BUB-1 and CENP-C recruit PLK1 to Control Chromosome Alignment and Segregation 1 During Meiosis I in C . elegans Oocytes 2 3

Phosphorylation is a key post-translational modification that is utilised in many biological processes for the rapid and reversible regulation of protein localisation and activity. Polo-like kinase 1 (PLK-1) is essential for both mitotic and meiotic cell divisions, with key functions being conserved in eukaryotes. The roles and regulation of PLK-1 during mitosis have been well characterised. However, the discrete roles and regulation of PLK-1 during meiosis have remained obscure. Here, we used Caenorhabditis elegans (C. elegans) oocytes to show that PLK-1 plays distinct roles in meiotic spindle assembly and/or stability, chromosome alignment and segregation, and polar body extrusion during meiosis I. Furthermore, by a combination of live imaging and biochemical analysis we identified the chromosomal recruitment mechanisms of PLK-1 during C. elegans oocyte meiosis. The spindle assembly checkpoint kinase BUB-1 directly recruits PLK-1 to the kinetochore and midbivalent while the chromosome arm population of PLK-1 depends on a direct interaction with the centromeric-associated protein CENP-CHCP-4. We found that perturbing both BUB-1 and CENP-CHCP-4 recruitment of PLK-1 leads to severe meiotic defects, resulting in highly aneuploid oocytes. Overall, our results shed light on the roles played by PLK-1 during oocyte meiosis and provide a mechanistic understanding of PLK-1 targeting to meiotic chromosomes.

PLK-1 plays distinct roles in meiotic spindle assembly and/or stability, chromosome 23 alignment and segregation, and polar body extrusion during meiosis I. Furthermore, by a 24 combination of live imaging and biochemical analysis we identified the chromosomal 25 recruitment mechanisms of PLK-1 during C. elegans oocyte meiosis. The spindle assembly 26 checkpoint kinase BUB-1 directly recruits PLK-1 to the kinetochore and midbivalent while 27 the chromosome arm population of PLK-1 depends on a direct interaction with the 28 centromeric-associated protein CENP-C HCP-4 . We found that perturbing both BUB-1 and 29 CENP-C HCP-4 recruitment of PLK-1 leads to severe meiotic defects, resulting in highly 30 aneuploid oocytes. Overall, our results shed light on the roles played by PLK-1 during oocyte 31 meiosis and provide a mechanistic understanding of PLK-1 targeting to meiotic 32 chromosomes. 33 . CC-BY-NC 4.0 International license made available under a (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 The copyright holder for this preprint this version posted March 28, 2023. In C. elegans, PLK-1 localisation in meiosis and mitosis is similar to other organisms (Chase 88 et al., 2000). In mitosis, roles of PLK-1 include nuclear envelope breakdown (NEBD) 89 elegans. However, meiotic roles of PLK-1 in C. elegans oocytes have remained obscure, as 94 PLK-1 depletion results in severely defective NEBD and fertilised oocytes with a whole 95 nucleus rather than condensed chromosomes (Chase et al., 2000). In the PLK-1-depleted 96 oocytes that 'escaped' the NEBD defect, chromosome congression, segregation, and polar 97 body extrusion were severely disrupted. However it is unclear whether these phenotypes are 98 indirect effects of the severe early meiotic defects or whether they result from specific 99 functions of PLK-1 throughout meiosis (Chase et al., 2000). Furthermore, while PLK-1 was 100 shown to localise broadly to chromosomes and the spindle during meiosis (Chase et al., 101 essential for meiosis I. 114 . CC-BY-NC 4.0 International license made available under a (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 The copyright holder for this preprint this version posted March 28, 2023. ; https://doi.org/10.1101/2022.10.07.511262 doi: bioRxiv preprint

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PLK-1 plays roles in spindle assembly and/or stability, chromosome alignment and 116

