Plasmodium ARK1 regulates spindle formation during atypical mitosis and forms a divergent chromosomal passenger complex

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
Mitosis in Plasmodium spp., the causative agent of malaria, is fundamentally different from model eukaryotes, proceeding via a bipartite microtubule organising centre (MTOC) and lacking canonical regulators such as Polo kinases. During schizogony, asynchronous nuclear replication produces a multinucleate schizont, while rapid male gametogony generates an octaploid nucleus before gamete formation. Here, we identify Aurora-related kinase 1 (ARK1) as a key component of inner MTOC and spindle formation, controlling kinetochore dynamics and driving mitotic progression. Conditional ARK1 depletion disrupts spindle biogenesis, kinetochore segregation, karyokinesis and cytokinesis in both stages, and affects parasite transmission. Interactome analysis shows that ARK1 forms the catalytic core of a non-canonical chromosomal passenger complex (CPC) containing two highly divergent inner centromere proteins (INCENPs), which we term INCENP-A and INCENP-B, and lacking the canonical chromatin-targeting subunits Survivin and Borealin. Comparative genomics suggest that apicomplexan INCENPs arose through recurrent lineage-specific duplications, reflecting an evolutionary rewiring of CPC architecture in this eukaryotic lineage. Together, these findings reveal key adaptations in Plasmodium mitosis involving ARK1 and its INCENP scaffolds, and identify the ARK1–INCENP interface as a potential multi-stage target for antimalarial intervention.

Introduction
Plasmodium spp., the causative agent of malaria, is a haploid unicellular eukaryote of the phylum Apicomplexa, which is evolutionarily distant from most model organisms and has some unusual divergent features of cell division. During its life cycle, mitosis is closed and occurs during two distinct forms of cell division: schizogony within vertebrate host cells, and male gametogony within the female mosquito gut (Fig. 1a). During asexual proliferation within host red blood cells (schizogony), repeated asynchronous nuclear division, without chromosome condensation, produces a multinucleate schizont. Subsequent budding produces invasive merozoites to continue the cycle.

In contrast, during male gametogony three rounds of rapid genome replication to octaploid (1N to 8N) occur in 8 min within the intact nucleus. Karyokinesis and cytokinesis are synchronised with axoneme dynamics by replication and separation of a bipartite microtubule-organising centre (MTOC), chromosome condensation, and complementary de novo basal body formation, leading to the budding of eight flagellated male gametes. These events occur within 12–15 min of gametocyte activation, which is probably the most rapid mitosis amongst eukaryotes1. Compared to schizogony, mitosis in male gametogony is unusual, with atypical continuous spindles and a bipartite MTOC that spans the nuclear envelope and coordinates the simultaneous assembly of spindle microtubules and the flagellar axoneme. Male gametes are the only flagellated stage in the parasite’s life cycle, and therefore the only stage where centriole formation and de novo replication of the basal body occur 1–4.

Aurora-related kinases (ARKs) are multi-functional mitotic regulators that coordinate spindle assembly, chromosome segregation, and late mitotic events across eukaryotes. They act on spindle pole duplication, spindle assembly, kinetochore-microtubule attachment, mitotic checkpoints, and finally cytokinesis 5–8. Budding yeast and the amoeba Dictyostelium discoideum9 have a single ARK to execute all functions, suggesting this state to be ancestral to all eukaryotes7. However, multiple different lineages (e.g., vertebrates, plants, kinetoplastids, and others) have one or more paralogues that resulted from parallel duplications, which subdivided functions over the different paralogues7,10. One paralogue (Aurora B in humans) is the catalytic centre of the chromosomal passenger complex (CPC), a key regulator of mitotic events at the cell equator, which initially hitchhikes on the inner centromere and finally remains at the central spindle to orchestrate cytokinesis. In canonical model systems, the CPC consists of Aurora B, inner centromere protein (INCENP), Survivin, and Borealin 11,12 that guide its localisation to kinetochores, spindle and the central spindle. It is also critical for mitotic histone phosphorylation, fixing errors in kinetochore-spindle attachment, and correct cytokinesis (Fig. 1b).

Apicomplexans, including Plasmodium and Toxoplasma encode three ARKs, most of which lack canonical localisation and activation motifs found in model eukaryotes 10,13,14. ARK1, 2, and 3 are refractory to gene disruption and indispensable during asexual blood stage schizogony in both P. berghei and P. falciparum 13–15. Consistent with this, a recent study has chemically validated Aurora kinases as promising targets for the development of multi-stage antimalarials 16,17. ARK1 has been observed at the MTOC during schizogony and in P. falciparum gametocytes 18,19. However, its precise function in asexual division and its behaviour during sexual development have remained unclear. Our recent studies on ARK2 revealed its association with spindle microtubules (MTs) forming a divergent protein complex with EB1, myosin K and MISFIT10, but little is known of ARK1 and 3 functions during male gametogony or whether they are essential for parasite transmission.

ARK1 function has also been explored in another apicomplexan, Toxoplasma gondii, where it interacts with two INCENP paralogues and is important for asexual replication. Whether Plasmodium INCENPs are direct orthologues of Toxoplasma INCENPs is not clear. The function of Toxoplasma ARK1 during male gametogony is difficult to study because the sexual cycle of these parasites only takes place in the gut of cats (Fig. 1b)20.

Here, we have investigated Plasmodium ARK1, integrating studies of location, function and molecular interactions across two life-cycle stages using P. falciparum (Pf, for asexual blood-stage schizogony) and P. berghei (Pb, for male gametogony). High-resolution live-cell imaging and expansion microscopy revealed ARK1 associated with the mitotic spindle, kinetochores, and the inner MTOC. Conditional gene knockout using rapamycin-DiCre in P. falciparum21 and gene knockdown using a promoter-trap approach in P. berghei22 revealed that ARK1 regulates spindle biogenesis, karyokinesis and cytokinesis during both schizogony and male gametogony, suggesting that ARK1 is a critical regulator of Plasmodium mitosis. Interactome analysis identifies ARK1 as the catalytic component of a non-canonical Chromosomal Passenger Complex (CPC) comprising two divergent INCENP paralogues – here termed INCENP-A and INCENP-B – in the absence of Survivin and Borealin. Together, these findings reveal how ARK1 and its INCENP scaffolds contribute to the distinctive mechanisms of apicomplexan nuclear division.

Results
ARK1 is present in asexual stage parasites undergoing mitotic division and kinetochore segregation
We studied the location of PfARK1 in asexual blood stages, using a rapamycin-inducible dimerizable Cre recombinase DiCre-based strategy to tag and knockout PfARK1 in the 1G5DC parasite line (Supplementary Fig. 1a)21. These parasites (HA-PfARK1-loxP) were used to locate PfARK1 during parasite division using an antibody to the HA-tag included at its N-terminal. Indirect immunofluorescence (IFA) and Western blot indicated that ARK1 is expressed only in trophozoite and schizont stages during asexual division, but was not detectable in ring and segmentor (Fig. 2a, Supplementary Fig. 1e). ARK1 was present as a punctum proximal to the MTOC, as identified by staining with an anti-centrin antibody, present only in cells with two associated centrin puncta, indicative of ongoing mitosis (Fig. 2a, c). These observations were largely consistent with previous studies of PfARK118,19.

To gain a deeper insight into ARK1 dynamic organisation, Ultra-structure Expansion Microscopy (U-ExM) was performed, and visualized ARK1 along with spindle microtubules using an anti-tubulin antibody to track spindle biogenesis. As reported previously23, parasites possessed hemispindles prior to the onset of division, but PfARK1 was not expressed in these parasites or parasites containing interpolar spindles (Fig. 2b, d). Strikingly, PfARK1 was found specifically associated with mitotic spindles and PfARK1-positive double puncta seemed to be present at the ends of the growing mitotic spindles (Fig. 2b, d). These data strongly indicate that ARK1 is expressed transiently, and only when parasite nuclei are undergoing mitotic division. These observations were also validated using the rodent malaria parasite, P. berghei, a well-established model for studying male gametogony and parasite development within the mosquito host10. The subcellular location of PbARK1 in asexual stages was examined by generating an endogenous, C-terminal GFP-tagged PbARK1 (PbARK1-GFP) transgenic parasite line (Supplementary Fig. 2a–c and Supplementary Note 1). Live cell imaging of PbARK1-GFP in asexual blood stages revealed a diffuse location in early ring stages. During trophozoite and early schizont stages, PbARK1-GFP re-localised to discrete focal points in the nucleus (Supplementary Fig. 2d). As schizogony progressed, the focal points of fluorescence divided into two closely associated puncta, which followed subsequent asynchronous nuclear divisions and eventually became diffuse during the later stages (Supplementary Fig. 2d, e). These observations are consistent with the ARK1 location in P. falciparum as described above (Fig. 2a).

PbARK1 is located in the nucleoplasm, at the spindle poles and along microtubules during male gametogony
In gametocytes both before and immediately after activation, PbARK1-GFP had a diffuse distribution predominantly within the nucleoplasm. Within one-min post-activation, part of this was concentrated to a single discrete focal point adjacent to the DNA (Fig. 3a), and then the distribution rapidly extended to form an arc-like structure, which within three-min post-activation divided into two distinct puncta positioned at opposite sides of the nucleus (Fig. 3a and Supplementary Movie 1). The two PbARK1-GFP foci elongated again, forming two arc-like structures along the periphery of the nucleus (Fig. 3a and Supplementary Movie 2). By 6- to 8-min post-activation, a subsequent round of extension and division had produced eight discrete PbARK1-GFP foci, coincident with the completion of three successive rounds of mitosis (Fig. 3a and Supplementary Movie 3). 3D-structured illumination microscopy (SIM) at 1.5–2 min post-activation clearly resolved the arc-like localisation of PbARK1-GFP across the nucleus, with distinct foci visible at both ends of the arc (Fig. 3b).

The arc-like pattern of PbARK1 observed in live cell imaging and 3D-SIM coincided with the pattern of anti-tubulin staining in U-ExM, suggesting an association with the mitotic spindle and spindle poles or inner MTOCs (Fig. 3c and Supplementary Fig. 3a). To further investigate ARK1 location, dynamics, and association with mitotic spindles and kinetochore segregation, we compared the location of PbARK1 with that of the spindle marker ARK2, the kinetochore marker NDC80, and cytoplasmic axonemal protein kinesin-8B. We generated dual fluorescent parasite lines expressing PbARK1-GFP (green) and either PbARK2-mCherry (magenta), PbNDC80-mCherry (magenta), or Pbkinesin-8B-mCherry (magenta) by genetic cross as described previously4. Live-cell imaging showed that both PbARK1-GFP and PbARK2-mCherry were co-located at the spindle (Fig. 3d and Supplementary Movie 5). Further 3D-SIM at 1.5–2 min post-activation showed an association of PbARK1-GFP with PbARK2-mCherry at the spindle, with PbARK1-GFP more outside at the spindle poles (Fig. 3e and Supplementary Fig. 3b). In parasite lines expressing PbARK1-GFP and PbNDC80-mCherry, these proteins were located next to the Hoechst-stained DNA, partially overlapping at the spindle but not at the spindle poles (Fig. 3f and Supplementary 4). 3D-SIM at 1.5–2 min post-activation showed a clear association of PbNDC80-mCherry with PbARK1-GFP at the spindle but not at the spindle poles (Fig. 3g and Supplementary Fig. 3c). However, in a parasite line expressing PbARK1-GFP and Pbkinesin-8B-mCherry (a cytoplasmic marker) there was no overlap between these two proteins (Fig. 3h), an observation which was further substantiated by 3D-SIM imaging (Fig. 3i).

During meiotic stages of development post-fertilisation, PbARK1-GFP was diffuse as seen during schizogony, and male gametogony in the cytoplasm, with transient nuclear foci forming in zygotes and disappearing in mature ookinetes (Supplementary Fig. 4a and Supplementary Note 2). A diffuse cytosolic signal with some foci was also observed throughout oocyst and sporozoite stages, including those from salivary glands (Supplementary Fig. 4b and Supplementary Note 2). In liver stages (in HepG2-infected cells), the PbARK1-GFP signal remained diffuse with faint foci even at 48 h post infection (hpi), with slight perinuclear enrichment at 64 hpi (Supplementary Fig. 4c and Supplementary Note 2).

PfARK1 regulates spindle formation and karyokinesis
The functional role of PfARK1 in asexual blood stage development was assessed by examining the effect of deleting the gene. Rapamycin (RAP) treatment of HA-PfARK1-loxP parasites efficiently excised the floxed sequence (Supplementary Fig. 1c) and caused depletion of PfARK1 protein (Supplementary Fig. 1d). Synchronised ring stage cultures of 1G5DC-SkipFlox (control) (Supplementary Fig. 5a) or HA-PfARK1-loxP parasite lines (Fig. 4a and Supplementary Fig. 5b) were treated with RAP or DMSO (control) and parasite growth was monitored every 24 h. In cycle 0 (RAP-treatment) there was little effect on the parasitaemia of HA-PfARK1-loxP parasites. However, a significant growth defect was observed at the beginning of the next cycle (cycle 1) as indicated by a marked reduction in parasitaemia (Fig. 4a and Supplementary Fig. 5b), whilst there was almost no effect of RAP treatment on 1G5DC-SkipFlox (Supplementary Fig. 5a). This growth defect was further accentuated in subsequent cycles. Microscopic examination of Giemsa-stained blood smears suggested normal ring to trophozoite development in cycle 1 of DMSO-treated parasites (Supplementary Fig. 5c). However, following RAP treatment and PfARK1 depletion, a population of parasites were pyknotic (at ~96 and 144 hpi) and fewer rings were found in cycle 1 and 2 (Fig. 4b and Supplementary Fig. 5c). Schizont development following treatment with E64 to block late schizont maturation and merozoite egress was analysed: a significant number of PfARK1-depleted cycle 1 schizonts had abnormal morphology, with unsegregated nuclei (Fig. 4c and Supplementary Fig. 5d), which is consistent with the role of PfARK1 in nuclear division. This suggestion, that the decrease in parasitaemia resulting from PfARK1 depletion was due to its role in nuclear division and/or schizont maturation, was investigated further.

Next, efforts were made to investigate whether PfARK1 regulates MTOC duplication and spindle formation during asexual division of the parasite. U-ExM revealed the formation of various forms of spindles as described earlier with one or two centrin-labelled foci on the cytoplasmic side of the nuclear envelope (Fig. 4d) and associated with NHS-ester labelled MTOCs or associated with mitotic spindles23,24. In PfARK1-depleted parasites, a very few had associated spindles indicated by either absent or speckled tubulin staining (Fig. 4d, grey arrow and Supplementary Fig. 5e) and as mentioned above the nuclei appeared unsegregated. Also, multiple centrin foci were associated with these unsegregated nuclei, and in some cases centrin aggregates were seen away from the nuclei and disorganised (Fig. 4d and Supplementary Fig. 5e, square). Collectively, these data suggested that while the MTOC duplication was unaltered, it was disorganised and spindle formation and karyokinesis were impaired, indicating that PfARK1-depletion lead to defective mitosis.

PfARK1 depletion impairs segmentation of daughter merozoites
At the end of schizogony, segmentation of the schizont by invagination of the parasite plasma membrane leads to formation of merozoites prior to their egress25. IFA swere performed using antibody to PfMSP1, a parasite surface marker, to assess segmentation following ARK1 depletion. PfMSP1 fluorescence with a bunch of grape-like contours, characterised the membrane of control parasites, but in contrast, PfARK1-depleted parasites either lacked PfMSP1 staining, or contained large aggregations that encompassed multiple merozoites or unsegregated nuclei (Fig. 4e, cyan arrow).

