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1 2 TRIM37 prevents formation of centriolar protein assemblies by regulating 3 Centrobin stability 4 5 Fernando R. Balestra1, 2 Benita Wolf3, 4, Andrés Domínguez-Calvo1, 2, Alizée Buff3, 6 Tessa Averink3, Marita Lipsanen-Nyman5, Coralie Busso3, Pablo Huertas1, 2, Rosa 7 M. Ríos2, Pierre Gönczy3 8 9 1 Departamento de Genética, Universidad de Sevilla, Sevilla, 41080, Spain 10 2 Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, 11 Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Sevilla, 41092, Spain 12 3 Swiss Institute for Experimental Cancer Research (ISREC), School of Life 13 Sciences, Swiss Federal Institute of Technology Lausanne (EPFL) 14 Lausanne, Switzerland 15 4 Present address: Department of Oncology, Lausanne University Hospital, and 16 Ludwig Institute for Cancer Research, University of Lausanne, 1066, Lausanne, 17 Switzerland. 18 5 Department of Endocrinology, Children's Hospital, University of Helsinki and 19 Helsinki University Hospital, FI-00290 Helsinki, Finland. 20 21 22 23 24 Correspondence: [email protected], [email protected] 25 26 27 28 Keywords: TRIM37 E3 ligase, Mulibrey nanism, centriole, microtubule 29 organizing center (MTOC), CLEM, U-ExM, Centrobin, PLK1, PLK4

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30 ABSTRACT 31 32 TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease 33 characterized by impaired growth and increased tumorigenesis, whose cellular 34 etiology is poorly understood. TRIM37 depletion from tissue culture cells results 35 in supernumerary foci bearing the centriolar protein Centrin. Here, we 36 characterized these centriolar protein assemblies (Cenpas) to uncover the 37 mechanism of action of TRIM37. We established that an atypical de novo 38 assembly pathway is notably involved in forming Cenpas, which can 39 nevertheless trigger further centriole assembly and act as MTOCs. We found also 40 that Cenpas are present and act similarly in Mulibrey patient cells. Through 41 correlative light electron microscopy, we uncovered that Cenpas correspond to 42 centriole related structures and elongated electron-dense structures with 43 stripes. Importantly, we established that TRIM37 regulates the stability and 44 solubility of the centriolar protein Centrobin. Our findings suggest that elongated 45 Centrobin assemblies are a major constituent of the striped electron dense 46 structures. Furthermore, we established that Cenpas formation upon TRIM37 47 depletion requires PLK4 activity, as well as two parallel pathways relying 48 respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 49 prevents the formation of Cenpas that would otherwise threaten genome 50 integrity, including possibly in Mulibrey patients. 51 52 53 54 55 56 57

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58 59 INTRODUCTION 60 61 Centrioles are tiny evolutionarily conserved cylindrical organelles 62 characterized by nine triplets of microtubules (MTs) arranged with a striking 9- 63 fold radial symmetry (reviewed in (Gönczy, 2012; Gönczy and Hatzopoulos, 64 2019)). In addition to MTs, centrioles contain multiple copies of tens of distinct 65 proteins that contribute to their assembly, structure and function (Andersen et 66 al., 2003; Jakobsen et al., 2011). Centrioles are essential for the formation of cilia 67 and also recruit pericentriolar material (PCM), including the MT nucleator γ- 68 tubulin ring complex, thus forming the centrosome of animal cells (reviewed in 69 (Bornens, 2012)). Probably because of such important roles, centriole number is 70 tightly regulated, with most cycling cells having two units at the cell cycle onset 71 and four units by the time of mitosis (reviewed in (Sullenberger et al., 2020)). 72 Alterations in centriole number control can have an adverse impact on cell 73 physiology and genome integrity. Thus, supernumerary centrioles lead to extra 74 cilia and centrosomes (Duensing et al., 2007; Habedanck et al., 2005; Mahjoub 75 and Stearns, 2012), which can be observed also in several human disease 76 conditions, including certain cancer types (reviewed in (Bettencourt-Dias et al., 77 2011; Chavali et al., 2014; Gönczy, 2015; Nigg and Holland, 2018; Nigg and Raff, 78 2009). Despite their importance, the mechanisms that prevent the formation of 79 excess centriolar structures remain incompletely understood. 80 The two centrioles present at the onset of the cell cycle differ in age: 81 whereas the older, mother, centriole is at least two cell generations old, the 82 younger, daughter, centriole was formed in the previous cell cycle. The mother 83 centriole bears distinctive distal and sub-distal appendages that the daughter 84 centriole acquires only later during the cell cycle (reviewed in (Sullenberger et 85 al., 2020)). In human cells, the proximal region of both mother and daughter 86 centrioles in the G1 phase of the cell cycle is encircled by a torus bearing the 87 interacting proteins CEP57/CEP63/CEP152 (Brown et al., 2013; Lukinavicius et 88 al., 2013) (reviewed in (Banterle and Gönczy, 2017)). The Polo-like-kinase PLK4 89 is recruited to this torus, where it focuses to a single location towards the G1/S 90 transition, owing notably to a protective interaction with its substrate STIL, thus 91 marking the site of procentriole assembly (Klebba et al., 2015; Moyer et al., 2015;

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92 Ohta et al., 2014) (reviewed in (Arquint and Nigg, 2016)). The onset of 93 procentriole assembly entails formation of a 9-fold radially symmetric cartwheel 94 thought to act as a scaffold for the organelle (reviewed in (Guichard et al., 2018; 95 Hirono, 2014)). The fundamental building block of the cartwheel is HsSAS-6, 96 which self-assembles in vitro into structures akin to those found in vivo 97 (Guichard et al., 2017; Kitagawa et al., 2011b; Strnad et al., 2007; van Breugel et 98 al., 2011). During S/G2, the emerging procentriole remains closely associated 99 with the resident centriole and elongates through the contribution notably of the 100 centriolar proteins CPAP/SAS-4, SPICE as well as C2CD3 (Balestra et al., 2013; 101 Comartin et al., 2013; Kohlmaier et al., 2009; Schmidt et al., 2009; Tang et al., 102 2009; Thauvin-Robinet et al., 2014). During mitosis, the procentriole disengages 103 from the resident centriole in a manner that requires the activity of the Polo-like- 104 kinase PLK1, with increased PLK1 levels during S/G2 leading to premature 105 centriole disengagement and centriole reduplication (Loncarek et al., 2010; Tsou 106 et al., 2009). Normally, disengagement during mitosis generates two centriolar 107 units that are then licensed to recruit PCM and trigger a new round of centriole 108 assembly in the following cell cycle. 109 Centrioles can also assemble independently of a resident centriole. Such 110 de novo assembly can occur in physiological conditions, for instance when the 111 protist Naegleria gruberii transitions from an acentriolar amoeboid life form to a 112 flagellated mode of locomotion (Fritz-Laylin et al., 2016; Fulton and Dingle, 113 1971). Likewise, centrioles assemble de novo at the blastocyst stage in rodent 114 embryos (Courtois et al., 2012). De novo assembly of centrioles can also be 115 triggered experimentally in human cells following removal of resident centrioles 116 through laser ablation or chronic treatment with the PLK4 inhibitor Centrinone 117 (Khodjakov et al., 2002; Wong et al., 2015). Therefore, in human cells, de novo 118 assembly is normally silenced by resident centrioles. In contrast to the situation 119 in physiological conditions, experimentally provoked de novo centriole assembly 120 in human cells is error prone and lacks number control (La Terra et al., 2005; 121 Wong et al., 2015). Moreover, de novo assembly of foci that contain some 122 centriolar proteins and which can function as MTOCs forms in human cells upon 123 depletion of the intrinsically disordered protein RMB14 or the Neuralized 124 Homology repeat containing protein Neurl4 (Li et al., 2012; Shiratsuchi et al.,

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125 2015). Such extra foci, although not bona fide centrioles as judged by electron- 126 microscopy, threaten cell physiology and could conceivably contribute to 127 disease. 128 TRIM37 is a RING-B-box-coiled-coil protein with E3 ubiquitin ligase 129 activity (Kallijarvi et al., 2002; Kallijarvi et al., 2005), which somehow prevents 130 the formation of foci bearing centriolar markers (Balestra et al., 2013). 131 Individuals with loss of function mutations in both alleles of TRIM37 are born 132 with a rare disorder known as Mulibrey nanism (Muscle-liver-brain-eye nanism). 133 The main features of this disorder are growth failure with prenatal onset, as well 134 as characteristic dysmorphic features and impairment in the organs that give 135 rise to the name of the condition (Avela et al., 2000). In addition, Mulibrey 136 patients have a high probability of developing certain tumor types (Karlberg et 137 al., 2009). Mice lacking Trim37 recapitulate several features of Mulibrey nanism, 138 including a higher propensity to form tumors (Kettunen et al., 2016). However, 139 the cellular etiology of Mulibrey nanism remains unclear, partially because of the 140 many roles assigned to this E3 ubiquitin ligase. In tissue culture cells, TRIM37 141 mono-ubiquitnates and thereby stabilizes PEX5, promoting peroxisomal function 142 (Wang et al., 2017). However, Trim37 knock out mice and mouse cell lines 143 depleted of Trim37 do not exhibit peroxisomal associated phenotype (Wang et 144 al., 2017), suggesting that the conserved pathological features exhibited also by 145 the mouse disease model must have a different cellular etiology. Furthermore, 146 the chromosomal region 17q23 where TRIM37 resides is amplified in ~40% of 147 breast cancers (Sinclair et al., 2003). TRIM37 mono-ubiquitinates histone H2A in 148 the MCF-7 breast cancer cell line dampening the expression of thousands of 149 genes, including tumors suppressors, thus offering a potential link between 150 TRIM37 overexpression and tumorigenesis (Bhatnagar et al., 2014). 151 Furthermore, TRIM37 overexpression has been linked to increased cell invasion 152 and metastasis in colorectal and hepatocellular carcinoma (Hu and Gan, 2017; 153 Jiang et al., 2015). Therefore, both the depletion and the excess of TRIM37 are 154 accompanied by detrimental consequences. 155 We previously performed a genome wide siRNA-based screen in human 156 cells to identify regulators of centriole assembly, using the number of foci 157 harboring the centriolar marker Centrin-1:GFP as a readout. In this screen, we

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158 identified TRIM37 as a potent negative regulator of Centrin-1:GFP foci number. 159 Our initial characterization of the TRIM37 depletion phenotype revealed that 160 ~50% of cells possess supernumerary foci harboring the centriolar proteins 161 Centrin and CP110, as well as instances of multipolar spindle assembly and 162 chromosome miss-segregation. Additionally, we found that inhibition of PLK1 163 partially suppressed supernumerary foci formation upon TRIM37 depletion, 164 leading to the suggestion that such foci occur through centriole reduplication 165 (Balestra et al., 2013), although the fact that suppression was partial suggested 166 that an additional explanation was to be found. Here, we set out to further 167 explore the nature of such supernumerary foci to uncover the mechanism of 168 action of TRIM37, and perhaps thereby also provide novel insights into Mulibrey 169 nanism. 170 171

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172 RESULTS 173 174 TRIM37 prevents formation of centriolar protein assemblies (Cenpas) 175 To further decipher the origin of supernumerary foci containing Centrin and 176 CP110 following TRIM37 depletion, we investigated where in the cell they first 177 appeared. We reasoned that appearance of supernumerary foci close to resident 178 centrioles could indicate centriole reduplication, whereby premature 179 disengagement would license resident centrioles and procentrioles to 180 prematurely seed centriole assembly. By contrast, appearance of supernumerary 181 foci away from resident centrioles would suggest some type of de novo process. 182 We performed live imaging of HeLa cells expressing Centrin-1:GFP (referred to 183 as HC1 cells hereafter) and depleted of TRIM37 by siRNAs. As shown in Figure 184 1A, we found that extra Centrin-1:GFP foci can appear in the vicinity of resident 185 centrioles (yellow arrows, 8/13 foci), but also far from them (orange arrows, 186 5/13 foci). These results suggest that extra Centrin-1:GFP foci upon TRIM37 187 depletion may form both through centriole reduplication and some type of de 188 novo process. 189 To further investigate this question, we analyzed fixed S/G2 HC1 cells 190 with antibodies against GFP to monitor Centrin-1:GFP foci, as well as against 191 CEP63 to mark the proximal region of resident centrioles and HsSAS-6 to mark 192 procentrioles. As expected, we found that control cells harbored four Centrin- 193 1:GFP foci, two of which were CEP63 positive and two of which were HsSAS-6 194 positive (Fig. 1B). Strikingly, in cells depleted of TRIM37, we found that in 195 addition to the normal four Centrin-1:GFP foci accompanied by two Cep63 foci 196 and two HsSAS-6 foci, ~90% of extra Centrin-1:GFP foci did not harbor CEP63 or 197 HsSAS-6 (Fig. 1B, 1C). For comparison, we likewise analyzed cells arrested in G2 198 following treatment with the CDK1 inhibitor RO3306, which induces PLK1- 199 dependent centriole reduplication (Loncarek et al., 2010). In this case, >90% of 200 extra Centrin-1:GFP foci harbored CEP63 and/or HsSAS-6 (Fig. 1B, 1C), in 201 contrast to the situation upon TRIM37 depletion, further indicating that TRIM37 202 does not act solely to prevent centriole reduplication. 203 Overall, we conclude that TRIM37 depletion results in extra Centrin- 204 1:GFP foci both near and far from resident centrioles, suggestive of centriole

