An evolutionary perspective on the relationship between kinetochore size and CENP-E dependence for chromosome alignment

At the onset of mitosis, DNA is packed into chromosomes, the nuclear envelope breaks down, and scattered chromosomes start to interact with spindle microtubules via proteinaceous structures that localize on the centromeric region of each sister chromatid, known as kinetochores (Musacchio and Desai, 2017). In vertebrates, when chromosomes are favorably positioned between the two spindle poles, they establish end-on kinetochore–microtubule attachments and align soon after bi-orientation, a pathway known as ‘direct congression’ (Auckland and McAinsh, 2015; Barisic et al., 2014; Klaasen et al., 2022; Maiato et al., 2017). In contrast, more peripheral chromosomes are first brought to the vicinity of spindle poles along laterally attached astral microtubules by the microtubule minus-end-directed motor protein dynein at kinetochores (Li et al., 2007; Vorozhko et al., 2008; Yang et al., 2007). Subsequently, chromosomes are transported towards the equator by the dominant role of the microtubule plus-end-directed kinesin motor CENP-E (kinesin-7), also at kinetochores, that prevents random polar-ejection forces (PEFs) assisted by chromokinesins on chromosome arms (Barisic et al., 2014; Kapoor et al., 2006). Thus, at least in vertebrates, CENP-E at kinetochores is the critical motor driving the alignment of peripheral chromosomes to the cell equator.

One often underappreciated aspect underlying chromosome interactions with mitotic spindle microtubules is kinetochore size. Kinetochore size varies among different animal and plant species (Comings and Okada, 1971; Maddox et al., 2004; Maiato et al., 2006; Malheiros et al., 1947; McEwen et al., 1998; Neumann et al., 2015; Ribeiro et al., 2009), between different chromosomes within a given species (including humans) (Cherry et al., 1989; Cherry and Johnston, 1987; Drpic et al., 2018; Logsdon et al., 2024; McEwen et al., 1998; Nixon et al., 2017; Parl, 2024; Peretti et al., 1986; Sacristan et al., 2024; Sanchez et al., 1991; Tomkiel et al., 1994), and in response to microtubule attachments throughout mitosis (Hoffman et al., 2001; Magidson et al., 2015; Sacristan et al., 2018). How kinetochore size impacts the mechanisms of chromosome alignment remains poorly understood. Based on previous live-cell recordings of Indian muntjac cells with only three large monocentric chromosome pairs and distinct/resolvable kinetochore sizes, we speculated that chromosomes with larger kinetochores depend less on CENP-E activity to bi-orient and align because a larger kinetochore surface area (and possibly a more favorable shape) favors the interaction with spindle microtubules (Drpic et al., 2018). Interestingly, despite the presence of CENP-E in branches of all major eukaryotic lineages suggesting that it was present in the last eukaryotic common ancestor (van Hooff et al., 2017), some metazoans with holocentric chromosomes, such as the Caenorhabditis elegans nematode, the kinetochores of which extend along the entire chromosome length, lack a bona fide CENP-E ortholog (Maddox et al., 2004). In this case, proper bi-orientation of sister kinetochores occurs via ‘direct congression’, where kinetochores on holocentric chromosomes initially capture microtubules from fully separated centrosomes soon after nuclear envelope breakdown (NEBD) (Redemann et al., 2017), in a process that is facilitated by the non-kinetochore chromokinesin KLP-19 (Kinesin-4) that assists PEFs required for the stabilization of end-on kinetochore–microtubule attachments (Powers et al., 2004). Whether this represents a C. elegans peculiarity or is a more general feature of species with holocentric chromosomes remained unclear. Altogether, these data raise the exciting possibility that CENP-E and the associated, motor-dependent, chromosome alignment pathway became dispensable for mitosis and were lost during evolution in species with relatively large kinetochores, such as those in holocentric chromosomes.