U-ExM of mature (segmented) schizonts with anti-GAP45 antibody-labelling revealed inner membrane complex (IMC) and tubulin labelled subpellicular MT, and rhoptries labelled with NHS-ester (Fig. 4f). However, for the PfARK1-depleted parasites, several unsegregated nuclei were present and these cells either lacked GAP45 staining, or the parasite pellicle (the plasma membrane/IMC) was disorganised (Fig. 4f, cyan arrow, square, zoom and Supplementary Fig. 5f). Although NHS-ester labelled rhoptries were found, their organisation seemed to be disorganised which may be due to aberrant segmentation. In addition, subpellicular MT were either not observed or were mislocalised (Fig. 4f, cyan arrow, square, zoom). These defects were possibly due to arrest in division of the parasites as also indicated by aberrant GAP45 staining. These observations suggested impaired cytokinesis of daughter merozoites upon PfARK1 depletion, the aberrant karyokinesis or nuclear division possibly contributes to these defects.

To study the effect of PfARK1 depletion on merozoite release and egress, time-lapse microscopy was performed26. For this purpose, parasites were treated with protein kinase G (PKG)-inhibitor ML-10 to allow schizont maturation but prevent egress, followed by its removal which allowed egress and was captured by time-lapse microscopy27,28. Egress and dispersal of merozoites for control parasites was observed (Fig. 4g and Supplementary Movie 6), and while egress from the erythrocyte initially appeared almost normal in PfARK1-depleted parasites, the merozoites either remained attached to the residual body or clumped together (Fig. 4g, magenta arrows, and Supplementary Movie 7, 8) even after an extended period post-egress. These defects likely resulted from the abnormal nuclear segregation and cytokinesis in PfARK1-depleted parasites.

PbARK1 knockdown reveals an essential role during male gametogony and parasite transmission
Previous analysis by gene disruption had shown that PbARK1 is probably essential for asexual blood-stage development14. To be able to manipulate its expression in sexual stages we used a promoter trap strategy, replacing the ark1 promoter with the promoter from ama1, resulting in P ark1 hereafter referred as (ark1PTD), a gene which is not transcribed in gametocytes but is highly expressed in asexual blood stages (Supplementary Fig. 6a)29. Correct genetic integration in the transgenic parasite line was confirmed by PCR (Supplementary Fig. 6b), and ark1 transcription was shown to be significantly downregulated in ark1PTD gametocytes by qRT-PCR (Fig. 5a). A phenotypic analysis of these ark1PTD parasites was then performed at different stages of parasite development within the mosquito.

Phenotypic analysis revealed that male gametogony (as scored by exflagellation centres) was severely affected in the ark1PTD line compared to GFP control (wild-type-GFP; WT-GFP) parasite (Fig. 5b)30, and therefore significantly fewer ookinetes (differentiated zygotes after fertilisation) were formed (Fig. 5c). In mosquitoes fed with ark1PTD parasites, we observed significantly fewer oocysts in the midgut at day 7 post infection, in contrast to the hundreds of oocysts in mosquitoes fed WT-GFP parasites (Fig. 5d). The number of ark1PTD oocysts was further reduced by day 14 and day 21 post infection compared to WT-GFP oocysts (Fig. 5d). The ark1PTD oocysts were also significantly smaller than the WT-GFP oocysts 7 days post infection and diminished further by day 21 post infection (Fig. 5e, f). The number of salivary glands sporozoites was significantly reduced in ark1PTD parasites on day 14 (Supplementary Fig. 6c). However, the ratio of salivary gland to midgut sporozoites on day 21 was comparable to that of WT-GFP parasites (Supplementary Fig. 6c), suggesting that the surviving ark1PTD sporozoites retain their invasive potential and ability to migrate to the salivary glands. To further confirm the absence of sporogony, mosquitoes that had been fed with ark1PTD or WT-GFP parasites were allowed to feed on susceptible naïve mice (bite-back experiments). Mosquitoes infected with WT-GFP parasites successfully transmitted the parasite, with blood-stage infection detected in naïve mice after 4 days. In contrast, ark1PTD parasites failed to transmit in most experiments (five out of eight) (Supplementary Fig. 6d). However, on three occasions, they were able to establish infection in these bite-back experiments, although the prepatent period was markedly prolonged, with blood-stage parasites first detected 14 days post-infection (Supplementary Fig. 6d). Collectively, these findings indicate that the observed transmission defect is primarily driven by the reduction in sporozoite numbers rather than a loss of sporozoite invasiveness.

To investigate the genome-wide transcriptional impact of PbARK1 depletion, we performed RNA-seq analysis. While few changes were observed in non-activated gametocytes, activated ark1PTD gametocytes (30 minute post-activation; mpa) exhibited a broad downregulation of genes involved in signalling, lipid biosynthesis, and ribosome biogenesis. This transcriptional repression is consistent with the observed developmental arrest (Supplementary Fig. 6e-g, and Supplementary Note 3).

ark1PTD parasites have defective spindle formation, MTOC separation, and axoneme formation during male gametogony
To further investigate the function of PbARK1 during male gametogony, WT-GFP and ark1PTD male gametocytes were observed during gametogony using U-ExM. In WT-GFP male gametocytes at 8 mpa, individual MTOCs were distinct and separated, whereas in ark1PTD male gametocytes multiple MTOCs were clumped and aggregated together (Fig. 5g and Supplementary Fig. 7a). At 15 mpa, WT-GFP male gametocytes were observed undergoing exflagellation, whereas in ark1PTD male gametocytes the MTOCs were still clumped together and no exflagellation was observed in the examined cells (Fig. 5g and Supplementary Fig. 7a).

We examined whether the lack of MTOC separation in the ark1PTD line was caused by a defect in spindle formation. At 8 mpa, long spindles with clearly separated MTOCs were present in WT-GFP parasites, but in ark1PTD lines, short spindles, with the two MTOCs remaining close together (Fig. 6a, b) were present. Measurement showed that ark1PTD spindles were significantly shorter (~0.8 μm) than WT-GFP spindles (~2.5 μm; Fig. 6c). These findings corroborated studies on PfARK1 during schizogony as depletion of the kinase resulted in abnormal spindle formation and accumulation of MTOCs around unsegregated nuclei (Fig. 4d).

TEM observations further revealed structural defects in ark1PTD male gametocytes. At 8 mpa, in the ark1PTD line, MTOCs formed a normal bipartite structure consisting of an inner spindle MTOC and an outer basal body MTOC, similar to those in WT-GFP male gametocytes, and normal axoneme formation was observed (Fig. 6dA–D, I–L and Supplementary Fig. 7bA–D), However, we also observed basal body MTOCs that appeared detached from the nucleus in the ark1PTD line (dBB; Fig. 6dI, J and Supplementary Fig. 7bA, B, D). As revealed by expansion microscopy, multiple basal body MTOCs were often aggregated and clumped together (Fig. 6dL). Within the cytoplasm, we observed singlet or doublet MT that may have been axoneme precursors and likely represent failures in axoneme formation (Fig. 6dK, L).

At 15 mpa, WT-GFP gametocytes had started to form gametes with flagellum and nucleus (Fig. 6dE, F, G, H), while in the ark1PTD line there was incomplete axoneme formation and exflagellation rarely occurred (Fig. 6dM–P and Supplementary Fig. 7dE–H). In addition, two basal body MTOCs remained close together without proper separation (Fig. 6dM, N, P), with short spindle MT visible between them (Fig. 6dP and Supplementary Fig. 7c). These results suggest that PbARK1 is essential for successful mitosis during male gametogony.

PbARK1 is part of a divergent chromosome passenger complex (CPC) with two INCENP paralogues
To understand whether ARK1 localisation could be explained by recruitment to specific regulatory scaffolds, we investigated which interacting proteins might direct ARK1 activity and positioning during both asexual and sexual stages of the parasite life cycle. We prepared lysates from P. berghei schizonts and male gametocytes expressing PbARK1-GFP, performed GFP-Trap immunoprecipitation, and identified co-purifying proteins by mass spectrometry.

At both stages two divergent inner centromere protein paralogues—here termed INCENP-A and INCENP-B—were the predominant ARK1 interactors (Fig. 7a and Supplementary Fig. 8). This physical interaction is further supported by a recent study demonstrating that PbARK1 co-localises with both INCENP-A and INCENP-B at spindle poles and kinetochores in P. berghei31. Although replicate numbers were limited (n=2), ARK1 pulldowns from male gametocytes also showed coordinated enrichment of several spindle and kinetochore proteins amongst the pulldowns (Supplementary Data 2) including components of the ARK2-associated spindle complex (MISFIT-MyoK-EB1, multiple kinetochore proteins (AKiT1, 3, 5 and 7)) and the condensin-II-subunits (CapH2-G2-D3).

No Survivin- or Borealin-like proteins were detected, supporting earlier predictions that Plasmodium spp., and more generally apicomplexans, lack these conserved components of the CPC (Fig. 7c). Given the distinct localisation dynamics of ARK1 suggesting that it may not act as the classic passenger complex on chromosomes, it is possible that the chromatin-directing function of Survivin and Borealin has become obsolete for apicomplexans and that the structural motifs for localisation and activation of ARK1 by INCENP-A and -B might be revealed upon detailed comparative sequence analyses.

Because T. gondii also encodes two INCENP paralogues that interact with TgARK120, we examined INCENP evolution with a focus on alveolates and apicomplexans (Fig. 7b, c). Phylogenetic reconstructions did not reveal a clear scenario supporting either a single ancestral duplication or multiple recurrent duplication events. Instead, the observed patterns are more consistent with parallel, lineage-specific duplications, rather than a conserved INCENP1/INCENP2 duplication inherited from the apicomplexan ancestor—hence we named the paralogue in Plasmodium INCENP-A/-B to prevent any misinterpretation of the predicted functional equivalence of INCENP between the Toxoplasma and Plasmodium paralogues. We found parallel but separate duplications in other different alveolate lineages such as apicomplexan-like Squirmida, Chrompodellida and dinoflagellate-related lineages concomitant with the loss of Borealin/Survivin (Fig. 7c), suggesting INCENP duplications might compensate for loss of these chromatin-directing subunits. Notably, INCENP-B is absent from Piroplasmida, which also lack ARK2 and ARK3, suggesting functional co-evolution among specific Aurora and INCENP paralogues.

AlphaFold3—based co-folding of PbARK1 with INCENP-A or INCENP-B predicted a conserved IN-box-ARK1 kinase interaction flanked in all apicomplexan proteins by N- and C-terminal extensions that form a beta-sheet—folding INCENP-A/-B around ARK1 (Fig. 7b and Supplementary Fig. 9). In addition, the C-terminal extension has a conserved CxC motif, possibly involved in posttranslational modification (Fig. 7b). We could only predict a coiled-coil in the N-terminus of INCENP-B, and no further structural features could be discerned for INCENP-A using AlphaFold3. Such absence of any feature makes it hard to predict the binding mode of INCENP-A either as a single ARK1-INCENP-A interaction or forming an INCENP-A-ARK1-INCENP-B trimeric complex.

Finally, we examined whether non-canonical sequence features in ARK1 or its INCENP partners might point to alternative models of CPC regulation in Plasmodium spp., given that autophosphorylation sites in both Aurora B and INCENP are critical for CPC function in vertebrates32. Both the broadly conserved Aurora-kinase activation-loop motif (RxTxCGT) and the canonical INCENP activation site (RTSS in vertebrates) are absent from the Plasmodium proteins: ARK1 instead carries a divergent GxSH motif, and INCENP-A and INCENP-B lack an RTSS-equivalent position. However, INCENP-A and INCENP-B retain Aurora consensus motifs in adjacent regions (Fig. 7b, green boxes). For comparison, ARK2 paralogues retain a canonical Aurora activation loop motif, whereas ARK3 possesses a serine-rich activation loop containing multiple candidate phosphorylation sites (Fig. 7c). The absence of clear conserved auto/trans-phosphorylations sites between ARK1 and INCENP-A/-B in Plasmodium spp. suggest that CPC kinase activity may rely on a distinct regulatory mechanism, potentially involving input from an additional kinase.

Methods
P. falciparum cultures
Plasmodium falciparum strain 1G5DC was obtained from European Malaria Reagent Repository (EMRR) and DSM1 was obtained from BEI resources, Malaria Research and Reference Reagents Resource Centre (MR4), American Type Culture Collection (ATCC). Parasites were maintained in O+ human erythrocytes (5% haematocrit) in RPMI-1640 supplemented with 0.5% Albumax II and 50 μg/mL hypoxanthine41. Cultures were maintained at 37°C under a mixture of 5% CO2, 3% O2, and 91.8% N2 or 5% CO2. Fresh erythrocytes and culture medium were used to dilute parasites to maintain 3–5% parasitaemia and 5% haematocrit. Relevant drugs were used during the culture of various transgenic lines, as described below. Parasite synchronisation was carried out using 5% sorbitol as described previously42.

Generation of transgenic parasite lines
All PCR primers used in this study were synthesized by Sigma and are indicated in Supplementary Data 3.

1G5DC SkipFlox
A dimerisable Cre recombinase (diCre) expressing parasite line was generated by transfecting pSkipFlox plasmid, a kind gift from Tobias Spielmann (Addgene Plasmid #85797) into the parent line 1G5DC21. Parasites were selected in the presence of 2.5 μg/ml of blasticidin. For simplicity of representation, this parasite line is referred to as 1G5DC in the text and figures.

HA-PfARK1-loxP To generate a conditional knockout transgenic parasite, a selection-linked integration (SLI) approach was used. To generate the pSLI-HA-ARK1-loxP construct, a 592 bp homology region corresponding to the 5’-end of the PfARK1 gene was amplified using primers (P1 and P2) derived from 3D7 genomic DNA. The PCR product was cloned into NotI and PmeI sites upstream of loxP and a Myc-tag coding sequence in the pSLI-N-sandwich-loxP (K13) vector, which was a gift from Tobias Spielmann (Addgene Plasmid #85793). A recodonised sequence version of full-length PfARK1 with a N-terminal 3xHA tag and followed by a loxP site was custom synthesised as a G-block (Genscript). The G-block was cloned in the above construct downstream of yDHODH using NheI and XhoI restriction sites.

P. falciparum transfection
The pSLI-HA-PfARK1-loxP construct was transfected in 1G5DC SkipFlox parasites at the ring stage using ~100 μg of purified plasmid DNA. Transgenic parasites were selected by using 2 nM WR99210 and integrants were then enriched using 1.5 μM DSM-1. The parasites that appeared after drug selection were genotyped using PCR primers (Supplementary Data 3 and Supplementary Fig. 1a). The desired integration was confirmed by sequencing of PCR products.

Conditional knockout of PfARK1
HA-PfARK1-loxP parasites were treated with 250 nM rapamycin at ring stage for 24 h. The genomic DNA was isolated from DMSO/RAP-treated parasites and excision of the PfARK1 locus flanked by loxP sites in the transgenic parasite was confirmed by PCR using primers (P7/P8).

