bioRxiv preprint doi: https://doi.org/10.1101/433466; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 TgCep250 is dynamically processed through the division cycle and

2 essential for structural integrity of the Toxoplasma

3

4

5 Chun-Ti Chen*† and Marc-Jan Gubbels*†

6

7 *Corresponding authors: [email protected] and [email protected]

8 †Department of Biology, Boston College, Chestnut Hill, MA 02467

9

10 Running title: TgCep250 stabilizes bipartite centrosome

11

12 Key words: Apicomplexa; Toxoplasma gondii; centrosome, cell division, Cep250

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13 Abstract 14 15 The Toxoplasma centrosome is a unique bipartite structure comprising an inner- 16 and outer-core responsible for the nuclear cycle () and budding cycles 17 (cytokinesis), respectively. These two cores remain associated during the 18 but have been proposed to function independently. Here, we describe the function 19 of a large coiled-coil , TgCep250, in connecting the two centrosomal cores 20 and promoting their structural integrity. Throughout the cell cycle TgCep250 21 localizes to the centrosome inner-core but resides on both inner- and outer-cores 22 during the onset of cell division. This dynamic localization pattern is associated with 23 proteolysis: the processed version residing on the inner-core. In the absence of 24 TgCep250, stray centrosome inner- and outer-core foci were observed; detachment 25 of the inner- outer-core connection resulted in nuclear partitioning defects. The 26 detachment between centrosome inner- and outer-core was found in only one of the 27 during cell division, indicating distinct states of mother and daughter 28 centrosomes. We further dissected the hierarchical organization of centrosome and 29 kinetochore complex through depletion of kinetochore component TgNuf2, which 30 resulted in dissociation of the intact bipolar centrosome from the nuclear periphery. 31 Together, these data suggest that TgCep250 bridges the interaction between the 32 centrosome cores but not between the inner-core and kinetochore.

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33 Short Summary 34 35 The opportunistic apicomplexan parasite Toxoplasma gondii uses a bipartite 36 centrosome to independently regulate mitosis and cytokinesis. Here we report a 37 large coiled-coil protein that functions to integrate the two centrosomal cores for 38 faithful cell division. This study also reveals the layered structural organization of 39 the centrosome/kinetochore complex.

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40 Introduction 41 42 The phylum Apicomplexa consists of almost exclusively obligate intracellular 43 parasites, which have significant impact on public health. Among these single-celled 44 organisms, Toxoplasma gondii is one of the most successful zoonotic pathogens that 45 can be transmitted and replicate in many different host species. T. gondii has 46 developed flexible replication strategies allowing efficient survival in distinct tissue 47 types. Tachyzoites are the acute replication form of T. gondii and are responsible for 48 most of the clinical burden in infected individuals. They undergo a binary 49 replication mode termed endodyogeny where two daughter cells assemble 50 internally and emerge from the mother cell. The cell cycle follows the sequence of 51 G1, S and M phases, but the G2 phase is absent or too short to be detected 1, and is 52 driven by a set of unconventional CDK-related kinases (Crks) and cyclins 2. Mitosis is 53 (semi-)closed and tightly spatially and temporally coordinated with cytokinesis as 54 these two events occur concomitantly. The 14 are clustered at the 55 centromeric region throughout the cell cycle at the ‘centrocone’, a unique 56 membranous structure that houses spindle microtubule during mitosis 3. The tight 57 organization of chromosomes is thought to ascertain each daughter cell receives a 58 full set of genetic material when the parasite undergoes more complex division cycle 59 in other cell cycle stages 4. In its definitive feline host, endopolygeny consists of 60 several rounds of DNA synthesis and mitosis in the same cytoplasmic mass followed 61 by synchronized cytokinesis in accordance with the last round of mitosis, producing 62 8-16 uni-nucleated progeny per replication cycle 5. 63 The Toxoplasma centrosome is divergent from mammalian cells in 64 architecture and composition. For example, the are composed of nine 65 singlet microtubules, smaller in size than mammalian centrioles, and the 66 pair is arranged in parallel rather than perpendicular 4. Orthologs of many key 67 components in the mammalian centrosome cannot be found in the Toxoplasma 68 genome 6. Even without canonical protein orthologs, recent studies have shown that 69 a group of coiled-coil localize to the centrosome and support proper 70 karyokinesis and cytokinesis 7-8. This work identified a distinctive architecture of