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205 reduplication happening together with some de novo process. Moreover, we find 206 that such foci harbor some centriolar proteins but usually not others. We will 207 hence refer hereafter to these entities as Centriolar protein assemblies, or 208 Cenpas in short. 209 210 TRIM37 regulates Cenpas formation from outside the nucleus and localizes to 211 centrosomes 212 TRIM37 can regulate transcription through nuclear association with the 213 polycomb repressive complex 2 (PRC2) (Bhatnagar et al., 2014). To explore 214 whether TRIM37 may function as a transcriptional regulator in preventing 215 Cenpas formation, we addressed whether rescue of the TRIM37 depletion 216 phenotype depended on the presence of the protein in the nucleus. We 217 generated a version of TRIM37 forced to exit the nucleus via fusion to a nuclear 218 export signal (NES), finding that both TRIM37:GFP and TRIM37:NES:GFP equally 219 rescued the TRIM37 depletion phenotype (Fig. 1D, 1E). This indicates that 220 TRIM37 acts outside the nucleus to prevent Cenpas formation. 221 Given the TRIM37 depletion phenotype, we explored whether the protein 222 localizes to centrioles. Since antibodies did not prove suitable to address this 223 question (Balestra et al., 2013; Meitinger et al., 2016), we instead expressed 224 TRIM37:GFP, finding it to be present weakly in the nucleus and more so in the 225 cytoplasm (Fig. S1A, S1B). Intriguingly, in some cells, TRIM37:GFP also localized 226 to centrosomes marked by γ-tubulin (Fig. S1A, S1B). To investigate whether this 227 might reflect a cell cycle restricted distribution, TRIM37:GFP expressing cells 228 were probed with antibodies against GFP and Centrobin, which localizes 229 preferentially to the resident daughter centriole and to procentrioles (Zou et al., 230 2005). Therefore, G1 cells bear a single Centrobin focus while S/G2 cells bear 2 231 or 3 (Fig. S1C), which enabled us to establish that whereas only ~10% of G1 cells 232 harbored centrosomal TRIM37:GFP, ~60% of S/G2 cells did so (Fig. S1D). We 233 also localized the fusion protein with respect to CEP63, Centrin-2 and the distal 234 appendage protein CEP164, finding that TRIM37:GFP partially overlapped with 235 CEP164 (Fig. S1E, S1F). Overall, we conclude that TRIM37 localizes to the distal 236 part of centrioles, and it will be interesting to investigate in the future whether

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237 TRIM37 acts from this location to prevent the formation of at least some Cenpas, 238 perhaps those in the vicinity of resident centrioles. 239 240 Cenpas can act as MTOCs, are present in Mulibrey patient cells and trigger new 241 rounds of centriolar assembly 242 TRIM37 depleted cells exhibit an increased incidence of multipolar 243 spindles and chromosome miss-segregation (Balestra et al., 2013), suggesting 244 that Cenpas can nucleate microtubules and serve as extra microtubule 245 organizing centers (MTOCs). To thoroughly test this possibility, we performed 246 microtubule depolymerization-regrowth experiments. We found that whereas 247 most control mitotic cells harbored two MTOCs, TRIM37 depletion resulted in an 248 increased frequency of cells with more than two MTOCs, which often differed in 249 size (Fig. 2A, Fig. S2A, Fig.2B). In addition, we found that ~40% of Cenpas did not 250 nucleate microtubules, indicative of some composition heterogeneity (Fig. 2A, 251 siTRIM37, inset 1). We conclude that microtubules nucleated from Cenpas 252 contribute to the defective spindle assembly and chromosome miss-segregation 253 phenotype of TRIM37 depleted cells. 254 To further explore the importance of Cenpas, we addressed whether they 255 are also present in Mulibrey patient cells. Using healthy donor fibroblasts as 256 controls, we analyzed fibroblasts derived from two patients bearing the Finnish 257 founder mutation, the most frequent TRIM37 disease alteration, which results in 258 a frame shift of the coding sequence generating a premature stop codon (Avela et 259 al., 2000). As shown in Figure 2C, Western blot analysis showed essentially no 260 detectable TRIM37 protein in patient cells. We immunostained control and 261 patients fibroblast with antibodies against Centrin-2 to monitor the presence of 262 Cenpas, as well as against γ-tubulin to probe their ability to recruit PCM and, 263 thereby, to nucleate microtubules. Echoing the results in tissue culture cells 264 depleted of TRIM37, we found that patient cells in mitosis harbored 265 supernumerary Centrin-2 foci, some of which were positive for γ-tubulin (Fig. 266 2D, 2E). Patient cells also exhibited evidence of chromosome miss-segregation, 267 as would be expected from multipolar spindle assembly (Fig. 2D). We conclude 268 that Cenpas are present and active also in Mulibrey patient cells.

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269 We set out to address whether Cenpas in tissue culture cells are also 270 active in triggering further rounds of centriole assembly, potentially in a 271 subsequent cell cycle to the one in which they formed. To this end, we 272 transfected cells with TRIM37 siRNAs and monitored the presence of Cenpas 24, 273 48 and 72 hours thereafter using antibodies against Centrin-2 and CP110. 274 Control cells harbored two individual Centrin-2/CP110 foci in G1 and two pairs 275 of such foci in S/G2, corresponding to two pairs of resident centriole plus 276 procentriole (Fig. S2B). Upon TRIM37 depletion, we found that supernumerary 277 Centrin-2/CP110 foci appeared principally as individual units at the 24 hours 278 time point, but that pairs of foci became more frequent at the 48 and 72 hours 279 time points (Fig. 2F, 2G). We conclude that Cenpas can trigger further rounds of 280 centriole assembly. 281 282 Ultra expansion microscopy and electron microscopy reveal aberrant centriole- 283 related structures upon TRIM37 depletion 284 We set out to address whether Cenpas exhibit further hallmarks of 285 centrioles. We thus tested whether Cenpas harbor microtubules characteristic of 286 centrioles by staining cells depleted of TRIM37 with antibodies against 287 acetylated tubulin, a signature modification of centriolar microtubules, finding 288 that ~23% such cells possessed extra acetylated tubulin foci (Fig. 3A, 3B). To 289 examine this feature at higher resolution, we analyzed cells using ultrastructure 290 expansion microscopy (U-ExM) coupled to confocal imaging (Gambarotto et al., 291 2019). RPE-1 cells expressing Centrin1:GFP were immunostained for GFP to 292 identify Cenpas, for CEP152 to mark mature centrioles and for acetylated 293 tubulin. Control cells contained two mature centrioles positive for all three 294 markers (Fig. 3C). We found that some Cenpas formed upon TRIM37 depletion 295 harbored merely Centrin1:GFP, but neither acetylated tubulin or CEP152 (Fig. 296 3D-3F, yellow arrows). By contrast, other Cenpas were positive for all three 297 markers (Fig. 3E-3G), with the acetylated tubulin signal being smaller than 298 normal in some cases (Fig. 3E, 3F, white arrows). Moreover, some Cenpas 299 appeared to have matured into entities with regular looking acetylated tubulin 300 and CEP152 signals (Fig. 3G). Together, these findings support the notion that 301 Cenpas are heterogeneous in nature with partially overlapping composition.

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302 To uncover the ultrastructure of Cenpas, we conducted correlative light 303 and electron microscopy (CLEM). Using fluorescence microscopy, we screened 304 HeLa and RPE-1 cells expressing Centrin-1:GFP depleted of TRIM37 to identify 305 Cenpas, using a gridded coverslip to acquire information regarding GFP foci 306 position, before proceeding with serial section transmission electron microscopy 307 (TEM). In addition to control cells (Fig. S3A-S3B), we analyzed 8 cells depleted of 308 TRIM37 (Fig. 3H, Fig. S3C-F). From a total of 47 Centrin-1:GFP foci observed by 309 light microscopy in TRIM37 depleted cells, serial section TEM analysis 310 established that in addition to those corresponding to normal looking resident 311 centrioles or procentrioles, 20 corresponded to unusual structures described 312 hereafter (Fig. S3F). We found the expected number of resident centrioles (15 313 found/16 expected, see Fig. S3F; Fig. 3I, 3J), as well as two extra centriole-like 314 structures in one cell (Fig. 3M; Fig. S3D). Eight of the other unusual structures 315 were centriole-related electron-dense assemblies that harbored microtubules 316 but only partially resembled centrioles (Fig. 3K, 3N; Fig. S3C, S3D). Strikingly, the 317 remaining 12 other unusual structures were elongated electron-dense striped 318 entities, hereafter referred as “tiger" structures (Fig. 3L, 3O; Fig. S3C, S3E). We 319 noted also that an individual tiger structure sometimes correlated with more 320 than one Centrin-1:GFP focus (Fig. S3E). Overall, we conclude that Cenpas 321 forming upon TRIM37 depletion are heterogeneous in nature, only sometimes 322 bearing resemblance to centrioles, perhaps reflecting different pathways or 323 steps in their assembly. 324 325 TRIM37 depletion triggers formation of elongated Centrobin assemblies 326 Because TRIM37 is an E3 ligase, the activity of which is important for preventing 327 Cenpas formation (Balestra et al., 2013), we reasoned that a protein implicated 328 in centriole assembly might accumulate in an aberrant manner upon TRIM37 329 depletion, causing the observed phenotype. Therefore, we conducted a small 330 screen by immunostaining cells depleted of TRIM37 with antibodies against >20 331 centriolar and centrosomal proteins (Fig. S4A and data not shown). This analysis 332 revealed that Centrobin, which normally localizes tightly to the daughter 333 centriole and to procentrioles (Zou et al., 2005), is present in striking elongated 334 cytoplasmic assemblies upon TRIM37 depletion (Fig. 4A, 4B). We found that

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335 ~80% of TRIM37 depleted cells bear usually one or two such Centrobin 336 assemblies (Fig. S4B, S4C). Furthermore, SPICE, which is involved in centriole 337 biogenesis, was also present in Centrobin assemblies upon TRIM37 depletion, 338 even though SPICE was not needed for their formation (Fig. S4D, S4E). 339 Remarkably, all cells with Cenpas were positive for Centrobin assemblies 340 (n=150) with Cenpas often colocalizing with them (Fig. 4A, 4B). In summary,

341 aberrant Centrobin assemblies are invariably present in cells with Cenpas and 342 are often associated with them. 343 How could TRIM37 regulate Centrobin? Performing real time quantitative 344 PCR experiments showed a mere slight diminution in Centrobin mRNA levels 345 upon TRIM37 depletion (Fig. S4F), suggesting that regulation is not at the 346 transcriptional level. By contrast, Western blot analysis uncovered that 347 Centrobin protein levels were increased upon TRIM37 depletion (Fig. 4C). Given 348 the elongated Centrobin assemblies identified by immunostaining, we speculated 349 that the overall increase in Centrobin protein level might reflect an accumulation 350 into such structures, potentially in an insoluble form. Accordingly, fractionating 351 cell lysates into soluble and insoluble fractions, we found that the increase in 352 Centrobin protein levels was most pronounced in the latter (Fig. 4D). We noted 353 also that the insoluble pool of Centrobin migrated slower in the gel upon TRIM37 354 depletion, suggesting that TRIM37 not only restricts Centrobin levels, but also 355 regulates its posttranslational state in some manner. 356 Since TRIM37 is an E3 ubiquitin ligase, we reasoned that its activity might 357 modulate Centrobin protein degradation and, thereby, stability. Therefore, we 358 assayed the stability of the Centrobin protein pool over time in the presence of 359 the translation inhibitor Cycloheximide. As reported in Figure 4E and 4F, we 360 found that TRIM37 depletion significantly increased Centrobin protein stability. 361 One possibility would be that TRIM37 ubiquitinates Centrobin, thus targeting it 362 for degradation, such that increased Centrobin levels upon TRIM37 depletion 363 would trigger formation of Centrobin assemblies and Cenpas. However, although 364 Centrobin overexpression generates aggregates (Jeong et al., 2007), we found 365 that such aggregates did not resemble the elongated Centrobin assemblies nor 366 did they trigger Cenpas formation (Fig. S4G). In addition, TRIM37 367 overexpression did not alter Centrobin centrosomal distribution (Fig. S4H).