P. falciparum growth rate assays
HA-PfARK1-loxP parasites were cultured in the presence of 10 nM WR99210, 2.5 μg/ml blasticidin and 1.5 μM DSM-1. Cultures were maintained at 2% haematocrit and 7-8% parasitaemia. To perform the growth rate assay, 1G5DC-SkipFlox and HA-PfARK1-LoxP parasites were synchronised at ring stage for two cycles using 5% sorbitol for 20 min at 37°C, and the rings were seeded at 0.5% parasitaemia and 2% haematocrit. To deplete PfARK1, parasites were treated with either DMSO (vehicle control) or 250 nM rapamycin for 24 h. Thin Giemsa-stained blood smears were examined periodically under a microscope to quantify the parasitaemia at different parasite stages. For assessment of parasitaemia by flow cytometry, cells were fixed by incubation in a solution containing 1% paraformaldehyde and 0.0075% glutaraldehyde at ambient temperature for 15 min on an end-to-end rocker. Post-fixation, samples were either stored at 4°C or stained immediately with Hoechst 33342 dye at 37°C for 10 min. After staining, samples were washed with FACS buffer and then analysed on either BD verse (BD Biosciences) or BD FACSymphony A1 Flow Cytometers, acquiring between 10,000 and 100,000 events per sample. Data analysis was carried out using FlowJo software version 10.10.0.

Purification of P. berghei schizonts and gametocytes
Blood-stage parasites were obtained from infected mice on day 4 post-infection and cultured at 37°C with gentle rotation (100 rpm) for 11 or 24 h. Schizonts were purified the following day using a 60% v/v NycoDenz gradient prepared in phosphate-buffered saline (PBS). The NycoDenz stock solution consisted of 27.6% w/v NycoDenz in 5 mM Tris-HCl (pH 7.2), 3 mM KCl, and 0.3 mM EDTA.

Gametocyte purification was performed by first infecting phenylhydrazine-treated mice43, followed by sulfadiazine treatment two days post-infection to enrich for gametocytes. Blood was collected on day 4 post-infection, and gametocyte-infected cells were purified using a 48% v/v NycoDenz gradient in PBS, prepared from the same NycoDenz stock solution. Gametocytes were collected from the interface and subsequently activated for downstream assays44.

Generation of P. berghei transgenic parasites
GFP-tagging constructs were generated using the p277 plasmid vector and transfected into parasites as previously described45. A schematic representation of the wild-type ark1 locus, the GFP-tagging constructs, and the recombined ark1 locus is shown in Supplementary Fig. 2a. Correct integration of the gfp sequence at the ark1 locus was confirmed by diagnostic PCR using primer 1 (intArk1tg) and primer 2 (ol492), as indicated in Supplementary Fig. 2b. The expression of native ARK1-GFP was confirmed by Western blot analysis (Supplementary Fig. 2c).

To investigate the function of ARK1, a promoter trap strategy was employed using double homologous recombination to generate a conditional knockdown line (ark1PTD). The knockdown construct was derived from the Pama1 plasmid (pSS368), in which ark1 was placed under the control of the ama1 promoter, as previously described22. A schematic representation of the endogenous ark1 locus, the targeting constructs, and the recombined ark1 locus is shown in Supplementary Fig. 6a. Diagnostic PCR was performed to confirm successful integration, as outlined in Supplementary Fig. 6a. Integration at the 5′ locus was verified using primers intPTD31_5 (Primer 1) and 5′-intPTD (Primer 2), while integration at the 3′ end was confirmed using primers intPTD31_3 (Primer 3) and 3′-intPTama1 (Primer 4) (Supplementary Fig. 6b). Primer sequences are listed in Supplementary Data 3. Transfections were carried out by electroporation using P. berghei ANKA line 2.34 for GFP-tagging experiments and ANKA line 507cl1 expressing GFP for generation of the knockdown line30.

Generation of dual tagged parasite lines (P. berghei)
To generate dual-labelled parasites, ARK1-GFP parasites were mixed in equal proportions with either NDC80-mCherry, ARK2-mCherry, or kinesin-8B-mCherry labelled parasites and co-injected into mice. Four to five days post-infection, when gametocytaemia was high, Anopheles mosquitoes were allowed to feed on the infected mice. Mosquitoes were examined for oocyst development and sporozoite formation at days 14 and 21 post-feeding. Infected mosquitoes were then used to infect naïve mice via mosquito bite-back. Blood-stage infection was assessed 4–5 days later by Giemsa-stained blood smear microscopy. In this way, parasites expressing both ARK1-GFP and either NDC80-mCherry, ARK2-mCherry, or kinesin-8B-mCherry were obtained. Gametocytes from these mixed lines were purified, and fluorescence microscopy was performed as described below to examine protein co-localisation.

P. berghei phenotype analyses
Infections were initiated by intraperitoneal injection of approximately 50,000 P. berghei parasites from WT-GFP or ark1PTD lines into mice. Asexual stages and gametocyte production were monitored on Giemsa-stained thin smears. Four to five days post-infection, exflagellation and ookinete conversion were assessed using a Zeiss Axio Imager M2 microscope with an AxioCam ICc1 digital camera. For mosquito transmission assays, 30–50 Anopheles stephensi (SD 500) mosquitoes were allowed to feed for 20 min on anaesthetised, infected mice with ~15% asexual parasitaemia and comparable gametocyte levels (as determined by Giemsa-stained smears). To evaluate midgut infection, ~15 mosquito guts were dissected on day 7, 14 and 21 post-feeding, and oocysts were counted using a 63× oil immersion objective on the same microscope setup. Sporozoites in oocysts were counted on day 14 and day 21 post feeding. Salivary glands were isolated on day 21 to count the sporozoites. Bite-back experiments were performed 21 days post-feeding using naïve mice, and blood smears were examined after 3–4 days. All experiments were conducted at least three times to assess the phenotype.

Immunofluorescence assay (IFAs)
Immunofluorescence assays (IFA) were conducted on thin blood smears following a previously described protocol46. In brief, air-dried smears were fixed for 2 min using ice cold 1:1 mixture of methanol and acetone. The smears were dried to remove any traces of methanol/acetone followed by blocking with 3% BSA for 45 min at room temperature. Samples were incubated with primary antibodies for 12 h at 4°C. After thorough washing with 1X PBS, Alexa Fluor-conjugated secondary antibodies were added and incubated for 2 h at room temperature. Smears were then mounted with VectaShield medium (Vector Laboratories Inc.) containing DAPI for nuclear staining. Microscopy was performed using a LSM980 AR confocal microscope (Carl Zeiss) or Zeiss Axio Observer microscope and image processing was carried out using Zeiss ZEN Blue (versions 3.1 or 3.9). Representative z-stack images were selected for figure preparation unless indicated otherwise. IFA or U-ExM (described below) with MSP1 or GAP45 antibodies was performed to assess segmentation in schizonts that had been treated with 10 μM E64 at ~42 hpi (cycle 1) for 4–5 h to prevent egress and allow complete maturation.

P. falciparum live-cell imaging
Live imaging was carried out with minor modifications to previously described protocols28. HA-PfARK1-loxP parasites were synchronised at the ring stage and treated with either DMSO or 250 nM rapamycin for 24 h. Schizonts (cycle 1) were subsequently purified and co-cultured with fresh erythrocytes at 2% haematocrit in RPMI1640 complete medium in the presence of 25 nM PKG inhibitor ML1026,27 (a gift from LifeArc). ML10 was removed after 4–6 h and parasites were plated onto µ-slide, 2-well ibiTreat chambers (ibidi, 80286) pre-coated with 0.5 mg/mL concanavalin A. The slides were maintained at 37°C in a humidified chamber with 5% CO2. Imaging was performed using a Zeiss Axio Observer microscope, capturing frames every 3 seconds for a minimum duration of 30 min and the acquired time-lapse videos were processed and analysed using AxioVision software (version 4.8.2) or ZEN blue 3.1 or 3.9.

P. berghei live cell imaging
To examine PbARK1-GFP expression during erythrocytic development, parasites cultured in schizont medium were imaged at various stages, including ring, trophozoite, schizont, and merozoite. Purified gametocytes were assessed for GFP expression and localisation at multiple timepoints (0 and 1–15 min) following activation in ookinete medium. For nuclear staining, Hoechst dye was used during live-cell imaging. Zygote and ookinete stages were analysed over a 24-h culture period. Images were acquired using a Zeiss Axio Imager M2 microscope equipped with a 63× oil immersion objective and an AxioCam ICc1 digital camera (Carl Zeiss) using auto exposure settings. Fluorescence signals were adjusted using Axiovision (Rel. 4.8) software to reduce background fluorescence while maintaining detection sensitivity.

P. berghei structured illumination microscopy
Formaldehyde-fixed (4%) gametocytes were stained with Hoechst dye, and 2 μl of the cell suspension was placed on a microscope slide and covered with a 50×34 mm coverslip to form a thin, immobilised cell monolayer. Imaging was performed using structured illumination microscopy (SIM) on a Zeiss Elyra PS.1 microscope with either a Zeiss Plan-Apochromat 63×/1.4 oil immersion or Zeiss C-Apochromat 63×/1.2 W Korr M27 water immersion objective, as previously described36.

Ultrastructure expansion microscopy
U-ExM was performed largely following previously published protocols23,47–49. In brief, to prepare U-ExM samples of P. falciparum schizonts, 12 mm coverslips were placed in a 24-well plate and coated with poly-D-lysine (Gibco, A3890401, 0.1 mg/mL) for 1 h at 37°C, followed by three washes with ultrapure water. P. falciparum schizonts were seeded onto the coverslips and incubated for 30 min at 37°C. Unattached cells were removed gently, and the remaining cells were fixed using 4% (v/v) paraformaldehyde in PBS for 15 min at 37°C. After fixation, coverslips were rinsed with prewarmed PBS and incubated for 12 h at 37°C in 1.4% (v/v) formaldehyde and 2% (v/v) acrylamide in PBS. A monomer solution consisting of 19% (w/w) sodium acrylate (Sigma-Aldrich, 408220), 10% (v/v) acrylamide (Sigma-Aldrich, A3553), and 2% (v/v) N,N′-methylenebisacrylamide (Sigma-Aldrich, M7279) in PBS was prepared 24 h in advance and stored at −20°C. For polymerisation, 5 μL of 10% (v/v) TEMED (Sigma-Aldrich, T9281) and 5 μL of 10% (w/v) APS (Sigma-Aldrich, A3678) were mixed with 90 μL of the thawed monomer solution. The gel mix (35 μL) was pipetted onto parafilm squares pre-cooled at −20°C and the coverslip was placed over the drop. Gels were allowed to polymerise for 1 h at 37°C. Polymerised gels were transferred to denaturation buffer (200 mM SDS, 200 mM NaCl, 50 mM Tris, pH 9) and shaken for 15 min at room temperature, then incubated at 95°C for 90 min. Denatured gels were expanded by sequential incubations in water (30 min each, repeated thrice) with gentle shaking. Expanded gels were equilibrated in PBS for 15 min and blocked in 3% BSA/PBS for 30 min at room temperature. Incubation with primary antibody (details in Supplementary Data 4) was carried out for 12 h at room temperature on a shaker, followed by three 10 min washes with PBST. Gels were then incubated with secondary antibodies, 405 NHSEster (ThermoFisher, A30000) and SYTOX/Hoechst in PBS for 2.5 h in the dark with gentle shaking followed by three PBST washes and final equilibration in MilliQ water (30 min, repeated thrice). For imaging, µ-slide 2-well ibiTreat chambers (ibidi, 80286) were coated with poly-D-lysine (0.5 mL, 1 h at 37°C), rinsed with MilliQ water, and used to mount the gels cell side down. Imaging was performed using a Zeiss LSM980 confocal microscope with a 63X oil immersion objective (NA 1.4). Z-stacks (0.5 μm intervals) were captured and processed using ZEN Blue (versions 3.1 or 3.9). Deconvolution was performed using Zen Deconvolution toolkit. U-ExM images are displayed as maximum intensity projection of full z-slices unless indicated otherwise.

To prepare U-ExM samples of P. berghei male gametocytes, purified and 8 min- and 15 min-activated gametocytes were fixed in 4% formaldehyde in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl2, pH 6.9) at room temperature for 15 min. Fixed samples were attached to 10 mm round Poly-D-Lysine coated coverslips for 15 min. Coverslips were incubated overnight at 4°C in 1.4% formaldehyde (FA)/ 2% acrylamide (AA). Gelation was performed in ammonium persulfate (APS)/TEMED (10% each)/monomer solution (23% sodium acrylate; 10% AA; 0,1% BIS-AA in PBS) on ice for 5 min and at 37°C for 30 min. Gels were denatured for 15 min at 37°C and for 90 min at 95°C in denaturation buffer (200 mM SDS, 200 mM NaCl, 50 mM Tris, pH 9.0, in water). After denaturation, gels were incubated in distilled water overnight for complete expansion. The following day, circular gel pieces with a diameter of ~13 mm were excised, and the gels were washed in PBS three times for 15 min to remove excess water. The gels were then incubated in blocking buffer (3% BSA in PBS) at room temperature for 30 min, incubated with mouse monoclonal anti-a-tubulin antibody (T9026, Sigma-Aldrich, 1:500 dilution) and/or anti-GFP antibody (A11122, Invitrogen, 1:500 dilution) in blocking buffer (1:500 dilution) at 4°C overnight and washed three times for 15 min in wash buffer (0.5% v/v TWEEN-20 in PBS). The gels were incubated with 8 μg/ml Atto 594 NHS ester (08741, Merck), 10 μg/ml Hoechst 33342 (H1399, Invitrogen), Alexa Fluor 488 goat anti-mouse IgG (A11001, Invitrogen, 1:500 dilution) and Alexa Fluor 568 goat anti-mouse IgG (A11004, Invitrogen, 1:500 dilution) in PBS at 37°C for 3 h followed by three washes of 15 min each in wash buffer (blocking and all antibody incubation steps were performed with gentle shaking). The gels were then washed three times for 15 min with wash buffer and expanded overnight in ultrapure water. The expanded gel was placed in a 35 mm glass bottom dish (MATTEK) with the 14 mm glass coated with Poly-D-Lysine and mounted with an 18×18 mm coverslip to prevent the gel from sliding and to avoid drifting while imaging. High-resolution confocal microscopy images were acquired using a Zeiss Cell discoverer 7 with Airyscan using a Plan-Apochromat 50 ×/1.2NA Water objective, with 405, 488 and 561 nm lasers. Confocal z-stacks were acquired using line scanning and the following settings: 55×55 nm pixel size, 170 nm z-step, 2.91 μs/pixel dwell time, 850 gain and 3.5% (405 nm), 4.5% (488 nm) and 5.0% (561 nm) laser powers. The z-stack images were processed and analysed using Fiji (Version 1.54f)50.

Calculation of the expansion factor for the U-ExM of male gametocytes
The expansion factor for the U-ExM of male gametocytes was calculated by comparing axoneme diameters from two types of images (U-ExM and TEM). The diameters were measured using Fiji (Version 1.54f)50. The average diameter of the axoneme in NHS-stained U-ExM images of male gametocytes activated for 8 min was 933.83 nm (n=12), while the average diameter in TEM images of male gametocytes activated for 8 min was 188.50 nm (n=8). We used the calculated expansion factor of 4.954 for the analysis.

Electron microscopy of male gametocytes
P. berghei gametocytes activated for 8 min and 15 min were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer and processed for electron microscopy51. Briefly, samples were post-fixed in 1% aqueous osmium tetroxide, treated en bloc with 2% aqueous uranyl acetate, dehydrated in graded ethanol series and embedded in Spurr’s epoxy resin. Thin sections were stained with lead citrate prior to examination in a Tecnai G2 12 BioTwin (FEI UK, UK) or a Jeol JEM-1400Flash (JEOL, Japan) electron microscope.