Because absolute kinetochore sizes remain largely unknown for most species, here, we set out to test this hypothesis by systematically investigating the presence of CENP-E orthologs in phylogenetically diverse metazoan taxa with either holocentric or monocentric chromosomes for which high-quality genome sequence data are available. Strikingly, we found a marked propensity for CENP-E loss in taxa with holocentric chromosomes and relatively larger kinetochores compared to taxa with monocentric chromosomes. We then focused our functional analysis on two closely related Nematoda with holocentric chromosomes, in which a CENP-E ortholog was either absent (C. elegans) or present (Pristionchus pacificus). Remarkably, targeted expression of human CENP-E to C. elegans kinetochores did not affect the normal course of mitosis and it was sufficient to partially rescue chromosome alignment defects imposed by the experimental attenuation of PEFs in this system. In turn, CRISPR/Cas9-mediated inactivation of CENP-E in P. pacificus had no clear detrimental effects on mitosis during embryogenesis and larval stages, giving rise to fully viable animals. These data strengthen the notion that CENP-E is dispensable even in holocentric species in which it is normally present and provide evidence supporting an inverse relationship between kinetochore size and CENP-E dependence for mitotic chromosome alignment across metazoans.

To systematically investigate the relationship between kinetochore size and the essentiality of CENP-E in a phylogenetic setting, we profiled the presence or absence of CENP-E in a selection of holocentric and monocentric metazoan taxa through highly sensitive hidden Markov model-based homology searches coupled with phylogenetic analysis. Our species selection comprised taxa at two taxonomic levels of comparable age for which there are high-quality sequence data and for which information on kinetochore type is available (Table S1). We compared the conservation of CENP-E in the predominantly holocentric phylum Nematoda [∼586 million years (Ma) old] to the monocentric phylum Vertebrata (∼462 Ma old). At relatively closer evolutionary timescales and between more similar organisms, we compared CENP-E conservation between the holocentric insect order Hemiptera (true bugs, ∼290 Ma old) and the monocentric insect order Diptera (flies, ∼241 Ma old). We additionally included species from the holocentric order Psocodea (lice, ∼200 Ma old) (Pittendrigh et al., 2006), the sister clade of Hemiptera. This framework allowed us to reconstruct the evolutionary history of CENP-E in these taxa with disparate kinetochore architectures. Our analysis shows that CENP-E is present in member species of all four taxa (Fig. 1A–D; Fig. S1). We found no evidence for horizontal gene transfer in our phylogenetic tree of CENP-E homologs on account of its strong congruency with the consensus phylogeny of our selected species. Therefore, we infer the presence of CENP-E in the last common ancestors of the included taxa, in line with the broad distribution of CENP-E orthologs across eukaryotes (van Hooff et al., 2017). This allowed us to identify subsequent losses along the phylogeny of each of these clades. We found that CENP-E is universally conserved across Vertebrata (Fig. 1B). In contrast, we inferred at least three independent losses of CENP-E in Nematoda (Fig. 1A). Two of these losses were in bona fide holocentric lineages of Nematoda, namely in the order Tylenchina and in the genus Caenorhabditis. The third loss was found in the order Trichinellida, which has monocentric chromosomes (Mutafova et al., 1982). Thus, loss of CENP-E appears not to be exclusive to species with holocentric chromosomes. However, we note that this lineage underwent extensive genomic streamlining (wholesale purging of genes) (Mitreva et al., 2011). When comparing the insect orders Hemiptera and Diptera, we found that CENP-E is absent in most Hemiptera lineages, as a result of at least three independent losses in this ancestrally holocentric clade (Fig. 1C,D). Furthermore, we found no CENP-E orthologs in its holocentric sister order Psocodea (Fig. 1C). In comparison, CENP-E is present throughout the monocentric Diptera, with the exception of Tephritidae (peacock flies) (Fig. 1D). Overall, although the type of centromere does not dictate the presence or absence of CENP-E, these analyses show a marked propensity for CENP-E loss in taxa with holocentric chromosomes and relatively larger kinetochores compared to taxa with monocentric chromosomes.

