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Plasmodium NEK1 coordinates MTOC organisation and kinetochore attachment during rapid mitosis in male gamete formation [1]

['Mohammad Zeeshan', 'University Of Nottingham', 'School Of Life Sciences', 'Nottingham', 'United Kingdom', 'Ravish Rashpa', 'University Of Geneva', 'Faculty Of Medicine', 'Geneva', 'David J. Ferguson']

Date: 2024-09

Mitosis is an important process in the cell cycle required for cells to divide. Never in mitosis (NIMA)-like kinases (NEKs) are regulators of mitotic functions in diverse organisms. Plasmodium spp., the causative agent of malaria is a divergent unicellular haploid eukaryote with some unusual features in terms of its mitotic and nuclear division cycle that presumably facilitate proliferation in varied environments. For example, during the sexual stage of male gametogenesis that occurs within the mosquito host, an atypical rapid closed endomitosis is observed. Three rounds of genome replication from 1N to 8N and successive cycles of multiple spindle formation and chromosome segregation occur within 8 min followed by karyokinesis to generate haploid gametes. Our previous Plasmodium berghei kinome screen identified 4 Nek genes, of which 2, NEK2 and NEK4, are required for meiosis. NEK1 is likely to be essential for mitosis in asexual blood stage schizogony in the vertebrate host, but its function during male gametogenesis is unknown. Here, we study NEK1 location and function, using live cell imaging, ultrastructure expansion microscopy (U-ExM), and electron microscopy, together with conditional gene knockdown and proteomic approaches. We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organising centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium spp. life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation, and kinetochore attachment. These data suggest that P. berghei NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation.

Funding: This work was supported by:ERC advance grant funded by UKRI Frontier Science (EP/X024776/1), Wellcome DBT India Alliance/Team Science (IA/TSG/21/1/600261), MRC UK (MR/K011782/1, MR/N023048/1) and BBSRC (BB/N017609/1) to RT; the Francis Crick Institute (FC001097), the Cancer Research UK (FC001097), the UK Medical Research Council (FC001097), and the Wellcome Trust (FC001097) to AAH; the Swiss National Science Foundation project grant 31003A_179321 to MB. RN, AKS and AP are supported by a faculty baseline fund (BAS/ 1/1020-01-01) granted to AP by King Abdullah University of Science and Technology (KAUST). ECT is supported by the Dutch Science Council (NWO-ENW: VI.Veni.202.223). This research was funded in whole, or in part, by the Wellcome Trust [FC001097]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Most information on NEKs was obtained using asexual stages of the parasite, and nothing is known about the spatiotemporal dynamics and function of NEK1 during the rapid mitoses of male gametogenesis. Here, using the rodent malaria Plasmodium berghei, with live cell imaging, expansion, super-resolution, and electron microscopy, together with proteomic and conditional gene knockdown approaches, we show the association of NEK1 with a bipartite MTOC that spans the nuclear membrane during male gamete formation. NEK1 function is essential for MTOC organisation, spindle formation, and kinetochore attachment, and hence male gamete formation. The absence of NEK1 blocks parasite transmission through the mosquito, with important implications for potential therapeutic strategies to control malaria.

Functional analysis of the Plasmodium spp. kinome by gene deletion, phylogenetic analysis and phenotypic approaches identified many unusual features of the cell cycle and revealed that the molecules that control it may differ significantly from those of other model eukaryotes [ 29 , 30 ]. Strikingly, there are no genes for polo-like kinases, typical cyclins, or many components of the anaphase promoting complex [ 29 – 32 ]. Genes for phosphatases involved in mitosis, such as Cdc25 and Cdc14, are absent [ 29 , 30 , 33 ]. However, 4 NIMA-like kinases were identified in Plasmodium spp., and all are expressed most highly in gametocytes [ 29 , 30 ]. These kinases have been named NEK1 to NEK4, but they are not directly analogous to mammalian NEK1 to NEK4. Previous gene knockout studies showed that Plasmodium NEK2 and NEK4 are required for zygote differentiation and meiosis, but not for mitotic division of parasite cells in mammalian red blood cells [ 34 , 35 ], whereas NEK1 is likely essential for blood stage schizogony and proliferation [ 30 , 36 ]. It has been shown that the recombinant PfNEK1 is able to autophosphorylate, and the FFXT consensus motif usually found in the NEK family is substituted by a SMAHS motif [ 37 ]. In addition PfNEK1 is able to phosphorylate PfMAP2 in vitro and hence may be involved in the modulation of MAPK pathway, in the absence of classical MEKK-MEK-MAPK signalling, which is missing from Plasmodium spp. [ 36 – 38 ]. Another study of asexual erythrocytic schizogony in Plasmodium falciparum demonstrated the inhibitory role of Hesparadin, a human aurora kinase inhibitor, targeting the kinase domain of PfNEK1 [ 39 ].