Liver stage parasite imaging
For P. berghei liver-stage imaging, 100,000 Hepatocelluar carcinoma cells (HepG2) were seeded on to glass coverslips in 48 well plate. Salivary glands from Anopheles stephensi mosquitoes infected with PbARK1-GFP parasites were dissected and homogenised using a pestle to release sporozoites. The sporozoites were gently pipetted onto the HepG2 cell monolayer and incubated at 37°C with 5% CO₂ in complete minimum Eagle’s medium The culture medium was replaced 2 h post-infection and subsequently changed daily. For live-cell imaging, Hoechst 33342 (Molecular Probes) was added to a final concentration of 1 μg/ml to stain host and parasite nuclei. Infected cells were imaged at, 48-, and 64 -hours post-infection using a Zeiss Axio Imager M2 microscope equipped with a 63× oil immersion objective and an AxioCam ICc1 digital camera (Carl Zeiss) using auto exposure settings. and Leica Application Suite X software. Each experiment was performed in triplicate, and 20–30 infected cells were analysed per time point to assess PbARK1-GFP localisation.

Immunoblotting
P. falciparum—Immunoblotting was performed as described previously52 using primary antibodies (Supplementary Data 4) and HRP-conjugated secondary antibodies and protein bands were visualized using either SuperSignal™ West Pico or Femto chemiluminescent substrates (Pierce, USA) using X-ray films.

P. berghei—Purified gametocytes were lysed in buffer containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, and 1% NP-40. After lysis, Laemmli buffer was added, and samples were boiled for 10 min at 95°C, followed by centrifugation at 13,000 × g for 5 min. The supernatants were separated on a 4–12% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Amersham Biosciences). Immunoblotting was performed using the Western Breeze Chemiluminescence Anti-Rabbit Kit (Invitrogen), following the manufacturer’s protocol. An anti-GFP polyclonal antibody (Invitrogen) was used at a 1:1,250 dilution to detect PbARK1-GFP.

Quantitative real-time PCR (qRT-PCR) analyses
P. falciparum—Total RNA was extracted using TRIzol reagent (G Biosciences) followed by purification with the Qiagen RNeasy Mini Kit (Cat. No. 74104). cDNA synthesis was performed using RevertAid H Minus Reverse Transcriptase (Thermoscientific, EP0451) and 1 μg of DNase-treated RNA as the template. Quantitative PCR was conducted using the CFX96 Real-Time PCR Detection System (Bio-Rad). Reactions were normalized using 18S rRNA as the internal control. Relative gene expression levels were calculated using the 2-ΔΔCt method. Primer sequences used for the qRT-PCR are listed in Supplementary Data 3.

P. berghei—Total RNA was extracted from gametocytes using the RNA Purification Kit (Stratagene), and cDNA was synthesised using the RNA-to-cDNA Kit (Applied Biosystems). qRT-PCR was performed with cDNA from 80 ng of RNA using SYBR Green Fast Master Mix (Applied Biosystems) on an Applied Biosystems 7500 Fast system. Cycling conditions were: 95°C for 20 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. Primers were designed using Primer3 (https://primer3.ut.ee/). Each gene was tested in three biological and technical replicates. hsp70 (PBANKA_081890) and arginyl-tRNA synthetase (PBANKA_143420) were used as reference genes. Primer sequences are listed in Supplementary Data 3.

Transcriptome analysis using RNA-seq
Total RNA was extracted from activated gametocytes and schizonts of P. berghei WT-GFP and ark1PTD parasites (three biological replicates each) using the RNeasy Kit (Qiagen), vacuum-concentrated, and transported in RNA-stable tubes (Biomatrica). Strand-specific mRNA libraries were prepared with the TruSeq Stranded mRNA Sample Prep Kit (Illumina) and sequenced on an Illumina HiSeq 4000 platform (paired-end, 150 bp reads). Read quality was assessed with FASTQC, and trimming of low-quality reads and adapter sequences was done using Trimmomatic53. Reads were mapped to the P. berghei ANKA genome (PlasmoDB release 40) using HISAT2 v2.1.054, and gene counts were generated with FeatureCounts55. Low-expression genes (CPM < 1) were excluded. Data were normalized using the TMM method (EdgeR56), transformed with voom (limma57), and analysed for differential expression using DESeq258. Statistical significance was determined using a two-sided Wald test, with P-values adjusted for multiple comparisons using the Benjamini-Hochberg procedure. Genes with fold-change > 2 and FDR < 0.05 were considered differentially expressed.

Immunoprecipitation and mass spectrometry
P. berghei purified schizonts and gametocytes (activated for 1.5–2 min) were crosslinked using formaldehyde 10-min incubation with 1% formaldehyde, followed by 5-min incubation in 0.125 M glycine solution and 3 washes with PBS (pH 7.5). Immunoprecipitation was performed using crosslinked protein and a GFP-Trap_A Kit (Chromotek) following the manufacturer’s instructions. Proteins bound to the GFP-Trap_A beads were digested using trypsin and the peptides were analysed by LC-MS/MS and subsequently visualised using both Principal Component Analysis (PCA) and for relative ratios of WT/POI using a volcano plot as published previously36,59. In short, significance of enrichment was assessed using the two-tailed Student t test (implemented in R), and p values were corrected for multiple testing using the Benjamini–Hochberg method. iBAQ values were normalized by the sum of all iBAQ values over one experiment. Only those proteins were considered with two or more unique peptides and at least 2 values in ARK1 pulldowns. Volcano plots were visualised using VolcaNoseR60.

Evolutionary bioinformatics
Our predicted proteome set was based on a version previously used (see Supplementary Data 5)61. Phyletic profiles for ARK1-3 and INCENP were partially derived from previous studies10,62. Sequence searches for missing orthologues were performed with the HHPred web server from MPI-toolkit63,64 and the hmmer package63, as previously described61. For the larger gene families like Aurora kinases, we used IQ-tree-based maximum-likelihood phylogenetics (standard setting-model finding, 1000 bootstraps65) to check the validity of bidirectional-best-blast (BBH) hits amongst our genome sets on an ad hoc basis. Off note, we could not reliably generate a single phylogenetic tree harbouring all three ARKs within the Aurora kinase orthologous group (OG), suggesting that ARK2 and ARK3 are either extremely divergent or not Aurora kinases. Similarly, for the INCENP gene family, multiple alignments were generally too divergent and phylogenetically informative stretches too short to infer any consistent phylograms. All sequences used for this study can be found in Supplementary Data 5.

