P-T121 H2A staining (C). 1046

The graphs show mean intensity of one (C, I), two (A) or four (B) experiments. The 1047

error bars indicate SEM and the mean values for DMSO-treated cells are set to 1. 1048

1049

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

kinetochore localization 1051



https://doi.org/10.1101/276204


34

A) Representative images of mitotic HeLa cells treated with 500 nM Hesperadin, 1052

showing that CENP-F kinetochore localization is strictly dependent on Aurora B kinase 1053

activity. Reduction in P-S10-H3 staining was used as a control for the Aurora B 1054

inhibition. Scale bar: 10 µm. B) Quantification of CENP-F kinetochore levels in cells 1055

treated as in A. The graph shows mean intensity of three experiments. The error bars 1056

indicate SEM and the mean values for DMSO-treated cells are set to 1. C) 1057

Representative images of GFP-expressing stable HeLa Flp-In T-REx cell lines mock 1058

treated or depleted of Bub1, showing that CENP-F kinetochore recruitment depends on 1059

the presence of Bub1 at kinetochores. D-G) Elution profiles and SDS-PAGE analysis of 1060

SEC experiments of mCherry-CENP-F2688-C with the Bub1FL:Bub3 complex; FL, full 1061

length (D), the Bub11-409:Bub3 complex (E), the Bub1 kinase domain (KinD) (F), and the 1062

BubR1:Bub3 complex (G). A shift in the elution volume is only observed for the Bub1 1063

constructs containing the C-terminal kinase domain (D, F) The interaction of CENP-F 1064

with Bub1 is specific, as no shift is observed for the BubR1:Bub3 complex (G). H) 1065

Representative images of stable HeLa Flp-In T-REx cell lines depleted of endogenous 1066

Bub1 and expressing GFP-Bub1 full length or lacking the kinase domain (Bub11-788). 1067

CENP-F kinetochore recruitment depends on the Bub1 kinase domain, as Bub11-788 does 1068

not rescue CENP-F localization, while full length Bub1 does. Scale bar: 10 µm. I) 1069

Quantification of CENP-F kinetochore levels in cells of panels C and H. The graph 1070

shows mean intensity of three independent experiments, the error bars indicate SEM. 1071

The mean value for non-depleted cells expressing GFP is set to 1. 1072

1073

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

A-E) Representative images and quantification of CENP-F kinetochore levels in mitotic 1075

Hela cells treated with the indicated type and concentrations of kinase inhibitors. Scale 1076

bar: 10 µm. CENP-F localization is sensitive to Mps1 (A) and partially to Plk1 (D) 1077

inhibition. Despite a requirement for the Bub1 kinase domain for CENP-F kinetochore 1078

recruitment, Bub1 catalytic activity is dispensable (B, C). Reduction in Bub1 localization 1079

was used as a control for Mps1 inhibition (A) and reduction in P-T121-H2A staining was 1080

used as a control for Bub1 inhibition (E).Loss of Plk1 localization was used as control 1081

for Plk1 inhibition (D). The graphs show mean intensity of one (B, C, E) or two (A, D) 1082

experiments. The error bars indicate SEM and the mean values for DMSO-treated cells 1083

are set to 1. F) Representative images of HeLa cells mock treated or depleted of CENP-1084



https://doi.org/10.1101/276204


35

F showing that CENP-F depletion does not affect the kinase activity of Bub1, as no 1085

changes are detected for the P-T121-H2A signal. Scale bar: 10 µm. 1086

1087

Figure 5. Requirements for CENP-F kinetochore localization 1088

A) Representative images of mitotic HeLa cells electroporated with mCherry, mCherry-1089

CENP-F2688-C WT or mCherry-CENP-F2688-C C3207A (farnesylation) mutant. Scale bar: 5 1090

µm. As for CENP-E, both the WT and the un-farnesylated mutant CENP-F constructs 1091

localize at kinetochore. B) mCherry-CENP-F2688-C sample was visualized by electron 1092

microscopy after glycerol spraying and low-angle platinum shadowing (right panel). The 1093

elongated shape of the observed particles is consistent with the secondary structure 1094

expected for the mCherry-tag coiled-coil construct (right panel). C-D) SEC elution 1095

profiles and SDS-PAGE analysis of binding experiments with 16 µM each of mCherry-1096