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71 two centrosome cores: the outer-core (distal to the nucleus) and the inner-core 72 (proximal to the nucleus). 73 The Toxoplasma centrosome is the key organelle coordinating mitotic and 74 cytokinetic rounds. The centrosome resides at the apical end of the nucleus during 75 interphase and remains closely associated with a specialized nuclear envelope fold 76 termed the centrocone that houses the spindle microtubules (MTs) during mitosis 9. 77 The centrosome also plays a critical role in partitioning the single copy Golgi 78 apparatus and the chloroplast-like organelle, the apicoplast, during cell division 10. 79 Late in G1, the centrosome rotates to the distal end of the nucleus where it 80 duplicates and then returns to the proximal end 11. At this point, the spindle MTs 81 assemble and are stabilized within the centrocone, whereas subsequently the 82 centrosome serves as a scaffold for assembly of daughter cell cytoskeletal 83 components 12. In mitosis, a striated fiber assemblin (SFA) protein emanates from 84 the centrosome and connects the daughter apical conoid to the centrocone. SFA 85 polymerization is thought to maintain the stoichiometric count of daughter cells and 86 replicating genomic materials. In SFA depleted cells, cell division is completely 87 disrupted 13. Throughout these timely regulated events, little is known about 88 centrosome biogenesis or regulation in Toxoplasma. Although orthologs in charge of 89 new centriole biogenesis, Polo-like kinase PLK4 and its substrate STIL/Ana2/SAS5, 90 are absent from the Toxoplasma genome , previous work has shown that a 91 conserved NIMA-kinase, TgNek1, is required for centrosome splitting 14 and a 92 TgMAPK-L1 place a role in inhibiting centrosome over-duplication 7. However, 93 exactly on what aspects of the centrosome these kinases act and what their 94 substrates are remains unclear. 95 Here we asked whether TgCep250 could be the substrate of TgNek1 required 96 for centrosome splitting in orthogology to the human HsNek2 – C-NAP1/Cep250 97 relationship in centrosomes splitting 15. Our data reject this hypothesis but instead 98 revealed that TgCep250 is required to connect the inner-core with the outer-core as 99 well as to maintain overall centrosome integrity. Moreover, our data are the first 100 report directly hinting at a differential status of mother versus daughter centrosome 101 in the Apicomplexa. In conclusion, TgCep250 is not the critical TgNek1 substrate,

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102 but its function in keeping centrosome complexes connected appears to be 103 orthologous to the role of human C-NAP1/Cep250. 104 105 106 Results 107 108 TgCep250 has a dynamic localization and is associated with proteolysis. 109 To start charting the function of previously reported Toxoplasma centrosome 110 protein TgCep250 (TGGT1_212880) 7 we first characterized the subcellular 111 localization of TgCep250 throughout the parasite’s tachyzoite division cycle. 112 TgCep250 was tagged endogenously with a C-terminal YFP tag 16 and the TgCep250- 113 YFP parasite line was transfected with a fosmid expressing TgCep250L1-HA as an 114 inner-core centrosome marker 7. During the G1 phase of the cell cycle TgCep250- 115 YFP is closely associated with the centrosome inner-core (highlighted by 116 TgCep250L1) whereas during cytokinesis we observed four TgCep250-YFP foci 117 where the upper two foci in close apposition to the outer-core (Fig. 1). 118 To dissect the function of TgCep250, we generated a conditional knockdown 119 line (TgCep250-cKD) using a tetracycline regulatable promoter. Simultaneously, we 120 inserted a single-Myc tag at the N-terminus of the (Fig. 2A). Correct integration 121 of the construct through single homologous recombination was validated by PCR 122 (Fig. 2B). In the presence of anhydrotetracycline (ATc), Myc-TgCep250 protein 123 becomes undetectable within six hours of incubation by immunofluorescence assay 124 (IFA) and western blot (Fig. 2C and D). Interestingly, the N-terminal Myc-tagged 125 TgCep250 only formed two foci co-localizing with α- throughout the cell 126 cycle. This suggested that the placement of the tag on TgCep250 either alters the 127 subcellular localization or that proteolytic processing is associated with this 128 differential localization pattern. To test this hypothesis, we added a C-terminal 3xTy 129 tag to the Myc-TgCep250-cKD locus (Fig. 3A) and analyzed TgCep250-3xTy by IFA 130 and western blot. As shown in Figure 3B and 3C, C-terminally 3xTy tagged 131 TgCep250 forms four foci like TgCep250-YFP. Moreover, we observed multiple 132 protein bands smaller than the full-length protein on the blot using α-Ty antibody

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133 (Fig. 3C; note that full-length TgCep250 is a large coil-coiled protein with a 134 molecular weight predicted to be 762 kDa). Thus, these data support the notion that 135 that only full-length TgCep250 localizes to the outer-core (during the onset of 136 cytokinesis) and a truncated version of TgCep250 localizes to the inner-core 137 (throughout all cell cycle stages; Fig. 3B and 3D). In line with this hypothesis, we 138 only detected a single band on western blot consistent with the full-length protein 139 using α-Myc antibody (Fig. 2D). Thus, we conclude that TgCep250 has dynamic 140 localization pattern during the cell cycle and that protein processing appears to 141 affect association of TgCep250 to the centrosome inner cores. 142 143 TgCep250 is essential and links the centrosome inner- and outer-cores 144 Next, we probed the function of TgCep250 with a series of experiments using the 145 TgCep250-cKD parasite line. In the presence of ATc, TgCep250-cKD parasites failed 146 to form plaques suggesting TgCep250 is essential for parasite survival (Fig. 4A). 147 Phenotypic analysis by IFA showed that in absence of TgCep250 the nucleus failed 148 to partition to the daughter cells (Fig. 4B, white arrow) and as a result anuclear 149 parasites were observed (Fig. 4B, white arrowheads). The stoichiometric balance of 150 1:1 daughter to (outer-)centrosome count was also disrupted resulting in three 151 Centrin foci accumulated in a single cell boundary highlighted by IMC3 (Fig. 4B, 152 double arrowhead). To examine the nuclear partitioning defect in more detail, we 153 used different sets of antibodies to analyze TgCep250 depleted parasites. As shown 154 in Figure 4C, we observed and quantified three different centrosomal defects in 155 TgCep250 depleted cells using centrosome inner- (TgCep250L1) and outer-core 156 (Centrin) markers. We observed detachment of inner-core from the outer-core in 157 the majority of mitotic, mutant parasites (marked in yellow). However, we also 158 observed parasites wherein the outer-core split into two foci while only two inner- 159 cores were present, suggestive of undergoing an additional replication cycle 160 (marked in cyan; 10% of the mitotic parasites). Finally, about 35% of the mitotic 161 parasites displayed, stray inner core foci (marked in magenta) which is a 162 combination of the two other phenotypes. Detachment of inner- and outer-cores 163 likely results in the nuclear partitioning defect observed in Figure 4B. An additional