AlphaFold3 modelling
Co-folds of 3D protein structures for PbARK1 and the extended IN-box of either PbINCENP-A or -B were modelled using the AlphaFold3 web server (https://alphafoldserver.com/) with standard settings (seed set to 100)66.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Nagar and colleagues evaluates the function of ARK1 in Plasmodium parasites. Using the rodent malaria, Plasmodium berghei, and the human Plasmodium falciparum, ARK1 is hypothesized to be part of the inner MTOC and to influence spindle/kinetochore dynamics. The findings are most striking during male gamete formation.\nThe manuscript combines elegant genetic, cell biology, and evolutionary biology to understand the function of the divergent ARK1 in Plasmodium. This is a nice study that combines findings in two Plasmodium species. The findings are largely well-supported. I have suggested some additions and changes that I think will improve the study.\nMajor\n1. Claims about the timing of ARK1 expression in line 124-125 are likely true but are only weakly supported by the provided data of two schizonts. It would be helpful for the reader to have some quantification of this phenotype. One could imagine counting the number of nuclei in each stage of their karyokinesis cycle and noting if ARK1 is present or absent. I do not doubt the result shown, but it would be stronger with quantification. Line 127-137, please explain why the localization of PbARK1 is diffuse when this localization is not mentioned or observed for PfARK1. This is likely due to method differences, but the claim that the localization is the similar is not fully supported without further explanation.\n2. The RNAseq paragraph (lines 279-300) does not seem to add any useful information. I think this is distracting and detracts from the more interesting story that is unfolding. This should be removed because the observations/hypotheses are not experimentally tested.\n3. The use of immunoprecipitation to identify INCENP1 and INCEP2 is a strong piece of data. It would be nice, if possible, to show colocalization of ARK1 and these proteins. While it seems extremely likely that they will colocalize, it is good technique to validate findings from mass spectrometry with additional experiments. Because these structures are stable and associated with microtubules, they may “interact” or immunoprecipitated over large distances.\nMinor\n1. Figure 1b is a nice figure to give the reader a map of the proposed localization and function. The small blue “nucleoplasm” dot is a bit odd and was confusing for me. I think that this would be better to move out of the center of the panel and into a legend box or something. This extra dot seems like it should be a structure when it is not really anything.\n2. Line 123 – the term “polar ends” should be clearly defined.\n3. Fig 2E – the GFP antibody staining has high background. Can a different cell be selected? Perhaps a different gel preparation and a different antibody preparation (or source of antibody) will help to reduce this unfortunate background.\n4. Line 197 – the statement “ a significant number” needs to be backed up by a statistical measurement or removed/changed.\n5. Line 234-236 – The primary defect is abnormal karyokinesis. The cytokinesis defect is likely entirely secondary to this primary defect. It should be stated very explicitly that ARK1 has little to do with cytokinesis. While this is implied, for the non-expert reader it should be stated clearly.\n6. In my opinion, the fig 6b could be moved to a supplement. The Alphafold3 server is publicly available and anyone could generate these models on their own.", "reviewer_2": "Summary:\nIn this manuscript, Nagar and colleagues present a comprehensive investigation of Aurora-related kinase 1 in P. falciparum schizogony and P. berghei male gametogony, combining live-cell imaging, expansion microscopy, and electron microscopy. The authors demonstrate that PbARK1 is essential for spindle formation, MTOC separation, exflagellation, and parasite transmission, while PfARK1 disruption impairs spindle assembly and normal karyokinesis/cytokinesis. Proteomics analysis suggests that PbARK1 interacts with INCENP1/2 to form a chromosome passenger complex.\nOverall, the study provides valuable insight into ARK1 function in both asexual and sexual stages of Plasmodium, contributing to a better understanding of how ARK1 regulates cell division through distinct mitotic mechanisms in schizogony and gametogony. However, several methodological and data presentation issues need to be addressed. Specially, the internal HA-tag insertion in PfARK1 requires further justification. Additionally, the PfAK1 expansion microscopy images lack sufficient resolution or stage comparability to convincingly support claims that PfARK1 deficiency causes centriolar plaque mis-segregation or separation from the nucleus, as well as altered subpellicular microtubule polymerization or localization. Therefore, additional clarification and higher-resolution data are required before publication.\nMajor comments:\n1) Line 112 and Extended Data Fig. 1a: The authors describing generating a HA-PfARK1-loxP parasite line, which appears to be N-terminally HA-tagged. However, Extended Data Fig. 1a shows that the HA tag was inserted internally within the PfARK1 coding sequence rather than at the N- or C-terminus. Although the localization pattern resembles PfARK1-GFP reported previously, and this line was used for conditional PfARK1 disruption, it remains unclear whether this internal tagging affects the protein’s native function or folding. Please clarify the rationale for this strategy and specify whether the HA tag lies in an unstructured or non-conserved region.\n2) Lines 113-125 and Fig. 2a-b: PfARK1 localization has been reported previously (PMID: 21166904; PMID: 39235260). The data here are confirmatory and should be stated as consistent with those earlier findings.\nFor Fig. 2a-b, please provide higher-magnification PfARK1/Tubulin/NHS-ester merged images to improve clarity and data presentation.\n3) Fig. 3d: Please provide higher-magnification merged images combining the tubulin, centrin, SYTOX, and NHS-ester channels to better illustrate the described phenotype. Current overview images lack resolution to convincingly support the statement that “the nuclei appeared unsegregated with associated multiple centrin foci, and centrin aggregates were seen away from the nuclei and disorganized” (line 211-212). In addition, the tubulin channel in RAP-treated parasites appears over-saturated. Please adjust intensity to accurate representation.\n4) Extended Data Fig. 4e: The control and PfARK1-KO schizonts appear at different developmental stages (control: early/mid schizonts; RAP-treated: late schizonts). Please clarify whether these images were acquired at comparable time points, as stage differences may confound phenotype interpretation.\n5) Fig. 3f: Please provide higher-magnification merged images showing GAP45, tubulin, SYTOX, and NHS-ester channels to better illustrate the defective merozoite phenotype in RAP-treated cells. The RAP-treated schizonts shown appear at an early segmentation stage, indicated by partially formed GAP45 signals and short subpellicular microtubules. In contrast, control schizonts are fully formed GAP45 sheaths surrounding individual merozoites with well-developed subpellicular microtubules. Because subpellicular microtubule length varies with stage, these images cannot be directly compared to support the statement that “subpellicular MT were either not observed in the cell or were mislocalised” (line 233 to 234). Please include representative fully segmented schizonts or clarify this limitation.\n6) Line 325 and Fig. 5dJ: The statement that “MTOCs that appeared detached from the nucleus in the ark1 PTD line” is not clearly supported. In Fig. 5dJ, the inner MTOC remains within the nucleus, and Extended Data Fig. 6bA, B and D, the inner MTOC is not evident. More convincing or annotated images are needed.\nMinor comments:\n1) Line 84: The localization of PfARK1-3 in gametocyte was previously reported (PMID: 39235260), showing PfARK1 is specific to male gametocytes.\n2) Figure 1b right panel: The localization of PfARK1-3 was previously described in PMID: 39235260 and PMID: 21166904.\n3) Line 114-115: The statement that “ARK1 is expressed in trophozoite and schizont stages, but not detectable in late schizonts and all developing merozoites (Fig. 2a)” is not fully supported. In Fig. 2a, the “trophozoite” image resembles an early schizonts (two nuclei), and the “late schizont” image resembles a segmented schizont. PfARK1 remains visible in late schizonts (Fig. 2b second panel) and was previously shown to persist (PMID: 39235260).\n4) Line 153-155, Fig. 2e: The data suggest that PbARK1 is not localized to spindle poles but instead associate with axonemal structures, as indicated by co-localization with α-tubulin. Please provide more representative images to support this interpretation.\n5) Extended Data Fig 2d: The “late schizont” image actually presents a segmented schizont. Additionally, strong background fluorescence in Extended Data Fig. 2d-e obscures PbAKR1 foci. Highlighting these regions would improve clarity.\n6) Extended Data Fig. 2f: The localization of PbARK1 relative to the mitotic spindle or spindle poles remains ambiguous. Higher-quality representative images are needed.\n7) Line 165 and Fig. 2f-g right panels: In Fig. 2f right panel, the PbARK1-GFP signal is broadly distributed within the nucleoplasm and does not clearly associate with the spindle or PbNDC80-mCherry. Similarly, in line 169 and Fig. 2g right panel, PbARK1-GFP is not distinctly localized to the spindle. Additional higher-resolution images or z-sections are recommended.\n8) Extended Data Fig. 6a: The highlighted squares do not match the zoom-in regions.\n9) Line 417-418: The claim that “Its colocalisation with kinetochore NDC80 and ARK2 markers further supports a dual role in spindle organization, kinetochore segregation, and spindle pole integrity” is not directly demonstrated. Consider revising to “potentially supports” rather than asserting it as proven.\n10) Line 427: The statement “In P. falciparum, ARK1 is required for proper spindle elongation” is unclear. Long spindle microtubules resembling hemispindles remain visible in RAP-treated parasites (Fig. 3d, third panel), suggesting elongation is not fully abolished. Please clarify or provide quantitative evidence.", "reviewer_3": "Summary: This study comprehensively defines the role of a key protein called ARK1 during mitosis of malaria parasites both in the asexual blood-stage, and during male gamete formation. Mitosis is perhaps the most well studied process in all of biology, but by contrast our understanding of mitosis and the proteins that control it in malaria parasites is relatively poor. The role of ARK1 is explored using a range of microscopy modalities (live-cell, TEM, expansion microscopy), transcriptomics, pull-downs, and an evolutionary/bioinformatic analysis. This study represents one of the more comprehensive protein-characterisation projects in malaria parasites and contributes to a growing body of literature on aurora-like kinases in apicomplexan parasites. These findings will definitely be of interest to the parasite cell biology community, and the evolutionary biology findings may be useful to the broader study of mitotic kinases. While not discussed extenstively by the authors, the aurora-like kinases do represent attractive targets for the development of novel antimalarials, and so findings from this study may also be useful in that context.\nOverall the experiments in this study are sound, and in my opinion could likely be published without the addition of new experiments. The way in which some of the data has been presented, or referred to, however could be improved. Below I have provided a number of comments that I have deemed as major (essential to address), minor (would be good to address), and some comments on spelling, syntax etc. Even though there are many comments, it is likely that these could be completely or mostly addressed in a reasonably short time-frame with changes to the text, figure presentation, or data analysis.\nMajor Comments\n• Throughout the text a range of terms for different biological processes are conflated with each other. Individually, these are not major problems, but collectively the make it confusing to determine the interpretation of some of the experiments. These should all be easily fixable with text changes. Below I will provide a few examples of this:\no Line 49 describes schizogony as a form of mitosis, when schizogony refers to the mode of cell division\no Lines 124 – 125: refers to parasites undergoing mitotic division, when the data regards nuclei undergoing karyokinesis\no Line 205 mentions looking at asexual cell division when karyokinesis is assessed\no Line 393, the first line of the discussion describes ARK1 as a central organiser of mitosis, but the term mitosis is not used at all when describing the phenotype of ARK1\no Line 399 implies ARK1 has a phenotype upstream of chromosome segregation. I assume this is meant to be chromatid segregation, but in either case, this is not directly assessed in any experiment.\no Lines 487-490 mention an atypical mechanism of cell division when it seems to be discussing mitosis.\n• The statistical analyses performed in Figures 3a, Extended data Figure 1f, 4a, and 4b are not appropriate. Because the control is normalised to 1, its standard deviation = 0. I don’t think this impacts the interpretation of the results hugely, but they should either be presented without this statistical test, or be presented without normalisation with a statistical test.\n• The interpretation of ARK1 localisation seems inconsistent across the manuscript. In the P. falciparum imaging, it was determined that ARK1 was only present alongside a mitotic spindle with no significant fluorescence outside this. Based on this, it seems reasonable that the diffuse cytosolic expression seen in the P. berghei-ARK1-GFP parasites is likely background. In some lifecycle stages like oocysts, sporozoites, and liver-stages this diffuse cytoplasmic staining is inferred as representing the localisation of ARK1, which in asexual and activated gametocytes it is ignored in favour of the punctate staining. The authors should be clear on when and why they think the observed fluorescence represents true ARK1 localisation vs off-target/background signal. Also, the observation of ARK1 in the Golgi of salivary gland sporozoites should be discussed.\n• The authors conclude that rhoptries were not formed properly or were absent following ARK1 iKO. The images in Figure 3F don’t seem to support this conclusion, as almost every merozoite has a visibly recognisable rhoptry pair.\n• I feel that the transcriptomic results are slightly over-interpreted. The only consistently downregulated group of proteins are ribosome-related, and none of them are mentioned individually (only as part of the GO analysis). The authors interpret the lack of ribosome biogenesis as indicative of cell cycle arrest and translational inhibition, but an alternative interpretation is that immediately post-fertilisation the parasites prepare to make new ribosomes.\nMinor Comments\n• The abstract says Plasmodium lacks a Bub1 homologue, but the Bub1 orthogroup on OrthoMCL contains the P. falciparum protein Pf3D7_1112100.\n• Lines 59-61 describes gametogeny as atypical because of an MTOC that spans the nuclear envelope and makes microtubules on both sides. With the exception of making an axoneme, this is also what happens in both asexual blood-stage parasites and during gametocytogenesis.\n• The naming of the centriolar plaque are very confusing across the manuscript. The structure is variously referred to as the MTOC (generally), the inner and outer MTOC, the centrosome, centriolar plaque, and spindle pole body. My suggestion for the least ambiguous way to define this structure is to call the overall structure the centriolar plaque, and specifically refer to the inner (intranuclear) and outer (cytoplasmic) regions of it. In any case, the naming conventions should be harmonised across the manuscript.\n• In the introduction it should also be stated that the Wyss et al study (ref 17) determined the localisation of ARK1 in P. falciparum gametocytes\n• Line 142 should specify activated gametocytes\n• It seems likely that impaired centriolar plaque segregation is the earliest occurring phenotype observed following ARK1 KD/iKO. All the other phenotypes, like impaired cytokinesis, karyokinesis, etc. are therefore most likely to be downstream consequences of impaired centriolar plaque segregation. It isn’t clear in the text that these are likely a related series of events.\n• Did the authors check to see if there is a compensatory ARK2 upregulation in the ark1PTD cell line, like there is following ARK1 iKO?\n• Line 310 says no exflagellation was observed, while line 333 says exflagellation was rarely observed.\n• Line 434-435 discusses a failure in MTOC-basal body segregation. Previous studies of inner centriolar plaque proteins have seen separation of the inner and outer centriolar plaque following knockdown. This seems similar enough to me to be worth mentioning here.\n• Line 481 says ARK1 and ARK2 are both indispensable, but this was not shown in this study.\n• Figures 2 and 3 could probably each be broken up into two figures. Figure 2 could logically be broken up into a falciparum and berghei figure. If not, the figure legends should be condensed as they’re currently over a full page of text.\n• For the microscopy analyses of aberrant nuclear, MSP1 and GAP45 staining, it should be better described what was defined as ‘normal’ and whether these were blinded.\n• For Figure 1a and 2a: The outer centriolar plaque is attached to the parasite plasma membrane from the onset of mitosis until at least the start of segmentation, but the diagram implies they float freely in the cytoplasm\n• Figure 1a says two hemispindles but depicts an interpolar spindle\n• In Figure 1b it says cytokinesis (central spindle), but this structure doesn’t exist when the parasite is doing cytokinesis. Also the diagram includes astral microtubules, which we don’t have evidence of in Plasmodium.\n• I don’t think it is fair to say ARK1-3 are absent in Cryptosporidium there are 6 C. parvum proteins in the ARK Orthogroup (Cdg1_1220, Cgd1_2630, Cgd8_750, Cgd3_3040, Cgd2_1830, Cgd6_5240). Comparing these proteins against the consensus sequences in Figure 6, it seems likely that Cgd3_3040 is the C. parvum homologue of ARK2.\n• Considering the strength and frequency of all the phenotypes quantified in Figure 3, it seems surprising that there is a <50% growth defect at this point in time. I think this is worth discussing in more detail, and also mentioning whether the reasonably modest growth defect might be due to the compensatory upregulation of ARK2.\n• In Extended Data Figure 2d, the Hoechst staining seems to be almost fully excluded from the forming merozoites. Is there a channel alignment issue or something there?\n• The expression profiles of ARK1 and AMA1 are very different. Did the authors determine if the ark1-PTD parasites have approximately normal blood-stage growth? This is important for interpreting the results of the bite-back experiment. Also, cause AMA1 is expressed in sporozoites, would it be expected to get a second wave of ARK1 expression during late oocyst development?\n• If I am interpreting Extended data Figure 5 correctly, roughly 50% of sporozoites produced sporozoites make it to the salivary gland in both WT and ark1-PTD parasites. From this, can it be inferred that the sporozoites that get produced are likely still invasive?