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368 Moreover, no evidence for TRIM37 mediated Centrobin ubiquitination was found 369 in cell free assays (data not show), such that the detailed mechanisms of 370 Centrobin modulation by TRIM37 remain to be deciphered. Regardless, we 371 conclude that TRIM37 normally regulates Centrobin stability, preventing the 372 protein from forming elongated assemblies invariably present in cells with 373 Cenpas. 374 375 Centrobin assemblies may serve as platforms for Cenpas formation 376 We set out to further characterize the elongated Centrobin assemblies formed 377 upon TRIM37 depletion and assay their role in Cenpas generation. We used U- 378 ExM coupled to STED super-resolution microscopy to analyze the distribution of 379 Centrobin upon TRIM37 depletion at higher resolution. We immunostained RPE- 380 1 cells expressing Centrin-1:GFP with antibodies against GFP, CEP152 and 381 Centrobin. In control conditions, centrioles viewed in cross section exhibited a 382 clear localization of Centrobin between the outer CEP152 and the inner Centrin- 383 1:GFP signals (Fig. 5A). Cells depleted of TRIM37 exhibited analogous 384 distributions at resident centrioles (Fig. 5A), but also harbored elongated 385 Centrobin assemblies often abutting Centrin-1:GFP foci (Fig. 5A, arrows). 386 Strikingly, the superior resolution afforded by U-ExM coupled to STED revealed 387 that such Centrobin assemblies are striated (Fig. 5A). Suggestively, the inter- 388 stripe distances of these Centrobin assemblies were analogous to those of the 389 tiger structures unveiled through CLEM (Fig. 5B). In summary, U-ExM analysis 390 strongly suggests that Centrobin is a constituent of the electron-dense tiger 391 structures observed by TEM upon TRIM37 depletion, and raises the possibility 392 that such structures serve as platforms for Cenpas formation. 393 To investigate the potential role of Centrobin in Cenpas formation, we 394 tested whether Centrobin depletion reduces Cenpas numbers in cells depleted of 395 TRIM37. Although Centrobin depletion was reported initially to impair centriole 396 assembly in HeLa cells (Zou et al., 2005), more recent work with Centrobin 397 knock out cells (Centrobin-ko) demonstrates that the protein is dispensable for 398 this process in RPE-1 cells (Ogungbenro et al., 2018). In our hands, siRNA- 399 mediated depletion of Centrobin did not impact centriole assembly either in 400 HeLa Kyoto cells, despite near-complete protein depletion (Fig. S5A-C). As

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401 anticipated, Centrobin assemblies disappeared entirely from cells doubly 402 depleted of Centrobin and TRIM37 (Fig. 5C). Importantly, we found that Cenpas 403 number was significantly lowered in such doubly depleted cells compared to 404 cells depleted of TRIM37 alone (Fig. 5D). Interestingly, however, even if 405 Centrobin depletion was complete as judged by Western blot analysis (Fig. S5C), 406 Cenpas formation upon TRIM37 depletion was only partially prevented by 407 Centrobin siRNA treatment (Fig. 5D). To test whether this might have been due 408 to residual Centrobin or TRIM37 in the double siRNA depletion setting, we 409 performed a similar experiment with RPE-1 Centrobin-ko cells (Ogungbenro et 410 al., 2018), reaching similar conclusions (Fig. 5E, 5F, Fig. S5D). Together, these 411 results support the view that upon TRIM37 depletion Centrobin assemblies act 412 as platform seeding the formation of some, but not all, Cenpas. 413 414 Centrobin and PLK1 together promote Cenpas assembly upon TRIM37 depletion 415 To further understand the mechanisms of Cenpas formation upon 416 TRIM37 depletion, we tested if select centriolar proteins that are critical for 417 canonical centriole duplication were also needed for Cenpas generation. To test 418 the role of PLK4, HeLa cells were grown in the presence of Centrinone for 5 days 419 and then depleted of TRIM37 for 3 days in the continued presence of Centrinone. 420 We found that Cenpas did not form under these conditions, demonstrating an 421 essential role for PLK4 kinase activity (Fig. 6A, 6B). We also tested the 422 requirement for HsSAS-6, STIL, CPAP and SPICE. As anticipated, single depletion 423 of these components resulted in decreased centriole number (Fig. 6A). However, 424 depletion of STIL, CPAP or SPICE did not dramatically modify the number of 425 Cenpas upon TRIM37 depletion (Fig. 6A). By contrast, HsSAS-6 depletion greatly 426 reduced Cenpas number (Fig. 6A). To further explore the impact of HsSAS-6, we 427 depleted TRIM37 from RPE-1 p53-/- HsSAS-6 knock out cells (HsSAS-6-ko) 428 (Wang et al., 2015). Although HsSAS-6-ko cells invariably lacked centrioles (Fig. 429 6A, 6B), some Cenpas nevertheless formed upon TRIM37 depletion, although to a 430 lesser extent than following depletion of TRIM37 alone (Fig. 6A, 6B). Moreover, 431 we found that elongated Centrobin assemblies were generated unabated upon 432 TRIM37 depletion in cells treated with Centrinone or lacking HsSAS-6 (Fig. 6C;

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433 Fig. S6). We conclude that PLK4 and HsSAS-6 act downstream of Centrobin in the 434 pathways leading to Cenpas formation upon TRIM37 depletion. 435 To further uncover requirements for Cenpas generation, considering that 436 PLK1 contributes only partially to their formation (Balestra et al., 2013), and that 437 we found here the same to be true for Centrobin, we set out to investigate 438 whether the combined removal of PLK1 and Centrobin may fully prevent Cenpas 439 generation. To avoid the negative impact of PLK1 inhibition on cell cycle 440 progression, we performed these experiments in synchronized cells depleted of 441 TRIM37, and monitor Cenpas appearance during G2 after release from an S 442 phase arrest. These cells were also subjected to Centrobin depletion and/or BI- 443 2536 treatment to inhibit PLK1. Importantly, we found that simultaneous 444 Centrobin depletion and PLK1 inhibition completely prevented Cenpas 445 formation (Fig. 6E), indicating that PLK1 and Centrobin act in parallel to promote 446 Cenpas formation upon TRIM37 depletion. 447 Further evidence supporting the existence of two parallel pathways 448 towards Cenpas generation was obtained by examining the distribution of 449 HsSAS-6 in cells depleted of TRIM37 plus either PLK1 or Centrobin. Indeed, we 450 found that Cenpas generated upon combined TRIM37 depletion and PLK1 451 inhibition, which thus rely strictly on Centrobin, rarely harbored HsSAS-6 (Fig. 452 6F, 6G). By contrast, Cenpas generated upon double depletion of TRIM37 and 453 Centrobin, which thus rely strictly on PKL1, frequently harbored HsSAS-6 (Fig. 454 6F, 6G). Taken together, our findings indicate that two pathways are triggered 455 when TRIM37 is lacking: one that relies on Centrobin assemblies that act as a 456 platform to assemble Cenpas, which at the least is initially independent of 457 HsSAS-6, and another one mediated by PLK1 that operates through HsSAS-6 458 recruitment (Fig. 6G, see discussion). 459

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460 DISCUSSION 461 462 Centriole number control is critical for proper cell physiology, including 463 genome integrity. Assemblies of centriolar proteins that can recruit PCM and 464 nucleate microtubules despite not being bona fide centrioles must likewise be 465 kept in check. Here, we identify the TRIM37 E3 ligase, which is mutated in 466 Mulibrey nanism, as a critical component that prevents the formation of 467 centriolar protein assemblies (Cenpas) through two parallel pathways relying on 468 PLK1 and Centrobin. Of particular interest, we uncover that TRIM37 regulates 469 the stability of Centrobin, which upon TRIM37 depletion forms striated 470 structures that we propose serve as platforms for Cenpas generation. 471 472 Two pathways together result in Cenpas upon TRIM37 depletion 473 What are the mechanisms leading to Cenpas formation upon TRIM37 depletion? 474 We previously hypothesized that TRIM37 could act by restricting centriole 475 reduplication in G2, since PLK1 inhibition in TRIM37 depleted cells reduced 476 Cenpas formation (Balestra et al., 2013). However, although blocking PLK1 477 activity in TRIM37 depleted cells reduced Cenpas numbers, some remained 478 despite such inhibition (Balestra et al., 2013). Moreover, while Cenpas form upon 479 TRIM37 depletion as early as 4 h after the G1/S transition (Balestra et al., 2013), 480 PLK1-mediated centriole reduplication occurs only 24 h after G2 arrest 481 (Loncarek et al., 2010). It may even be that the role exerted by PLK1 following 482 TRIM37 depletion is not linked to its known function in regulating licensing. 483 Regardless, we obtained further evidence here that Cenpas do not form solely 484 through a reduplication mechanism. First, some Cenpas appear away from 485 resident centrioles. Second, most Cenpas do not harbor the procentriolar protein 486 HsSAS-6, at the least initially, which is in contrast to the situation during 487 centriole reduplication during G2 arrest. Furthermore, analysis with CLEM 488 revealed that Cenpas are usually either centriole-related structures or novel 489 striped electron-dense structures. Together, these findings indicate that Cenpas 490 do not form solely through centriole reduplication, but also through an 491 alternative novel de novo pathway. 492 Our findings indicate that this alternative pathway relies on Centrobin: 493 whereas the sole removal of Centrobin also merely decreases Cenpas number,

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494 the joint removal of PLK1 and Centrobin entirely prevent their generation (Fig. 495 6E). 496 The heterogeneity in Cenpas ultrastructure uncovered by CLEM might 497 also reflect the co-existence of these independent assembly pathways. Such 498 heterogeneity may reflect in addition a step-wise nature of the de novo 499 generation process. This possibility is in line with the fact that more extra HsSAS- 500 6 foci are present 72 hours after transfection with TRIM37 siRNAs (Balestra et 501 al., 2013), compared to the 48 hours post-transfection analyzed here (Fig. 1C). 502 Therefore, HsSAS-6 might not be present or required for the onset of de novo 503 Cenpas formatoin, but could contribute later to their consolidation. In line with 504 this view, HsSAS-6-k-o cells depleted of TRIM37 can assemble some Cenpas, 505 perhaps more rudimentary ones. Interestingly in addition, this observation 506 further suggests that de novo Cenpas generation upon TRIM37 depletion must in 507 some way differ from the classical de novo centriole assembly, which is fully 508 reliant on HsSAS-6 (Wang et al., 2015). 509 We found that one protein that is essential for forming all Cenpas upon 510 TRIM37 depletion is PLK4, which is also required for centriole reduplication and 511 de novo centriole assembly following Centrinone treatment (Habedanck et al., 512 2005; Wong et al., 2015). How could PLK4 be required for Cenpas generation 513 stemming from the Centrobin assemblies formed upon TRIM37 depletion? PLK4 514 condensates forming away from resident centrioles have been observed in RPE- 515 1 TRIM37 knock out (TRIM37-ko) cells (Meitinger et al., 2016). However, we did 516 not detect such PLK4 localization, perhaps reflecting differences between 517 chronic versus acute TRIM37 depletion. Another difference potentially related to 518 distinct depletion regimes is that upon Centrinone treatment, TRIM37-ko cells 519 form centrosome-like structures harboring notably PLK4 and HsSAS-6, and 520 which recruit PCM components, behaving as MTOCs (Meitinger et al., 2016). 521 This is in contrast to our findings whereby no Cenpas forms upon treatment with 522 TRIM37 siRNAs and Centrinone. Regardless, it is interesting to note that in 523 Xenopus extracts, PLK4 self-assembles into condensates that recruit γ-tubulin 524 and behave as MTOCs (Montenegro Gouveia et al., 2018), raising the possibility 525 that Centrobin assemblies may serve as platforms to recruit such condensates. 526

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527 Centrobin as a TRIM37 target 528 What are the targets of TRIM37 that are relevant for restricting Cenpas 529 formation? Our work suggests that a critical target is Centrobin, since upon 530 TRIM37 depletion Centrobin protein levels increase and elongated Centrobin 531 assemblies form in the cytoplasm. U-ExM coupled to STED super-resolution 532 microscopy reveals that these Centrobin assemblies have a striped pattern akin 533 to the structures uncovered by CLEM, and are often intimately linked with 534 Cenpas. Given the cytoplasmic localization of these assemblies, this role of 535 TRIM37 is likely to be exerted by the cytoplasmic protein pool rather than the 536 centrosomal or the nuclear ones. Centrobin contributes to several aspects of 537 centriole assembly and growth, as well as ciliogenesis (Gudi et al., 2011; 538 Ogungbenro et al., 2018; Zou et al., 2005). These functions might be linked to 539 Centrobin’s ability to stabilize and promote microtubule nucleation (Gudi et al., 540 2011; Jeong et al., 2007; Shin et al., 2015). Future work will undoubtedly clarify 541 the molecular connection between TRIM37 and Centrobin. Because Centrobin 542 depletion does not fully prevent Cenpas formation upon TRIM37 depletion, other 543 TRIM37 targets must be invoked, and PLK1 or a protein regulating its activity, is 544 an attractive possibility in this respect. 545 546 Cenpas form through different routes but similarly threaten cell physiology 547 Although with different molecular origins, centriolar protein assemblies 548 have been reported in other contexts (Li et al., 2012; Shiratsuchi et al., 2015). 549 Thus, the centriolar protein Neurl4 interacts with CP110 and promotes its 550 destabilization, such that Neurl4 depletion results in increased CP110 protein 551 levels and formation of ectopic MTOCs (Li et al., 2012). Likewise, depletion of 552 RMB14 triggers the formation of centriolar protein complexes that do not 553 initially require HsSAS-6 for their assembly. RBM14 normally limits formation of 554 the STIL/CPAP complex, which upon RMB14 depletion triggers aberrant 555 centriolar protein complex formation (Shiratsuchi et al., 2015). In both cases, 556 however, Centrobin distribution was inspected and no elongated structures as 557 the ones reported here were observed (Li et al., 2012; Shiratsuchi et al., 2015), 558 suggesting different assembly routes. Although these previously reported 559 centriolar protein assemblies and the ones analyzed here do not share a clear