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164 observation is that detachment of inner- and outer-cores occurred in most instances 165 on only one of the two centrosomes. The most logical explanation is that the 166 detached inner/outer-core pair represents the daughter centrosome and the intact 167 pair represents the mother centrosome 17. In this scenario, during centrosome 168 biogenesis, TgCep250 cannot be incorporated into the newly formed daughter 169 centrosome resulting in a detached inner- and outer-core. However, the TgCep250 170 in the mother centrosome is stably incorporated thereby maintaining inner- and 171 outer-core adhesion in this centrosome. 172 Interestingly, the ability to form daughter buds remained intact in TgCep250 173 depleted cells (Fig. 4B; daughter buds marked with asterisk). Thus, we sought to 174 determine whether the detached centrosome cores remain functional in daughter 175 bud assembly. For this purpose, an IMC-subcompartment protein ISP1 was used as a 176 marker to determine early daughter formation near the centrosome 18. As shown in 177 Figure 4D, α-ISP1 signal was detected surrounding the outer-core in the presence 178 and absence of ATc, even when the outer-core is detached from the inner-core (Fig. 179 4D 4x zoom, asterisk). These results indicate that the function of the outer-core in 180 daughter bud assembly remains intact when it is detached from the inner-core. This 181 observation is in line with the proposed working model predicting that the inner- 182 core and outer-core functions can act independently 7, 19. 183 184 TgCep250 is not required for the connection of the inner-core with the 185 kinetochore 186 Since the anuclear phenotype observed in TgCep250-cKD parasites was very similar 187 to the anuclear daughters observed in parasites depleted in kinetochore component 188 TgNuf2 20 we tested how these two observations relate to each other. We performed 189 IFA assays using mitotic markers to reveal the interactions between centrosome 190 outer-core (α-Centrin), the kinetochore (α-TgNdc80) 20 and the spindle 191 microtubules (EB1-YFP) 11. As shown in Figure 5, in the absence of TgCep250, both 192 kinetochore complex and spindle microtubules became detached from the 193 centrosome outer-core (Fig. 5A, white arrowhead). To determine whether 194 TgCep250 plays a role in interaction between the inner-core and the kinetochore we

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195 co-stained the parasite with inner-core (TgCep250L1) and kinetochore (TgNdc80) 196 markers. Surprisingly, although TgCep250L1 fragmented into multiple foci, the 197 majority of TgNdc80 foci co-localized with TgCep250L1 foci in the absence of 198 TgCep250, even though in some cases the connection with the nucleus was gone 199 (Fig. 5B). Thus, the inner-core and the kinetochore remained associated in the 200 absence of TgCep250 and indicates that the nuclear function of the inner core is also 201 maintained when the inner/outer-core association is lost. 202 203 TgNuf2 anchors the centrosome to the nuclear periphery 204 To further dissect the association between the inner-core and the kinetochore, we 205 expressed the inner-core marker TgCep250L1 in a kinetochore-depleted parasite 206 line, TgNuf2-cKD 20. It has been reported that in the absence of TgNuf2, the 207 association between the centrosome and spindle pole is abolished and the 208 centrosome is pulled away from the nucleus 20. In Figure 6, we observed that the 209 centrosome inner- and outer-core co-migrate away from the nucleus (Fig. 6 210 arrowhead). In this scenario, association between outer-core and inner-core 211 remains intact in the absence of TgNuf2. These data suggest that the kinetochore is 212 required to anchor the centrosome cores to the nuclear periphery but not required 213 for the connection between the inner- and outer-cores. 214 215 TgNek1 promotes separation of only the outer-cores 216 It has been shown that a Toxoplasma ortholog of NIMA kinases, TgNek1, is 217 responsible for centrosome separation in tachyzoites 14. To dissect the regulation of 218 centrosome core separation in a TgNek1 depleted background we constructed a 219 conditional knockdown line of TgNek1 (TgNek1-cKD) (Fig. S1A). Plaque assays 220 showed that in the presence of ATc, TgNek1-cKD parasites display a severe growth 221 defect (Fig. S1B) consistent with the temperature sensitive TgNek1 phenotype 222 previously reported 14. Moreover, IFA with a-TgNek1 confirmed that TgNek1 223 expression is depleted in the presence of ATc (Fig. S1C). Interestingly, upon 224 depletion of TgNek1 the duplicated outer-cores remain connected whereas the