\n• Figure 4e is missing some scale bars and the ark1PTD oocysts take up less of the FOV than the WT, making them look much smaller than the quantification would indicate.\n• Figure 3f, DMSO Tubulin is hugely over-exposed\n• Figure 2h the Hoechst looks very strange in the 3D-SIM image, is this expected?\nSpelling, grammar, syntax, etc.\n• Lines 57 and 63: De novo is not italicised.\n• Line 125 should say nuclei, or parasite nuclei, instead of parasites\n• Line 140: I think “along microtubules” rather than “at microtubules” is probably a better description of the localisation\n• Line 275: ‘bite-back experiments’ should be explained in more detail\n• Line 312-313: “led us to examine whether this” can be deleted\n• Line 339-340: I don’t understand “to confirm the logic of the observed subcellular localisations”.\n• Line 353-358 is more discussion rather than results\n• Extended data Figure 2c: What do the two annotated sizes correspond to?", "reviewer_author_response": "Response to reviewers\nWe thank all the reviewers for their comprehensive evaluation of our manuscript and\nconstructive feedback.\nWe have performed additional experiments and further quantification, revised the manuscript’s\ntext and figures to improve clarity and accuracy, and addressed most comments of the reviewers.\nBelow is a point-by-point response to the reviewers’ comments.\nAs suggested by the reviewers, the original Figure 2 is now divided into two separate figures\n(Figure 2 for P. falciparum results and 3 for P. berghei results, respectively). Please note that\nthe figure numbers mentioned below refer to this revised numbering. The line numbers\nmentioned below refer to those in the clean version of the manuscript.\nReviewer comments\nReviewer #1 (Remarks to the Author):\nThe manuscript by Nagar and colleagues evaluates the function of ARK1 in Plasmodium\nparasites. Using the rodent malaria, Plasmodium berghei, and the human Plasmodium\nfalciparum, ARK1 is hypothesized to be part of the inner MTOC and to influence\nspindle/kinetochore dynamics. The findings are most striking during male gamete formation.\nThe manuscript combines elegant genetic, cell biology, and evolutionary biology to understand\nthe function of the divergent ARK1 in Plasmodium. This is a nice study that combines findings\nin two Plasmodium species. The findings are largely well-supported. I have suggested some\nadditions and changes that I think will improve the study.\nMajor\n1. Claims about the timing of ARK1 expression in line 124-125 are likely true but are only\nweakly supported by the provided data of two schizonts. It would be helpful for the reader to\nhave some quantification of this phenotype. One could imagine counting the number of nuclei\nin each stage of their karyokinesis cycle and noting if ARK1 is present or absent. I do not doubt\nthe result shown, but it would be stronger with quantification. Line 127-137, please explain\nwhy the localization of PbARK1 is diffuse when this localization is not mentioned or observed\nfor PfARK1. This is likely due to method differences, but the claim that the localization is the\nsimilar is not fully supported without further explanation.\nThe quantification of parasites with ARK1 staining has been added to Fig. 2c.\nMethodological differences and different reporters (GFP for P. berghei and HA for P.\nfalciparum) probably are the reason. Live cell imaging of GFP was done to localize PbGFP-\nARK1, whereas indirect immunofluorescence (IFA) with fixed cells was used to locate HA-\nPfARK1. These two methodologies may have different sensitivities and IFA may give some\ncytoplasmic autofluorescence.\n2. The RNAseq paragraph (lines 279-300) does not seem to add any useful information. I think\nthis is distracting and detracts from the more interesting story that is unfolding. This should be\nremoved because the observations/hypotheses are not experimentally tested.\nWe have removed this paragraph from the main text as suggested, and relocated it to the\nsupplementary note. A brief paragraph describing the RNA-seq analysis is now included (Lines\n301-306).\n3. The use of immunoprecipitation to identify INCENP1 and INCENP2 is a strong piece of\ndata. It would be nice, if possible, to show colocalization of ARK1 and these proteins. While it\nseems extremely likely that they will colocalize, it is good technique to validate findings from\nmass spectrometry with additional experiments. Because these structures are stable and\nassociated with microtubules, they may “interact” or immunoprecipitated over large distances.\nWe strongly agree with the reviewer on the importance of validating mass spectrometry\ninteractions with colocalization studies to rule out indirect associations. In fact, a preprint by\nRoques et al. (2025)1, published subsequent to our submission, explicitly demonstrates that\nPbARK1 co-localises with INCENP1 and INCENP2 at spindle poles, spindles, and\nkinetochores in Plasmodium berghei. These independent imaging studies provide the requested\nvisual confirmation of co-localisation, strongly supporting our immunoprecipitation results.\nWe have opted to discuss the findings of Roques et al. in the main text to corroborate our\ninteraction data (Lines 356-358), rather than repeating these specific experiments with\nadditional mice and thereby adhering to the ethical principle of minimising animal usage.\nCorrection of terminology (INCENP2 → INCENP-A; INCENP1 → INCENP-B): In our\ninitial submission, we referred to the two Plasmodium INCENP proteins as INCENP1 and\nINCENP2, following the nomenclature from studies on Toxoplasma gondii, where two\nINCENP paralogs had been identified. We hypothesised that these were direct orthologs\nbetween Plasmodium and Toxoplasma.\nHowever, further phylogenetic inspection, in the light of the recent Roques et al independent\nstudy, suggested that a duplication giving rise to two INCENP paralogs likely occurred\nindependently in different apicomplexan lineages (i.e. there is no clear evidence for an INCENP\nduplication in the shared ancestor of Coccidia and Hematozoa). Thus, the two Plasmodium\nINCENPs are not orthologs of the Toxoplasma INCENP1/2 pair. Most importantly, both our\ndataset and that of Roques et al. show that the Plasmodium INCENP originally called\nINCENP2 in our manuscript (INCENP1 in Roques et al.) is the constitutive ARK1-interacting\nparalog, whereas the protein originally named INCENP1 interacts with ARK1 only during\nmeiosis. To avoid confusion and incorrect evolutionary inference, we have renamed\nPlasmodium INCENP2 as INCENP-A and Plasmodium INCENP1 as INCENP-B throughout\nthe manuscript. This naming reflects functional roles rather than unsupported assumptions of\northology. All text, figures, legends, and Supplementary Tables have been updated accordingly\n(Lines 346-409, 486-503, Figure 7, Extended Data Fig. 8 and 9, and Supplementary Table 5).\nMinor\n1. Figure 1b is a nice figure to give the reader a map of the proposed localization and function.\nThe small blue “nucleoplasm” dot is a bit odd and was confusing for me. I think that this would\nbe better to move out of the center of the panel and into a legend box or something. This extra\ndot seems like it should be a structure when it is not really anything.\nWe thank the reviewer for pointing out this confusion. In the revised Figure 1b, we have\nremoved the “nucleoplasm” dot that could be interpreted as a biological structure, and\nredesigned the panel to portray more clearly the locations of various Aurora kinases.\n2. Line 123 – the term “polar ends” should be clearly defined.\nWe have replaced the term \"polar ends\" with \"growing mitotic spindles\" for improved clarity.\n3. Fig 2E – the GFP antibody staining has high background. Can a different cell be selected?\nPerhaps a different gel preparation and a different antibody preparation (or source of antibody)\nwill help to reduce this unfortunate background.\nWe have prepared new gels and re-acquired the data using a different anti-GFP antibody. As a\nresult, the background was reduced, and we saw a clearer localization of ARK1-GFP to the\nspindle and spindle poles (Figure 3c).\n4. Line 197 – the statement “ a significant number” needs to be backed up by a statistical\nmeasurement or removed/changed.\nQuantification and statistics are now provided in revised Extended Data Fig. 5c to support this\nstatement.\n5. Line 234-236 – The primary defect is abnormal karyokinesis. The cytokinesis defect is likely\nentirely secondary to this primary defect. It should be stated very explicitly that ARK1 has little\nto do with cytokinesis. While this is implied, for the non-expert reader it should be stated clearly.\nWe agree with the reviewer that this is a critical distinction, and that the observed cytokinesis\ndefects are most likely the consequences of the primary failure in karyokinesis. To ensure that\nthis distinction is evident we have revised the text, stating explicitly that the impairment in\ncytokinesis is likely a downstream effect of a primary defect in nuclear division, rather than\nthe result of a direct role of ARK1 in cytokinesis (line 251-253).\n6. In my opinion, the fig 6b could be moved to a supplement. The Alphafold3 server is publicly\navailable and anyone could generate these models on their own.\nWe appreciate the reviewer’s suggestion; however we would prefer to retain this figure in the\nmain paper. Our argument is that although AlphaFold3 is publicly accessible, the specific co-\nfold predictions of ARK1 with INCENP-A/B structure presented in Fig. 6b required custom\nmodelling tailored to this study, and are not available elsewhere. Although single-protein\nAlphaFold2 predictions are widely accessible, these interaction-focused AF3 models provide\nnovel structural insight into the apicomplexan CPC architecture, which directly supports our\nsuggestions about conserved IN-box–kinase interfaces and the divergent CxC motifs. These\nstructural features are integral to our mechanistic interpretation and are discussed explicitly in\nthe main text, therefore we feel that they should be retained in the primary figure to improve\nclarity and strengthen the narrative.\nReviewer #2 (Remarks to the Author):\nSummary:\nIn this manuscript, Nagar and colleagues present a comprehensive investigation of Aurora-\nrelated kinase 1 in P. falciparum schizogony and P. berghei male gametogony, combining live-\ncell imaging, expansion microscopy, and electron microscopy. The authors demonstrate that\nPbARK1 is essential for spindle formation, MTOC separation, exflagellation, and parasite\ntransmission, while PfARK1 disruption impairs spindle assembly and normal\nkaryokinesis/cytokinesis. Proteomics analysis suggests that PbARK1 interacts with\nINCENP1/2 to form a chromosome passenger complex.\nOverall, the study provides valuable insight into ARK1 function in both asexual and sexual\nstages of Plasmodium, contributing to a better understanding of how ARK1 regulates cell\ndivision through distinct mitotic mechanisms in schizogony and gametogony. However, several\nmethodological and data presentation issues need to be addressed. Specially, the internal HA-\ntag insertion in PfARK1 requires further justification. Additionally, the PfAK1 expansion\nmicroscopy images lack sufficient resolution or stage comparability to convincingly support\nclaims that PfARK1 deficiency causes centriolar plaque mis-segregation or separation from the\nnucleus, as well as altered subpellicular microtubule polymerization or localization. Therefore,\nadditional clarification and higher-resolution data are required before publication.\nMajor comments:\n1) Line 112 and Extended Data Fig. 1a: The authors describing generating a HA-PfARK1-loxP\nparasite line, which appears to be N-terminally HA-tagged. However, Extended Data Fig. 1a\nshows that the HA tag was inserted internally within the PfARK1 coding sequence rather than\nat the N- or C-terminus. Although the localization pattern resembles PfARK1-GFP reported\npreviously, and this line was used for conditional PfARK1 disruption, it remains unclear\nwhether this internal tagging affects the protein’s native function or folding. Please clarify the\nrationale for this strategy and specify whether the HA tag lies in an unstructured or non-\nconserved region.\nWe hereby confirm that the HA-tag was introduced at the N-terminus of ARK1 as indicated in\nthe schematic in Extended Fig. 1a. A small homology arm from the 5'-region of the PfARK1\ngene was used for recombination to insert a loxP site, followed by sequence for a 2A skip\npeptide spanning yDHODH, and downstream a recodonized sequence coding for full-length\nARK1 with an HA-tag at its N-terminus and another loxP site was introduced. Following\nrecombination, N-terminal HA-tagged ARK1 was expressed, and following rapamycin-\nmediated excision, this gene sequence was deleted. A similar Selection-Linked Integration\n(SLI) approach has been used in the past by us2 and others3 . We have modified the figure\nlegend to clarify the construction and the methods section also has these details (Lines 551-560\nand Extended Data Fig 1).\n2) Lines 113-125 and Fig. 2a-b: PfARK1 localization has been reported previously (PMID:\n21166904; PMID: 39235260). The data here are confirmatory and should be stated as\nconsistent with those earlier findings.\nFor Fig. 2a-b, please provide higher-magnification PfARK1/Tubulin/NHS-ester merged\nimages to improve clarity and data presentation.\nWe include now a magnified PfARK1/Tubulin/NHS-ester image and cite the mentioned papers.\n3) Fig. 3d: Please provide higher-magnification merged images combining the tubulin, centrin,\nSYTOX, and NHS-ester channels to better illustrate the described phenotype. Current overview\nimages lack resolution to convincingly support the statement that “the nuclei appeared\nunsegregated with associated multiple centrin foci, and centrin aggregates were seen away from\nthe nuclei and disorganized” (line 211-212). In addition, the tubulin channel in RAP-treated\nparasites appears over-saturated. Please adjust intensity to accurate representation.\nWe include now the magnified tubulin/centrin/ SYTOX/NHS-ester image, and a new image\npanel (revised Figure 4d) that displays this defect better has been included. These images,\ntogether with additional images in Extended Fig 5e, collectively show the presence of multiple\ncentrin foci associated with nuclear \"aggregates\". In RAP-treated parasites spindles are not\nformed and residual tubulin is visualised as random \"aggregates or speckles\". This distribution\ngives an impression of being over-saturated; however as suggested by the reviewer we have\nadjusted the brightness and replaced one panel of the figure (revised Figure 4d).\n4) Extended Data Fig. 4e: The control and PfARK1-KO schizonts appear at different\ndevelopmental stages (control: early/mid schizonts; RAP-treated: late schizonts). Please clarify\nwhether these images were acquired at comparable time points, as stage differences may\nconfound phenotype interpretation.\nThe experiments were performed simultaneously with control and PfARK1-KO schizonts,\nhowever the growth of the KO parasites was impaired and the stage of schizont development\nwas skewed. Therefore, for better comparison we now include additional images of late\nschizonts for control parasites and early schizonts for RAP-treated parasites. It is clear from\nthese data that in late schizonts, which have undergone more rounds of nuclear division and\nCP duplication, the defects due to lack of nuclear division are even more apparent as the number\nof centrin foci is much higher.\n5) Fig. 3f: Please provide higher-magnification merged images showing GAP45, tubulin,\nSYTOX, and NHS-ester channels to better illustrate the defective merozoite phenotype in\nRAP-treated cells. The RAP-treated schizonts shown appear at an early segmentation stage,\nindicated by partially formed GAP45 signals and short subpellicular microtubules. In contrast,\ncontrol schizonts are fully formed GAP45 sheaths surrounding individual merozoites with\nwell-developed subpellicular microtubules. Because subpellicular microtubule length varies\nwith stage, these images cannot be directly compared to support the statement that\n“subpellicular MT were either not observed in the cell or were mislocalised” (line 233 to 234).\nPlease include representative fully segmented schizonts or clarify this limitation.\nWe now include a merged image of GAP45/tubulin/ SYTOX/NHS-ester channels (revised Fig.\n4f). In PfARK1-depleted parasites, proper schizont segmentation was largely not observed,\nand these parasites \"arrested\" prior to segmentation. These parasites did not undergo proper,\ncomplete segmentation, which either caused or was a consequence of disrupted SMTs. This\npoint is now clarified in the manuscript (Lines 243-253). Some additional images of parasites\nthat showed some segmentation were selected and these are included in Extended Fig. 5f.\n6) Line 325 and Fig. 5dJ: The statement that “MTOCs that appeared detached from the nucleus\nin the ark1 PTD line” is not clearly supported. In Fig. 5dJ, the inner MTOC remains within the\nnucleus, and Extended Data Fig. 6bA, B and D, the inner MTOC is not evident. More\nconvincing or annotated images are needed.\nWe apologise for the confusion caused by an incomplete description of the images. The\nintention had been to demonstrate that some basal body MTOCs appear detached from the\nnucleus. We have now included additional annotation (\"dBB\") to indicate basal body MTOCs\nthat appear to be detached and provided an additional image (Extended Data Fig. 7bC),\nalthough since TEM provides only two-dimensional images, it does not prove definitively that\nthese basal body MTOCs are truly detached from the nucleus. Using 3D optical sectioning of\nark1PTD gametocytes we have clearly seen that some basal body MTOCs are located away\nfrom the nucleus (Figure 5g and Extended Data Fig. 7a), and these TEM observations provide\nsupporting evidence for this observation.\nMinor comments:\n1) Line 84: The localization of PfARK1-3 in gametocyte was previously reported (PMID:\n39235260), showing PfARK1 is specific to male gametocytes.\nWe have revised the text and Figure 1b to clarify the known locations and cite previous work4,\n5.\n2) Figure 1b right panel: The localization of PfARK1-3 was previously described in PMID:\n39235260 and PMID: 21166904.\nThe subcellular locations of ARK1-3 determined previously are shown in Figure 1b. The\nitalicised text refers to putative complexes that are known to scaffold specific aurora kinases.\nTo avoid confusion, we have attempted to clarify this point.\n3) Line 114-115: The statement that “ARK1 is expressed in trophozoite and schizont stages,\nbut not detectable in late schizonts and all developing merozoites (Fig. 2a)” is not fully\nsupported. In Fig. 2a, the “trophozoite” image resembles an early schizonts (two nuclei), and\nthe “late schizont” image resembles a segmented schizont. PfARK1 remains visible in late\nschizonts (Fig. 2b second panel) and was previously shown to persist (PMID: 39235260).\nWe agree that the image is of an early schizont; we have corrected the figure and its labelling.\n4) Line 153-155, Fig. 2e: The data suggest that PbARK1 is not localized to spindle poles but\ninstead associate with axonemal structures, as indicated by co-localization with α-tubulin.\nPlease provide more representative images to support this interpretation.\nWe have prepared new expansion gels and acquired new data, which are incorporated in Figure\n3. These images, including NHS-ester and α-tubulin staining, show more clearly that PbARK1\nis located in particular at the spindle poles and not axonemal structures (white arrows in Figure\n3c).