Given the tendency of CENP-E loss in species with holocentric chromosomes, we next directly investigated the relationship between kinetochore size and CENP-E dependence for chromosome alignment. To this end, we targeted codon-optimized human CENP-E (hCENP-E) expression to C. elegans kinetochores that span over 4 µm in length along the entire chromosome in early embryos (Ladouceur et al., 2015; Rocha et al., 2023) and lack a CENP-E ortholog. In vertebrates, CENP-E localizes at the expandable kinetochore fibrous corona throughout mitosis (Cooke et al., 1997; Craske and Welburn, 2020; Yen et al., 1991). Taking this into account, while ensuring the correct kinetochore localization of transgene-encoded mKate2::hCENP-E in C. elegans embryos, we fused it to the Spc25 ortholog KBP-3 (Cheeseman et al., 2004). A construct containing mKate2::KBP-3 was used as a control (Fig. 2A). Both constructs were expressed from the germline promoter Pmex-5. Kinetochore localization of mKate2-tagged KBP-3 and hCENP-E::KBP-3 during the first embryonic division was confirmed by live-cell imaging (Fig. 2B). Despite codon optimization, the expression levels of mKate2::CENP-E::KBP-3 were reduced to approximately 40% and 95% of mKate2::KBP-3 levels at the kinetochores and cytoplasm, respectively (Fig. 2C,D), perhaps due to protein folding issues or transgene silencing mechanisms operating in the C. elegans germline (Aljohani et al., 2020).

To characterize mitotic progression in the transgenic worms, animals stably expressing mKate2::CENP-E::KBP-3 and mKate2::KBP-3 were crossed with animals expressing GFP::H2B and GFP::γ-tubulin, thus enabling tracking of chromosomes and the spindle poles, respectively. Live-cell imaging of one-cell embryos unveiled that the expression of neither mKate2 fusion constructs perturbed mitosis or compromised embryo viability (Figs 3A and 4A). Two parameters were quantified over time: spindle length, defined as the distance between the two spindle poles; and chromosome span, defined as the maximal distance between the outermost chromosomes along the spindle axis (Fig. 3B). Both parameters remained unaltered in hCENP-E-expressing embryos compared with control embryos (Fig. 3C,D), thus ruling out unspecific effects associated with hCENP-E expression.

To generate embryos with defective chromosome alignment without directly interfering with end-on kinetochore–microtubule attachments, we attenuated PEFs on chromosome arms by knocking down the C. elegans chromokinesin KLP-19 (Powers et al., 2004). As expected, under these conditions, chromosomes became scattered in the cytoplasm after NEBD and took longer to align at the spindle equator, ultimately leading to lagging chromosomes in anaphase (Figs 3A,D,F and 4B). In the first mitotic division, premature spindle pole separation prior to anaphase is indicative of impaired formation of load-bearing kinetochore–microtubule attachments capable of moving chromosomes (Desai et al., 2003; Gassmann et al., 2008; Oegema et al., 2001). As predicted, klp-19-depleted embryos displayed premature spindle pole separation starting around 30 s after NEBD. The spindle length decreased around 110 s after NEBD and remained shorter than that in controls at anaphase onset (Fig. 3E). Premature spindle pole separation roughly overlapped with the moment when chromosome span increased, suggesting that chromosomes had not established proper bipolar attachments, with spindle microtubules capable of resisting cortical pulling forces exerted on the poles (Fig. 3F). To test whether hCENP-E could rescue the observed chromosome alignment defects, we imaged one-cell embryos stably expressing mKate2::hCENP-E::KBP-3 after depletion of klp-19 by RNAi (Fig. 3A). Under these conditions, the delay in establishing load-bearing microtubule attachments was still present (premature pole separation observed 30–40 s after NEBD), suggesting the incapacity of CENP-E to rescue attachment defects. However, the extended chromosome span normally observed after depletion of klp-19 was partially rescued in hCENP-E-expressing embryos (chromosomes remained closer to the spindle equator) (Fig. 3F). The partial rescue might be explained by the fact that, compared with KBP-3 alone, hCENP-E::KBP-3 expression and the respective kinetochore association in C. elegans embryos were significantly lower. Moreover, we found that detyrosinated α-tubulin, which enhances hCENP-E processivity and plays a role in the alignment of pole-proximal chromosomes in human cells (Barisic et al., 2015), was almost exclusively localized to the centrosome region in one-cell C. elegans embryos (Fig. S2) and might compromise optimal hCENP-E function in the engineered animals. Of note, although hCENP-E expression did not rescue the viability of klp-19-depleted embryos (Fig. 4A), likely owing to enduring lagging chromosomes during anaphase (Fig. 4B), it rescued the brood size to control levels (Fig. 4A). These data provide in vivo evidence that hCENP-E is functional when expressed in C. elegans embryos and is able to overcome defects associated with other congression pathways that would normally benefit from having holocentric chromosomes with larger kinetochores. Moreover, they open the exciting possibility that species with holocentric chromosome architecture have lost CENP-E due to its dispensability for chromosome alignment and mitosis in general.