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225 inner-cores separate along with the kinetochores (Fig. 7). The distance between 226 separated kinetochores in TgNek1-cKD and wild type parasites is comparable, 227 which in turn is comparable with previously reported distances 7. This indicates that 228 the spindle MT polymerization is not affected when outer-core splitting is blocked. 229 In all, we concluded that the centrosome outer-core, inner-core and kinetochore are 230 hierarchically organized and that TgCep250 is responsible for connecting the inner- 231 and outer-core, but not the inner-core and the kinetochore. 232 233 234 Discussion 235 In mammalian cells, duplicated centrosomes are tethered together by a fiber 236 complex composed of C-NAP1/Cep250 21, 22 and LRRC45 23. 237 Phosphorylation of C-NAP1/Cep250 by NIMA kinase Nek2 promotes degradation of 238 C-NAP1/Cep250 and results in splitting of the linked centrosomes 21. Reciprocal 239 BLAST analysis suggests that C-NAP1/Cep250 and rootletin orthologs are absent 240 from the Toxoplasma genome 6, however, the regulatory machinery might be 241 conserved. 242 We showed here that the functional ortholog of human Nek2 in Toxoplasma, 243 TgNek1, is essential for outer-core splitting (Fig. 7) but not the inner-core. This 244 observation implies that substrate of TgNek1 only tethers the outer-core and the 245 mechanism that physically separates the inner- and outer-core is different. Both 246 TgNek1 and TgCep250 are closely associated with the centrosome outer-core, 247 however, their timing is different. TgNek1 is only transiently recruited to the 248 centrosome in G1/S phase transition and disappears after cell cycle progresses into 249 mitosis 14, which is just when TgCep250 shows up in the outer-core. Thus, although 250 we cannot exclude the possibility of TgCep250 is a TgNek1 substrate, the proteolytic 251 event of TgCep250 that leads to both inner- and outer-core localization is likely 252 independent from TgNek1 activity since TgNek1 is already absent from the 253 centrosome during cytokinesis. Phenotypic analysis of the TgCep250-cKD and 254 TgNek1-cKD phenotypes also showed distinct cell cycle defects indicating the roles 255 of TgCep250 and TgNek1 are functionally distinct. It is worth noticing that

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256 TgCep250 is phosphorylated at S252 in whole organism phopho-proteome data 24. 257 However, the function of this TgCep250 phosphorylation and the kinase that 258 phosphorylates it remains to be determined. 259 In mammalian cells, the differential decoration of centriole appendages 260 defines the difference in age and ability to nucleate microtubules between the 261 centriole pairs 17. Toxoplasma’s annotated genome does not contain orthologs of 262 mammalian centriole appendage and no clear appendage structure was 263 observed by transmission election microscopy. However, the observation that the 264 detachment of outer-core and inner-core occurs mostly in one pair of centrosomes 265 upon TgCep250 depletion provides the first evidence that daughter and mother 266 centrosomes can be differentiated in apicomplexan parasites. 267 Overduplication of outer-cores has been reported in parasites expressing a 268 temperature sensitive allele of PCM component MAPK-L1 7, whereas outer-core 269 fragmentation was observed in TgCep530 depleted parasites 8. In TgCep250 270 depleted cells we observed multiple outer-cores, which could either be the result of 271 overduplication or fragmentation (Fig. 4C). The TgCep250 phenotypes differs from 272 the MAPK-L1 defective cells as an accumulation of inner- and outer-cores aligned in 273 a “dumbbell form” was not observed. On the other hand, the TgCep250 phenotype is 274 also dramatically different from the multiple outer-cores of various intensity 275 observed in TgCep530 deficient parasites. The multiple outer-cores observed upon 276 TgCep250 depletion appear clearly organized and are of equal intensity, which is 277 more consistent with an over duplication than with fragmentation. In addition, we 278 observed normal initiation of daughter buds in absence of TgCep250 (Fig. 4D), 279 suggestive of fully functional outer-cores. 280 The above observation provides further support for the hypothesis of 281 independent function and regulation of inner- and outer-cores 7. In addition, the 282 formation of spindle microtubules on disconnected inner-cores (Fig. 5A) is also in 283 line with the independent functionality of the bifunctional centrosome cores. We 284 further tested this model in an IFA assay combining SFA, Centrin, TgCep250L1 and 285 TgNdc80 (Fig. 8), as the integrity of each of these detected structures is essential 286 and critical for the endowment of each daughter cell with a single copy of genetic

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287 material 4, 18. Indeed, we observed a hierarchical organization of these structures 288 required in wild type parasites (Fig. 8, upper panels). However, in TgCep250-cKD 289 cells, the connection between the inner-(green) and outer-core (blue) is lost while 290 association between the SFA and outer-core as well as between the kinetochore and 291 the inner-core remain intact (Fig. 8, bottom panels). 292 Our previous work showed that the kinetochore is required for Centrin 293 (outer-core) association with nucleus 20 by locking the MTs emanating from the 294 centrosome on the nuclear envelope 11. Using the novel tools developed here, we 295 now expanded these insights by showing that in TgNuf2-cKD cells the intact 296 inner/outer-core pair dissociates in its entirety from the nucleus (Fig. 6). Thus, we 297 can conclude that the kinetochore serves as a molecular anchor to lock the complete 298 centrosome cores, and that spindle MTs anchoring to the nuclear periphery is not 299 required to maintain the connection between the inner- and outer-core. 300 Taken together, our data support the notion of functional independency of 301 centrosomal cores as previous hypothesized. Furthermore, the observed differential 302 states of mother and daughter centrosomes provide support for the non-geometric 303 daughter expansion numbers observed in the Plasmodium spp. red blood cell cycle: 304 it was proposed that the mother centrosome could be sooner ready for another 305 round of duplication, associated with S-phase and mitosis, than the daughter 306 centrosome, which still had to mature 25. 307 TgCep250 is essential for structurally stability of the connection between the 308 centrosome cores and two different forms of Cep250 are present in tachyzoites: a 309 full-length form localizing to the outer-core during the transition between mitosis/ 310 cytokinesis and a proteolytically processed form localizing to the inner-core 311 throughout the tachyzoite cell cycle. TgNek1 is unlikely the kinase triggering 312 proteolytic cleavage. However, the tethering function of TgCep250 appears to be 313 maintained between human and Toxoplasma orthologs, but the structures 314 connected are tailored to each organism. The phosphorylation controls and the 315 proteolytic mechanisms remain unknown in Toxoplasma but provide an exciting 316 area for future research. 317