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560 common molecular composition or assembly pathway, we propose to group 561 them jointly under the acronym Cenpas, reflecting the fact that they similarly 562 form following a de novo process and entail centriole-related structures that 563 behave as active MTOCs. 564 To our knowledge, our work is the first example in which Cenpas have 565 been reported in a human genetic disorder. The fact that Cenpas are present in 566 Mulibrey derived patient cells raises the possibility that some disease features 567 could be due to Cenpas formation, perhaps owing to the extra MTOCs and 568 resulting chromosome miss-segregation phenotype. As one of the characteristics 569 of Mulibrey nanism is the propensity to develop tumors, we speculate that the 570 presence of Cenpas could contribute to this phenotype since extra centrioles can 571 promote tumorigenesis (Ganem et al., 2009; Godinho et al., 2014; Levine et al., 572 2017; Sercin et al., 2016). We further speculate that some of the instances in 573 which extra centriole numbers are observed in solid and hematological tumors 574 may in reality correspond to Cenpas. 575 576

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577 ACKNOWLEDGMENTS 578 579 We thank Graham Knott and Marie Croisier (BioEM platform of the School of Life 580 Sciences, EPFL) for assistance with CLEM and TEM, Isabelle Flückiger for 581 technical support, as well as Niccolò Banterle for advice with U-ExM. Anna-Elina 582 Lehesjoki (Department of Medical and Clinical Genetics, University of Helsinki, 583 Finland) is acknowledged for her contribution towards securing patient and 584 control fibroblast, Daniel Gerlich (Vienna BioCenter, Austria), Ciaran Morrison 585 (Centre for Chromosome Biology, Galway, Ireland), Bryan Tsou (Memorial Sloan 586 Kettering Cancer Center, New York, US) and George Hatzopoulos (EPFL, 587 Lausanne, Switzerland) for cell lines, Michel Bornens (Institut Curie, Paris, 588 France), Andrew Holland (Johns Hopkins University School of Medicine, 589 Baltimore, USA) and Laurence Pelletier (Lunenfeld-Tanenbaum Research 590 Institute, Toronto, Canada) for antibodies. We are also grateful to Niccolò 591 Banterle and Fernando Monje Casas for critical reading of the manuscript. This 592 work has been supported in part by the Swiss Cancer league (KFS-3388-02- 593 2014, to P.G.). A.B. was supported by the National Centre for Competence in 594 Research (NCCR) Chemical Biology, funded by the Swiss National Science 595 Foundation. FRB thanks PG for having hosted and supported him at the 596 beginning of the project. F.R.B. was funded by a Marie Curie IEF Postdoctoral 597 Fellowship (PIEF_GA-2013-629414; CSIC-Cabimer, Seville, Spain) and by the 598 University of Seville through a postdoctoral contract of the V PPIT-US (US- 599 Cabimer, Seville, Spain). CABIMER is supported by the regional government of 600 Andalucia (Junta de Andalucía). 601 602 603 604 605 606 607 608 609

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610 611 FIGURE LEGENDS 612 613 Figure 1. Supernumerary centriolar protein assemblies (Cenpas) form 614 upon TRIM37 depletion 615 A. Relevant images from wide-field time-lapse recordings of HeLa cells 616 expressing Centrin-1:GFP and depleted of TRIM37 for 48h before imaging onset 617 (10 min. time frame). Yellow arrows point to two foci appearing close to resident 618 centrioles (8/13 extra foci in 11 cells), orange arrow to one focus appearing 619 away from resident centrioles (5/13 extra foci). Solid arrows indicate first 620 occurrence of foci, dashed arrows their continued presence. Time is indicated in 621 h:min since imaging onset. Note that the intensity of extra Centrin-1:GFP foci is 622 typically weaker than that of regular centrioles, especially in the early assembly 623 stages. Note also resident centriole and procentriole appearing in the field of 624 view at the bottom right in Cell 1, 9:20. In this and other Figure panels, scale bars 625 correspond to 5 μm, unless indicated otherwise. 626 B. HeLa cells expressing Centrin-1:GFP upon treatment with control or TRIM37 627 siRNAs, or upon RO3306 addition for 48h. Cells were immunostained for GFP, 628 HsSAS-6 and CEP63. Nuclear contours are drawn with dashed yellow lines. In 629 this and subsequent figures, magnified images from indicated regions are shown. 630 C. Corresponding percentage of cells with extra Centrin-1:GFP foci that also 631 harbor CEP63 and/or HsSAS-6. Unless otherwise indicated, all graphs report 632 averages from two or more independent experiments (n = 50 cells each), along 633 with SDs; P<0.01 here. Note that extra Centrin-1:GFP foci could be positive for 634 both Cep63 and HsSAS-6 in RO3306 treated cells. 635 D. HeLa cells expressing TRIM37:GFP or TRIM37 tagged with a nuclear export 636 signal and GFP (TRIM37:NES:GFP) immunostained for GFP. 637 E. Quantification of extra number of CP110 foci in HeLa cells treated with control 638 or TRIM37 siRNAs and transfected with indicated plasmids (pcDNA3: parental 639 vector). Cells were immunostained for GFP and CP110. The difference between 640 TRIM37:GFP and. TRIM37:NES:GFP is not significant; P=0.7327. 641

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642 Figure 2. Cenpas can behave as extra MTOCs, including in Mulibery patient 643 cells, and trigger new rounds of centriole duplication 644 A. Microtubule depolymerization-regrowth experiment in mitotic HeLa cells 645 treated with control or TRIM37 siRNAs. Microtubules were depolymerized by a 646 30 min cold shock followed by 1-2 min at room temperature before fixation of 647 cells and immunostaining for Centrin-2 and α-tubulin. 648 B. Corresponding percentage of mitotic cells with >2 MTOCs; P<0.01. Note that 649 ~40% of the extra Centrin-2 foci observed in mitosis did not nucleate 650 microtubules, as is the case for two of them in inset 1 (siTRIM37). N= 40 Cenpas 651 each scored in three independent experiments, SD 12.6%. 652 C. Western blot of cell lysates from control and patient (P-1, P-2) fibroblasts 653 probed with antibodies against TRIM37 (top) or α-tubulin as loading control 654 (bottom). The arrow indicates TRIM37, the asterisk a non-specific band. Select 655 molecular weight markers are indicated in kDa in this and other Western blot 656 panels. 657 D. Control and patient-1 (P-1) fibroblasts in mitosis immunostained for Centrin- 658 2 and γ-tubulin. 659 E. Corresponding percentage of mitotic cells with extra number of Centrin-2 or 660 γ-tubulin foci in control and patient (P-1 and P-2) fibroblasts. Data from a total of 661 57 (P-1) and 54 (P-2) mitotic cells obtained from three independent 662 experiments. 663 F. HeLa cells depleted of TRIM37 and immunostained for Centrin-2 plus CP110, 664 illustrating a case with an extra single focus (left, inset 1) and one with an extra 665 pair of foci (right). 666 G. Corresponding percentage of interphase cells with extra single focus or extra 667 pairs of foci. 668 669 670 671 672 673

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674 Figure 3. Cenpas are structures related to centrioles or electron-dense 675 striped structures 676 A. RPE-1 cells treated with control or TRIM37 siRNAs, and immunostained for 677 CEP152 plus acetylated tubulin. 678 B. Corresponding percentage of cells with extra foci of acetylated tubulin; 679 P<0.05. 680 C-G. Ultrastructure expansion microscopy (U-ExM) confocal images of control 681 (C) or TRIM37 (D-G) depleted RPE-1 cells expressing Centrin-1:GFP, and 682 immunostained for GFP, CEP152 as well as acetylated tubulin. Yellow arrows 683 point to Cenpas lacking CEP152 and acetylated tubulin, white arrows to those 684 harboring both proteins, but with an unusual distribution. Scale bar 500 nm. 685 H-L. CLEM analysis of HeLa cell (cell 3 in Fig. S3F) expressing Centrin-1:GFP and 686 depleted of TRIM37. Maximal intensity projection of wide-field microcopy image 687 covering the entire cell volume (H), and magnified insets from the light 688 microscopy images above the corresponding EM images (I-L), with white arrows 689 pointing to relevant Centrin-1:GFP focus. Scale bars: 5 μm in F, 500 nm in G. Here 690 and in panels M-O, orange, green and pink dashed lines surround respectively 691 centrioles-like, centriole-related and tiger structures. Filled orange lines 692 surround resident centrioles. 693 M-O. Centriole-like (M, cell 7 in Fig. S3F), centriole-related (N, cell 7 in Fig, S3F), 694 and tiger (O, cell 2 in Fig, S3F) structures. Scale bar is 500 nm. 695 696 697 698

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699 Figure 4. TRIM37 regulate Centrobin protein stability and levels 700 A. HeLa cells in G2 or mitosis, as indicated, treated with control or TRIM37 701 siRNAs, and immunostained for CP110 plus Centrobin. 702 B. High magnification confocal view of cells treated with control or TRIM37 703 siRNAs immunostained for Centrobin and CP110. Arrow points to elongated 704 Centrobin assembly. Scale bar 1 μm. 705 C. Western blot of lysates from HeLa cells treated with control or TRIM37 siRNAs 706 probed with antibodies against Centrobin (top) or HSP70 as loading control 707 (bottom). 708 D. Western blot of soluble (S) or insoluble (P, for pellet) fractions of lysates from 709 HeLa cells treated with control or TRIM37 siRNAs, probed with antibodies 710 against Centrobin (top) or α-tubulin as loading control (bottom). Note that 711 Centrobin present in the insoluble fraction migrates slower upon TRIM37 712 depletion, suggestive of some posttranslational modification. 713 E. Western blot of total Centrobin protein levels in control and TRIM37 depleted 714 HeLa cells treated with cycloheximide (CHX) for indicated time in hours (h), 715 probed with antibodies against Centrobin (top) or α-tubulin as loading control 716 (bottom). Note that the amount of lysate loaded for the TRIM37 depleted sample 717 was ~50% of that loaded for the siControl condition. 718 F. Quantification of relative Centrobin protein levels from the Western blot 719 shown in E. 720 721

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722 Figure 5. Centrobin promotes Cenpas formation 723 A. U-ExM coupled to STED super-resolution microscopy of RPE-1 cells 724 immmunostained for CEP152, Centrin-1 and Centrobin. White arrows point to 725 Cenpas in close proximity to Centrobin assembly. Scale bars are 250nm. 726 B. Box-and-whisker plot of inter stripe distances in TEM tiger structures (n = 53 727 from 5 tiger structures) and U-ExM Centrobin structures (n = 30 from 3 728 Centrobin structures); P=0.38, not significant. 729 C. HeLa cells depleted of TRIM37 alone or simultaneously of TRIM37 and 730 Centrobin, and immunostained for CP110 plus Centrobin. 731 D. Corresponding percentages of mitotic cells treated with the indicated siRNAs 732 harboring >4 CP110 foci. siTRIM37 versus siTRIM37+siCentrobin: P < 0.05. 733 E. Control and Centrobin-ko RPE-1 cells transfected with TRIM37 siRNAs 734 immunostained for Centrin-2 and CP110. 735 F. Corresponding percentages of mitotic cells with >4 CP110 foci. siTRIM37 736 versus siTRIM37+Centrobin-KO: P < 0.01. 737 738 739

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740 Figure 6. Two pathways contribute to Cenpas formation upon TRIM37 741 depletion 742 A. Box-and-whisker Tukey plot of Centrin-2 foci number per cell in indicated 743 conditions. All cells were analyzed in mitosis with the exception of HsSAS-6-ko 744 conditions. Centrinone versus centrinone + siTRIM37: P < 0.01, HsSAS-6-ko vs 745 HsSAS-6 ko + siTRIM37: P < 0.0001. 746 B. HeLa cells grown with centrinone for 8 days (top) or RPE-1 HsSAS-6-ko cells 747 (bottom), both treated with control or TRIM37 siRNA, before immunostaining 748 for CP110 and Centrin-2 and CP110. 749 C-D. HeLa cells grown with centrinone for 8 days (C) or RPE-1 HsSAS-6-ko cells 750 (D), both treated with control or TRIM37 siRNA, before immunostaining for 751 Centrobin and Centrin-2. 752 E. HeLa cells were synchronized with a double thymidine block, released and 753 transfected with control, TRIM37, Centrobin, or both TRIM37 and Centrobin 754 siRNAs, as indicated. Additionally, DMSO or BI-2536 was added to the cells, 755 which were fixed at time 0 h or 8vh after release, before immunostaining with 756 antibodies against CP110 and Centrobin. The percentage of cells with extra 757 CP110 foci was quantified in each condition. siTRIM37+siCentrobin versus 758 siTRIM37+siCentrobin+BI2536: P = 0.063, non significant. 759 F. HeLa cells were synchronized with a double thymidine block, released and 760 transfected with control, TRIM37, Centrobin, or both TRIM37 and Centrobin 761 siRNAs, as indicated. Additionally, DMSO or BI-2536 was added to the cells, 762 which were fixed at time 0 h or 8 h after release, before immunostaining with 763 antibodies against CP110 and HsSAS-6. 764 G. Corresponding percentage of cells with extra CP110 foci, with an indication of 765 the fraction of them bearing HsSAS-6. The percentage of cells bearing extra 766 HsSAS-6 foci between siTRIM37+siCentrobin versus siTRIM37+BI2536 was 767 significant: P value < 0.05. 768 H. Working model of TRIM37 role in preventing formation of supernumerary 769 MTOCs. Our findings lead us to propose that TRIM37 prevents the formation of 770 supernumerary Centrin foci through two independent pathways mediated by 771 Centrobin (top) and PLK 1 (bottom). The Centrobin pathway relies on the 772 assembly of tiger Centrobin assemblies that act as platforms for PLK4-dependent