\n5) Extended Data Fig 2d: The “late schizont” image actually presents a segmented schizont.\nAdditionally, strong background fluorescence in Extended Data Fig. 2d-e obscures PbAKR1\nfoci. Highlighting these regions would improve clarity.\nWe agree that this stage is a segmented schizont. We have revised the label (“segmentor”) and\nreplaced the image with a more representative one. We also highlight the PbARK1 foci in\nExtended Data Fig. 3d-e to improve clarity.\n6) Extended Data Fig. 2f: The localization of PbARK1 relative to the mitotic spindle or spindle\npoles remains ambiguous. Higher-quality representative images are needed.\nThese data were from an IFA study, and the location of PbARK1-GFP was not as clearly\ndemonstrated as in live-cell or U-ExM imaging studies. Therefore, we have decided to remove\nthese IFA data, and we have revised the manuscript to refer instead to the U-ExM data.\n7) Line 165 and Fig. 2f-g right panels: In Fig. 2f right panel, the PbARK1-GFP signal is broadly\ndistributed within the nucleoplasm and does not clearly associate with the spindle or\nPbNDC80-mCherry. Similarly, in line 169 and Fig. 2g right panel, PbARK1-GFP is not\ndistinctly localized to the spindle. Additional higher-resolution images or z-sections are\nrecommended.\nWe have re-acquired these data and added additional higher-solution SIM and U-ExM images\nto the main and supplementary figures that show more clearly the co-localisation of PbARK1-\nGFP with PbNDC80-mCherry and PbARK2-mCherry (Figure 3e, g and Extended Data Fig. 3b,\nc). We have also moved to the main figures the live-cell imaging data that show the co-\nlocalisation in activated gametocytes. As described above, the original Figure 2 has been\ndivided into two separate figures (revised Figures 2 and 3) to distinguish the P. falciparum\nresults from those of P. berghei.\n8) Extended Data Fig. 6a: The highlighted squares do not match the zoom-in regions.\nWe have corrected the positions of the highlighted squares (revised Extended Data Fig 7a).\n9) Line 417-418: The claim that “Its colocalisation with kinetochore NDC80 and ARK2\nmarkers further supports a dual role in spindle organization, kinetochore segregation, and\nspindle pole integrity” is not directly demonstrated. Consider revising to “potentially supports”\nrather than asserting it as proven.\nAs suggested by the reviewer, we have revised this sentence and use \"potentially supports” to\nreflect a more cautious interpretation (Lines 437-440).\n10) Line 427: The statement “In P. falciparum, ARK1 is required for proper spindle elongation”\nis unclear. Long spindle microtubules resembling hemispindles remain visible in RAP-treated\nparasites (Fig. 3d, third panel), suggesting elongation is not fully abolished. Please clarify or\nprovide quantitative evidence.\nWe agree that there is residual staining of tubulin, which we have referred to as “speckled\ntubulin staining” in the text (Lines 225-228). Although RAP-treated parasites show a loss of\nspindles, a few parasites still exhibit aberrant or speckled tubulin staining. For clarity, we have\nreplaced the parasite image in the revised Fig. 4d (last two panels), which represent these\ndefects with more clarity. It is clear from Fig. 3d (now Figure 4 and Extended Data Fig. 5e that\nRAP-treated parasites largely lack proper spindles.\nReviewer #3 (Remarks to the Author):\nSummary: This study comprehensively defines the role of a key protein called ARK1 during\nmitosis of malaria parasites both in the asexual blood-stage, and during male gamete formation.\nMitosis is perhaps the most well studied process in all of biology, but by contrast our\nunderstanding of mitosis and the proteins that control it in malaria parasites is relatively poor.\nThe role of ARK1 is explored using a range of microscopy modalities (live-cell, TEM,\nexpansion microscopy), transcriptomics, pull-downs, and an evolutionary/bioinformatic\nanalysis. This study represents one of the more comprehensive protein-characterisation projects\nin malaria parasites and contributes to a growing body of literature on aurora-like kinases in\napicomplexan parasites. These findings will definitely be of interest to the parasite cell biology\ncommunity, and the evolutionary biology findings may be useful to the broader study of mitotic\nkinases. While not discussed extenstively by the authors, the aurora-like kinases do represent\nattractive targets for the development of novel antimalarials, and so findings from this study\nmay also be useful in that context.\nOverall the experiments in this study are sound, and in my opinion could likely be published\nwithout the addition of new experiments. The way in which some of the data has been presented,\nor referred to, however could be improved. Below I have provided a number of comments that\nI have deemed as major (essential to address), minor (would be good to address), and some\ncomments on spelling, syntax etc. Even though there are many comments, it is likely that these\ncould be completely or mostly addressed in a reasonably short time-frame with changes to the\ntext, figure presentation, or data analysis.\nMajor Comments\n• Throughout the text a range of terms for different biological processes are conflated with each\nother. Individually, these are not major problems, but collectively the make it confusing to\ndetermine the interpretation of some of the experiments. These should all be easily fixable with\ntext changes. Below I will provide a few examples of this:\no Line 49 describes schizogony as a form of mitosis, when schizogony refers to the mode of\ncell division\nWe agree with the reviewer’s distinction between mitosis and cell division, and that schizogony\nrefers to a mode of cell division rather than a form of mitosis. We have revised the sentence to\nclarify this point (Lines 52-54).\no Lines 124 – 125: refers to parasites undergoing mitotic division, when the data regards nuclei\nundergoing karyokinesis\nIn these experiments, PfARK1 localisation was assessed during parasite division. Since it was\nonly localised specifically in parasites that possessed mitotic spindles, we mention that its\nexpression was \" only when parasites undergo mitotic division\" (Lines 136-137).\no Line 205 mentions looking at asexual cell division when karyokinesis is assessed\nThe statement now reads \"Next, efforts were made to investigate if PfARK1 regulates MTOC\nduplication and spindle formation during asexual division of the parasite\" (Lines 221-222).\no Line 393, the first line of the discussion describes ARK1 as a central organiser of mitosis, but\nthe term mitosis is not used at all when describing the phenotype of ARK1\nWhile we described specific phenotypic defects such as impaired spindle formation, MTOC\nseparation, and karyokinesis, we did not explicitly categorise these as defects in \"mitosis\"\nwithin the Results section. To address this point and to support more fully our conclusions in\nthe Discussion, we have revised the Results section to explicitly state that ARK1 depletion\nleads to defects in mitosis (Lines 230-232, 343-344).\no Line 399 implies ARK1 has a phenotype upstream of chromosome segregation. I assume this\nis meant to be chromatid segregation, but in either case, this is not directly assessed in any\nexperiment.\nWe agree that \"sister chromatid segregation\" is the appropriate terminology. We also\nacknowledge that while our data show that ARK1 depletion leads to failures in spindle\nformation and MTOC separation—thereby preventing segregation—we did not strictly assess\nits hierarchical position upstream of the segregation machinery itself. We have revised the text\nto replace \"chromosome segregation\" with \" sister chromatid segregation\" and to state more\naccurately that ARK1 is essential for the spindle and MTOC functions required for these\nprocesses (Line 418-420).\no Lines 487-490 mention an atypical mechanism of cell division when it seems to be discussing\nmitosis.\nWe agree that given the focus of the work on the molecular machinery regulating chromosome\nsegregation and the divergence of canonical regulators like the CPC, the term \"mitosis\" is more\nappropriate than the broader term \"cell division\". We have revised the text accordingly (Lines\n526-529).\n• The statistical analyses performed in Figures 3a, Extended data Figure 1f, 4a, and 4b are not\nappropriate. Because the control is normalised to 1, its standard deviation = 0. I don’t think this\nimpacts the interpretation of the results hugely, but they should either be presented without this\nstatistical test, or be presented without normalisation with a statistical test.\nTo clarify the statistical analyses in these figures (in the revised Figure 4a and Extended data\nFigure 1f, 4a, and 4b), the purpose of the quantitation was to compare change in parasitemia\nin RAP-treated and DMSO-treated (control) parasites at the time points indicated in three\nreplicate experiments. For this purpose, the control parasitemia at each time point was treated\nas 1 and the fold difference in parasitemia was calculated for RAP-treated parasites. For\ninstance, if the control parasitemia was 4 % and the RAP-treated parasitemia was 2%, then the\nfold difference would be 0.5, and at the following time point if it was 7% versus 2% the fold\ndifference would be 0.28. We think that the statistical analysis of these data for 3 replicates\nprovides an appropriate comparison, and we and others have used this method in previous\nstudies6 .\n• The interpretation of ARK1 localisation seems inconsistent across the manuscript. In the P.\nfalciparum imaging, it was determined that ARK1 was only present alongside a mitotic spindle\nwith no significant fluorescence outside this. Based on this, it seems reasonable that the diffuse\ncytosolic expression seen in the P. berghei-ARK1-GFP parasites is likely background. In some\nlifecycle stages like oocysts, sporozoites, and liver-stages this diffuse cytoplasmic staining is\ninferred as representing the localisation of ARK1, which in asexual and activated gametocytes\nit is ignored in favour of the punctate staining. The authors should be clear on when and why\nthey think the observed fluorescence represents true ARK1 localisation vs off-\ntarget/background signal. Also, the observation of ARK1 in the Golgi of salivary gland\nsporozites should be discussed.\nWe attribute the differences in \"diffuse\" signal primarily to the imaging methodology used: P.\nfalciparum parasites were analysed by IFA on fixed cells, where it is possible that soluble\ncytosolic protein may be washed away, whereas P. berghei parasites were analysed using live-\ncell GFP imaging, which detects all the GFP-tagged protein within the cell.\nWe agree that the distinct focal staining at the spindle represents the location of active ARK1\nduring mitosis. We interpret the diffuse signal observed in several life cycle stages of P. berghei\nas a cytosolic pool of the protein, rather than non-specific background, although we cannot rule\nout some level of cellular autofluorescence or background. To ensure consistency and clarity\nthroughout the manuscript, we have revised the text for oocyst, sporozoite, and liver stages to\ndescribe the signal as a \"diffuse cytosolic distribution\" without inferring a specific location.\nFor the signal in salivary gland sporozoites, we use the same description, and have refrained\nfrom speculating on a Golgi location since we lack specific Golgi markers.\n• The authors conclude that rhoptries were not formed properly or were absent following ARK1\niKO. The images in Figure 3F don’t seem to support this conclusion, as almost every merozoite\nhas a visibly recognisable rhoptry pair.\nWe agree with the reviewer that rhoptry formation was largely unaffected but their cellular\nlocation seemed to be disorganized, which may be due to aberrant cellular segmentation. We\nhave made this correction (Lines 243-253).\n• I feel that the transcriptomic results are slightly over-interpreted. The only consistently\ndownregulated group of proteins are ribosome-related, and none of them are mentioned\nindividually (only as part of the GO analysis). The authors interpret the lack of ribosome\nbiogenesis as indicative of cell cycle arrest and translational inhibition, but an alternative\ninterpretation is that immediately post-fertilisation the parasites prepare to make new\nribosomes.\nWe agree that the transcriptomic results should be interpreted with caution and so we have\nmoved the paragraph describing these results from the main text to the Supplementary Note.\nAs suggested, in the revised text, we include specific examples of downregulated genes for\nribosome-related proteins. In addition, we have softened the conclusion to state that a\ndownregulation of ribosome biogenesis genes is consistent with developmental arrest, rather\nthan claiming that it indicates stress sensing or translational inhibition.\nMinor Comments\n• The abstract says Plasmodium lacks a Bub1 homologue, but the Bub1 orthogroup on\nOrthoMCL contains the P. falciparum protein Pf3D7_1112100.\nWe thank the reviewer for spotting this error. Previously, we had concluded that Plasmodium\nlacked a Bub1 ortholog because candidate kinases lacked a clearly identifiable N-terminal TPR\ndomain, a hallmark of the Bub1/BubR1 family in model eukaryotes. However, in a recent study\nusing centromere-proximity proteomics combined with AlphaFold/FoldSeek analyses, a Bub1\northolog has been identified in Plasmodium\n(https://www.biorxiv.org/content/10.1101/2025.09.23.678001v1.full), which contains a highly\ndivergent TPR-like region. Although the sequence of this Plasmodium Bub1 is unconventional\nand appears to lack several motifs associated with a canonical spindle-assembly checkpoint\nresponse, its presence invalidates our original claim. We have, therefore, removed this sentence\nfrom the abstract and introduction, and now focus solely on the absence of polo-like kinases.\n• Lines 59-61 describes gametogeny as atypical because of an MTOC that spans the nuclear\nenvelope and makes microtubules on both sides. With the exception of making an axoneme,\nthis is also what happens in both asexual blood-stage parasites and during gametocytogenesis.\nWe agree that an MTOC spanning the nuclear envelope is a feature shared with other stages,\nincluding asexual blood-stage parasites. Our intention was to highlight the unique feature of\nmale gametogony in which the MTOC connects spindle microtubules specifically with the\naxoneme, a structure absent in schizogony. To avoid any confusion, we have revised the text to\nstate explicitly that what is unique is the coordination with axoneme formation (Lines 63-68).\n• The naming of the centriolar plaque are very confusing across the manuscript. The structure\nis variously referred to as the MTOC (generally), the inner and outer MTOC, the centrosome,\ncentriolar plaque, and spindle pole body. My suggestion for the least ambiguous way to define\nthis structure is to call the overall structure the centriolar plaque, and specifically refer to the\ninner (intranuclear) and outer (cytoplasmic) regions of it. In any case, the naming conventions\nshould be harmonised across the manuscript.\nWe apologise for the confusing terminology regarding the microtubule organising centre and\nagree that use of terms such as \"centrosome,\" \"spindle pole body,\" and \"centriolar plaque\"\ninterchangeably is confusing. For consistency across asexual stages and male gametogony\n(where basal bodies are involved), we now use a harmonised terminology throughout the\nmanuscript. We now use the term \"microtubule organising centre (MTOC)\" for the overall\nstructure and \"inner MTOC\" and \"outer MTOC\" to distinguish the intranuclear and cytoplasmic\nregions.\n• In the introduction it should also be stated that the Wyss et al study (ref 17) determined the\nlocalisation of ARK1 in P. falciparum gametocytes\nWe have revised the Introduction to acknowledge that Wyss et al. had determined the location\nof ARK1 in P. falciparum gametocytes, and clarify that our study builds on this work to\ninvestigate the dynamics and function of ARK1 in both P. falciparum and P. berghei (Line 91-\n92).\n• Line 142 should specify activated gametocytes\nWe describe the location of PbARK1-GFP both before and immediately after gametocyte\nactivation. We have revised the text to specify clearly the activation status of the gametocytes\nin the relevant descriptions (Line 154).\n• It seems likely that impaired centriolar plaque segregation is the earliest occurring phenotype\nobserved following ARK1 KD/iKO. All the other phenotypes, like impaired cytokinesis,\nkaryokinesis, etc. are therefore most likely to be downstream consequences of impaired\ncentriolar plaque segregation. It isn’t clear in the text that these are likely a related series of\nevents.\nThe MTOC/CP duplication was unaltered in PfARK1-depleted parasites. However, several\nMTOC/CPs were found associated with unsegregated nuclei, likely due to defects in\nkaryokinesis or nuclear segregation caused by loss of spindles (lines 230-232). We agree that\ndefects in karyokinesis likely impact the downstream process of cytokinesis (lines 251-253)\nand we now mentioned this in the Discussion as well (lines 446-450).\n• Did the authors check to see if there is a compensatory ARK2 upregulation in the ark1PTD\ncell line, like there is following ARK1 iKO?\nWe examined the expression levels of ark2 and ark3 in the ark1PTD line using qRT-PCR.\nInterestingly, we found that both ark2 and ark3 were actually downregulated in ark1PTD\ngametocytes compared to wild type gametocytes (Figure below), which is in contrast to the\ncompensatory upregulation observed in P. falciparum upon ARK1 depletion.\n• Line 310 says no exflagellation was observed, while line 333 says exflagellation was rarely\nobserved.\nWe thank the reviewer for pointing out this apparent inconsistency. As noted in Line 341 and\nFigure 5b, exflagellation occurs rarely in the total population. However, in Line 316 we refer\nspecifically to a small subset of ark1PTD gametocytes which we examined by expansion\nmicroscopy, and among which we found no exflagellating cells. We have revised the text to\nclarify that no exflagellation was observed in the cells examined by expansion microscopy in\nthis specific experiment, distinguishing them from the total population (Line 316).\n• Line 434-435 discusses a failure in MTOC-basal body segregation. Previous studies of inner\ncentriolar plaque proteins have seen separation of the inner and outer centriolar plaque\nfollowing knockdown. This seems similar enough to me to be worth mentioning here.\nWe agree that the defect in the ark1PTD line resembles the phenotype reported following\ndepletion of other inner MTOC proteins, where the coupling between inner and outer components\nis compromised. The Discussion has been revised to mention this similarity explicitly and we have\ncited the relevant literature (Lines 460-463).\n• Line 481 says ARK1 and ARK2 are both indispensable, but this was not shown in this study.\nThe reviewer is correct, the indispensability of ARK2 was shown in previous studies on asexual\nblood stages7, 8, 9, not in this study. To avoid confusion, the sentence has been revised to cite\nthe prior literature for ARK2, and clarify that this study demonstrates the essential role of\nARK1.\n• Figures 2 and 3 could probably each be broken up into two figures. Figure 2 could logically\nbe broken up into a falciparum and berghei figure. If not, the figure legends should be\ncondensed as they’re currently over a full page of text.