Plasmodium spp., the causative agent of malaria, is an alveolate Apicomplexa parasite that exhibits extensive plasticity in mitosis and cell division during its life cycle, with proliferation within the 2 hosts, the Anopheles spp. mosquito, and a vertebrate. These parasites display some atypical aspects of cell division, for example, in terms of cell cycle, MTOC organisation, chromosome segregation, and nuclear division, presumably to facilitate genome replication and cell proliferation in the varied environments [ 16 – 18 ]. In a mammalian host, asexual division (schizogony) occurs first in hepatocytes followed by cyclic schizogony and replication in erythrocytes (the blood stage); parasites progress through ring, trophozoite and the multinucleate schizont stage, finally releasing merozoites to infect new cells. In the schizont, division occurs by closed mitosis with asynchronous division of individual nuclei, and cytokinesis only occurs at the end of the schizont stage. In the mosquito, sexual stages occur within the gut. Male and female gametes (gametogenesis) are produced from gametocytes in the blood meal, and following fertilisation the zygote develops into a motile ookinete in which meiosis begins. The ookinete migrates through the mosquito gut wall and forms an oocyst, where further asexual replication occurs to produce the sporozoites that migrate to the mosquito’s salivary glands prior to transfer to the vertebrate host in a further blood meal. During male gamete formation (microgametogenesis), mitosis is unconventional: 3-fold genome replication from 1N to 8N is accompanied by 3 rapid successive cycles of spindle formation and chromosome segregation (Mitosis I, II, and III) and followed by subsequent karyokinesis, all within 8 min [ 16 , 19 , 20 ]. Neither centrosomes nor centrioles have been observed during the proliferative schizont stages within the mammalian host [ 21 ]. Plasmodium spp. contains a bipartite MTOC that consists of a cytoplasmic part (outer MTOC) and a nuclear component (inner MTOC). During erythrocytic schizogony, the MTOC is also known as the centriolar plaque and contains an acentriolar cytoplasmic MTOC and a nuclear component called the intranuclear body [ 17 , 22 , 23 ]. The centriolar plaque is located at the nuclear membrane during mitosis and appears morphologically more similar to the yeast SPB than the human centrosome [ 24 ]. During male gametogenesis, the bipartite MTOC has a cytoplasmic centriolar part where basal bodies are organised and an inner nuclear component called a nuclear pole [ 19 , 25 – 28 ]. A centriole is found in the life cycle only during male gametogenesis and is associated with formation of the flagellated gamete [ 19 , 20 ].

NEKs are located at MTOCs in many organisms, including Aspergillus, yeast, and human cells [ 3 , 10 , 11 ]. Of the 11 mammalian NEKs, NEK2, NEK6, NEK7, and NEK9 localise to centrosomes, playing roles in mitotic spindle assembly [ 12 ]. The centrosome is an MTOC in mammalian cells composed of 2 microtubule-based barrel-shaped structures, termed centrioles, surrounded by a meshwork of mainly coiled-coil proteins that form the pericentriolar matrix [ 13 , 14 ]. The MTOC equivalent in yeast, the spindle pole body (SPB), lacks centrioles and is normally embedded in the nuclear membrane [ 15 ]. The SPB largely controls spindle dynamics during the so-called closed mitosis that occurs in these organisms, since the nuclear envelope does not break down. Here, the SPB acts not only to nucleate and anchor microtubules, but also to form a signalling hub for cell cycle-dependent kinases and phosphatases [ 1 ].