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773 Cenpas assembly. Thereafter, Cenpas could evolve into centriole-related and 774 then centriole-like structures with the stepwise incorporation of other centriolar 775 proteins such as HsSAS-6. We propose that the PLK1 pathway might reflect its 776 role in promoting centriole disengagement. Note that only extra MTOCs are 777 represented. See text for details. 778 779 780 781

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782 MATERIAL AND METHODS 783 784 Cell culture, cell lines and cell treatments 785 HeLa Kyoto (generous gift from Daniel Gerlich) and U2OS (ATCC) cells were 786 grown in high glucose DMEM medium (Sigma-Aldrich), hTERT-RPE-1(ATCC) 787 cells in high glucose DMEM/F-12 medium (Sigma-Aldrich). Fibroblast cultures 788 were established from skin biopsy samples of two Mulibrey nanism patients 789 homozygous for the Finnish founder mutation, as well as a control individual, 790 with approval by the Institutional Review Board of the Helsinki University 791 Central Hospital (183/13/03/03/2009). The patients signed an informed 792 consent for the use of fibroblast cultures. Fibroblasts were grown in RPMI 793 medium (Sigma-Aldrich). Other cell lines used were HeLa cells carrying an 794 integrated plasmid expressing Centrin-1:GFP (Piel et al., 2000), RPE-1 p53 -/- 795 cells carrying an integrated plasmid (pCW57.1) expressing Centrin-1:eGFP under 796 a doxycycline inducible promoter (generous gift from George Hatzopoulos), RPE- 797 1 p53 -/- Centrobin knock out cells (Ogungbenro et al., 2018) (generous gift from 798 Ciaran Morrison), and RPE-1 p53 -/- HsSAS-6 knock out cells (Wang et al., 2015) 799 (generous gift from Bryan Tsou). All media were supplemented with 10% fetal 800 bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml 801 streptomycin (all from Sigma-Aldrich) and grown at 37°C in 5% CO2. HeLa Kyoto 802 cells were synchronized using a double-thymidine block and release protocol as 803 follows: cells were incubated in medium with 2 mM thymidine (Sigma Aldrich, 804 T9250) for 17 h, released for 8 h and again incubated with 2 mM Thymidine for 805 17 h. For single transfection experiments, control or TRIM37 siRNAs 806 transfections were performed during the 8 h period between the two thymidine 807 treatments. For double transfection experiments, in addition to the above, either 808 control or Centrobin siRNAs were transfected before the first Thymidine 809 treatment. Drugs used in this work were 10 μM BI-2536 (S1109, Selleck 810 Chemicals), 10 μM RO-3306 (Sigma-Aldrich, SML0569), 125 nM Centrinone 811 (MCE, Hy-18682) and 150 μg/ml Cycloheximide (Sigma-Aldrich, C7698). 812 813 Transfections, plasmids and siRNAs

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814 For siRNA treatments, cells were typically transfected in a 6 well plate format 815 with 20 μM siRNAs and 4 μL Lipofectamine RNAiMAX (Thermo Fisher Scientific); 816 the depletion phenotype was inspected 72 h after transfection unless otherwise 817 indicated in the text or the legends. siRNAs sequences were as follows: TRIM37 818 (5‘-UUAAGGACCGGA GCAGUAUAGAAAA-3’) (Balestra et al., 2013), Centrobin (5‘- 819 AGUGCCAGACUGCAGCAACGGGAAA-3’) (Zou et al., 2005), SPICE (5‘- 820 GCAGCUGAGAACAAAUGAGUCAUUA-3’) (Archinti et al., 2010), HsSAS6 (5‘- 821 GCACGUUAAUCAGCUACAAUU-3’) (Strnad et al., 2007), STIL (5‘- 822 AACGUUUACCAUACAAAGAAA-3’) (Kitagawa et al., 2011a), CPAP (5‘- 823 AGAAUUAGCUCGAAUAGAA-3’) (Kitagawa et al., 2011a), and Stealth RNAi™ 824 siRNA Negative Control Lo GC (Ref: 12935200; Invitrogen). For plasmid 825 transient transfection, FuGENE 6 Transfection Reagent (Promega) was used 826 according to the manufacturer’s protocol and the phenotype inspected 24 or 48 827 h after transfection. Transfected plasmids were as follows: pEBTet-TRIM37:GFP 828 (Balestra et al., 2013), pGFP-Centrobin:GFP (pGFP-NIP2) (Shin et al., 2015) 829 (generous gift from Kunsoo Rhee, Seoul National University, Korea) pEGFP:SPICE 830 (Archinti et al., 2010) (generous gift from Jens Lüders, IRB, Barcelona, Spain) 831 pcDNA3-TRIM37:GFP and pcDNA3-TRIM37:NES:GFP were generated by cloning 832 the TRIM37 ORF (964 aa) fused to GFP or to the HIV-Rev NES sequence 833 (LQLPPLERLTLD) (Wen et al., 1995) and GFP. 834 835 Immunoblotting and Cycloheximide chase assay 836 For Western blot analysis, cells were lysed either in 2× Laemmli buffer (4% SDS, 837 20% glycerol, 125 mM Tris-HCl, pH 6.8) and passed 10 times through a 0.5 mm 838 needle–mounted syringe to reduce viscosity, or in NP40 lysis buffer [10 mM 839 Tris/HCl (pH 7.4)/150 mM NaCl/10% (v/v) glycerol/1% (v/v) Nonidet P40/1 840 mM PMSF and 1 μg/ml of each pepstatin, leupeptin, and aprotinin (Sigma- 841 Aldrich) for 20 min at 4°C and then for 3 min at 37°C, before centrifugation at 842 20,000 g for 20 min. In this manner, the soluble fraction was separated from the 843 insoluble pellet, which was then solubilized in 1x Laemmli buffer. Protein 844 concentration was determined with a NanoDrop Spectrophotometer. Lysates 845 were resolved by SDS-PAGE on a 10% polyacrylamide gel and immunoblotted on 846 Immobilon-P transfer membrane (IPVH00010; 21 Millipore Corporation).

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847 Membranes were first blocked with TBS containing 0.05% Tween-20 (TBST) and 848 5% non-fat dry milk (TBST-5% milk) for 1h at room temperature, and then 849 incubated with primary antibodies diluted in PBST-5% milk. Primary antibodies 850 were 1:1000 rabbit anti-TRIM37 (A301-174A; Bethyl Laboratories), 1:30,000 851 mouse anti- α -tubulin (DM1a; Sigma-Aldrich), 1:500 rabbit anti-Centrobin 852 (HPA023321; Atlas), and 1:20,000 anti-HSP70 (sc-24; Santa Cruz). Membranes 853 were washed and incubated for 1h in secondary antibodies prepared also in 854 TBST-5% milk. Secondary antibodies were 1:5,000 HRP-conjugated anti-rabbit 855 (W4011; Promega) or mouse (W4021; Promega) IgGs. The signal was detected 856 by standard chemiluminescence (34077; Thermo Scientific). Alternatively, 857 polyacrylamide gels were immunoblotted on low fluorescence PVDF membranes 858 (Immobilon-FL, Millipore), membranes blocked with Odyssey Blocking Buffer 859 (LI-COR) and blotted with appropriate primary antibodies and 1:5,000 860 secondary antibodies IRDye 680RD anti-mouse IgG (H+L) Goat LI-COR (926- 861 68070) and IRDye 800CW anti-rabbit IgG (H+L) Goat LI-COR (926-32211). 862 Membranes were then air-dried in the dark and scanned in an Odyssey Infrared 863 Imaging System (LI-COR), and images analyzed with ImageStudio software (LI- 864 COR). In all cases, membrane washes were in TBST. For the cycloheximide chase 865 experiment, HeLa Kyoto cells were treated with fresh DMEM containing 150 866 μg/ml cycloheximide (CHX). Cells were collected 0, 2, 4, 6 and 8 h after CHX 867 addition, and protein extracts prepared in 2× Laemmli buffer as described above. 868 40 μg of siControl lysate and 20 μg of siTRIM37 lysate were resolved by SDS- 869 PAGE, analyzed by immunoblotting with Centrobin and α-tubulin antibodies 870 before quantification with ImageStudio. The siControl and siTRIM37 conditions 871 at time 0 were normalized as 100%, and the other conditions for the same siRNA 872 treatment expressed relative to this. Centrobin expression was quantified as the 873 Centrobin signal divided by the α-tubulin signal. 874 875 RNA isolation, reverse transcription and real-time PCR 876 RNA was extracted using the RNeasy Mini kit according to the manufacturer’s 877 instruction (QIAGEN), including DNase I to avoid potential contaminations with 878 DNA. 3 μg of total RNA, random hexamers and SuperScript III Reverse 879 Transcriptase (InvitrogenTM) were used to obtain complementary DNA (cDNA).

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880 Quantitative PCR from cDNA was performed to assess siRNA-mediated knock- 881 down of TRIM37 and Centrobin, using iTaq Universal SYBR Green Supermix 882 following the manufacturer’s instructions (Bio-Rad) in an Applied Biosystems 883 7500 Fast Real-time PCR System (Thermo Fisher Scientific). Relative mRNA 884 levels of the indicated genes were calculated by the 2-DDCT method (Bulletin 885 5279, Real-Time PCR Applications Guide, Bio-Rad), using GAPDH expression as 886 endogenous control. The primer sequences used were: Centrobin:_CNTROB-FW 887 5‘-GTCTCCATCTAGCTCAGCCC-3’, CNTROB-RV 5‘-AGGCTCTGAATATGGCGCT C-3’, 888 TRIM37: TRIM37-FW 5‘-TGCCATCTTACGATTCAGCTAC-3’, TRIM37-RV 5‘- 889 CGCACAACTCCATTTCCATC-3’. GAPDH: GAPDH-FW 5‘-GGAAGGTGA 890 AGGTCGGAGTC-3’, GAPDH-RV 5‘-GTTGAGGTCAATGAAGGGGTC-3’ 891 892 Indirect immunofluorescence and microtubule-regrowth assay 893 Cells were grown on glass coverslips and fixed for 7 min in -20°C methanol, 894 washed in PBS, and blocked for 30 min in PBS 0.05% Tween 20 (PBST) with 1% 895 bovine serum albumin. Cells were incubated overnight at 4°C with primary 896 antibodies, washed three times for 5 min with PBST, incubated for 1 h at room 897 temperature with secondary antibodies, washed three times for 5 min in PBST 898 and mounted in Vectashield mounting medium with DAPI (H-1200; Vector 899 Laboratories). Primary antibodies used for immunofluorescence were: 1:50 900 human anti-GFP from the recombinant antibody platform of Institut Curie (hVHH 901 antiGFP-hFc, A-R-H#11), 1:1000 rabbit anti-GFP (RGFP-45ALY-Z; ICL), 1:500 902 mouse anti-HsSAS-6 (sc-81431; Santa Cruz), 1:1000 rabbit anti-CEP63 (06-1292; 903 Millipore), 1:2000 rabbit anti-CEP152 (HPA039408; Sigma-Aldrich), 1:1000 904 mouse anti-acetylated tubulin (T6793; Sigma-Aldrich), 1:1000 mouse anti-γ- 905 tubulin (GTU88, T5326; Sigma-Aldrich), 1:1000 mouse anti-Centrin2 (20H5; 906 Sigma-Aldrich), 1:2000 rabbit anti-CEP164 (45330002; Novus Biologicals), 907 1;1000 mouse anti-α-tubulin (T6199; Sigma-Aldrich), 1:1000 rabbit anti-CP110 908 (12780-1-AP; Proteintech), 1:1000 mouse anti-Centrobin (ab70448; Abcam), 909 1:1000 rabbit anti-Centrobin (HPA023321; Atlas Antibodies), 1:1000 rabbit anti- 910 CEP135 (ab75005; Abcam), 1:500 rabbit anti-CPAP (Kohlmaier et al., 2009), 911 1:500 rabbit anti-SPICE (HPA064843, Sigma-Aldrich), 1:8,000 rabbit anti-Ninein 912 (L77), 1:1000 rabbit anti-hPOC5 (Azimzadeh et al., 2009) and 1:1000 rabbit anti-