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318 Material and Methods 319 320 Parasite strains 321 Toxoplasma gondii tachyzoites of RHΔHX 26, RHΔKu80ΔHX 16, TATiΔKu80 27 and 322 their transgenic derivatives were grown in human foreskin fibroblast (HFF) as 323 described 28. 1 μM pyrimethamine, 20 μM of chloramphenicol, or 25 μg/ml 324 mycophenolic acid in combination with 50 μg/ml xanthine were used to select 325 stable transgenic parasite lines and cloned by serial dilution. 1.0 μg/ml of 326 anhydrotetracycline was used to repress specific of conditional 327 knockdown lines. Cep250Like-HA fosmid was kindly provided by Dr. Michael White, 328 University of South Florida 29. The tub-EB1-YFP/sagCAT plasmid has been described 329 before 11. 330 331 Plasmid constructs 332 All primers used are listed in Table S1. Endogenous tagging parasite strains were 333 generated as described 27. Briefly, 1 kb and 1.9 kb region upstream of the stop codon 334 of the TgCep250 gene were PCR amplified using RH genomic DNA as template and 335 were LIC-cloned into pYFP-LIC-DHFR (kindly provided by Dr. Vern Carruthers, 336 University of Michigan). NarI was used to linearize pCep250-YFP-LIC-DHFR for site- 337 specific homologous recombination. 338 Tetracycline regulatable parasite strains were generated by single-crossover 339 homologous recombination using a plasmid kindly provided by Dr. Wassim Daher 30. 340 The plasmid was re-designed by adding a single Myc-tag between the minimum 341 promoter and the N-terminal genomic region of the target genes. All the inserts 342 were amplified by PCR and digested with enzyme pairs BglII and NotI. To construct 343 Myc-Cep250-cKD line, the 5’ end of the TgCep250 was amplified, digested with BglII 344 and NotI enzyme pairs, and ligated into the DHFR-TetO7sag4-Myc vector. The 345 resulting construct was linearized using NcoI, electroporated into the TATiΔKu80 346 line by electroporation, and selected for pyrimethamine resistance. The Myc-Nek1- 347 cKD line was generated following the same strategy.

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348 The Myc-Cep250-3xTy-cKD line was generated by site-specific insertion of a 349 PCR product carrying the tag and HXGPRT selectable marker using CRISPR/Cas9. 350 The pU6 plasmid expressing the guide RNA and Cas9 nuclease was kindly provided 351 by Dr. Lourido from the Whitehead Institute 31. The CRISPR construct targeted the 352 3’UTR of TgCep250 near the stop codon. A primer pair was designed to amplify the 353 3xTy tag and the selectable marker (HXGPRT) flanking with 40 bp of homologous 354 region prior to the stop codon and after the protospacer-adjacent motif (PAM) site. 355 Amplicon generated from the primer pair was gel purified and co-transfected with 356 the protospacer and Cas9 encoding plasmid. 357 358 Immunofluorescence assay and microscopy 359 Parasites were seeded overnight (~16-18 h) and fixed with methanol as described 9. 360 The following antibodies were used in this study: Myc (MAb 9E10, mouse, 1:50; 361 Santa Cruz); HA (3F10, rat; 1:3000; Roche); IMC3 (rat, 1:2000 32); TgNuf2 and 362 TgNdc80 (guinea pig, 1:2000 20); SFA (rabbit, 1:1000 13 kindly provided by Dr. Boris 363 Striepen, University of Georgia); TgNek1 (1:1000, 14), HsCentrin (rabbit, 1:1000; 364 kindly provided by Dr. Iain Cheeseman, Whitehead Institute); Ty (mouse, 1:1000; 365 kindly provided by Dr. Lourido). Alexa Fluor A488, A568, A594, A633, and A647 366 secondary antibodies were used. 4’,6’-diamidino-2-phenylindole (DAPI) was used to 367 stain nuclear material. Images were acquired using a Zeiss Axiovert 200M wide-field 368 fluorescent microscope equipped with DAPI, FITC, YFP and TRITC filter sets, and a 369 Plan-Fluar 100X/1.45 NA oil objective and a Hamamatsu Orca-Flash 4.0LT camera. 370 For SIM super-resolution microscopy a Zeiss Elyra S.1 microscope equipped with a 371 Plan-Apochromat 63x/1.40 oil objective and PCO-Tech Inc. pco.edge 4.2 sCMOS 372 camera in the Boston College Imaging Core was used in consultation with Bret Judson. 373 Images were acquired and processed in Zeiss ZEN 2.3 software using standard 374 mode. 375 376 Western blot analysis 377 Freshy lysed parasites were collected after 3 μm filtration by centrifugation and 378 washed twice in 1X PBS. Parasite pellets were lysed by resuspension in 50 mM Tris-

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379 HCl (pH7.8), 150 mM NaCl, 1% SDS containing 1X protease inhibitor cocktail 380 (Sigma-Aldrich) and heating at 95°C for 10 min. Parasite lysates were analyzed by 381 SDS-PAGE on a 3-8% Tris-acetate gradient gel. Prior to PVDF membrane transfer the 382 gel was subjected to 100 mM acetic acid for 5 hr to break up the large TgCep250 383 protein. Transfer buffer contained 25 mM Tris pH 8.3, 195 mM glycine, 0.025% 384 (w/v) SDS and 15% methanol. Blots were hybridized with α-Myc-HRP, α- Ty or α-α- 385 tubulin (MAb 12G10; developmental studies hybridoma bank). 386 387 388 Acknowledgements 389 We thank William Jacobus for technical assistance, Drs. Carruthers, Cheeseman, 390 Daher, Lourido, Striepen, Suvorova, and White and for sharing reagents. We thank 391 Bret Judson and the Boston College Imaging Core for infrastructure and support and 392 Drs. White and Suvorova for fruitful suggestions and discussion. 393 This study was supported by National Science Foundation Major 394 Instrumentation Grant 1626072 and through support from National Institute of 395 Health grants AI110690, AI110638, and AI128136.