Budding and fission yeast express a single NEK kinase [ 6 ], but the family is expanded to 11 members in mammals, which may reflect the increased complexity of microtubule-dependent processes [ 3 ]. Several NEKs are essential to form functional cilia and flagella in mammals and other organisms, including Chlamydomonas, Trypanosoma, and Tetrahymena [ 7 – 9 ]. Other NEKs are more directly implicated in mitotic cell division, with defects in NEK expression likely to contribute to aberrant chromosome segregation in cancer cells [ 3 , 10 ].

Mitosis has a key role in the eukaryotic cell cycle when the cell divides and produces daughter cells. During mitosis, centrosomes act as microtubule organising centres (MTOCs) coordinating spindle dynamics with chromosome congression and segregation. They also act as signalling hubs for regulators of mitosis, including cyclin-dependent kinases (CDKs), and Aurora, Polo and NIMA (Never In Mitosis)-related (NEK) kinases [ 1 ]. NIMA-related kinases were first identified in Aspergillus nidulans during a screen for regulators of mitosis [ 2 ], and members of this family are found in a wide range of eukaryotes where they are crucial for cell cycle progression and microtubule organisation [ 3 – 5 ].

Results

Plasmodium berghei NEK1 has a punctate location, partially overlapping with the kinetochore and MTOC during blood stage schizogony To study the expression and subcellular location of NEK1, we generated a transgenic parasite line by single crossover recombination at the 3′ end of the endogenous nek1 locus to express a C-terminal GFP-tagged fusion protein (S1A Fig). PCR analysis of genomic DNA using locus-specific diagnostic primers indicated correct integration of the GFP tagging construct (S1B Fig). Western blot showed NEK1-GFP protein expression of the correct size (approximately 134 kDa) (S1C Fig). NEK1-GFP parasites completed the full life cycle, with no detectable phenotype resulting from the GFP tagging. Live cell imaging was done using a NEK1-GFP line to study the location of NEK1 during various stages of parasite life cycle. Blood was collected from mice infected with NEK1-GFP parasites and incubated in schizont culture medium to analyse the location of NEK1 in the parasite erythrocytic stages. NEK1-GFP was undetectable in early stages (ring stages) by live cell imaging but was visible with a diffuse cytoplasmic location during later stages (trophozoites) (Fig 1A). At the start of schizogony, NEK1-GFP re-located in the cytoplasm initially to single focal points near individual nuclei (Fig 1A), which later divided into 2 tightly associated punctae. A schematic of the first round of nuclear division during schizogony is portrayed at the top of Fig 1A, showing different mitotic components. The NEK1-GFP punctae splitting follows the subsequent asynchronous nuclear divisions. NEK1-GFP signal disappeared at the end of nuclear division and was undetectable in free merozoites (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Location of NEK1 during asexual blood stage schizogony and its association with kinetochore (NDC80) and centrin. (A) The schematic on the upper panel illustrates structures associated with mitosis during erythrocytic stage. Live cell imaging of NEK1-GFP (green) showing its location during different stages of intraerythrocytic development and in a free merozoite. DIC: differential interference contrast; Hoechst: stained DNA (blue); Merge: green and blue images merged; Guide: schematic of NEK1-GFP and nuclear DNA location at different stages of development. More than 30 images were analysed in more than 3 different experiments for each time point. Scale bar = 5 μm. (B) Live cell imaging of centrin-4-GFP (green) location in relation to the kinetochore marker NDC80-mCherry (red) and DNA (Hoechst, blue). Merge: green, red, and blue images merged. Guide: schematic of centrin-4, NDC80, and DNA location at 2 stages of schizogony. The dotted line indicates the periphery of the cell. More than 30 images were analysed in more than 3 different experiments for each time point; the