31 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

913 PLK4(KD) (Sillibourne et al., 2010) (both generous gifts from Michel Bornens, 914 Institut Curie, Paris, France), 1:1000 rabbit anti-P-PLK4 (Moyer and Holland, 915 2019) (generous gifts from Andrew Holland), 1:400 mouse anti-C-Nap (611374; 916 BD Biosciences) 1:2000 rabbit anti-STIL (ab222838; Abcam), 1:1000 rabbit anti- 917 PCNT (ab4448; Abcam), 1:1000 mouse anti-AKAP450 (611518; BD Biosciences), 918 1:1000 rabbit anti-CDK5Rap2 (06-1398; Millipore), 1:1000 rabbit anti-CEP192 919 (a generous gift from Laurence Pelletier), 1:1000 rabbit anti-CEP170 920 (HPA042151; Sigma-Aldrich), 1:1000 mouse anti-P-T210-PLK1 (558400; BD 921 Bioscience). Secondary antibodies were 1:500 mouse Alexa-488, 1:3000 rabbit 922 Cy3, 1:3000 human Alexa-633, 1:1000 mouse Alexa-649, and 1:500 human 923 Alexa-488, all from Jackson ImmunoResearch. For microtubule 924 depolymerization-regrowth experiments, cells were first incubated at 4°C for 30 925 min, then rinsed in pre-warmed medium (37°C), followed by incubation at room 926 temperature for 1-2 min to allow microtubule regrowth. Thereafter, cells were 927 fixed and stained as described above. 928 929 Live imaging, ultrastructure expansion microscopy and confocal microscopy 930 HeLa Centrin-1:GFP cells were transfected with control or TRIM37 siRNAs for 48 931 hours, transferred to 35mm imaging dishes (Ibidi, cat.no 81156), and imaged at 932 37°C and 5% CO2 in medium supplemented with 25mM HEPES (Thermofisher) 933 and 1% PenStrep (Thermofisher). Combined DIC and GFP-epifluorescence time- 934 lapse microscopy was performed on a motorized Zeiss Axio Observer D1 using a 935 63x 1.4 NA plan-Apochromat oil immersion objective, equipped with an Andor 936 Zyla 4.2 sCMOS camera, a piezo controlled Z-stage (Ludl Electronic Products), 937 and an LED light source (Lumencor SOLA II). Imaging was conducted every 10 938 minutes, capturing Z-stacks of optical sections 0.5um apart, covering a total 939 height of 8 um. Ultrastructure expansion microscopy was conducted essential as 940 reported (Gambarotto et al., 2019). For imaging, the sample was mounted on a 941 25 mm round poly-D-lysine coated precision coverslip. STED imaging was 942 performed on a Leica TCS SP8 STED 3X microscope with a 100x 1.4 NA oil- 943 immersion objective. Secondary antibodies were 1:500 Alexa-488 (A-11039; 944 Thermofisher) Alexa-594 (ab150072; Abcam) and Atto647N (2418; Hypermol). 945 Confocal images were captured on a Leica TCS SP5 with a HCX PL APO Lambda

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946 blue 63× 1.4 NA oil objective. All images shown are maximal intensity 947 projections. Image processing was carried out using Image J and Adobe 948 Photoshop (Adobe). 949 950 Correlative light electron microcopy (CLEM) 951 HeLa and RPE-1 cells expressing Centrin-1:GFP were cultured in glass-bottom 952 Petri dishes (MatTek, Cat. No. P35G-1.5-14-CGRD), with an alpha-numeric grid 953 pattern, and transfected with control or TRIM37 siRNAs. Cells were chemically 954 fixed 72 h after transfection with a buffered solution of 1 % glutaraldehyde 2 % 955 paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. Dishes were then 956 screened with a wide-field fluorescent microscope (Zeiss Observer D1, using a 957 63x 1.4 NA oil objective) to identify cells of interest, which were imaged with 958 both transmitted and fluorescence microscopy to register the position of each 959 cell on the grid, as well as the location of their GFP foci, capturing optical slices 960 500 nm apart. The cells were then washed thoroughly with cacodylate buffer 961 (0.1M, pH 7.4), postfixed for 40 min in 1.0 % osmium tetroxide 1.5% potassium 962 ferrocyanide, and then for 40 min in 1.0% osmium tetroxide alone. Finally, cells 963 were stained for 40 min in 1% uranyl acetate in water before dehydration 964 through increasing concentrations of alcohol and then embedding in Durcupan 965 ACM resin (Fluka, Switzerland). The coverslips were then covered with 1 mm of 966 resin, which was hardened for 18 hours in a 65° C oven. The coverslips were 967 removed from the cured resin by immersing them alternately into hot (60° C) 968 water followed by liquid nitrogen until the coverslips parted. Regions of resin 969 containing the cells of interest were then identified according to their position on 970 the alpha-numeric grid, cut away from the rest of the material and glued to blank 971 resin block. Ultra-thin (50 nm thick) serial sections were cut through the entire 972 cell with a diamond knife (Diatome) and ultramicrotome (Leica Microsystems, 973 UC7), and collected onto single slot grids with a pioloform support film. Sections 974 were further contrasted with lead citrate and uranyl acetate and images taken in 975 a transmission electron microscope (FEI Company, Tecnai Spirit) with a digital 976 camera (FEI Company, Eagle). To correlate the light microscopy images with the 977 EM images and identify the exact position of the Centrin-1:GFP foci, fluorescent

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978 images were overlaid onto the electron micrographs of the same cell using 979 Photoshop. 980 981 Statistical analysis 982 Statistical significance was determined with a Student’s t-test using PRISM 983 software (Graphpad Software Inc.). Statistically significance of pair-wise 984 comparisons are indicated in the figure legends with P < 0.05, P < 0.01 or P < 985 0.001. 986 987

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988 989 990 BIBLIOGRAPHY 991 992 Andersen, J.S., Wilkinson, C.J., Mayor, T., Mortensen, P., Nigg, E.A., and Mann, M. 993 (2003). Proteomic characterization of the human centrosome by protein 994 correlation profiling. Nature 426, 570-574. 995 Archinti, M., Lacasa, C., Teixido-Travesa, N., and Luders, J. (2010). SPICE--a 996 previously uncharacterized protein required for centriole duplication and 997 mitotic chromosome congression. Journal of cell science 123, 3039-3046. 998 Arquint, C., and Nigg, E.A. (2016). The PLK4-STIL-SAS-6 module at the core of 999 centriole duplication. Biochemical Society transactions 44, 1253-1263. 1000 Avela, K., Lipsanen-Nyman, M., Idanheimo, N., Seemanova, E., Rosengren, S., 1001 Makela, T.P., Perheentupa, J., Chapelle, A.D., and Lehesjoki, A.E. (2000). Gene 1002 encoding a new RING-B-box-Coiled-coil protein is mutated in mulibrey nanism. 1003 Nature genetics 25, 298-301. 1004 Azimzadeh, J., Hergert, P., Delouvee, A., Euteneuer, U., Formstecher, E., 1005 Khodjakov, A., and Bornens, M. (2009). hPOC5 is a centrin-binding protein 1006 required for assembly of full-length centrioles. The Journal of cell biology 185, 1007 101-114. 1008 Balestra, F.R., Strnad, P., Fluckiger, I., and Gönczy, P. (2013). Discovering 1009 regulators of centriole biogenesis through siRNA-based functional genomics in 1010 human cells. Developmental cell 25, 555-571. 1011 Banterle, N., and Gönczy, P. (2017). Centriole Biogenesis: From Identifying the 1012 Characters to Understanding the Plot. Annual review of cell and developmental 1013 biology 33, 23-49. 1014 Bettencourt-Dias, M., Hildebrandt, F., Pellman, D., Woods, G., and Godinho, S.A. 1015 (2011). Centrosomes and cilia in human disease. Trends Genet 27, 307-315. 1016 Bhatnagar, S., Gazin, C., Chamberlain, L., Ou, J., Zhu, X., Tushir, J.S., Virbasius, C.M., 1017 Lin, L., Zhu, L.J., Wajapeyee, N., et al. (2014). TRIM37 is a new histone H2A 1018 ubiquitin ligase and breast cancer oncoprotein. Nature 516, 116-120. 1019 Bornens, M. (2012). The centrosome in cells and organisms. Science 335, 422- 1020 426. 1021 Brown, N.J., Marjanovic, M., Luders, J., Stracker, T.H., and Costanzo, V. (2013). 1022 Cep63 and cep152 cooperate to ensure centriole duplication. PloS one 8, e69986. 1023 Chavali, P.L., Putz, M., and Gergely, F. (2014). Small organelle, big responsibility: 1024 the role of centrosomes in development and disease. Philosophical transactions 1025 of the Royal Society of London Series B, Biological sciences 369. 1026 Comartin, D., Gupta, G.D., Fussner, E., Coyaud, E., Hasegan, M., Archinti, M., 1027 Cheung, S.W., Pinchev, D., Lawo, S., Raught, B., et al. (2013). CEP120 and SPICE1 1028 cooperate with CPAP in centriole elongation. Current biology : CB 23, 1360-1366. 1029 Courtois, A., Schuh, M., Ellenberg, J., and Hiiragi, T. (2012). The transition from 1030 meiotic to mitotic spindle assembly is gradual during early mammalian 1031 development. The Journal of cell biology 198, 357-370. 1032 Duensing, A., Liu, Y., Perdreau, S.A., Kleylein-Sohn, J., Nigg, E.A., and Duensing, S. 1033 (2007). Centriole overduplication through the concurrent formation of multiple 1034 daughter centrioles at single maternal templates. Oncogene 26, 6280-6288.

35 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1035 Fritz-Laylin, L.K., Levy, Y.Y., Levitan, E., Chen, S., Cande, W.Z., Lai, E.Y., and Fulton, 1036 C. (2016). Rapid centriole assembly in Naegleria reveals conserved roles for both 1037 de novo and mentored assembly. Cytoskeleton 73, 109-116. 1038 Fulton, C., and Dingle, A.D. (1971). Basal bodies, but not centrioles, in Naegleria. 1039 The Journal of cell biology 51, 826-836. 1040 Gambarotto, D., Zwettler, F.U., Le Guennec, M., Schmidt-Cernohorska, M., Fortun, 1041 D., Borgers, S., Heine, J., Schloetel, J.G., Reuss, M., Unser, M., et al. (2019). Imaging 1042 cellular ultrastructures using expansion microscopy (U-ExM). Nature methods 1043 16, 71-74. 1044 Ganem, N.J., Godinho, S.A., and Pellman, D. (2009). A mechanism linking extra 1045 centrosomes to chromosomal instability. Nature 460, 278-282. 1046 Godinho, S.A., Picone, R., Burute, M., Dagher, R., Su, Y., Leung, C.T., Polyak, K., 1047 Brugge, J.S., Thery, M., and Pellman, D. (2014). Oncogene-like induction of 1048 cellular invasion from centrosome amplification. Nature 510, 167-171. 1049 Gönczy, P. (2012). Towards a molecular architecture of centriole assembly. 1050 Nature reviews Molecular cell biology 13, 425-435. 1051 Gönczy, P. (2015). Centrosomes and cancer: revisiting a long-standing 1052 relationship. Nature reviews Cancer 15, 639-652. 1053 Gönczy, P., and Hatzopoulos, G.N. (2019). Centriole assembly at a glance. Journal 1054 of cell science 132. 1055 Gudi, R., Zou, C., Li, J., and Gao, Q. (2011). Centrobin-tubulin interaction is 1056 required for centriole elongation and stability. The Journal of cell biology 193, 1057 711-725. 1058 Guichard, P., Hamel, V., and Gonczy, P. (2018). The Rise of the Cartwheel: Seeding 1059 the Centriole Organelle. BioEssays : news and reviews in molecular, cellular and 1060 developmental biology 40, e1700241. 1061 Guichard, P., Hamel, V., Le Guennec, M., Banterle, N., Iacovache, I., Nemcikova, V., 1062 Fluckiger, I., Goldie, K.N., Stahlberg, H., Levy, D., et al. (2017). Cell-free 1063 reconstitution reveals centriole cartwheel assembly mechanisms. Nat Commun 1064 8, 14813. 1065 Habedanck, R., Stierhof, Y.D., Wilkinson, C.J., and Nigg, E.A. (2005). The Polo 1066 kinase Plk4 functions in centriole duplication. Nature cell biology 7, 1140-1146. 1067 Hirono, M. (2014). Cartwheel assembly. Philosophical transactions of the Royal 1068 Society of London Series B, Biological sciences 369. 1069 Hu, C.E., and Gan, J. (2017). TRIM37 promotes epithelialmesenchymal transition 1070 in colorectal cancer. Molecular medicine reports 15, 1057-1062. 1071 Jakobsen, L., Vanselow, K., Skogs, M., Toyoda, Y., Lundberg, E., Poser, I., Falkenby, 1072 L.G., Bennetzen, M., Westendorf, J., Nigg, E.A., et al. (2011). Novel asymmetrically 1073 localizing components of human centrosomes identified by complementary 1074 proteomics methods. The EMBO journal 30, 1520-1535. 1075 Jeong, Y., Lee, J., Kim, K., Yoo, J.C., and Rhee, K. (2007). Characterization of 1076 NIP2/centrobin, a novel substrate of Nek2, and its potential role in microtubule 1077 stabilization. Journal of cell science 120, 2106-2116. 1078 Jiang, J., Yu, C., Chen, M., Tian, S., and Sun, C. (2015). Over-expression of TRIM37 1079 promotes cell migration and metastasis in hepatocellular carcinoma by 1080 activating Wnt/beta-catenin signaling. Biochemical and biophysical research 1081 communications 464, 1120-1127. 1082 Kallijarvi, J., Avela, K., Lipsanen-Nyman, M., Ulmanen, I., and Lehesjoki, A.E. 1083 (2002). The TRIM37 gene encodes a peroxisomal RING-B-box-coiled-coil