segregation, and polar body extrusion in meiosis I 117
A previous study showed that PLK-1 localises to the meiotic spindle and chromosomes in C. 118 elegans oocytes and depletion of PLK-1 using RNAi led to several defects including defective 119 NEBD, chromosome congression, segregation, and polar body extrusion (Chase et al., 2000). 120 While this suggested that PLK-1 plays several roles during meiosis, the use of RNAi presents 121 a limitation to addressing them independently. In particular, the strong NEBD defect 122 complicates delineation of the roles of PLK-1 at later stages of meiosis. Therefore, we sought 123 to understand the distinct localisation and roles of PLK-1 during meiosis I. 124 To assess PLK-1 localisation during meiosis with high spatial and temporal resolution, we 125 imaged endogenously tagged PLK-1::sfGFP in dissected oocytes. PLK-1 localises to the 126 spindle poles ( Figure 1C that renders it sensitive to chemically modified derivatives of PP1, a Src family inhibitor 138 (Bishop et al., 2000). We reasoned that acute PLK-1 as inhibition for a short period of time 139 . CC-BY-NC 4.0 International license made available under a (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 8 would allow us to study the post-NEBD effects ( Figure 1D). We tested a variety of analogues 140 and all of them led to embryonic lethality of the plk-1 as strain without severely affecting a 141 wild type strain ( Figure 1-figure supplement 2). We decided to continue our experiments with 142 the 3-substituted benzyl PP1 derivative 3IB-PP1 (Figure 1-figure supplement 2). A wild type 143 strain in the presence of 3IB-PP1 and plk-1 as in the presence of vehicle control ('EtOH') 144 behaved normally during meiosis ( Figure 1E,F & Figure 1-figure supplement 3). Addition of 145 10 µM 3IB-PP1 between 5 and 15 min before dissection and imaging of oocytes allowed us to 146 bypass the NEBD defect and 6 bivalents were easily identifiable within the newly fertilised 147 oocyte ( Figure 1E, yellow arrows). Under these conditions PLK-1 as inhibition led to drastic 148 spindle defects with no observable bipolar spindle formation and no consequent chromosome 149 segregation was observed, indicating that PLK-1 is involved in spindle assembly and/or 150 stability during oocyte meiosis ( Figure 1E). We sought to minimise the spindle defects upon 151 PLK-1 inhibition by reducing the concentration of 3IB-PP1 to 1 μM and omitting the pre-152 treatment step prior to dissection. Under these conditions, ~62% of oocytes had seemingly 153 bipolar spindles and chromosomes remained somewhat associated with the spindle, although 154 chromosome alignment was still affected in 78% of oocytes (2 misaligned chromosomes, 155 Figure 1F,G). A more detailed analysis of chromosome dynamics after PLK-1 inhibition is 156 presented in Figure 1-figure supplement 3, where individual chromosomes are followed every 157 20 seconds and, as opposed to wild type, chromosomes from PLK-1-inhibited oocytes show a 158 highly dynamic behaviour whereby they seem to briefly align and then become misaligned 159 again (Figure 1-figure supplement 3 and arrows therein). We then used the pole marker 160 ASPM-1 to allow proper characterisation of spindle bipolarity under these conditions and 161 confirmed that even when two ASPM-1 poles are clearly discerned ( Figure 1H, blue arrows), 162 chromosome alignment fails ( Figure 1H, yellow arrows). Hence, it appears that PLK-1 163 . CC-BY-NC 4.0 International license made available under a (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 The copyright holder for this preprint this version posted March 28, 2023. ; https://doi.org/10.1101/2022.10.07.511262 doi: bioRxiv preprint participates in chromosome alignment in a manner that is at least partially independent of its 164 roles in overall spindle assembly and/or stability. 165 166 BUB-1 recruits PLK-1 to the midbivalent during oocyte meiosis 167 To further understand the role of PLK-1 during meiosis, we sought to identify the PLK-1 168 recruitment mechanism(s). In mammalian mitosis, the kinase BUB-1 and its paralog BUBR1 169 directly recruit PLK-1 to the kinetochore via STP motifs that are phosphorylated by Cdk1 170  Figure 2C). Therefore, we sought to identify whether C. elegans BUB-1 179 directly interacts with PLK-1 to mediate its recruitment to the midbivalent region. 180

BUB-1 directly interacts with PLK-1 through a Cdk1-dependent STP motif 182
To test whether BUB-1 directly interacts with PLK-1 in vitro, we purified a recombinant 183 fragment of BUB-1 encompassing the intrinsically disordered region between the TPR and 184 kinase domains that contains the putative STP motif ('BUB-1 190-628' , Figure 2D (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 The copyright holder for this preprint this version posted March 28, 2023. ; https://doi.org/10.1101/2022.10.07.511262 doi: bioRxiv preprint higher volume ( Figure 3C). Together, these data indicate that there is a Cdk1 214 phosphorylation-dependent interaction between BUB-1 and PLK1 PBD that requires 215 phosphorylation of T527 within the STP motif of BUB- 1. 216 To further confirm that the STP motif of BUB-1 can interact with PLK1 PBD in a phT527-217 dependent manner, FITC-labelled peptides containing the BUB-1 STP motif were incubated 218 with either wild type MBP-PLK1 PBD or a mutant that cannot interact with phSTP motifs 219 Altogether, these data indicate that C. elegans BUB-1 can directly bind to PLK1 PBD in vitro in 224 a phospho-dependent manner via a newly characterised STP motif. 225