scale bar is 5 μm. (C) Live cell imaging of NEK1GFP (green) location in relation to the kinetochore marker NDC80-mCherry (red) and DNA (Hoechst, blue). Merge: green, red, and blue images merged. Guide: schematic of NEK1, NDC80, and DNA location at 3 stages of schizogony. The dotted line indicates the periphery of the cell. More than 30 images were analysed in more than 3 different experiments for each time point; the scale bar is 5 μm. (D) Live cell imaging of NEK1-mCherry (red) location in relation to the kinetochore marker Centrin-4-GFP (green) and DNA (Hoechst, blue). Merge: green, red, and blue images merged. Guide: schematic of NEK1, centrin, and DNA location at 2 stages of schizogony. The dotted line indicates the periphery of the cell. More than 30 images were analysed in 2 different experiments for each time point; the scale bar is 5 μm. (E) Arrow in the representative image demonstrates an example region used for fluorescence intensity profiles (right) for DNA, NEK1, and centrin-4. The data underlying this figure can be found in S1 Data. (F) Arrow in the representative image demonstrates an example region used for fluorescence intensity profiles (right) for DNA, NEK1, and NDC80. The data underlying this figure can be found in S1 Data. (G) Cartoon diagram showing the respective location of DNA, NDC80 (kinetochore), NEK1, and centrin (MTOC). https://doi.org/10.1371/journal.pbio.3002802.g001 To investigate further the subcellular location of NEK1, we compared its location with that of the kinetochore marker, NDC80 and MTOC marker, centrin-4. Parasite lines expressing NEK1-GFP and NDC80-mCherry or centrin-4-mCherry were crossed, and the progeny were analysed by live cell imaging to establish the spatiotemporal relationship of the 2 tagged proteins. We also crossed previously published Centrin-4-GFP and NDC80-Cherry lines to observe the respective locations of these proteins in reference to DNA. Live cell images of schizonts at different time points showed NDC80-mCherry signal adjacent to the nuclear DNA and Centrin-4GFP was further away without any overlap (Fig 1B). The location of both NEK1-GFP and NDC80-mCherry was adjacent to the nuclear DNA, and with a partial overlap, although NDC80-mCherry was always closer to the DNA (Fig 1C). Next, we investigated NEK1-GFP co-localisation with centrin (MTOC marker) by live cell imaging and indirect immunofluorescence assay (IFA), using antibodies to GFP and centrin, and schizonts fixed at different time points. We observed an overlap of NEK1 and centrin signals but in this case NEK1 was closer than centrin to the DNA (Figs 1D and S1D). We measured the overlap of DNA with NEK1, NDC80 and centrin to demonstrate their respective locations. The Pearson’s correlation coefficient was high for NDC80 compared to NEK1 showing NDC80 is closer to DNA (S1E Fig). Again, the Pearson’s correlation coefficient was high for NEK1 compared to centrin showing NEK1 is closer to DNA (S1F Fig). We also measured the distance between the intensity peaks of Hoechst (DNA) signal and NEK1 and NDC80 signals, which suggested NDC80 is closer to DNA than NEK1 (Figs 1E and S1G). Similar analysis for NEK1 and centrin intensity peaks showed that NEK1 is closer to DNA than centrin (Figs 1F and S1H). Together, these data suggest that NEK1 is located close to the nuclear DNA, between the kinetochore, as marked by NDC80, and the MTOC, as marked by centrin (Fig 1G). The location of NEK1-GFP was also studied during other asexual stages like liver schizogony and mosquito gut sporogony. In liver and oocyst stages, there are thousands of progeny and therefore an accurate study of the temporal dynamics of GFP expression is very difficult. We observed that NEK1-GFP is located at focal points within the nucleus, together with a diffuse cytoplasmic location in these stages, which is a similar location to that in asexual blood and male gametocyte stages. These nuclear foci were only observed in proliferative stages in cells undergoing active endomitosis during both liver schizogony (S1I Fig) and sporogony (S1J Fig).

