Saccharomyces cerevisiae deficient in the early anaphase release of Cdc14 can traverse anaphase I without ribosomal DNA disjunction and successfully complete meiosis

ABSTRACT Eukaryotic meiosis is a specialized cell cycle of two nuclear divisions that give rise to haploid gametes. The phosphatase Cdc14 is essential for meiosis in the yeast Saccharomyces cerevisiae. Cdc14 is sequestered in the nucleolus, a nuclear domain containing the ribosomal DNA, by its binding partner Net1, and released in two distinct waves, first in early anaphase I, then in anaphase II. Current models posit that the meiosis I release is required for ribosomal DNA disjunction, disassembly of the anaphase spindle, spindle pole re-duplication and counteraction of cyclin-dependent kinase, all of which are essential events. We examined Cdc14 release in net1-6cdk mutant cells, which lack six key Net1 CDK phosphorylation sites. Cdc14 release in early anaphase I was partially inhibited, and disjunction of the rDNA was fully inhibited. Failure to disjoin the rDNA is lethal in mitosis, and we expected the same to be true for meiosis I. However, the cells reliably completed both meiotic divisions to produce four viable spores. Therefore, segregation of the rDNA into all four meiotic products can be postponed until meiosis II without decreasing the fidelity of chromosome inheritance.

meiosis II. The early anaphase release is thought to be important for disjunction of the ribosomal 48 DNA, disassembly of the anaphase I spindle, spindle pole re-duplication and the counteraction of 49 CDK, all of which are required for progression into meiosis II. The release of Cdc14 from its 50 nucleolar binding partner Net1 is stimulated by phosphorylation of cyclin-dependent kinase sites 51 in Net1, but the importance of that phospho-regulation in meiosis is not well understood. We 52 induced net1-6cdk mutant cells to enter meiosis and examined the localization of Cdc14 and 53 various indicators of meiotic progression. The net1-6cdk mutations inhibit, but don't fully 54 prevent Cdc14 release, and they almost completely prevent disjunction of the ribosomal DNA 55 during meiosis I. Failure to disjoin the ribosomal DNA is lethal in mitosis, and we expected the 56 same to be true in meiosis. However, the cells were able to complete meiosis II, yielding the 57 expected four meiotic products as viable spores. Therefore, all ribosomal DNA disjunction 58 required for meiosis can occur in meiosis II. We discuss the implications of these findings for 59 our understanding of meiotic chromosome segregation. 60 61  INTRODUCTION  62  63 In eukaryotes, the formation of gametes through meiosis requires the execution of a reductional 64 chromosome segregation (meiosis I) and a subsequent equational division (meiosis II) (1). 65 Diploid cells of the yeast S. cerevisiae can undergo meiosis to produce an ascus containing four 66 spores, each with the necessary haploid chromosome content. A variety of mutations studied in 67 S. cerevisiae, including severe mutations and deletions of the SLK19, SPO12 and CDC14 genes, 68 prevent cells from fully completing two meiotic chromosome divisions (2-6). 69 Cdc14 protein undergoes a distinctive localization cycle during cell division, and the 70 dynamics of its localization are important for regulating its activity. From G1 to metaphase, 71 Cdc14 is stored within the nucleolus, attached to its binding partner Net1 in a chromatin 72 silencing complex termed the RENT (7-9). In early anaphase of mitosis, Cdc14 is released from 73 the RENT in a process that requires the Slk19 and Spo12 proteins and phosphorylation of Net1 74 by cyclin-dependent kinase (CDK) (10,11). Slk19 and Spo12 are also required for the release of 75 Cdc14 during anaphase I of meiosis (Buonomo et al. 2003;Marston et al. 2003). The early 76 anaphase release of Cdc14, abbreviated FEAR (Fourteen Early Anaphase Release), is seen 77 cytologically in both mitosis and meiosis I as a brief redistribution of the protein from the 78 nucleolus throughout the nucleus without its export to the cytoplasm, followed by its return to 79 the nucleolus near the end of anaphase (12). While non-essential for mitosis, FEAR is thought to 80 be absolutely required for the completion of two rounds of meiotic division. 81 A separate, and essential pathway, the mitotic exit network (MEN), releases Cdc14 again at 82 the end of anaphase of mitosis (7,9). The MEN, in addition to releasing Cdc14, drives its export 83 from the nucleus to the cytoplasm (13). The MEN is also active in meiosis II, when Cdc14 is 84