In contrast to C. elegans, the P. pacificus genome contains a CENP-E ortholog that is annotated in the current version of the genome as PPA38637 (ElPaco at http://pristionchus.org/). We named this gene Ppa-cenp-E following standard nematode nomenclature rules. We generated three independent alleles (tu1915, tu1916 and tu1917) with a 4 bp deletion and 20 bp and 13 bp insertions, respectively, all of which resulting in frameshift mutations. All three mutant lines were viable and grew normally with no obvious difference in lifespan and/or accumulating lethality over generations. These data indicate that CENP-E is dispensable for mitosis even in holocentric species in which it is normally present, but the kinetochores of which are one order of magnitude larger (up to 2 µm in length) than in most monocentric species (Rillo-Bohn et al., 2021). However, we note that brood size counts revealed a higher variability than what is typically observed in P. pacificus wild-type PS312 animals (normally between 120 and 180 progeny per hermaphrodite). Specifically, we found 55–213 (tu1915), 75–269 (tu1916) and 93–217 (tu1917) progeny in the three mutant lines (Table S2). These numbers suggest that, although dispensable for mitosis, CENP-E has been retained in P. pacificus due to other roles, such as the ‘sperm–oocyte switch’ that determines progeny number and might confer a fitness advantage.

We recognize that our study has limitations in breadth, imposed mostly by the lack of information on absolute kinetochore size in the majority of the species analyzed. Nevertheless, we contend that comparing species with holocentric chromosomes in which the kinetochore extends along the entire chromosome length with species with localized kinetochores that normally cannot be resolved by light microscopy (i.e. they are less than 200 nm in length) is a reasonable first approximation to the problem, even taking into consideration that chromosome (and kinetochore) size scales with a reduction in cell and nuclear size during embryonic development (Ladouceur et al., 2015). For instance, kinetochore sizes in the two nematode species used in the present study for experimental follow-up after phylogenetic analyses correspond to 4 µm and 2 µm in C. elegans (Rocha et al., 2023) and P. pacificus early embryos (Rillo-Bohn et al., 2021), respectively. Therefore, the tendency to lose CENP-E during eukaryotic evolution appears to correlate with a larger kinetochore size, and this is particularly evident among species with holocentric chromosomes. For instance, C. elegans embryos rapidly bi-orient and align all chromosomes within approximately 120 s after NEBD, and only ∼1% of anaphases show lagging chromosomes (Stear and Roth, 2002). However, when experimentally challenged, C. elegans embryos can have high rates of chromosome alignment defects and missegregation events (Cheeseman et al., 2005; Desai et al., 2003; Gassmann et al., 2008; Oegema et al., 2001; Powers et al., 2004; Stear and Roth, 2002). Remarkably, C. elegans engineered to express hCENP-E fused to the kinetochore protein KBP-3 did not show any problems throughout mitosis or in animal viability, whereas the related holocentric worm P. pacificus, which harbors a bona fide CENP-E ortholog, was able to survive and grow normally without CENP-E. These experiments provide compelling evidence that CENP-E is dispensable for chromosome alignment during mitosis in well-established model systems with holocentric chromosomes, possibly due to a compensatory mechanism associated with their larger kinetochore size. Nevertheless, CENP-E appears to be important for the sperm–oocyte switch in P. pacificus, possibly reflecting recently uncovered roles of CENP-E during spermatogenesis (She et al., 2021, 2020; Zhang et al., 2024).

We assembled a dataset of predicted proteomes from holocentric and monocentric species. The species were selected to span the known diversity of the holocentric taxa Nematoda and Hemiptera, and the monocentric taxa Vertebrata and Diptera. The divergence times of these taxa were estimated with https://timetree.org/ (last accessed 19 June 2024) (Kumar et al., 2017). To measure the quality of the predicted proteomes prior to inclusion for this study, we performed a BUSCO (v5.7.1) completeness analysis using the metazoan_odb10 dataset (Manni et al., 2021). The final dataset comprised 12 Nematoda species, six Vertebrata species, ten Hemiptera species, two Psocodea species and eight Diptera species, totaling 38 species. We filtered out isoforms of the human proteome by annotation in the original sequence identifiers. The sources of the used proteomes and BUSCO completeness scores are listed in Table S1.