The spatiotemporal location of NEK1 with respect to kinetochore, spindle, and axoneme markers during male gametogenesis To assess more fully the association of NEK1 with the mitotic spindle during male gametogenesis, we compared its location in real time with that of kinetochore marker, NDC80 [42], spindle microtubule binding protein, EB1 [25], spindle-associated aurora kinase 2 (ARK2) [25], and cytoplasmic axonemal protein, kinesin-8B [43]. A parasite line expressing NEK1-GFP (green) was crossed with lines expressing mCherry (red) tagged NDC80, EB1, ARK2, or kinesin-8B, and progeny were examined by live cell imaging to establish the spatiotemporal relationships of the tagged proteins. First, we analysed the expression and location of these proteins in male gametocytes before activation that showed a diffused location in nucleus (NDC80, ARK2) or cytoplasm (kinesin-8B) or in both compartments (NEK1 and EB1) (S3A Fig). As soon as gametocytes were activated, these proteins relocated making focal points that are discussed below. Within 30 to 60 s after activation, both NEK1-GFP and NDC80-mCherry (kinetochore) appeared as focal points, close to but not overlapping each other and adjacent to the nuclear DNA as shown by merge images and individual channel images, respectively (Figs 3A and S3B). Later, within 1 to 2 min after activation, the NEK1-GFP signal split into 2 strong focal points in the cytoplasm, while NDC80-mCherry extended to form a bridge-like structure within the nucleus as shown by merge images and individual channel images, respectively (Figs 3B and S3C and S3 Video). After reaching the opposing sides of the nucleus, the 2 NEK1-GFP focal points remained stationary, while the NDC80-mCherry bridge was split and the signal retracted towards NEK1-GFP, forming 2 focal points adjacent to but not overlapping the NEK1-GFP signal. There were 2 further rounds of this splitting of NEK1-GFP and extension and rupture of NDC80-mCherry signal, resulting in 8 focal points of NEK1 in the cytoplasm adjacent to NDC80 in the nucleus (Fig 3A) at the end of mitosis. NDC80 is a kinetochore marker with a centromeric location [42] and the dynamics of NDC80 and NEK1 suggest a role of NEK1 in chromosome segregation. A similar dynamic pattern of localisation was observed for NEK1-GFP with the nuclear spindle microtubule-associated protein, EB1-mCherry, which also formed an extended bridge as shown by merge and individual channel images, respectively (Figs 3C, 3D and S3D). These proteins were adjacent but did not overlap throughout male gametogenesis when EB1 was present at focal points (spindle poles) (Figs 3C and S3E and S4 Video). A similar pattern was observed with spindle-associated kinase, ARK2-mCherry; NEK1-GFP and ARK2-mCherry were in a similar location at spindle poles (Fig 3E). The NEK1-GFP signal was observed at the ends of ARK2-mCherry decorated extending spindles, but again with no overlap throughout male gametogenesis as shown by merge and individual channel images, respectively (Figs 3E, 3F, S3F and S3G and S5 Video). PPT PowerPoint slide