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Yeast strains 126 All strains of S. cerevisiae used in this study were of the W303 background. Strain names and 127 their genotypes are listed in Table 1. Mutations and epitope fusion alleles that were not part of 128 the background genotype are described in detail in Table 2  CDC14-7MYC-HIS3MX6 C-terminal 7MYC fusion as per (36) Media and growth conditions 132 YPAD was used for pre-sporulation growth of S. cerevisiae cultures, and SC dropout media were 133 used to score the genetic markers of dissected tetrads. YPAD, SC dropouts and liquid 134 sporulation media were standard and have been previously described (39). A growth 135 temperature of 30 o C was used for both routine growth and during induction of meiosis. 136 137

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The net1-6cdk mutations impair the quantitative release of Cdc14 during meiosis I 196 During anaphase of meiosis I, S. cerevisiae cells release Cdc14 into the nucleus from its storage 197 location within the nucleolus. Since the net1-6cdk mutations severely inhibit FEAR in mitosis 198 (12), we wanted to observe the phenotypes of these mutations in meiosis. 199 Our wild-type cells behaved as expected, quantitatively releasing Cdc14 and reaching 200 maximal release in early and mid-anaphase I ( Figures 1A and 1B). In contrast, less than half of 201 net1-6cdk cells observed in anaphase I detectably released Cdc14, and only one sixth released 202 Cdc14 as robustly as wild-type. The quantitative data derived from scoring individual images 203 are in Table S1. The release was often partial, with a substantial amount of the protein 204 remaining in a concentrated region. By late anaphase I, both wild-type and net1-6cdk cells had 205 begun to relocalize Cdc14 efficiently to the nucleolus. 206 For comparison, we analyzed mutants lacking the Slk19 and Spo12 proteins, both of which 207 have been extensively studied and are known to be required for the release of Cdc14. Both 208 slk19Δ and spo12Δ mutations severely inhibited the release of Cdc14 in early anaphase ( Figures  209   1A and 1B). Unlike wild-type and net1-6cdk cells, however, cells of both deletion genotypes 210 proceeded to release some Cdc14 during late anaphase, and many cells reached telophase with 211 Cdc14 partially released ( Figure 1A and Table S1). To our knowledge, this delayed release has 212 not previously been reported, and would be difficult to see except in the type of intensive single-213 cell imaging we carried out. The meiotic phenotypes of slk19Δ and spo12Δ cells are more 214 complex and extensive than what we have observed for net1-6cdk, including the execution of 215 chromosome segregation patterns that are a mix of MI and MII (4,6). It is possible that the late 216 meiosis I release of Cdc14 we observed in slk19Δ and spo12Δ cells represents partial 217 biochemical progression into meiosis II, during which the MEN is active (16). 218 In summary, the net1-6cdk mutations impaired the quantitative anaphase I release of Cdc14, 219 although less severely than slk19Δ and spo12Δ. We don't know how much released Cdc14 220 might be required for its meiosis I activities. Therefore, we must consider that the hypomorphic 221 net1-6cdk allele may compromise different meiosis I activities of Cdc14 to varying degrees. Since net1-6cdk cells had efficiently returned Cdc14 to the nucleolus by late anaphase I, we 230 were able to infer the position of the nucleolus from Cdc14 localization. In striking contrast to 231 wild-type, the majority of net1-6cdk cells failed to disjoin the nucleolus (Figures 2A and 2B). 232 During the late stages of spindle elongation and into telophase, when spindle breakdown was 233 under way, Cdc14 remained in a single mass positioned between the divided chromosomal DNA. 234 Because slk19Δ and spo12Δ cells had mostly released Cdc14 during anaphase spindle elongation, 235 we were unable to observe the positions of their nucleoli, but previous findings indicate they also 236 fail to disjoin the rDNA (5). By meiotic metaphase II, the vast majority of net1-6cdk cells still 237 maintained the nucleolus in a single mass. The quantitative data derived from scoring individual 238 images are in Table S2. There was some re-grouping of the nucleoli into a single mass in 239 metaphase II wild-type cells, probably due to the relaxation of spindle tension. 240 For additional confirmation of rDNA positioning, we examined the localization of the 241 nucleolar protein Nop1 ( Figure 2C). All of the wild-type or net1-6cdk cells that we observed up 242 to mid-anaphase I had a single mass of Nop1 as expected, since nucleolar segregation occurs in 243 late anaphase I. When we looked at cells in late anaphase or telophase I, however, all seven 244 wild-type cells that we observed had separated Nop1 into two masses, while none of the seven 245 net1-6cdk cells had done so . In summary, localization of the Nop1 protein gave results 246 consistent with Cdc14; the vast majority of net1-6cdk cells traversed meiosis I without disjoining 247 their rDNA. 248 249