To find homologs of CENP-E in each of the selected species, we ran the hmmsearch method from the HMMER3 package (v3.1b2; http://hmmer.org/) with all heuristic filters turned off for maximum sensitivity (--max option). Specifically, we queried the CENP-E motor domain-specific hidden Markov model from Tromer et al. (2019) against each predicted proteome in our dataset. The hits were sorted on ascending domain e-value, and hits with identical domain e-values were filtered to remove redundant hits. Then, the best 20 hits were selected for further phylogenetic analysis.

Delineating orthologs from a set of homologs requires robust phylogenetic analysis. Here, we took the best 20 homology hits from each of the selected proteomes and these were collated. Then, a multiple sequence alignment (MSA) was created from the homologs using MAFFT E-INS-i (Katoh and Standley, 2013). The resulting MSA was filtered manually by excluding poorly aligned sequences and truncated to exclusively contain the conserved motor domain. Additionally, the alignment was trimmed to remove positions with ≤70% occupancy using trimAl (Capella-Gutiérrez et al., 2009). A phylogenetic tree was inferred from the MSA with IQ-TREE using ModelFinder to find the best-fitting substitution model (Kalyaanamoorthy et al., 2017; Nguyen et al., 2015). IQ-TREE was run ten independent times to maximize the likelihood of the topology inference, in accordance with Zhou et al. (2018), and UFBoot2 was used to get bootstrap support for branches with 1000 replicates (Hoang et al., 2018). The inferred phylogeny was visualized in iTOL (Letunic and Bork, 2021). To annotate the orthologous clades in the phylogeny, a blastp search was conducted for each of the hit sequences against the Swiss-Prot database (Altschul et al., 1990; Sayers et al., 2022). An interactive version of our phylogenetic tree is available via https://itol.embl.de/tree/145909325331361721030804, hosted by the iTOL website.

In order to increase the probability of human gene expression in C. elegans, we performed a codon bias optimization of the human CENP-E sequence (isolated from the 883-CENP-E 620 plasmid, a gift from Michael Lampson, University of Pennsylvania, Philadelphia, USA; Zhang et al., 2017) using the GenScript OptimumGene service. Accordantly, the codon adaptation index and the frequency of optimal codons were calculated to enhance gene expression in this organism. The optimized CENP-E sequence used for this study is given in Table S5 (optimized sequence length: 1906; GC content: 36.70%). The sequence was cloned into a standard cloning pUC57 vector (GenScript) flanked by two EcoRV (GATATC) cutting sites.

The Pmex5_mKate2_KBP-3 and Pmex5_mKate2_CENP-E_KBP-3 plasmids were generated in this study from genomic DNA isolated from C. elegans, pCFJ151 containing mKate2 (Frokjaer-Jensen et al., 2008) and pUC57-CENP-E (codon optimized). Gibson assembly was used to assemble the two or three DNA fragments, respectively (Gibson, 2009). The primers used for PCR amplification and Gibson assembly are described in Table S3. Gibson Assembly Master Mix (New England Biolabs) was added to the DNA fragments and incubated at 50°C for 1 h. After transforming Top10 competent bacteria with 4–6 µl of the Gibson assembly reaction products, the bacteria were plated in Luria–Bertani (LB) agar plates with ampicillin (ForMedium, AMP25). The following day, colonies were picked to perform a colony PCR. Positive colonies were selected and DNA was extracted using the NZYMiniprep kit (NZYtech). The Sanger sequencing reaction was performed by GenCore (i3S) according to the manufacturer's instructions with the following components: BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), BigDye Terminator v1.1 (Thermo Fisher), v3.1 5× Sequencing Buffer (Applied Biosystems), primer (10 µM), nuclease-free water (Ambion) and plasmid (∼100 ng). The results were then analyzed using SnapGene.