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TIFF original image Download: Fig 3. The location of NEK1 in relation to kinetochore (NDC80), nuclear spindle (ARK2 and EB1), and basal body (kinesin-8B) markers during chromosome segregation in male gametogenesis. The guide illustrates structures associated with mitosis and axoneme formation. (A) Live cell imaging showing the dynamics of NEK1-GFP (green) in relation to kinetochore marker (NDC80-mCherry [red]) at different time points during gametogenesis. DNA is stained with Hoechst dye (blue). More than 50 images were analysed in more than 3 different experiments. Scale bar = 5 μm. (B) Stills from time lapse imaging showing the dynamics of NEK1-GFP (green) in relation to kinetochore/spindle marker (NDC80-mCherry [red]) after 1 to 2 min activation. DNA is stained with Hoechst dye (blue). More than 30 time lapses were analysed in more than 3 different experiments. Scale bar = 5 μm. (C) Live cell imaging showing the dynamics of NEK1-GFP (green) in relation to spindle marker (EB1-mCherry [red]) at different time points during gametogenesis. DNA is stained with Hoechst dye (blue). More than 50 images were analysed in more than 3 different experiments. Scale bar = 5 μm. (D) Stills from time lapse imaging showing the dynamics of NEK1-GFP (green) in relation to spindle marker (EB1-mCherry [red]) after 1 to 2 min activation. DNA is stained with Hoechst dye (blue). More than 30 time lapses were analysed in more than 3 different experiments. Scale bar = 5 μm. (E) Live cell imaging showing the dynamics of NEK1-GFP (green) in relation to spindle associated marker (ARK2-mCherry [red]) at different time points during gametogenesis. More than 50 images were analysed in more than 3 different experiments. Scale bar = 5 μm. (F) Stills from time lapse imaging showing the dynamics of NEK1-GFP (green) in relation to spindle associated marker (ARK2-mCherry [red]) after 1 to 2 min activation. DNA is stained with Hoechst dye (blue). More than 30 time lapses were analysed in more than 3 different experiments. Scale bar = 5 μm. (G) Live cell imaging showing location of NEK1-GFP (green) in relation to the basal body and axoneme marker, kinesin-8B-mCherry (red) at different time points during gametogenesis. NEK1, like kinesin-8B, has a cytoplasmic location and remains associated with basal bodies during their biogenesis and axoneme formation throughout gamete formation. More than 50 images were analysed in more than 3 different experiments. Scale bar = 5 μm. (H) Stills from time lapse imaging showing the dynamics of NEK1-GFP (green) in relation to kinesin-8B-mCherry (red) after 1- to 2-min activation. DNA is stained with Hoechst dye (blue). More than 30 time lapses were analysed in more than 3 different experiments. Scale bar = 5 μm. (I) Super-resolution 3D imaging of NEK1-GFP and NDC80-mCherry in gametocytes fixed at 1- to 2-min post activation. More than 10 images were analysed in more than 3 different experiments. Scale bar = 1 μm. The inset is a higher magnification of the boxed area on the main panel; the guide is a cartoon of the cell. (J) Super-resolution 3D imaging of NEK1-GFP and EB1-mCherry in gametocytes fixed at 1- to 2-min post activation. More than 10 images were analysed in more than 3 different experiments. Scale bar = 1 μm. The inset is a higher magnification of the boxed area on the main panel; the guide is a cartoon of the cell. (K) Super-resolution 3D imaging of NEK1-GFP and ARK2-mCherry in gametocytes fixed at 1- to 2-min post activation. More than 10 images were analysed in more than 3 different experiments. Scale bar = 1 μm. The inset is a higher magnification of the boxed area on the main panel; the guide is a cartoon of the cell. (L) Super-resolution 3D imaging of NEK1-GFP and kinesin-8B-mCherry in gametocytes fixed at 1- to 2-min post activation. More than 10 images were analysed in more than 3 different experiments. Scale bar = 1 μm. The inset is a higher magnification of the boxed area on the main panel; the guide is a cartoon of the cell. https://doi.org/10.1371/journal.pbio.3002802.g003 We next compared the spatiotemporal dynamics of NEK1-GFP with those of the basal body and axoneme protein, kinesin-8B-mCherry that localises in the cytoplasm [43]. Live cell imaging showed that both NEK1 and kinesin-8B were located in the cytoplasm (Figs 3G and S3H). Within 30 to 60 s after activation, kinesin-8B appeared as a tetrad marking the basal bodies with NEK1 located in the middle of the kinesin-8B tetrad but extending more towards the nucleus (Fig 3G). The duplication of both NEK1-GFP and kinesin-8B-mCherry–labelled tetrads occurred within 1 min before the duplicates moved to opposite sides of the nucleus (Figs 3G and S3I and S6 Video). In later stages, NEK1-GFP was duplicated 2 more times and remained associated with MTOCs, while kinesin-8B-mCherry had a distinct axonemal location in the cytoplasm (Figs 3G, 3H and S3H). The dynamic spatiotemporal distribution of these proteins demonstrates that chromosome segregation and spindle dynamics in the nucleus (tagged with NDC80, ARK2, and EB1) and basal body/MTOC formation in the cytoplasm (tagged with kinesin-8B and NEK1) begin very early in gametogenesis, continuing in parallel and synchronously within different compartments of the male cell. To investigate further at higher resolution the location of NEK1 with respect to the kinetochore, spindle, and basal body, 3D-SIM was performed on fixed gametocytes expressing NEK1-GFP/NDC80-mCherry, NEK1-GFP/EB1-mCherry, NEK1-GFP/ARK2-mCherry, and NEK1-GFP/kinesin-8B-mCherry at 1- to 2-min post activation, when the first spindles had formed. Gametocytes expressing NEK1-GFP/NDC80-mCherry had elongated focal points of NEK1 in close vicinity to nucleus and punctate NDC80 labelled-kinetochores distributed across the nucleus to form a bridge (Fig 3I). There was no overlap between NEK1 and NDC80 but the beaded NDC80 signals (kinetochores) were aligned closely at the ends with dumbbell shaped NEK1 signals (Fig 3I). Some representative images of NDC80 at a single focal point during spindle formation are shown in S3J (NEK1-NDC80) and S3K (NDC80 alone). Similarly, the SIM images showed that EB1 and ARK2 were distributed on nuclear spindle microtubules with the elongated focal points of NEK1 close to the nucleus at both ends of the spindle and without any overlap with EB1 and ARK2 (Fig 3J and 3K). SIM images of gametocytes expressing kinesin-8B-mCherry and NEK1-GFP showed that their location was in the cytoplasm close to the nucleus, with NEK1 located in the centre of kinesin-8B-marked tetrads but without any overlap (Fig 3L). The nonoverlapping NEK1 signal in the centre of cytoplasmic kinesin-8B tetrads suggest that the basal bodies are assembled at the outer MTOC in close proximity to the nucleus.