\nAs stated above, Figure 2 has been split into two separate figures: one for P. falciparum and\none for P. berghei as suggested (revised Figure 2 and 3).\n• For the microscopy analyses of aberrant nuclear, MSP1 and GAP45 staining, it should be\nbetter described what was defined as ‘normal’ and whether these were blinded.\nThe revised Figure 4f legend defines normal MSP1/GAP45 staining as staining at the cell\nperiphery, encompassing individual merozoites.\n• For Figure 1a and 2a: The outer centriolar plaque is attached to the parasite plasma membrane\nfrom the onset of mitosis until at least the start of segmentation, but the diagram implies they\nfloat freely in the cytoplasm\nWe agree that the outer centriolar plaque (the outer MTOC) is anchored to the parasite plasma\nmembrane during mitosis, and we have revised the diagrams in the revised Figure 1a and Figure\n2a to show that the MTOC is in close proximity to the plasma membrane, correcting the\nprevious representation where they appeared to be floating in the cytoplasm. We opted to not\nshow a physical connection because the current image lacked the space to accommodate such\ndetail.\n• Figure 1a says two hemispindles but depicts an interpolar spindle\nRecent high-resolution U-ExM studies (Liffner et al., eLife 2023) have clearly distinguished\nthree intranuclear microtubule architectures during schizogony in Plasmodium:\n(1) Hemispindles, which are unipolar arrays extending from a single MTOC before division;\n(2) Mitotic spindles, formed after MTOC duplication and separation; and\n(3) Interpolar spindles, long microtubules connecting two fully separated MTOCs after\nchromatid segregation.\nHemispindles do not arise as symmetric pairs that fuse into a spindle. Instead, a single\nhemispindle forms, the MTOC duplicates, and then after a short delay the first bipolar spindle\nappears. Interpolar microtubules form later, after chromosome segregation. Thus, the formation\nof interpolar spindles and hemispindles is mechanistically distinct. To reflect this, we have\nrevised Figure 1a so that it now correctly illustrates one hemispindle, followed by bipolar\nspindle formation and interpolar microtubules, in accordance with the data of Liffner et al.\n(2023) and other recent U-ExM studies.\n• In Figure 1b it says cytokinesis (central spindle), but this structure doesn’t exist when the\nparasite is doing cytokinesis. Also the diagram includes astral microtubules, which we don’t\nhave evidence of in Plasmodium.\nWe thank the reviewer for highlighting these inconsistencies. Our original intention in Figure\n1b was to illustrate the range of Aurora kinase locations known from model eukaryotes, in\nwhich open mitosis includes astral microtubules and a well-defined central spindle during\ncytokinesis. We agree that this approach may be confusing in the context of Plasmodium, where\nmitosis is closed and neither astral microtubules nor a canonical central spindle have been\ndemonstrated.\nIn the revised Figure 1b, we have therefore:\n(i) removed astral microtubules,\n(ii) depicted a closed nucleus with the MTOC embedded in the nuclear envelope, and\n(iii) avoided implying canonical midzone structures.\nWe chose to retain a generalised “cytokinesis-associated zone” to indicate that some Aurora\nparalogs, particularly ARK3 in Toxoplasma, are located at structures involved in\nkaryokinesis/cytokinesis. This is meant to imply a functional analogy, not a homologous central\nspindle, and is now presented more cautiously in the revised panel and legend.\n• I don’t think it is fair to say ARK1-3 are absent in Cryptosporidium there are 6 C. parvum\nproteins in the ARK Orthogroup (Cdg1_1220, Cgd1_2630, Cgd8_750, Cgd3_3040,\nCgd2_1830, Cgd6_5240). Comparing these proteins against the consensus sequences in Figure\n6, it seems likely that Cgd3_3040 is the C. parvum homologue of ARK2.\nWe thank the reviewer for raising this point. In part the difficulty is that automated clustering\napproaches differ markedly in their ability to capture orthology for highly divergent kinase\nfamilies—a situation also encountered with Bub1. In that case, early analyses concluded that\nPlasmodium lacked Bub1 because candidate kinases were extremely divergent and lacked the\ncharacteristic N-terminal TPR domain. However, as mentioned above, recent analyses have\nidentified a highly divergent, Bub1 ortholog in Plasmodium. This underscores a general point:\nfor rapidly evolving kinase families, inference of orthology requires explicit phylogenetic and\nstructural analysis rather than reliance on clustering-based orthogroups alone. There is a similar\nchallenge for the Aurora/ARK kinases. Here, inference of orthology is complicated by multiple\nparallel gene family expansions and extensive lineage-specific divergence, particularly in\napicomplexans. Because the Aurora family is much closer to other eukaryotic kinase families\nthan is Bub1, incorrect grouping by automated tools is more likely.\nThis is precisely what occurs between OrthoDB version 6 and version 7:\nOrthoDB v6 (correct reconstruction)\n- OrthoDB v6 clusters Cgd2_3190 together with ARK1–2–3 from Plasmodium and\nToxoplasma.\n- This grouping reflects the true phylogenetic unity of the apicomplexan Aurora/ARK\nfamily.\n- In other words, Cgd2_3190 is the only bona fide Aurora-related kinase in\nCryptosporidium.\nOrthoDB v7 (source of the confusion)\n- OrthoDB v7 expanded clustering thresholds, which caused the apicomplexan Aurora\nparalogs to be split across multiple orthogroups.\n- OG7_0000004 contains Cgd2_3190 together with ARK1 orthologues — this is the\ncorrect orthogroup.\n- OG7_0000103, referenced by the reviewer, contains ARK2 and ARK3 mixed with\nunrelated kinase families.\no This heterogeneous composition indicates a clustering artefact, not true\northology.\nThus, the presence of multiple C. parvum kinases in OG7_0000103 does not imply that\nCryptosporidium possesses ARK2 or ARK3 homologues. Rather, it reflects the same issue we\nsaw with Bub1: when kinase families are highly divergent, automated orthogroup clustering is\ninsufficient and must be complemented by targeted phylogenetic and structural analysis.\nOur own analyses (based on phylogenetic reconstruction, active-site motif comparison, domain\narchitecture) consistently show that:\n- Cgd2_3190 is the sole Aurora/ARK-like kinase in Cryptosporidium,\n- ARK2 and ARK3 are apicomplexan-specific expansions, restricted to hematozoans and\ncoccidians. The Cryptosporidium IDs that are mentioned by the reviewer are different\nkinases altogether.\n• Considering the strength and frequency of all the phenotypes quantified in Figure 3, it seems\nsurprising that there is a <50% growth defect at this point in time. I think this is worth\ndiscussing in more detail, and also mentioning whether the reasonably modest growth defect\nmight be due to the compensatory upregulation of ARK2.\nWe agree with the reviewer that upregulation of ARK2 may compensate in part for the loss of\nPfARK1 and have mentioned this possibility in the Discussion (Lines 514-518).\n• In Extended Data Figure 2d, the Hoechst staining seems to be almost fully excluded from the\nforming merozoites. Is there a channel alignment issue or something there?\nWe have replaced the image with a higher-quality version that clearly shows Hoechst staining\nin the forming merozoites (Extended Data Fig. 2d).\n• The expression profiles of ARK1 and AMA1 are very different. Did the authors determine if\nthe ark1-PTD parasites have approximately normal blood-stage growth? This is important for\ninterpreting the results of the bite-back experiment. Also, cause AMA1 is expressed in\nsporozoites, would it be expected to get a second wave of ARK1 expression during late oocyst\ndevelopment?\nWe appreciate these insightful comments. During blood stage asexual growth, although the\ntiming of expression of ARK1 and AMA1 differs slightly, the ama1 promoter is strongly active\nduring schizogony. We monitored the asexual growth of ark1-PTD parasites and found it to be\ncomparable to that of the WT control, confirming that ARK1 expression driven by the ama1\npromoter is sufficient to support blood-stage replication. This implies that the delayed patency\nin the bite-back experiment is due to the low number of transmitted sporozoites rather than a\nslow parasite growth rate in the mouse.\nWe agree that given the expression profile of AMA1, ARK1 is likely to be expressed in\nsporozoites of the ark1-PTD line. However, since the primary defect occurs during male\ngametogony, there is a severe reduction in oocyst formation and the absolute number of\nsporozoites is very low. Thus, any potential expression of ARK1 at this late stage of\ndevelopment in the mosquito is insufficient to rescue the transmission phenotype.\n• If I am interpreting Extended data Figure 5 correctly, roughly 50% of sporozoites produced\nsporozoites make it to the salivary gland in both WT and ark1-PTD parasites. From this, can it\nbe inferred that the sporozoites that get produced are likely still invasive?\nWe agree that the absolute number of parasites is reduced, but the proportion of sporozoites\nsuccessfully migrating to the salivary glands is comparable to that of the WT line. Thus, the\nfew successfully differentiated ark1-PTD sporozoites are likely functional and invasive. This\nsuggestion is supported by our bite-back experiments, since infection was established (albeit\nwith a delay), confirming that the sporozoites are indeed invasive. We have added a sentence\nto the Results section to highlight the fact that the transmission defect is primarily driven by\nthe reduction in sporozoite numbers rather than a loss of invasiveness (Lines 298-300).\nWe also have corrected Extended Data Fig. 6c, as we identified an error in the plotting of this\ngraph. The revised figure now accurately reflects the data. This change does not affect our\ninterpretation of the results.\n• Figure 4e is missing some scale bars and the ark1PTD oocysts take up less of the FOV than\nthe WT, making them look much smaller than the quantification would indicate.\nWe apologise for this oversight and agree that the size of the scale bars in the original Figure\n4e (now Figure 5e) was incorrect. The appropriate scale bars are now included in Figure 4e\n(now Figure 5e).\n• Figure 3f, DMSO Tubulin is hugely over-exposed\nWe have reprocessed the image and included it in the figure (revised Figure 4f).\n• Figure 2h the Hoechst looks very strange in the 3D-SIM image, is this expected?\nThe unusual appearance of the Hoechst signal in the 3D-SIM image was due to the selection\nof the focal plane. In this image, the focus was optimized to resolve the PbARK1-GFP signal\nin the apical region of the nucleus. However, due to the shallow depth of field in 3D-SIM, the\nsignal from the bulk of the nuclear DNA (the Hoechst signal), which lies beneath this focal\nplane, was not fully captured, resulting in a weaker signal. To prevent confusion, we have added\na clarification to the Figure Legend (revised Figure 3i)\nSpelling, grammar, syntax, etc.\n• Lines 57 and 63: De novo is not italicised.\nWe revised the manuscript (Lines 61 and 68).\n• Line 125 should say nuclei, or parasite nuclei, instead of parasites\nWe revised the manuscript (Line 137).\n• Line 140: I think “along microtubules” rather than “at microtubules” is probably a better\ndescription of the localisation\nWe revised the manuscript (Line 152).\n• Line 275: ‘bite-back experiments’ should be explained in more detail\nWe have revised the text to explain bite back experiments (Lines 291-298).\n• Line 312-313: “led us to examine whether this” can be deleted\nWe revised the text (Line 319).\n• Line 339-340: I don’t understand “to confirm the logic of the observed subcellular\nlocalisations”.\nOur intent was to state that the subcellular location of ARK1 is expected to reflect its\ninteractions with specific scaffolding or regulatory proteins, such as INCENP-A/-B. However,\nsince this causal link was not explicit in the original text, so the phrasing was unclear. We have\nrephrased this sentence to articulate this assumption directly:\nLines 348-350: To understand whether ARK1 localisation results from recruitment to specific\nregulatory scaffolds, we investigated which interacting proteins might direct ARK1 activity\nand positioning during both asexual and sexual stages of the parasite life cycle.\n• Line 353-358 is more discussion rather than results\nWe agree that the interpretation of the interactome data, specifically regarding the absence of\nSurvivin/Borealin and its evolutionary implications, is better suited to the Discussion.\nAccordingly, we have moved this text into the Discussion, where we address the composition\nof the CPC (Lines 465-484).\n• Extended data Figure 2c: What do the two annotated sizes correspond to?\nThe two annotated sizes correspond to full-length PbARK1-GFP fusion protein (~68 kDa) and\nthe control, PK2-GFP (~80 kDa). We have repeated this western blot analysis, using WT-GFP\nparasites as a control. In the revised figure, the ~27 kDa band in the control lane corresponds\nto GFP and in the ARK1-GFP lane, the ~68 kDa upper band corresponds to the full-length\nARK1-GFP protein, while the lower band likely represents truncated ARK1-GFP containing\nGFP sequence (Extended Data Fig. 2c).\nReferences\n1. Roques M, Niu C, Brochet M, Brusini L. A modular chromosomal passenger complex\nrewires chromosome segregation in Plasmodium berghei. bioRxiv, (2025).\n2. Rawat RS, et al. Protein kinase PfPK2 mediated signalling is critical for host\nerythrocyte invasion by malaria parasite. PLoS Pathog 19, e1011770 (2023).\n3. Birnbaum J, et al. A genetic system to study Plasmodium falciparum protein function.\nNat Methods 14, 450-456 (2017).\n4. Wyss M, Thommen BT, Kofler J, Carrington E, Brancucci NMB, Voss TS. The three\nPlasmodium falciparum Aurora-related kinases display distinct temporal and spatial\nassociations with mitotic structures in asexual blood stage parasites and gametocytes.\nmSphere 9, e0046524 (2024).\n5. Reininger L, Wilkes JM, Bourgade H, Miranda-Saavedra D, Doerig C. An essential\nAurora-related kinase transiently associates with spindle pole bodies during\nPlasmodium falciparum erythrocytic schizogony. Mol Microbiol 79, 205-221 (2011).\n6. Rawat A, et al. PfPPM2 signalling regulates asexual division and sexual conversion of\nhuman malaria parasite Plasmodium falciparum. Nat Commun 16, 4790 (2025).\n7. Bushell E, et al. Functional Profiling of a Plasmodium Genome Reveals an Abundance\nof Essential Genes. Cell 170, 260-272 e268 (2017).\n8. Solyakov L, et al. Global kinomic and phospho-proteomic analyses of the human\nmalaria parasite Plasmodium falciparum. Nat Commun 2, 565 (2011).\n9. Tewari R, et al. The systematic functional analysis of Plasmodium protein kinases\nidentifies essential regulators of mosquito transmission. Cell Host Microbe 8, 377-387\n(2010)."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed my previous concerns. This is a high-quality study that provides new insights into the MTOC of Plasmodium parasites.", "reviewer_2": "The authors have addressed most comments in this revised version, and the clarity of the manuscript has improved. To further strengthen data presentation, as noted in my previous major comments 2, 3 and 5, I encourage the inclusion of zoomed-in views of individual mitotic spindle structures in Fig. 2b and zoomed-in views of the highlighted phenotypic regions in Fig. 4d and 4f of P. falciparum, similar to Fig. 5g for P. berghei. Without such magnification, several described phenotypes-such as “centrin aggregates were seen away from the nuclei and disorganised” (line 229) in Fig. 4d and “subpellicular MT were either not observed or were mislocalised” (lines 249-250) in Fig. 4f- are difficult to discern. I was also unable to locate Extended Data Fig. 3d-e referenced in the response to minor comment 5.", "reviewer_3": "The authors have done a very thorough job responding to all of my comments, and these changes have no impact on the significance/novelty of the findings of this study. In my opinion this study is now ready for publication.\nMy only unaddressed comment is regarding the statistical analysis in Figure 4a. Here the authors use ANOVA to calulate what they're presenting as a pairwise analysis (which should be a t-test or similar instead). In either case, statistical tests where the algorithm includes sample variance (like ANOVA or t-tests) cannot be performed on this data because the variance of the DMSO treated sample = 0 due to normalisation. This means the statistical significance of these comparison gets inflated because true sample variances are not being compared."}, "author_response": "Response to reviewers\nWe thank the reviewers for their continued assessment of our work and their constructive feedback. We are pleased that Reviewers #1 and #3 find the manuscript ready for publication.\nWe have addressed the remaining suggestions from Reviewer #2 regarding figure presentation and the statistical correction pointed out by Reviewer #3. We also have corrected Figure 5a and 7a as we noticed an older version of these panels was inadvertently used.\nReviewer comments\nReviewer #1 (Remarks to the Author):\nThe authors have adequately addressed my previous concerns. This is a high-quality study that provides new insights into the MTOC of Plasmodium parasites.\nWe thank the reviewer for the positive evaluation and the time in reviewing our manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed most comments in this revised version, and the clarity of the manuscript has improved. To further strengthen data presentation, as noted in my previous major comments 2, 3 and 5, I encourage the inclusion of zoomed-in views of individual mitotic spindle structures in Fig. 2b and zoomed-in views of the highlighted phenotypic regions in Fig. 4d and 4f of P. falciparum, similar to Fig. 5g for P. berghei. Without such magnification, several described phenotypes-such as “centrin aggregates were seen away from the nuclei and disorganised” (line 229) in Fig. 4d and “subpellicular MT were either not observed or were mislocalised” (lines 249-250) in Fig. 4f- are difficult to discern.\nWe have included zoomed-in views in Figures 2b, 4d, 4f, as recommended and thank the reviewer for the suggestion. In order to accommodate extra panels, the indicated figures are slightly re-arranged.\nI was also unable to locate Extended Data Fig. 3d-e referenced in the response to minor comment 5.\nIn this response, we intended to refer to Extended Data Fig. 2d-e (Supplementary Fig. 2d-e). We apologise for the error.\nReviewer #3 (Remarks to the Author):\nThe authors have done a very thorough job responding to all of my comments, and these changes have no impact on the significance/novelty of the findings of this study. In my opinion this study is now ready for publication.\nMy only unaddressed comment is regarding the statistical analysis in Figure 4a. Here the authors use ANOVA to calulate what they're presenting as a pairwise analysis (which should be a t-test or similar instead). In either case, statistical tests where the algorithm includes sample variance (like ANOVA or t-tests) cannot be performed on this data because the variance of the DMSO treated sample = 0 due to normalisation. This means the statistical significance of these comparison gets inflated because true sample variances are not being compared.\nWe have redone the inset graphs and the related stats in Figure 4a and Extended Fig. 5a, b and performed pairwise t-test for each individual time point (48, 96 and 144 h) to compare the parasitemia between DMSO and RAP-treated parasites at these time points."}]

Decision letter