Meiosis I spindles appear normal in net1-6cdk cells 250
Cdc14 is required for microtubule spindle growth and stability in meiosis, and both Slk19 and 251 Spo12 are required for normal meiotic spindle morphology (5,44). Slk19 is required for spindle 252 midzone stability independent of its role in Cdc14 release (Havens et al. 2010), and in both slk19 253 Δ and spo12Δ mutants, the anaphase I spindle persists when progression into meiosis II requires 254 that it be disassembled (5). 255 We considered that the failure of net1-6cdk cells to disjoin the rDNA could result from 256 impairment of the spindle. In our observations of Cdc14 localization, the anaphase I spindles of 257 net1-6cdk cells had appeared normal, with spindle disassembly proceeding as the cells 258 approached telophase (Figures 1A and 2A). As previously reported, slk19Δ cells often had a 259 weakened spindle midzone and short spindles, a phenotype that was particularly evident in mid-260 anaphase I. We confirmed that this phenotype was unique to the slk19Δ cells, and that the 261 spindle midzones of net-6cdk and spo12Δ cells were similar to wild-type ( Figure S1). Our 262 analysis did not provide any insight into spindle dynamics in the mutants, since the data were 263 snapshots from asynchronous populations. In summary, net1-6cdk cells have an apparently 264 normal meiosis I spindle which should supply the force necessary to disjoin the rDNA, and is 265 disassembled as cells complete meiosis I.   ). Since the slk19Δ phenotype is difficult to assess and has previously been described, we did 285 not include it in the analysis. 286 After 36 hours in sporulation medium net1-6cdk cells had formed tetrad asci with an 287 efficiency similar to wild-type cells, although the mutant formed slightly more dyad asci than the 288 wild-type ( Figure 3B). It is hard to definitively distinguish triads from tetrads, but we informally 289 observed that the spores formed by slk19Δ were often of unequal sizes and appeared to contain a 290 high proportion of dyads and triads. As expected, spo12Δ cells almost exclusively formed dyads. 291 We examined spore viability by dissecting asci, and the data are reported in Table 3. The 292 viability of spores from homozygous net1-6cdk cells, and a heterozygous mutant strain that we 293 also dissected, were as high as wild-type cells at 97%. We also analyzed the viability of spores 294 from slk19Δ and spo12Δ dyad asci. The slk19Δ mutant produced 44% viable spores, while 295 spo12Δ spore viability was nearly normal at 86%. The simplest explanation for low spore 296 viability is inaccurate chromosome segregation and conversely, the formation of viable spores by 297 the net1-6cdk mutant indicated that all chromosomes were faithfully segregated. 298 299 and net1-6cdk cells (Figure 4). In summary, the net1-6cdk mutant produced only slightly 303 elevated levels of dyad asci, and otherwise was highly proficient in the completion of meiosis. 304 305 C-terminal epitope fusions to Cdc14 and Net1 compromise the activity of the proteins 306 During our investigation of dyad formation, we observed that a C-terminal Net1-6HA epitope 307 fusion caused elevated levels of dyads to form, an effect that was synergistic with the net1-6cdk 308 mutations, leading to even higher levels of dyads ( Figure 3B). Recent work has revealed a role 309 for the C-terminus of Net1 in activating RNA polymerase I transcription (46). The C-terminal 310 Cdc14-7MYC epitope fusion to Cdc14 was also synergistic with net1-6cdk for dyad formation, 311 while Cdc14-3MYC had only a very weak effect. It has been reported that a C-terminal Cdc14-312 3HA epitope fusion strongly inhibits meiosis in yeast cells of the SK-1 background (47). The 313 effects we observed were modest, indicating that the penetrance of such phenotypes depends 314 strongly on the genetic background. Nevertheless, it appears that the C-termini of both Cdc14 315 and Net1 have activities that affect meiotic chromosome segregation. The study of C-terminal 316 mutations of these proteins may yield additional insight into these sensitive functions. 317