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396 References 397 398 1. Gubbels, M. J.; White, M.; Szatanek, T., The cell cycle and Toxoplasma gondii 399 cell division: Tightly knit or loosely stitched? Int J Parasitol 2008, 38 (12), 400 1343-58. 401 2. Alvarez, C. A.; Suvorova, E. S., Checkpoints of apicomplexan cell division 402 identified in Toxoplasma gondii. PLoS Pathog 2017, 13 (7), e1006483. 403 3. Brooks, C. F.; Francia, M. E.; Gissot, M.; Croken, M. M.; Kim, K.; Striepen, B., 404 Toxoplasma gondii sequesters centromeres to a specific nuclear region 405 throughout the cell cycle. Proc Natl Acad Sci U S A 2011, 108 (9), 3767-72. 406 4. Francia, M. E.; Striepen, B., Cell division in apicomplexan parasites. Nature 407 reviews. Microbiology 2014, 12 (2), 125-36. 408 5. Ferguson, D. J.; Hutchison, W. M.; Dunachie, J. F.; Siim, J. C., Ultrastructural 409 study of early stages of asexual multiplication and microgametogony of 410 Toxoplasma gondii in the small intestine of the cat. Acta Pathol Microbiol 411 Scand [B] Microbiol Immunol 1974, 82 (2), 167-81. 412 6. Morlon-Guyot, J.; Francia, M. E.; Dubremetz, J. F.; Daher, W., Towards a 413 molecular architecture of the centrosome in Toxoplasma gondii. Cytoskeleton 414 (Hoboken) 2017, 74 (2), 55-71. 415 7. Suvorova, E. S.; Francia, M.; Striepen, B.; White, M. W., A novel bipartite 416 centrosome coordinates the apicomplexan cell cycle. PLoS Biol 2015, 13 (3), 417 e1002093. 418 8. Courjol, F.; Gissot, M., A coiled-coil protein is required for coordination of 419 karyokinesis and cytokinesis in Toxoplasma gondii. Cell Microbiol 2018, 20 420 (6), e12832. 421 9. Gubbels, M. J.; Vaishnava, S.; Boot, N.; Dubremetz, J. F.; Striepen, B., A MORN- 422 repeat protein is a dynamic component of the Toxoplasma gondii cell 423 division apparatus. J Cell Sci 2006, 119 (Pt 11), 2236-45. 424 10. Nishi, M.; Hu, K.; Murray, J. M.; Roos, D. S., Organellar dynamics during the cell 425 cycle of Toxoplasma gondii. J Cell Sci 2008, 121, 1559-1568. 426 11. Chen, C. T.; Kelly, M.; Leon, J.; Nwagbara, B.; Ebbert, P.; Ferguson, D. J.; 427 Lowery, L. A.; Morrissette, N.; Gubbels, M. J., Compartmentalized Toxoplasma 428 EB1 bundles spindle microtubules to secure accurate 429 segregation. Mol Biol Cell 2015, 26 (25), 4562-76. 430 12. Anderson-White, B. R.; Beck, J. R.; Chen, C. T.; Meissner, M.; Bradley, P. J.; 431 Gubbels, M. J., Cytoskeleton assembly in Toxoplasma gondii cell division. Int. 432 Rev. Cell Mol. Biol. 2012, 298, 1-31. 433 13. Francia, M. E.; Jordan, C. N.; Patel, J. D.; Sheiner, L.; Demerly, J. L.; Fellows, J. D.; 434 de Leon, J. C.; Morrissette, N. S.; Dubremetz, J. F.; Striepen, B., Cell division in 435 Apicomplexan parasites is organized by a homolog of the striated rootlet 436 fiber of algal flagella. PLoS biology 2012, 10 (12), e1001444. 437 14. Chen, C. T.; Gubbels, M. J., The Toxoplasma gondii centrosome is the platform 438 for internal daughter budding as revealed by a Nek1 kinase mutant. J Cell Sci 439 2013, 126 (Pt 15), 3344-55.