A Mos1 transposon-based strategy (MosSCI) was used to generate a strain stably expressing mKate2::KBP-3 and mKate2::CENP-E::KBP-3 under the control of the mex-5 promotor and tbb-2 3′ untranslated region for expression in germline cells (Frokjaer-Jensen et al., 2012, 2008). Other fluorescent markers were subsequently introduced by mating. The GCP826 (mKate2::KBP-3×GFP::H2B/GFP::TBG-1) and GCP815 (mKate2::CENP-E::KBP-3×GFP::H2B/GFP::TBG-1) strains created in this study were maintained at 20°C on standard nematode growth medium (NGM) plates seeded with Escherichia coli OP50 bacteria.

Double-stranded RNAs for klp-19 (gene ID: Y43F4B.6) were obtained using the following oligonucleotides – T3 promoter, 5ʹ-aattaaccctcactaaaggTGACCCAGAAGAACTCTCGC-3ʹ; T7 promoter, 5ʹ-taatacgactcactataggTCGGAGATCTTCACACAGCC-3ʹ – and were delivered by injecting L4 hermaphrodites. After injection, animals were incubated for 48 h at 20°C for a penetrant depletion before embryos were isolated for live-cell imaging.

Adult gravid hermaphrodite worms were dissected in a watch glass filled with Egg Salts medium (118 mM KCl, 3.4 mM MgCl₂, 3.4 mM CaCl₂ and 5 mM HEPES pH 7.4), and embryos were mounted on a fresh 2% agarose pad and covered with an 18 mm×18 mm coverslip (no. 1.5H, Marienfeld). Embryos co-expressing GFP::H2B, GFP::γ-tubulin and mKate2::KBP-3 or mKate2::CENP-E::KBP-3 for tracking of nuclei, centrosomes and kinetochores were imaged on an Axio Observer microscope (Zeiss) equipped with an Orca Flash 4.0 camera (Hamamatsu), a Colibri.2 light source, and controlled by ZEN software (Zeiss). All other imaging was performed on a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i, Andor Technology) and a CSU-X1 confocal scanner (Yokogawa Electric Corporation), controlled by Andor IQ3 software (Andor Technology). All imaging was performed in temperature-controlled rooms kept at 20°C. Time-lapse sequences were processed and analyzed with Fiji software (ImageJ version 2.0.0-rc-56/1.51h).

Embryos expressing GFP::H2B, GFP::γ-tubulin and mKate2::KBP-3 or mKate2::CENP-E::KBP-3 were imaged at 10 s intervals, with nine z-slices spaced 1.5 μm apart for the fluorescence channel, and a single central slice per time point for the differential interference contrast (DIC) channel, at 2×2 binning with a 63× NA 1.4 oil immersion objective (Zeiss), from just prior to NEBD in the one-cell embryo until the onset of cytokinesis. Embryo length was defined as the distance between the outermost points of the eggshell visible in the DIC image. After maximum intensity projection of GFP z-stacks, the x and y coordinates of the centrosomes and of the chromosomes closer to each centrosome were recorded over time using the MtrackJ plugin in ImageJ by manually clicking on the center of centrosomes and on the outer edge of the chromosomes.

Ten to 12 adult worms were dissected into 3 μl of M9 buffer (86 mM NaCl, 42 mM Na₂HPO₄, 22 mM KH₂PO₄ and 1 mM MgSO₄) on a poly-L-lysine-coated slide. A 18×24 mm coverslip was placed on the 3 μl drop, and slides were plunged into liquid nitrogen. After rapid removal of the coverslip (‘freeze-cracking’), embryos were fixed in −20°C methanol for 20 min. Embryos were re-hydrated two times for 5 min in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄ and 1.47 mM KH₂PO₄), blocked with AbDil (PBS with 2% BSA and 0.1% Triton X-100) in a humid chamber at room temperature for 30 min, and incubated with a rabbit monoclonal anti-detyrosinated α-tubulin (1:1000; a gift from Marin Barisic, Danish Cancer Institute, Copenhagen, Denmark; Liao et al., 2019) for 2 h at room temperature. After washing four times for 5 min in PBS, embryos were incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:1000; Thermo Fisher Scientific, A-11029; RRID: AB_143165) for 1 h at room temperature. Embryos were washed four times for 5 min in PBS and mounted in Prolong Gold with DAPI stain (Invitrogen, P36931). Images were recorded on an AxioImager Z1 (100× Plan-Apochromatic oil DIC objective lens, 1.46 NA, Carl Zeiss Microimaging) equipped with a CCD camera (ORCA-R2, Hamamatsu) operated by Zen software (Carl Zeiss). Image files were imported into Fiji for further processing.