CENP-F2688-C WT (C) or C2961S mutant (D) and 4 µM Bub1FL:Bub3 complex. The shift 1097

in elution volume of Bub1:Bub3 is observed with both wild type and mutant CENP-F, 1098

but it significantly less pronounced for the CENP-F mutant, suggesting that the C2961S 1099

mutation reduces the affinity of CENP-F for the Bub1 kinase domain without 1100

completely abolishing it. E) SEC elution profile and SDS-PAGE analysis of a binding 1101

experiment with 16 µM each of mCherry-CENP-F2688-C and eGFP-CENP-E2070-C. No 1102

shift is observed, indicating that the tested constructs do not interact. 1103

1104

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

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

MWobs, observed molecular weight; MWtheo, the predicted molecular weight of the 1107

monomer; Frict. ratio denotes the frictional ratio. AU, arbitrary units. mCherry-CENP-1108

F2866-2990 forms a dimer. B) Elution profiles and SDS-PAGE analysis of SEC experiments 1109

of the Bub1 kinase domain (KinD) with mCherry-CENP-F2866-2990. The shift in elution 1110

volume of Bub1KinD indicated binding. The red asterisk indicated a breakdown product of 1111

mCherry that is produced during boiling in sample buffer. C) As in (A) but with the 1112

CENP-F2922-2990construct, which is also dimeric. D) As in (B) but with the CENP-F2922-29901113

construct. Also in this case, an interaction with the kinase domain of CENP-F is clearly 1114

discernible. E) As in (A) but with the CENP-F2950-2990construct, which is monomeric. F) 1115

As in (B) but with the CENP-F2950-2990 construct. In this case, no interaction with the 1116



https://doi.org/10.1101/276204


36

kinase domain of CENP-F is discernible. G) Representative images of mitotic HeLa cells 1117

electroporated with the indicated constructs. mCherry-CENP-F2688-C (positive control), 1118

mCherry-CENP-F2866-2990, and mCherry-CENP-F2922-2990 localized to kinetochores, 1119

whereas mCherry (negative control) and mCherry-CENP-F2950-2990 did not. Scale bar: 5 1120

µm. 1121

1122

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

The schematic summarizes the interactions occurring at kinetochores between CENP-E, 1124

CENP-F, Bub1, and BubR1. Bub1 is recruited to the kinetochore subunit Knl1 (see 1125

Introduction) after phosphorylation by the SAC kinase Mps1. There, Bub1 recruits 1126

BubR1 through a pseudo-dimeric interaction (Musacchio, 2015). CENP-F kinetochore 1127

localization strictly depends on Bub1, while CENP-E recruitment requires a wider and 1128

still uncharacterized network of interactions, indicated by a question mark. RZZ and 1129

Mad1 recruitment, as well as the corona expansion, appear to be independent from 1130

CENP-E and CENP-F, and are not shown. An interaction of CENP-E and CENP-F has 1131

also been identified (grey arrow), but is not sufficient for CENP-F localization in absence 1132