36 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1084 protein: classification of mulibrey nanism as a new peroxisomal disorder. 1085 American journal of human genetics 70, 1215-1228. 1086 Kallijarvi, J., Lahtinen, U., Hamalainen, R., Lipsanen-Nyman, M., Palvimo, J.J., and 1087 Lehesjoki, A.E. (2005). TRIM37 defective in mulibrey nanism is a novel RING 1088 finger ubiquitin E3 ligase. Exp Cell Res 308, 146-155. 1089 Karlberg, N., Karlberg, S., Karikoski, R., Mikkola, S., Lipsanen-Nyman, M., and 1090 Jalanko, H. (2009). High frequency of tumours in Mulibrey nanism. The Journal of 1091 pathology 218, 163-171. 1092 Kettunen, K.M., Karikoski, R., Hamalainen, R.H., Toivonen, T.T., Antonenkov, V.D., 1093 Kulesskaya, N., Voikar, V., Holtta-Vuori, M., Ikonen, E., Sainio, K., et al. (2016). 1094 Trim37-deficient mice recapitulate several features of the multi-organ disorder 1095 Mulibrey nanism. Biology open. 1096 Khodjakov, A., Rieder, C.L., Sluder, G., Cassels, G., Sibon, O., and Wang, C.L. (2002). 1097 De novo formation of centrosomes in vertebrate cells arrested during S phase. 1098 The Journal of cell biology 158, 1171-1181. 1099 Kitagawa, D., Kohlmaier, G., Keller, D., Strnad, P., Balestra, F.R., Fluckiger, I., and 1100 Gönczy, P. (2011a). Spindle positioning in human cells relies on proper centriole 1101 formation and on the microcephaly proteins CPAP and STIL. Journal of cell 1102 science 124, 3884-3893. 1103 Kitagawa, D., Vakonakis, I., Olieric, N., Hilbert, M., Keller, D., Olieric, V., Bortfeld, 1104 M., Erat, M.C., Fluckiger, I., Gönczy, P., et al. (2011b). Structural basis of the 9-fold 1105 symmetry of centrioles. Cell 144, 364-375. 1106 Klebba, J.E., Buster, D.W., McLamarrah, T.A., Rusan, N.M., and Rogers, G.C. (2015). 1107 Autoinhibition and relief mechanism for Polo-like kinase 4. Proceedings of the 1108 National Academy of Sciences of the United States of America 112, E657-666. 1109 Kohlmaier, G., Loncarek, J., Meng, X., McEwen, B.F., Mogensen, M.M., Spektor, A., 1110 Dynlacht, B.D., Khodjakov, A., and Gönczy, P. (2009). Overly long centrioles and 1111 defective cell division upon excess of the SAS-4-related protein CPAP. Current 1112 biology : CB 19, 1012-1018. 1113 La Terra, S., English, C.N., Hergert, P., McEwen, B.F., Sluder, G., and Khodjakov, A. 1114 (2005). The de novo centriole assembly pathway in HeLa cells: cell cycle 1115 progression and centriole assembly/maturation. The Journal of cell biology 168, 1116 713-722. 1117 Levine, M.S., Bakker, B., Boeckx, B., Moyett, J., Lu, J., Vitre, B., Spierings, D.C., 1118 Lansdorp, P.M., Cleveland, D.W., Lambrechts, D., et al. (2017). Centrosome 1119 Amplification Is Sufficient to Promote Spontaneous Tumorigenesis in Mammals. 1120 Developmental cell 40, 313-322 e315. 1121 Li, J., Kim, S., Kobayashi, T., Liang, F.X., Korzeniewski, N., Duensing, S., and 1122 Dynlacht, B.D. (2012). Neurl4, a novel daughter centriole protein, prevents 1123 formation of ectopic microtubule organizing centres. EMBO reports 13, 547-553. 1124 Loncarek, J., Hergert, P., and Khodjakov, A. (2010). Centriole reduplication during 1125 prolonged interphase requires procentriole maturation governed by Plk1. 1126 Current biology : CB 20, 1277-1282. 1127 Lukinavicius, G., Lavogina, D., Orpinell, M., Umezawa, K., Reymond, L., Garin, N., 1128 Gonczy, P., and Johnsson, K. (2013). Selective chemical crosslinking reveals a 1129 Cep57-Cep63-Cep152 centrosomal complex. Current biology : CB 23, 265-270. 1130 Mahjoub, M.R., and Stearns, T. (2012). Supernumerary centrosomes nucleate 1131 extra cilia and compromise primary cilium signaling. Current biology : CB 22, 1132 1628-1634.

37 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1133 Meitinger, F., Anzola, J.V., Kaulich, M., Richardson, A., Stender, J.D., Benner, C., 1134 Glass, C.K., Dowdy, S.F., Desai, A., Shiau, A.K., et al. (2016). 53BP1 and USP28 1135 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic 1136 duration. The Journal of cell biology 214, 155-166. 1137 Montenegro Gouveia, S., Zitouni, S., Kong, D., Duarte, P., Ferreira Gomes, B., Sousa, 1138 A.L., Tranfield, E.M., Hyman, A., Loncarek, J., and Bettencourt-Dias, M. (2018). 1139 PLK4 is a microtubule-associated protein that self-assembles promoting de novo 1140 MTOC formation. Journal of cell science 132. 1141 Moyer, T.C., Clutario, K.M., Lambrus, B.G., Daggubati, V., and Holland, A.J. (2015). 1142 Binding of STIL to Plk4 activates kinase activity to promote centriole assembly. 1143 The Journal of cell biology 209, 863-878. 1144 Moyer, T.C., and Holland, A.J. (2019). PLK4 promotes centriole duplication by 1145 phosphorylating STIL to link the procentriole cartwheel to the microtubule wall. 1146 eLife 8. 1147 Nigg, E.A., and Holland, A.J. (2018). Once and only once: mechanisms of centriole 1148 duplication and their deregulation in disease. Nature reviews Molecular cell 1149 biology. 1150 Nigg, E.A., and Raff, J.W. (2009). Centrioles, centrosomes, and cilia in health and 1151 disease. Cell 139, 663-678. 1152 Ogungbenro, Y.A., Tena, T.C., Gaboriau, D., Lalor, P., Dockery, P., Philipp, M., and 1153 Morrison, C.G. (2018). Centrobin controls primary ciliogenesis in vertebrates. 1154 The Journal of cell biology 217, 1205-1215. 1155 Ohta, M., Ashikawa, T., Nozaki, Y., Kozuka-Hata, H., Goto, H., Inagaki, M., Oyama, 1156 M., and Kitagawa, D. (2014). Direct interaction of Plk4 with STIL ensures 1157 formation of a single procentriole per parental centriole. Nat Commun 5, 5267. 1158 Piel, M., Meyer, P., Khodjakov, A., Rieder, C.L., and Bornens, M. (2000). The 1159 respective contributions of the mother and daughter centrioles to centrosome 1160 activity and behavior in vertebrate cells. The Journal of cell biology 149, 317-330. 1161 Schmidt, T.I., Kleylein-Sohn, J., Westendorf, J., Le Clech, M., Lavoie, S.B., Stierhof, 1162 Y.D., and Nigg, E.A. (2009). Control of centriole length by CPAP and CP110. 1163 Current biology : CB 19, 1005-1011. 1164 Sercin, O., Larsimont, J.C., Karambelas, A.E., Marthiens, V., Moers, V., Boeckx, B., Le 1165 Mercier, M., Lambrechts, D., Basto, R., and Blanpain, C. (2016). Transient PLK4 1166 overexpression accelerates tumorigenesis in p53-deficient epidermis. Nature cell 1167 biology 18, 100-110. 1168 Shin, W., Yu, N.K., Kaang, B.K., and Rhee, K. (2015). The microtubule nucleation 1169 activity of centrobin in both the centrosome and cytoplasm. Cell cycle 14, 1925- 1170 1931. 1171 Shiratsuchi, G., Takaoka, K., Ashikawa, T., Hamada, H., and Kitagawa, D. (2015). 1172 RBM14 prevents assembly of centriolar protein complexes and maintains mitotic 1173 spindle integrity. The EMBO journal 34, 97-114. 1174 Sillibourne, J.E., Tack, F., Vloemans, N., Boeckx, A., Thambirajah, S., Bonnet, P., 1175 Ramaekers, F.C., Bornens, M., and Grand-Perret, T. (2010). Autophosphorylation 1176 of polo-like kinase 4 and its role in centriole duplication. Molecular biology of the 1177 cell 21, 547-561. 1178 Sinclair, C.S., Rowley, M., Naderi, A., and Couch, F.J. (2003). The 17q23 amplicon 1179 and breast cancer. Breast cancer research and treatment 78, 313-322. 1180 Strnad, P., Leidel, S., Vinogradova, T., Euteneuer, U., Khodjakov, A., and Gönczy, P. 1181 (2007). Regulated HsSAS-6 levels ensure formation of a single procentriole per

38 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1182 centriole during the centrosome duplication cycle. Developmental cell 13, 203- 1183 213. 1184 Sullenberger, C., Vasquez-Limeta, A., Kong, D., and Loncarek, J. (2020). With Age 1185 Comes Maturity: Biochemical and Structural Transformation of a Human 1186 Centriole in the Making. Cells 9. 1187 Tang, C.J., Fu, R.H., Wu, K.S., Hsu, W.B., and Tang, T.K. (2009). CPAP is a cell-cycle 1188 regulated protein that controls centriole length. Nature cell biology 11, 825-831. 1189 Thauvin-Robinet, C., Lee, J.S., Lopez, E., Herranz-Perez, V., Shida, T., Franco, B., 1190 Jego, L., Ye, F., Pasquier, L., Loget, P., et al. (2014). The oral-facial-digital 1191 syndrome gene C2CD3 encodes a positive regulator of centriole elongation. 1192 Nature genetics 46, 905-911. 1193 Tsou, M.F., Wang, W.J., George, K.A., Uryu, K., Stearns, T., and Jallepalli, P.V. 1194 (2009). Polo kinase and separase regulate the mitotic licensing of centriole 1195 duplication in human cells. Developmental cell 17, 344-354. 1196 van Breugel, M., Hirono, M., Andreeva, A., Yanagisawa, H.A., Yamaguchi, S., 1197 Nakazawa, Y., Morgner, N., Petrovich, M., Ebong, I.O., Robinson, C.V., et al. (2011). 1198 Structures of SAS-6 suggest its organization in centrioles. Science 331, 1196- 1199 1199. 1200 Wang, W., Xia, Z.J., Farre, J.C., and Subramani, S. (2017). TRIM37, a novel E3 ligase 1201 for PEX5-mediated peroxisomal matrix protein import. The Journal of cell 1202 biology 216, 2843-2858. 1203 Wang, W.J., Acehan, D., Kao, C.H., Jane, W.N., Uryu, K., and Tsou, M.F. (2015). De 1204 novo centriole formation in human cells is error-prone and does not require SAS- 1205 6 self-assembly. eLife 4. 1206 Wen, W., Meinkoth, J.L., Tsien, R.Y., and Taylor, S.S. (1995). Identification of a 1207 signal for rapid export of proteins from the nucleus. Cell 82, 463-473. 1208 Wong, Y.L., Anzola, J.V., Davis, R.L., Yoon, M., Motamedi, A., Kroll, A., Seo, C.P., 1209 Hsia, J.E., Kim, S.K., Mitchell, J.W., et al. (2015). Cell biology. Reversible centriole 1210 depletion with an inhibitor of Polo-like kinase 4. Science 348, 1155-1160. 1211 Zou, C., Li, J., Bai, Y., Gunning, W.T., Wazer, D.E., Band, V., and Gao, Q. (2005). 1212 Centrobin: a novel daughter centriole-associated protein that is required for 1213 centriole duplication. The Journal of cell biology 171, 437-445. 1214 1215 1216