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The early anaphase release of Cdc14 has been linked to a variety of events necessary for the 320 termination of mitosis (23,48) and meiosis I. However, it has been difficult to ascertain whether 321 FEAR is directly responsible for the coincident events of chromosome segregation, spindle 322 disassembly, spindle pole reduplication and CDK downregulation, particularly since the MEN 323 becomes active soon after anaphase and is functionally redundant for the completion of those 324 events. The use of highly specific mutations is one approach to untangling this problem, and we 325 have previously used the net1-6cdk allele to show that FEAR does not have a significant role in 326 mitotic nuclear division, spindle morphogenesis and mitotic exit -events closely associated with 327 cell cycle progression (12). With the caveat that net1-6cdk is hypomorphic for meiotic FEAR, 328 our current findings suggest that, in meiosis, bulk nuclear division, spindle morphogenesis, and 329 progression into meiosis II are likewise independent of FEAR. There are many additional CDK 330 candidate phosphorylation sites in Net1 (11), and in future investigations, it may be useful to test 331 different combinations of mutations of those sites to search for alleles that are more severely 332 inhibitory to meiotic FEAR. 333 We found only one strong phenotype for the net1-6cdk allele in meiosis, a phenotype it 334 shares with the classic FEAR mutants slk19Δ and spo12Δ -failure to disjoin the rDNA in 335 meiosis I. From studies of mitotic cells we know that rDNA disjunction depends on two 336 important activities of Cdc14 related to rDNA chromatin organization: condensin loading 337 (17,20,49,50) and control of transcription within the rDNA (41,51). Our current findings suggest 338 that the phospho-regulation of Net1, while it is important for the retention and release of Cdc14 339 from the RENT complex, primarily affects rDNA chromatin organization rather than cell cycle 340 progression per se. The slk19Δ and spo12Δ alleles delay the phosphorylation of Net1, at least at 341 the T212 CDK site (11). Therefore it is an open possibility that Slk19 and Spo12 similarly 342 promote rDNA disjunction by stimulating phosphorylation of Net1. 343 Disjunction of the rDNA occurs in meiosis I and was thought to be important for faithful 344 chromosome segregation. In mitosis, failure to disjoin the rDNA is lethal, so how can it be non-345 lethal in meiosis I, as we found it to be in net1-6cdk mutant cells? In yeast, both meiotic nuclear 346 divisions occur within the mother cell cytoplasm, without the accompanying division of the 347 nuclear membrane or cytokinesis that occurs in mitosis (52,53). In metazoans, the nuclear 348 envelope breaks down during meiosis (54). Therefore, until gametes are packaged at the end of 349 meiosis, there is no physical structure to sever the lagging chromosomal domains. 350 Cells with net1-6cdk, slk19Δ or spo12Δ mutations fail to disjoin the rDNA during meiosis I, 351 but they must eventually do so in order to form viable spores, and even the deletion mutants form 352 some viable spores. net1-6cdk cells proceed efficiently into meiosis II, and in late anaphase of 353 meiosis II they release Cdc14 similarly to wild-type. If Cdc14 release is the critical event that 354 drives rDNA disjunction, then its release in meiosis II seems to be fully redundant and able to 355 disjoin the rDNA loci of both bivalent homologous chromosomes (meiosis I disjunction) and 356 sister chromatids (meiosis II disjunction). Likewise, the late partial release of Cdc14 in slk19Δ 357 and spo12Δ cells may be responsible for some level of rDNA disjunction. 358 Our overall conclusions about the release of Cdc14 in early anaphase of meiosis parallel what 359 we previously found for mitosis (12). FEAR is not required for a variety of key cell cycle 360 events. Instead, it is critical for rDNA disjunction, and the MEN appears to be fully redundant 361 for cell cycle progression. Cell cycle regulation by Cdc14 has a long history of investigation 362 (55-57), but the general model for Cdc14 in counteraction of CDK activity has recently been 363 called into question (58). We hope our findings will help distinguish the roles of Cdc14 in the 364 cell cycle from other critical but indirectly related activities. 365 366 367 ACKNOWLEDGEMENTS 368 369 I wish to thank Jennifer Fung and Peter B. Yellman for advice about image processing. 370