of Bub1. 1133

1134



https://doi.org/10.1101/276204


SpindleMT

RZZ &SpindlyMAD1

MAD2SAC

activation

KT-MTattachment

SACinactivation

DyneinDynactin

Unattachedkinetochore

AttachedkinetochoreCorona

IPOP

Ciossani, Overlack et al. Figure 1

A

MTbinding

MTbinding

CENP-Ebinding

KTlocalization

321010

1CoiledCoil

KinesinDomain

CENP-F +KT localization

MTbinding

270110

1CoiledCoil

2688

CENP-E

CENP-F

2070 2701

3210

B

DN

AC

RE

ST

CE

NP

-ETubulin

DN

AC

RE

ST

CE

NP

-FTubulin

Inter-phase

Pro-phase

Prometa-phase

Meta-phase

Ana-phase

Telo-phase

earlyG1

Inter-phase

Pro-phase

Prometa-phase

Meta-phase

Ana-phase

Telo-phase

earlyG1

C D

CENP-F

CENP-EKT-MT

attachment



https://doi.org/10.1101/276204


DNA CREST Zwilch TubulinA

Inter-phase

Pro-phase

Prometa-phase

Meta-phase

Ana-phase

Telo-phase

earlyG1

Ciossani, Overlack et al. Figure 1S1

DNA CREST TubulinMad1

Inter-phase

Pro-phase

Prometa-phase

Meta-phase

Ana-phase

Telo-phase

earlyG1

B



https://doi.org/10.1101/276204


DN

AC

RE

ST

CE

NP

-FC

EN

P-E

DN

AC

RE

ST

CE

NP

-FM

ad1

0.0

0.5

1.0

CENP-F RNAi - +0.0

0.5

1.0

CENP-F RNAi - +

0.0

0.5

1.0

CENP-F RNAi - +

DN

AC

RE

ST

Zwilc

h

C

DN

AC

RE

ST

CE

NP

-EC

EN

P-F

CENP-E RNAi - +0.0

0.5

1.0

0.0

0.5

1.0

CENP-E RNAi - +

DN

AC

RE

ST

Mad

1


DN

AC

RE

ST

Zwilc

hC

EN

P-E

Zwilch RNAi- +

CE

NP

-E in

tens

ityno

rmal

ized

to C

RES

T

Zwilch RNAi - +0.0

0.5

1.0

A

DN

AC

RE

ST

CE

NP

-EZw

ilch

0.0

0.5

1.0

Zwilc

h in

tens

ityno

rmal

ized

to C

RES

T

CENP-E RNAi - +

CENP-E RNAi- +

DN

AC

RE

ST

CE

NP

-EZw

ilch

CENP-E + CENP-F RNAi- +

0.0

0.5

1.0

Zwilc

h in

tens

ityno

rmal

ized

to C

RES

T

CENP-E RNAi+ CENP-F RNAi

- +

B D

CE

NP

-F

CENP-F RNAi- +

Zwilc

h in

tens

ityno

rmal

ized

to C

RES

T

CENP-F RNAi- +

GCENP-F RNAi- +

F

CE

NP

-E in

tens

ityno

rmal

ized

to C

RES

T

MA

D1

inte

nsity

norm

aliz

ed to

CR

EST

CENP-E RNAi- +

HCENP-E RNAi- +

E

CE

NP

-E

CE

NP

-F in

tens

ityno

rmal

ized

to C

RES

T

MA

D1

inte

nsity

norm

aliz

ed to

CR

EST



https://doi.org/10.1101/276204


DN

AC

RE

ST

CE

NP

-E

CENP-E RNAi- +

CENP-E (316 kDa)

Tubulin (50kDa)

100 %

50 %

25 %

5 % 100 %

mockCENP-E

RNAi

-260

-50

MW(kDa)

0.0

0.5

1.0

CE

NP

-E in

tens

ityno

rmal

ized

to C

RES

T

CENP-E RNAi - +

B

C

A

DN

AC

RE

ST

CE

NP

-F

- +

CENP-F (368 kDa)

Tubulin (50 kDa)

100 %

50 %25

%10

%10

0 %

-260

-50

MW(kDa)

0.0

0.5

1.0

CE

NP

-F in

tens

ityno

rmal

ized

to C

RES

T

CENP-F RNAi - +

E

F

D CENP-F RNAimock

CENP-FRNAi


DN

AC

RE

ST

Zwilc

hM

ad1

- +

Mad1 in Zwilch RNAi

Zwilch RNAi efficiency(Cenp-E exp)

Zwilch RNAi efficiency(Mad1 exp)

ZwilchRNAi

- +

Zwilch RNAi - +

Zwilch RNAi - +

0.0

0.5

1.0

0.0

0.5

1.0

0.0

0.5

1.0

0.0 0.1 0.2 0.30.00

0.01

0.02

0.03

0.04

Mad

1 in

tens

ity (A

U)

Zwilch intensity (AU)