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1217 SUPPLEMENTARY FIGUE LEGENDS 1218 1219 Suppl. Figure 1. TRIM37:GFP localizes to centrioles 1220 A, B. HeLa (A) and U2OS (B) cell transfected with TRIM37:GFP, and 1221 immunostained for GFP plus γ-tubulin. In this and other supplementary figure 1222 panels, scale bars correspond to 5 μm, unless indicated otherwise. 1223 C. HeLa cells transfected with TRIM37:GFP, and immunostained for GFP plus 1224 Centrobin. Cells with a single Centrobin focus (left) were classified as being in 1225 G1, cells with two or three Centrobin foci (right) as being in S/G2. 1226 D. Corresponding percentage of cells in G1 or S/G2 exhibiting TRIM37:GFP at 1227 centrosomes. n=50 cells, single experiment. 1228 E, F. High magnification confocal images of TRIM37:GFP localization with respect 1229 to indicated centriolar markers; HeLa cells were fixed 24h after transfection in 1230 this case. Scale bar 500 nm. 1231 1232

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1233 Suppl. Figure 2. Cenpas can behave as extra MTOCs 1234 A. Microtubule depolymerization-regrowth experiment in mitotic HeLa cells 1235 treated with control or TRIM37 siRNAs. Microtubules were depolymerized by a 1236 30 min cold shock followed by 1-2 min at room temperature before fixation of 1237 cells and immunostaining for Centrin-2 and α-tubulin. 1238 B. HeLa cells immunostained for Centrin-2 and CP110. Left: G1 cell, with two 1239 resident centrioles, right: S/G2 cell with two centriole pairs, each with one 1240 resident centriole and one procentriole. 1241 1242 1243

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1244 Supplementary Figure 3. CLEM analysis of Cenpas 1245 A-E. CLEM analysis of HeLa and RPE-1 cells expressing Centrin-1:GFP and 1246 transfected with control (A-B) or TRIM37 (C-E) siRNAs. Left-most images show 1247 maximal intensity projection of wide-field microcopy image covering the entire 1248 cell volume; scale bar: 5 μm. Magnified insets from the light microscopy images 1249 are shown above the electron microscopy images taken from the corresponding 1250 position. C corresponds to cell 2, D to cell 7, and E to cell 6 in Fig. S3F. When 1251 present, white arrows indicate the Centrin-1:GFP focus that correlates with the 1252 EM imaged shown below. Scale bars in insets are 500 nm. Orange, green and 1253 pink dashed lines surround respectively centriole-like, centriole-related and 1254 tiger structures. Filled orange lines surround resident centrioles. 1255 F. Summary of CLEM analysis of HeLa or RPE-1 cells depleted of TRIM37, with 1256 number of GFP foci, as well as corresponding resident centriole/procentriole and 1257 Cenpas ultrastructure identified by CLEM. See main text for further details. Note 1258 that no distinct ultrastructure was found for 5 Centrin-1:GFP foci in cell 4, 1259 perhaps reflecting a technical issue in this case. 1260

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1261 Supplementary Figure 4. Analysis of Centrobin structures formed upon 1262 TRIM37 depletion 1263 A, Centriolar and centrosomal proteins analyzed by immunofluorescence upon 1264 TRIM37 depletion. See Materials and Methods for antibodies utilized. 1265 B, C. Quantification of frequency (B) and number per cell (C) of Centrobin 1266 assemblies in HeLa cells depleted of TRIM37. Unless otherwise indicated, in this 1267 and subsequent supplementary figures all graphs report averages from two or 1268 more independent experiments (n = 50 cells each), along with SDs. 1269 D. HeLa cell expressing SPICE:GFP immunostained for GFP and Centrobin. 1270 E. HeLa cells treated with control, TRIM37 or double TRIM37 and SPICE siRNAs, 1271 immunostained for SPICE and Centrobin. 1272 F. Quantitative real time PCR of TRIM37 and Centrobin mRNA in HeLa cells 1273 treated with control or TRIM37 siRNAs. Average of three independent 1274 experiments. 1275 G. HeLa cell (left) and HeLa cell overexpressing Centrobin:GFP (right), 1276 immunostained for GFP and CP110. 1277 H. Confocal images of HeLa cells overexpressing TRIM37:GFP immunostained 1278 with antibodies against GFP and Centrobin. 1279

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1280 Supplementary Figure 5. Centrobin is not required for canonical centriole 1281 duplication 1282 A. HeLa cells in G2 or mitosis, as indicated, treated with control or Centrobin 1283 siRNAs, aand immunostained for Centrobin plus CP110. 1284 B. Corresponding percentage of mitotic cells with indicated number of CP110 1285 foci. 1286 C. Western blot of soluble (S) and insoluble (P, for pellet) fractions of lysates 1287 from HeLa cells transfected with siRNAs against TRIM37 or against both TRIM37 1288 and Centrobin, probed with antibodies against Centrobin (top) or α-tubulin as 1289 loading control (bottom). 1290 D. Control or Centrobin-ko RPE-1 cells transfected with control siRNAs and 1291 immunostained for Centrin-2 plus CP110.

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1292 Supplementary Figure 6. Two pathways contribute to Cenpas formation 1293 upon TRIM37 depletion 1294 Percentages of cells with Centrobin structures in the indicated conditions. P 1295 value: not significant for both pair-wise comparisons (i.e. siTRIM37 versus 1296 Centrinone + siTRIM37 and siTRIM37 versus. HsSAS-6-ko + siTRIM37). 1297 1298

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A siTRIM37 - HeLa Centrin-1:GFP

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Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B 60 siControl siTRIM37 50 1 2 1 2 3 40 Merge 2 Merge 2 30 1 C2 1 C2 / Centrin-2 20 DNA 3 10 % mitotic cells with >2 MTOCs -tub -tub ᵅ ᵅ

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Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in A perpetuity. It is made available under aCC-BY 4.0 International licenseB . siControl siTRIM37 30 1 2 3 25

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Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. A Centrobin/CP110/DNA

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Centrin-2/CP110/DNA Centrobin/CP110/DNA Centrobin/CEP152/Centrin-1 bioRxiv preprint preprint (whichwasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplayin 1 U-ExM siTRIM37 siControl siTRIM37 2 3 1 3 doi: 2 https://doi.org/10.1101/2020.09.03.281097 2 1 3 2 1 perpetuity. Itismadeavailableundera 3 siTRIM37 CP110 C2 CP110 CTRB Centrobin-ko siCentrobin siTRIM37 siTRIM37 2 1 Figure 5 1 2 ; this versionpostedSeptember4,2020. CC-BY 4.0Internationallicense 2 1 2 1

CP110 C2 CP110 CTRB B D F

% miotic cells with >4 centrioles % miotic cells with >4 centrioles inter stripe distance (nm) 100 150 50 20 40 60 80 20 40 60 80 . 0 0 0 siControl siControl The copyrightholderforthis structures Tiger EM siTRIM37 siTRIM37

Centrobin-ko structures siCentrobin Centrobin U-ExM

Centrobin-kosiTRIM37 siCentrobinsiTRIM37 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in A perpetuity. It is made available under aCC-BYB 4.0 International license. CP110/Centrin-2/DNA 15 HeLa RPE-1 14 13 siControl siTRIM37 12 11 10 9 8 7 HeLa 6 Centrinone Centrin-2 foci 5 4 1 2 1 2 3 2 CP110 1 0 C2 RPE-1 HsSAS6-ko

siSTIL siCPAPsiSPICE iTRIM37iTRIM37 iTRIM37 siControlsiTRIM37 siControlsiTRIM37 CentrinonsiHsSAS-e 6 S-6 + sTIL + s siS siSPICE + s iHsSA siCPAP + siTTRIM37 Centrinones + siTRIM37 HsSAS-6-KOHsSAS-6-KO + siControl + siTRIM37

C Centrinone D HsSAS6-ko

siControl siTRIM37 siControl siTRIM37 1 2 1 CTRB CTRB / Centrin-2 / Centrin-2 C2 C2 DNA DNA 2 Centrobin Centrobin

E F siCentrobin siTRIM37 60 siTRIM37 siTRIM37 BI2536

2 50 3

1 3 40 1

DNA 2 2 30 1

20 / HsSAS-6 CP110 % cells with >4 centrioles 10 1 2 3 1 2 1 2 3 CP110

0 2x Thy-B&R S G2 S G2 S G2 S G2 S G2

siControl siTRIM37 siTRIM37 siTRIM37 siTRIM37 HsSAS-6 siCentrobin BI2536 siCentrobin BI2536

G 60 H All extra foci 50 HsSAS-6 positive extra foci Tiger structure Seeding Centriole-related Centriole-like Cepas 40 assembly Centrobin PLK4 HsSAS-6 Extra MTOCs 30

20 TRIM37 2 4 2 PLK4

% cells with extra foci 2 1 3 1 3 10 1 3 PLK1 HsSAS-6 Extra MTOCs 5 4 0 Premature 4 4 siTRIM37 + + + Resident disengagement centrioles siCentrobin - + -

BI2536 - - +

Figure 6 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B HeLa - TRIM37:GFP U2OS - TRIM37:GFP 1 2 ɣ- TUB T37:GFP ɣ- TUB T37:GFP 2 ɣ- TUB / DNA

ɣ- TUB / DNA 1 TRIM37:GFP TRIM37:GFP

C D HeLa - TRIM37:GFP 70 G1 S/G2 60 50

40

30 CTRB T37:GFP CTRB T37:GFP at centrosome 20 % cells with TRIM37 % cells with Centrobin

TRIM37:GFP 10

0 G1 S/G2 cell cycle stage E F TRIM37:GFP/Centrin-2/CEP63 TRIM37:GFP/CEP164 MERGE CEP164 MERGE T37:GFP Centrin-2 CEP63 T37:GFP

Supplementary Figure 1 bioRxiv preprint B A preprint (whichwasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplayin

CP110/Centrin-2 ᵅ -tubulin/Centrin-2 DNA DNA doi: G1 https://doi.org/10.1101/2020.09.03.281097 1 1 2 perpetuity. Itismadeavailableundera 2 3 1 1 Supplementary Figure2 2 2 Control 3 C2 CP110 siTRIM37 S/G2

-tub C2 Merge ;

ᵅ this versionpostedSeptember4,2020. 1 2 1 CC-BY 4.0Internationallicense 1 2 1 4 3 1 2 2 1

C2 CP110 . 3 The copyrightholderforthis 4

ᵅ-tub C2 Merge bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. HeLa Centrin-1:GFP RPE-1 Centrin-1:GFP 1 2 1 2 A B

2 1 2 1 siControl

HeLa Centrin-1:GFP 1 2 2 3 4 C

3 2 1

4 siTRIM37

RPE-1 Centrin-1:GFP D siTRIM37

1 1 2 E 2 Resident Centriole Centriole-like

Centriole-related 1 Tiger

F Cell Resident ultrastructures Cenpas ultrastructures

Number Type GFP foci Centrioles Procentrioles Centriole-like Centriole-related Tiger Not found

1 HeLa 5 2 0 0 0 3 0

2 HeLa 6 2 1 0 2 1 0

3 HeLa 5 2 0 0 1 2 0

4 HeLa 8 1 1 0 1 0 5

5 RPE-1 5 2 2 0 1 0 0

6 RPE-1 5 2 0 0 0 3 0

7 RPE-1 7 4 0 2 1 0 0

8 RPE-1 6 2 0 0 0 3 1

Total 47 17 4 2 6 12 6

Supplementary Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A

Centriolar and centrosomal proteins tested by immunostaining upon TRIM37 depletion

Centriole: CEP135, CPAP, CNTROB, SPICE, hPOC5, Centrin-2, CP110, CEP63, CEP152, C-Nap, CEP192. Procentriole: HsSAS6, PLK4, STIL. PCM: PCNT, AKAP450, CDK5Rap2, ɣ-tubulin. Appendages: CEP164, Ninein, CEP170

B C D SPICE:GFP/Centrobin

100 100 1 2 80 80

60 1 60

40 40 CTRB GFP 2 20 20

% cells with Centrobin structures 0 % cells with Centrobin structures 0 siControl siTRIM37 1 2 3 >3

number of Centrobin structures

E SPICE/Centrobin/DNA

siSPICE siControl siTRIM37 siTRIM37 1 2

1

CTRB SPICE

CTRB SPICE 2 CTRB SPICE

F G H HeLa + Centrobin:GFP HeLa + TRIM37:GFP

siControl 1.5 siTRIM37 GFP/CP110/DNA GFP/Centrobin/DNA CP110 21 1 1

1 GFP 2

2 1 CTRB 0.5 2 mRNA levels relative to control mRNA

0 TRIM37 Centrobin

Supplementary Figure 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A Centrobin/CP110/DNA

G2 G2 1 2

1 CP110 CTRB CP110 CTRB 2

1 2 1 2 M M siControl siCentrobin

1 2 1 2 CP110 CTRB CP110 CTRB

B C siControl siCentrobin siTRIM37 100 siTRIM37 siCentrobin 80 SPSP 60 Centrobin 40 100 KDa % miotic cells 20 αTUB 0 <3 3 or 4 >4 48 KDa CP110 foci

D siControl Centrobin-ko 1 2 1 2

1 1 2 2 CP110 C2 CP110 C2 / DNA / CP110 Centrin-2

Supplementary Figure 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.03.281097; this version posted September 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

HeLa RPE-1 100

90

80

70

60

50

40

30

20

% cells with Centrobin structures 10

0

siTRIM37 siTRIM37 siTRIM37 siTRIM37

Centrinone HsSAS6-ko

Supplementary Figure 6