BUB-1 directly recruits PLK-1 to the midbivalent in vivo 227
We then sought to determine whether the STP motif in BUB-1 is responsible for PLK-1 228 recruitment in vivo. Using CRISPR-Cas9, we generated the T527A mutation in the 229 endogenous bub-1 locus (bub-1 T527A ). bub-1 T527A mutant worms showed significant embryonic 230 and larval lethality so we generated a balanced strain in which the bub-1 T527A allele was 231 maintained as a heterozygote. Homozygous bub-1 T527A worms from heterozygous parents 232 develop to adulthood and produce oocytes that go through meiosis, which allowed us to study 233 the role of the STP motif in BUB-1 during meiosis. PLK-1 was absent from the midbivalent 234 in bub-1 T527A oocytes, reminiscent of the bub-1(RNAi) phenotype ( Figure 4A, blue 235 arrowheads, and 4B). Importantly, BUB-1 localisation to the midbivalent and kinetochore 236 was maintained in the bub-1 T527A strain ( Figure 4C), indicating that BUB-1 directly interacts 237 with PLK-1 via this STP motif in vivo to recruit PLK-1 to the midbivalent. To assess the 238 . CC-BY-NC 4.0 International license made available under a (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 The copyright holder for this preprint this version posted March 28, 2023.   To further confirm this STP motif binds to MBP-PLK1 PBD in a phT163-dependent manner, 296 we conducted fluorescence polarisation assays with FITC-labelled CENP-C HCP-4 STP motif 297 peptides ( Figure 6D). The phT163 peptide bound to MBP-PLK1 PBD with a KD of 104 ± 14 298 nM but did not interact with MBP-PLK1 PBD (H538A/K540M), while the unphosphorylated 299 peptide did not bind to MBP-PLK1 PBD at the concentrations tested ( Figure 6D). Collectively, 300 these data indicate that the putative STP motif in CENP-C HCP-4 binds to the PBDs of PLK-1 in 301 a Cdk1 phosphorylation-dependent manner, which led us to assess the importance of this STP 302 motif in vivo. 303

CENP-C HCP-4 recruits PLK-1 to chromosome arms in vivo through an STP motif 305
The T163A mutation in CENP-C HCP-4 was generated in the endogenous hcp-4 locus (hcp-306 4 T163A ) which, unlike bub-1 T527A , did not affect viability. When PLK-1::sfGFP was monitored 307 in dissected oocytes, hcp-4 T163A recapitulated the full CENP-C HCP-4 depletion with PLK-1 308 localising to the midbivalent and kinetochore but largely absent from chromosome arms 309 ( Figure 7A,B). This indicates that PLK-1 is targeted to chromosome arms directly through the 310 phospho-dependent STP motif in CENP-C HCP-4 . Importantly, CENP-C HCP-4 (T163A) displays 311 . CC-BY-NC 4.0 International license made available under a (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 The copyright holder for this preprint this version posted March 28, 2023. ; https://doi.org/10.1101/2022.10.07.511262 doi: bioRxiv preprint an indistinguishable localisation from wild type CENP-C HCP-4 ( Figure 7C) and the other PLK-312 1 receptor, BUB-1, also localises normally in the hcp-4 T163A strain ( Figure 7D). 313 These data indicate that CENP-C HCP-4 directly recruits PLK-1 to the chromosome arms during 314 meiosis I via a newly characterised N-terminal STP motif. These data indicate that recruitment of PLK-1 to the midbivalent and kinetochore by BUB-1 358 appears to be primarily responsible for the chromosomal roles of PLK-1 during meiosis I, as 359 disruption of this pathway leads to meiotic defects while perturbing CENP-C HCP-4 recruitment 360 of PLK-1 on its own does not. However, the fact that disruption of both BUB-1 and CENP-361 . CC-BY-NC 4.0 International license made available under a (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 The copyright holder for this preprint this version posted March 28, 2023. ; https://doi.org/10.1101/2022.10.07.511262 doi: bioRxiv preprint C HCP-4 recruitment pathways enhances the severity of the resulting meiotic defects indicates 362 that the CENP-C HPC-4 pathway does still play an active part in the roles of PLK-1 during 363 meiosis I. 364 . CC-BY-NC 4.0 International license made available under a (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 In the present manuscript we describe specific, post-NEBD roles played by PLK-1 during 366 oocyte meiosis. PLK-1 is important for spindle assembly and/or stability, chromosome 367 alignment and segregation, and polar body extrusion during meiosis I. We found that PLK-1 368 localises to spindle poles and chromosomes during metaphase I before localising to the 369 chromosomes and central spindle in anaphase. Furthermore, we characterised the mechanisms 370 of chromosomal PLK-1 targeting during oocyte meiosis, which rely on the centromere-371 associated protein CENP-C HCP-4 and the spindle assembly checkpoint kinase BUB-1. (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