R2: 0.8345y=0.08298*x+0.006827

Zwilch RNAi

Mad

1 in

tens

ityno

rmal

ized

to C

RES

TZw

ilch

inte

nsity

norm

aliz

ed to

CR

EST

Zwilc

h in

tens

ityno

rmal

ized

to C

RES

T

G H

I J



https://doi.org/10.1101/276204


Ciossani, Overlack et al. Figure 2S2D

NA

CR

ES

TZw

ilch

CE

NP

-E

DN

AC

RE

ST

Zwilc

hB

ubR

1

DN

AC

RE

ST

Zwilc

hC

EN

P-E

DN

AC

RE

ST

Zwilc

hB

ub1

DN

AC

RE

ST

CE

NP

-EC

EN

P-F

0.0

0.5

1.0

Cen

p-E/

-F in

tens

ityno

rmal

ized

to C

RES

T

CENP-E RNAi+ CENP-F RNAi

- + - +CENP-E CENP-F

CENP-E + CENP-F RNAi- +

A

Zwilch + Bub1 RNAi- + +

Zwilch + Bub1 RNAi- + +

Zwilch + BubR1 RNAi- +

Zwilch + BubR1 RNAi- +

D

B

E

C



https://doi.org/10.1101/276204


DNA CREST Zwilch Tubulin

Prometa-phase

Meta-phase

Prometa-phase

Meta-phase

Prometa-phase

Meta-phase

Prometa-phase

Meta-phase

moc

kC

EN

P-E

+ C

EN

P-F

RN

Ai

CE

NP

-E R

NA

iC

EN

P-F

RN

Ai


A

C

D

B



https://doi.org/10.1101/276204


DN

AC

RE

ST

Tubu

linC

EN

P-F

CENP-F RNAi - - + +

Metaphasealigned

Metaphase withmisalignment

Metaphasealigned

Metaphase withmisalignment

Metaph

ase

align

ed

Metaph

ase w

ith

misalig

nmen

t

mockCENP-F RNAi

mockCenp-F RNAi

Metaph

ase

align

ed

Metaph

ase w

ith

misalig

nmen

t

NO TREATMENT 10 uM MG-132 for 2 h

0.0

0.5

1.0

1.5

Dur

atio

n of

mito

sis (h

)

CENP-F RNAi - +

D


A

B C

0

20

40

60

80

100

perc

enta

ge o

f cel

ls

0

20

40

60

80

100

perc

enta

ge o

f cel

ls



https://doi.org/10.1101/276204


GFPCENP-E2070-C

GFPCENP-E2070-C

BubR1

Bub3

Elution volume (ml)

75

5037

25

100150

75

50

37

25

100150

7550

37

25

100150

BubR1

Bub3

0

10

20

30

40

1.0 1.5 2.0 2.5

BubR1KinD

BubR1KinD

Elution volume (ml)

75

5037

25

100150

755037

25

100150

7550

37

25

100150

0

10

20

30

40

1.0 1.5 2.0 2.5

C

DN

AC

RE

ST

CE

NP

-E

mock Bub1 BubR1 Bub1+BubR1

0.0

0.5

1.0

- +

Bub1

Bub3

Elution volume (ml)

755037

25

100150

75

50

37

25

100150

7550

37

25

100150

Bub1

Bub3

0

10

20

30

40

1.0 1.5 2.0 2.5

D


BGFPCENP-E2070-C +B1FL-B3GFPCENP-E2070-C

B1FL-B3

488

Superose 6 Increase 5/150280

Abso

rban

ce28

0/48

8 nm

(mAU

)

Abso

rban

ce28

0/48

8 nm

(mAU

)

Abso

rban

ce28

0/48

8 nm

(mAU

)GFPCENP-E2070-C +BR1KinD

GFPCENP-E2070-C

BR1KinD

488

Superose 6 Increase 5/150280GFPCENP-E2070-C +

BR1-B3GFPCENP-E2070-C

BR1-B3

488


CEN

P-E

inte

nsity

norm

aliz

ed to

CR

EST

Bub1RNAi

eGFP-CENP-E2070-C

eGFP-CENP-E2070-C

+ BubR1:Bub3

eGFP-CENP-E2070-C

MWtheo = 103 kDa(monomer)

BubR1-Bub3 (1:1)MWtheo= 182 kDa

RNAi

0 2 4 6 8 10 12 14sedimentation coefficient (S)

1.0

0.8

0.6

0.4

0.2

0.0

c(S)

(AU

/S)

