1
Kinetochore recruitment of CENP-F illustrates how paralog 1
divergence shapes kinetochore composition and function 2
3
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
7
(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
12
# Correspondence: [email protected] 13
* These authors contributed equally to this work 14
15
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
19
Short title: Mechanism of kinetochore recruitment of CENP-F 20
21
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2
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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21
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Yao, X., Anderson, K.L., and Cleveland, D.W. (1997). The microtubule-dependent 901motor centromere-associated protein E (CENP-E) is an integral component of 902kinetochore corona fibers that link centromeres to spindle microtubules. The Journal of 903cell biology 139, 435-447. 904
Yen, T.J., Compton, D.A., Wise, D., Zinkowski, R.P., Brinkley, B.R., Earnshaw, W.C., 905and Cleveland, D.W. (1991). CENP-E, a novel human centromere-associated protein 906required for progression from metaphase to anaphase. The EMBO journal 10, 1245-9071254. 908
Yen, T.J., Li, G., Schaar, B.T., Szilak, I., and Cleveland, D.W. (1992). CENP-E is a 909putative kinetochore motor that accumulates just before mitosis. Nature 359, 536-539. 910
Zhang, G., Lischetti, T., Hayward, D.G., and Nilsson, J. (2015). Distinct domains in 911Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint. Nature 912communications 6, 7162. 913
Zhu, X. (1999). Structural requirements and dynamics of mitosin-kinetochore interaction 914in M phase. Mol Cell Biol 19, 1016-1024. 915
Zhu, X., Chang, K.H., He, D., Mancini, M.A., Brinkley, W.R., and Lee, W.H. (1995a). 916The C terminus of mitosin is essential for its nuclear localization, 917centromere/kinetochore targeting, and dimerization. The Journal of biological chemistry 918270, 19545-19550. 919
Zhu, X., Mancini, M.A., Chang, K.H., Liu, C.Y., Chen, C.F., Shan, B., Jones, D., Yang-920Feng, T.L., and Lee, W.H. (1995b). Characterization of a novel 350-kilodalton nuclear 921
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29
phosphoprotein that is specifically involved in mitotic-phase progression. Mol Cell Biol 92215, 5017-5029. 923
924
925
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30
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
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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
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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
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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
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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
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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
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The copyright holder for this preprint (which wasthis version posted March 4, 2018. ; https://doi.org/10.1101/276204doi: bioRxiv preprint
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
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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
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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
Ciossani, Overlack et al. Figure 2
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
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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
Ciossani, Overlack et al. Figure 2S1
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
.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
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
.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
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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
Ciossani, Overlack et al. Figure 2S3
A
C
D
B
.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
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
Ciossani, Overlack et al. Figure 2S4
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
.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
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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
Ciossani, Overlack et al. Figure 3
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
Superose 6 Increase 5/150280
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
.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
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
Ciossani, Overlack et al. Figure 3S1
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
.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
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
)
Superose 6 Increase 5/150280
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
Ciossani, Overlack et al. Figure 4
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
Superose 6 Increase 5/150280
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
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The copyright holder for this preprint (which wasthis version posted March 4, 2018. ; https://doi.org/10.1101/276204doi: bioRxiv preprint
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
Ciossani, Overlack et al. Figure 4S1
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
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The copyright holder for this preprint (which wasthis version posted March 4, 2018. ; https://doi.org/10.1101/276204doi: bioRxiv preprint
Ciossani, Overlack et al. Figure 5
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
)
Superose 6 Increase 5/150
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
Superose 6 Increase 5/150
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
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Ciossani, Overlack et al. Figure 6
MWtheo= 43.5 kDaMWobs= 83.1 kDaFrict. ratio = 1.7
MWtheo= 36.8 kDaMWobs= 67.2 kDaFrict. ratio = 1.6
MWtheo= 33.6 kDaMWobs= 34.3 kDaFrict. ratio = 1.4
mCherry-CENP-F2866-2990 mCherry-CENP-F2922-2990 mCherry-CENP-F2950-2990
0 2 4 6 8 10 12 14sedimentation coefficient (S)
0 2 4 6 8 10 12 14sedimentation coefficient (S)
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)
0 2 4 6 8 10 12 14sedimentation coefficient (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
Superdex 75 Increase 5/150
280mChCENP-F2950-2990
+ B1KinD
mChCENP-F9250-2990
B1KinD
587
Superdex 75 Increase 5/150
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
* * *
.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
Bub3
TPR TPR
Bub1kinase
BubR1pseudokinase
Knl1
Mps1
P
Bub3
CEN
P-F
CEN
P-E
?
Kinetochore
Ciossani, Overlack et al. Figure 7
.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