Brood size and embryonic viability assays were performed at 20°C. L4 hermaphrodites, injected with klp-19 RNAi or untreated, were grown for 40 h on NGM plates containing OP50 bacteria. Single adults were then placed on new plates with a small amount of OP50, removed 8 h later, and the plates were incubated for another 16–24 h to give viable embryos time to hatch. Embryonic viability was calculated by dividing the number of hatched larvae by the total number of progenies on the plate. Replicate counts were averaged and plotted as the mean percentage ±95% confidence interval (c.i.).

CRISPR experiments were done with the P. pacificus wild-type strain PS312. Three alleles of Ppa-cenp-E were obtained. All worm cultures were propagated at room temperature (20°C) on NGM in 6 cm Petri dishes, as outlined before (Werner et al., 2017). In all experiments, E. coli OP50 was used as food source. Bacteria were grown overnight at 37°C in LB medium, and 400 μl of the overnight culture was pipetted on NGM agar plates and left for several days at room temperature to grow bacterial lawns. For brood size counts, single J4 juvenile worms were passed on these lawns and transferred daily over a 5-day period.

The procedure for CRISPR/Cas9 mutagenesis was based on previously published protocols for P. pacificus (Han et al., 2020; Witte et al., 2015). A target-specific CRISPR RNA (crRNA) of the sequence 5′-GCCTACGGACAGTCTGGCAG-3′ was synthesized to target 20 bp upstream of the protospacer adjacent motifs and fused to tracrRNA (1072534; Integrated DNA Technologies) at 95°C for 5 min, and subsequently allowed to cool down to room temperature and anneal. The hybridization product was combined with Cas9 protein (1081058; Integrated DNA Technologies). After a further 5 min incubation at room temperature, TE buffer was added for final concentration of 18.1 µM for the single guide RNA and 12.5 µM for Cas9. A plasmid carrying the Ppa-eft-3 promoter and modified TurboRFP sequences was used as a co-injection marker (Han et al., 2020). After 2 days, P0 plates containing the F1 animals with fluorescence signal of the co-injection marker were isolated, and eight to ten F1 progenies from these plates were singled out on individual plates. The genotypes of the F1 animals were subsequently analyzed via Sanger sequencing after PCR amplification of a Ppa-cenp-E fragment covering the targeted site using the forward primer (5′-GAAGCCTCTCGTTGATGGAACACTTCAGG-3′) and the reverse primer (5′-CACATATTGACATTCTTAGGGCGAAAGTC-3ʹ). Mutations were identified before isolation of homozygous mutant carriers. Detailed information of mutants generated in this study can be found in Table S2.

Statistical analysis was performed with GraphPad Prism 8.1.2 software. D'Agostinho–Pearson omnibus normality test was used to determine whether the data followed a normal distribution. If α=0.05, the difference between the population distributions was determined by two-tailed unpaired Student's t-test. Two-tailed unpaired t-test was used to compare the fluorescence intensity of KBP-3 at the kinetochores and at the cytoplasm in the transgenic worms, and to analyze the brood size, with and without klp-19 RNAi. Statistical differences were considered significant at P<0.05. To assess the overall effects of hCENP-E expression on klp-19-depleted embryos, we performed a repeated measures two-way ANOVA (from 0 to 200 s, NEBD to anaphase onset), followed by post hoc comparisons using Bonferroni correction to evaluate specific time points relevant for chromosome alignment (Table S4). All plots represent mean values and s.d. or 95% c.i. calculated for all replicates. Sample sizes were not predetermined but were obtained based on current best practice in the field.

The authors thank members of their laboratories for insightful comments throughout the project and the critical reading of the manuscript.

The peer review history is available online at https://journals.biologists.com/jcs/lookup/doi/10.1242/jcs.263466.reviewer-comments.pdf