A E

F G

eGFPCREST

eGFP

-C

EN

P-E

2070

-C

WT

eGFP

-C

EN

P-E

2070

-C

C26

97A

Con

trol

CRESTGFP

GFPCENP-E2070-C

GFPCENP-E2070-C GFPCENP-E2070-C

GFPCENP-E2070-C

0.0

0.5

1.0

BubR1RNAi

- +

CEN

P-E

inte

nsity

norm

aliz

ed to

CR

EST



https://doi.org/10.1101/276204


DNA CREST CENP-E P-T232 AurB

DM

SO

Hes

pera

din

500

nMH

espe

radi

n50

0 nM

DMSO50

0 nM

Hesp

0.0

0.5

1.0

CE

NP

-E in

tens

ityno

rmal

ized

to C

RES

T

CENP-E in HespD

MS

OR

ever

sine

500

nM

DNA CREST CENP-E Bub1 CENP-E in Rev

DMSO0.0

0.5

1.0


DM

SO

BI2

563

500

nM

DNA CREST CENP-E

DMSO50

0 nM

BI-256

3

0.0

0.5

1.0

1.5CENP-E in BI-2536D

DM

SO

BAY

-320

10 µ

M

DNA CREST CENP-E P-T121 H2A

DMSO10

µM

BAY-320

0.0

0.5

1.0

1.5

CENP-E in10 µM BAY-320

0.0

0.5

1.0

1.5

P-T121-H2A in10 µM BAY-320

pH2A

inte

nsity

norm

aliz

ed to

CR

EST

C

A

CE

NP

-E in

tens

ityno

rmal

ized

to C

RES

T

500 n

M

Revers

ine

B

CE

NP

-E in

tens

ityno

rmal

ized

to C

RES

T

DMSO10

µM

BAY-320

CE

NP

-E in

tens

ityno

rmal

ized

to C

RES

T



https://doi.org/10.1101/276204


mChCENP-F2688-C

mChCENP-F2688-C

Bub1

Bub3

0

10

20

30

40

1.0 1.5 2.0 2.5

mChCENP-F2688-C

+ B1FL-B3mChCENP-F2688-C

B1FL-B3

587

Abso

rban

ce28

0/58

7 nm

(mAU

)


Elution volume (ml)

755037

25

100150

7550

37

25

100150

7550

37

25

100150

Bub1

Bub3

mChCENP-F2688-C

mChCENP-F2688-C

Bub1KinD

Elution volume (ml)

755037

25

100150

7550

37

25

100150

7550

37

25

100150

Bub1KinD

0

10

20

30

40

1.0 1.5 2.0 2.5

Bub11-409

Bub3

Bub11-409

Bub3

Elution volume (ml)

755037

25

100150

7550

37

25

100150

7550

37

25

100150

0

10

20

30

40

1.0 1.5 2.0 2.5

mChCENP-F2688-C

mChCENP-F2688-C

BubR1

Bub3

BubR1

Bub3

Elution volume (ml)

75

5037

25

100150

7550

37

25

100150

7550

37

25

100150

0

10

20

30

40

1.0 1.5 2.0 2.5

DNA CREST CENP-F P-S10-H3

DM

SO

Hes

pera

din

500

nM

DMSOHes

p

(500 n

M)0.0

0.5

1.0

CEN

P-F

inte

nsity

norm

aliz

ed to

CR

EST

A B

DN

AC

RE

ST

CE

NP

-FG

FP

GFP- +

GFP-Bub1

+ +FL 1-788


0.0

0.5

1.0

Bub1 RNAi - + + +

GFPGFP

GFP-Bub

1FL

GFP-Bub

11-78

8

Bub1RNAi

Bub1RNAi

Cen

p-F

inte

nsity

norm

aliz

ed to

CR

EST

mChCENP-F2688-C

+ B11-409-B3mChCENP-F2688-C

B11-409-B3

587


Abso

rban

ce28

0/58

7 nm

(mAU

)

Abso

rban

ce28

0/58

7 nm

(mAU

)

Superose 6 Increase 5/150 Superose 6 Increase 5/150

Abso

rban

ce28

0/58

7 nm

(mAU

)

D E

F G

C

H

I

DN

AC

RE

ST

CE

NP

-FG

FP

mChCENP-F2688-C

+ B1KinD

mChCENP-F2688-C

B1KinD

587 280 mChCENP-F2688-C

+ BR1FL-B3mChCENP-F2688-C

BR1-B3FL

587 280

mChCENP-F2688-C

mChCENP-F2688-C



https://doi.org/10.1101/276204


DM

SOR

ever

sine

500

nMDNA CREST CENP-F Bub1

DMSO

500 n

M

Rev

0.0

0.5

1.0

CE

NP

-F in

tens

ityno

rmal

ized

to C

RE

ST

A


DM

SO

BAY

-320

10 µ

M

DNA CREST CENP-F

0.0

0.5

1.0

1.5C

DNA CREST CENP-F

DM

SOBA

Y-32

0 3

µM

DMSO3 µ

M

BAY-320

0.0

0.5

1.0

1.5

DM

SO

BI

500

nM

DNA CREST CENP-F Plk1

DMSO

500 n

M

BI-256

3

0.0

0.5

1.0

1.5

B

D

DM

SO

BAY

-320

10 µ

M

DNA CREST P-T121-H2A

DMSO10

µM

BAY-320

0.0

0.5

1.0

1.5

P-H

2A in

tens

ityno

rmal

ized

to C

RE

ST

E

DNA CREST P-T121-H2A

CE

NP

-F RN

Ai

-+

CE

NP

-F in

tens

ityno

rmal

ized

to C

RES

TC

EN

P-F

inte

nsity

norm

aliz

ed to

CR

EST

DMSO10

µM

BAY-320

CE

NP

-F in

tens

ityno

rmal

ized

to C

RES

T

F



https://doi.org/10.1101/276204



mCherry-CENP-F2688-C

50 nm

mCherrymCherryCREST

mCh

erry

CENP

-F26

88-C

WT

mCh

erry

CENP

-F26

88-C

C320

7Am

Che

rry

cont

rol

A B

MTKinetochorelocalisation

mCherry32102688

mChCENP-F2688-C

Bub1

Bub3

Elution volume (ml)