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440 15. Agircan, F. G.; Schiebel, E.; Mardin, B. R., Separate to operate: control of 441 centrosome positioning and separation. Philos Trans R Soc Lond B Biol Sci 442 2014, 369 (1650). 443 16. Huynh, M. H.; Carruthers, V. B., Tagging of endogenous genes in a Toxoplasma 444 gondii strain lacking Ku80. Eukaryot Cell 2009, 8 (4), 530-9. 445 17. Bornens, M.; Gonczy, P., Centrosomes back in the limelight. Philos Trans R Soc 446 Lond B Biol Sci 2014, 369 (1650). 447 18. Beck, J. R.; Rodriguez-Fernandez, I. A.; Cruz de Leon, J.; Huynh, M. H.; 448 Carruthers, V. B.; Morrissette, N. S.; Bradley, P. J., A novel family of 449 Toxoplasma IMC proteins displays a hierarchical organization and functions 450 in coordinating parasite division. PLoS Pathog 2010, 6 (9), e1001094. 451 19. Chen, C. T.; Gubbels, M. J., Apicomplexan cell cycle flexibility: centrosome 452 controls the clutch. Trends Parasitol 2015, 31 (6), 229-30. 453 20. Farrell, M.; Gubbels, M. J., The Toxoplasma gondii kinetochore is required for 454 centrosome association with the centrocone (spindle pole). Cellular 455 Microbiology 2014, 16 (1), 78-94. 456 21. Fry, A. M.; Mayor, T.; Meraldi, P.; Stierhof, Y. D.; Tanaka, K.; Nigg, E. A., C-Nap1, 457 a novel centrosomal coiled-coil protein and candidate substrate of the cell 458 cycle-regulated protein kinase Nek2. J Cell Biol 1998, 141 (7), 1563-74. 459 22. Bahe, S.; Stierhof, Y. D.; Wilkinson, C. J.; Leiss, F.; Nigg, E. A., Rootletin forms 460 centriole-associated filaments and functions in centrosome cohesion. J Cell 461 Biol 2005, 171 (1), 27-33. 462 23. He, R.; Huang, N.; Bao, Y.; Zhou, H.; Teng, J.; Chen, J., LRRC45 is a centrosome 463 linker component required for centrosome cohesion. Cell Rep 2013, 4 (6), 464 1100-7. 465 24. Treeck, M.; Sanders, J. L.; Elias, J. E.; Boothroyd, J. C., The phosphoproteomes 466 of Plasmodium falciparum and Toxoplasma gondii reveal unusual 467 adaptations within and beyond the parasites' boundaries. Cell Host Microbe 468 2011, 10 (4), 410-9. 469 25. Arnot, D. E.; Ronander, E.; Bengtsson, D. C., The progression of the intra- 470 erythrocytic cell cycle of Plasmodium falciparum and the role of the 471 centriolar plaques in asynchronous mitotic division during schizogony. Int J 472 Parasitol 2011, 41 (1), 71-80. 473 26. Donald, R. G.; Roos, D. S., Gene knock-outs and allelic replacements in 474 Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run 475 mutagenesis. Mol Biochem Parasitol 1998, 91 (2), 295-305. 476 27. Sheiner, L.; Demerly, J. L.; Poulsen, N.; Beatty, W. L.; Lucas, O.; Behnke, M. S.; 477 White, M. W.; Striepen, B., A systematic screen to discover and analyze 478 apicoplast proteins identifies a conserved and essential protein import 479 factor. PLoS Pathog 2011, 7 (12), e1002392. 480 28. Roos, D. S.; Donald, R. G.; Morrissette, N. S.; Moulton, A. L., Molecular tools for 481 genetic dissection of the protozoan parasite Toxoplasma gondii. Methods Cell 482 Biol 1994, 45, 27-63. 483 29. Vinayak, S.; Brooks, C. F.; Naumov, A.; Suvorova, E. S.; White, M. W.; Striepen, 484 B., Genetic manipulation of the Toxoplasma gondii genome by fosmid 485 recombineering. MBio 2014, 5 (6), e02021.

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486 30. Morlon-Guyot, J.; Berry, L.; Chen, C. T.; Gubbels, M. J.; Lebrun, M.; Daher, W., 487 The Toxoplasma gondii Calcium Dependent Protein Kinase 7 is involved in 488 early steps of parasite division and is crucial for parasite survival. Cellular 489 Microbiology 2014, 16 (1), 95-114. 490 31. Sidik, S. M.; Hackett, C. G.; Tran, F.; Westwood, N. J.; Lourido, S., Efficient 491 genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS ONE 492 2014, 9 (6), e100450. 493 32. Anderson-White, B. R.; Ivey, F. D.; Cheng, K.; Szatanek, T.; Lorestani, A.; 494 Beckers, C. J.; Ferguson, D. J.; Sahoo, N.; Gubbels, M. J., A family of 495 intermediate filament-like proteins is sequentially assembled into the 496 cytoskeleton of Toxoplasma gondii. Cell Microbiol 2011, 13 (1), 18-31. 497

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498 Figure 1. Dynamic localization of TgCep250 throughout the cell cycle. A) IFA of 499 TgCep250-YFP parasites co-stained with α-GFP (Cep250, shown in green), α-Centrin 500 (centrosome outer-core marker, shown in magenta), α-HA (centrosome inner-core 501 marker shown in red) and DAPI (in blue). Images acquired by super-resolution 502 microscopy (SIM). B) Single-colored panels showed dynamic localization of 503 TgCep250-YFP during asexual division cycle. Cell cycle stages as indicated.