75

5037

25

100150

75

50

37

25

100150

7550

37

25

100150

Bub1

Bub3

0102030405060

1.0 1.5 2.0 2.50

102030405060

1.0 1.5 2.0 2.5

mChCENP-F2688-C/C2961S

Bub1

Bub3

Elution volume (ml)

75

5037

25

100150

75

50

37

25

100150

75

50

37

25

100150

Bub1

Bub3

D Superose 6 Increase 5/150

mChCENP-F2688-C/mChC2961S

+ B1FL-B3mChCENP-F2688-C/C2961S

B1FL-B3

587 280

Abs

orba

nce

280/

587

nm (m

AU

)


mChCENP-F2688-C/WT

+ B1FL-B3mChCENP-F2688-C/WT

B1FL-B3

587 280

Abs

orba

nce

280/

587

nm (m

AU

)

C

GFPCENP-E2070-C

GFPCENP-E2070-C


Elution volume (ml)

75

50

37

25

100150

7550

37

25

100150

7550

37

25

100150

0

10

20

30

40

1.0 1.5 2.0 2.5

mChCENP-F2688-C

mChCENP-F2688-CmChCENP-F2688-C

mChCENP-F2688-C/C2961S

E

Abs

orba

nce

280/

488/

587

nm (m

AU

)

mChCENP-F2688-C

+ GFPCENP-E2070-C

mChCENP-F2688-C

GFPCENP-E2070-C

488 280

CREST



https://doi.org/10.1101/276204



MWtheo= 43.5 kDaMWobs= 83.1 kDaFrict. ratio = 1.7



mCherry-CENP-F2866-2990 mCherry-CENP-F2922-2990 mCherry-CENP-F2950-2990



1.5

1.0

0.5

0.0

c(S)

(AU

/S)

2.0

1.5

1.0

0.5

0.0

c(S)

(AU

/S)


1.00.80.60.40.20.0

c(S)

(AU

/S) 1.2

1.41.6

Bub1KinD

Bub1KinD

mChCENP-F2866-2990 mChCENP-F2950-2990

mChCENP-F2950-2990Bub1KD

Elution volume (ml)

755037

25

100

Elution volume (ml) Elution volume (ml)

01020304050607080

0.8 1.0 1.2 1.4 1.6 1.80

1020304050607080

0.8 1.0 1.2 1.4 1.6 1.80

102030405060

1.0 1.2 1.4 1.6 1.8 2.0

20

755037

25

100

20

Bub1KD

755037

25

100

20

755037

25

100

20

755037

25

100

20

755037

25

100

20

755037

25

100

20

75503725

100

20

755037

25

100

20

Abso

rban

ce28

0/58

7 nm

(mAU

) mChCENP-F2866-2990

+ B1KinD

mChCENP-F2866-2990

B1KinD

587

Superdex 75 Increase 5/150

280Ab

sorb

ance

280/

587

nm (m

AU)

Abso

rban

ce28

0/58

7 nm

(mAU

)mChCENP-F2922-2990

+ B1KinD

mChCENP-F2922-2990

B1KinD

587


280mChCENP-F2950-2990

+ B1KinD

mChCENP-F9250-2990

B1KinD

587


280

Bub1KinD

Bub1KinD

mChCENP-F2866-2990

* * *mChCENP-F2922-2990

mChCENP-F2922-2990

A C E

B D F

mC

herr

yC

RE

ST

CR

ES

Tm

Che

rry

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

* * *



https://doi.org/10.1101/276204


Bub3

TPR TPR

Bub1kinase

BubR1pseudokinase

Knl1

Mps1

P

Bub3

CEN

P-F

CEN

P-E

?

Kinetochore




https://doi.org/10.1101/276204


Date post:	01-Oct-2020
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Kinetochore recruitment of CENP-F illustrates how paralog ...3 54 Early in mitosis, prior to end-on...

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