504

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505 Figure 2. Generation and validation of a TgCep250-cKD line. A) Schematic 506 representation of TgCep250 conditional knockdown (cKD) construction by single 507 homologous recombination. B) PCR validation using TATi-ΔKu80 (parent) and Myc- 508 TgCep250-cKD genomic DNA as templates. Primer pairs as indicated in Fig. 2A. C) 509 IFA using α-Myc and α-Centrin showed that the Myc-tagged TgCep250 is depleted 510 with in 6-hour incubation of ATc. Boxed areas magnified in the 4x zoom panels. D) 511 Western blot using α-Myc showed that the protein expression level is down- 512 regulated in the presence of ATc. The purple arrowhead marks the full-length 513 Cep250 protein (note that TgCep250 has a predicted molecular weight of 762 kDa); 514 the smaller bands present in both the - and +ATc samples and considered aspecific 515 and serve as loading control. 516

517

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518 Figure 3. Differential localization of TgCep250 is regulated by proteolysis. A) 519 Schematic representation of Myc-TgCep250-3xTy-cKD construction using 520 CRISPR/Cas9. B) Myc-TgCep250-cKD localized only to the outer-core (stained with 521 α-Myc and α-Centrin) and TgCep250-3xTy localized to both inner- and outer-core 522 (stained with α-Ty and α-HA on endogenously-tagged TgCep250L1-HA as inner- 523 core marker). Left panels are conventional wide-field microscopy; right panels are 524 super-resolution (SIM) microscopy. C) Multiple protein bands were detected in 525 western blot using α-Ty antibody suggestive of extensive proteolytic processing. α- 526 tubulin antiserum (12G10) was used as loading control. Orange arrowheads mark 527 the specifically detected TgCep250 protein fragments. D) Schematic interpretation 528 of western blot and IFA data representing the relationship between TgCep250 529 proteolysis and subcellular localization. 530

531

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532 Figure 4. Loss of TgCep250 causes lethal centrosomal defects. A) Plaque assay 533 of TgCep250-cKD and the TATi-ΔKu80 parent line ±ATc for seven days shows that 534 TgCep250 is essential. B) IFA of TgCep250-cKD stained with α-Centrin (in red) and 535 α-IMC3 (in green) ±ATc. Arrowheads mark parasites with nuclear loss; arrow marks 536 a nucleus outside a parasite; double arrowhead marks a parasite with two nuclei 537 and 3 outer-cores.; asterisk marks a parasite with daughter buds out of sync with 538 the rest of the vacuole. C) TgCep250-cKD parasites co-expressing TgCep250L1-HA 539 were stained with α-Centrin (in red) and α-HA (in green) marking the outer- and 540 inner-cores, respectively. Top panel shows IFA images representative of observed 541 phenotypes: grey boxed: control wild-type nuclei displaying duplicated 542 centrosomes; cyan boxed: outer-core duplication independent of inner-core 543 duplication; magenta boxed: stray inner-core complex in presence of two normal 544 appearing inner/outer pairs; yellow boxed: complete detachment of inner- and 545 outer-cores. Lower panel: quantification of defective centrosome phenotypes; bar 546 colors match the colored boxes in the top panel. At least 100 nuclei with a 547 duplicated centrosome pair were counted; error bars represent standard deviation 548 from 3 independent experiments D) Super-resolution (SIM) IFA of TgCep250-cKD 549 ±ATc co-expressing TgCep250L1-HA stained with α-ISP1 (green: early daughter 550 scaffold marker), α-Centrin (red: outer-core) and α-HA (cyan: inner-core). 551 Numbered, boxed areas are magnified on the right. Note that outer-cores are 552 normally associated with daughter scaffolds (asterisk), even if inner-core 553 connection is lost (arrowhead).

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554

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555 Figure 5. The inner-core remains associated with the mitotic apparatus in 556 TgCep250 depleted parasites. A) IFA of TgCep250-cKD ±ATc stained with α- 557 Centrin (outer-core; red) and co-stained with either α-TgNdc80 (kinetochore; green; 558 left panels) or co-expression of TgEB1-YFP (spindle microtubules; green; right 559 panels). Arrowheads mark nuclei displaying loss of outer-core association with the 560 kinetochore; double arrowheads mark nuclei displaying loss of outer-core 561 association with the spindle. B) IFA of TgCep250-cKD ±ATc expressing TgCep250L1- 562 HA (inner-core: α-HA; green) and co-stained α-TgNdc80 (kinetochore; red) showed 563 that the interaction between the kinetochore and inner-core centrosome remains 564 intact in the absence of TgCep250. 565

566

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567 Figure 6. The kinetochore is required to anchor centrosome cores to the 568 nuclear periphery. TgNuf2-cKD parasites treated ±ATc stained with centrosome 569 outer-core (α-Centrin, in green) and inner-core (TgCep250L1-HA; α-HA, in red;) 570 markers. Asterisks mark intact nucleus-centrosome connections; arrowheads mark 571 intact inner/outer-core pairs strayed from the nucleus (the associated small DAPI 572 spot is the plastid genome); arrows mark mis-segregated nuclei. 573

574

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575 Figure 7. Loss of TgNek1 prevents outer-core splitting but does not interfere 576 with inner-core separation. Image acquired by super-resolution microscopy (SIM) 577 of TgNek1-cKD parasites ±ATc using kinetochore (α-Ndc80), centrosome inner-core 578 (α-HA marking Cep250L1-HA) and outer-core (α-Centrin) markers. Boxed areas 579 magnified on the right.

580

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581 Figure 8. Hierarchical organization of the nuclear and cell division roles of the 582 Toxoplasma centrosome inner- and outer-cores. Left panels: super-resolution 583 (SIM) IFA of TgCep250-cKD co-expressing TgCep250L1-HA stained with α-HA 584 (green: inner-core), α-Centrin (blue: outer-core), α-TgSFA (magenta: daughter 585 tether) and α-TgNdc80 (red: kinetochore). Dotted lines outline parasites. Right 586 panels represent schematic representation of the data with corresponding colors. 587 Cyan represents the connection between the inner- and outer-core that is lost in the 588 daughter centrosome upon depletion of TgCep250. 589

590

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