NEK1-GFP interacting proteins are components of the axoneme and flagellum Next we aimed to identify putative proteins interacting with NEK1. We used a GFP-specific nanobody to immunoprecipitate GFP-NEK1 and any associated proteins, from lysates of purified gametocytes prior to lysate preparation, the gametocytes had been activated for 1 to 2 min, when the mitotic spindles were fully extended to a bridge like structure, and cross-linked with 1% paraformaldehyde. The mild cross linking with formaldehyde helps to retain the loosely attached or indirectly interacting partners during the process of pull down. We analysed co-immunoprecipitates using an LC-MS/MS pipeline and performed enrichment analyses based on normalized iBAQ values (Fig 4 and S1 Table). We found 3 categories of proteins associated with NEK1: (1) DNA replication/repair; (2) cilium/flagellar/axoneme proteins (CFAPs); and (3) proteins of unknown function. Many of these broadly conserved proteins are not annotated in PlasmoDB, but in-depth sequence/alphafold-guided searches uncovered their homology with known ciliary and flagellar components (Fig 4, see annotations in S1 Table). Due to variability between NEK1 pulldown replicates and within the controls, probably due to differences in precise timing of when samples were harvested at between 1- to 2-min post activation of gametocytes, we found only a few interactors meeting our strict significance thresholds (correction for multiple testing; false discovery rate (FDR) = 5%). The only proteins we uncovered with an FDR = 5% were kinesin-8B and kinesin-13 (Fig 4), which indeed have been reported to be localised at the MTOC [44]. Furthermore, we find enrichment for proteins in the aforementioned categories: (1) the pre-replication complex subunits, CDC6, MCM4, and MCM5; (II) the CFAPs, CFAP157, CFAP73 (CCDC43), CFAP57 (WDR65), and the axoneme-associated kinesin-15 and 3 inner/outer arm dynein complex subunits DHC4A, DHC4B, and DHC3; and (III) 3 genes with unknown function LSA1 (PBANKA_1236600), the putative lysine decarboxylase UIS25 (PBANKA_1003400), and the unnamed gene PBANKA_0823000 (Fig 4 and S1 Table). Our data suggest a general association of NEK1 with axoneme/ciliary proteins as well as subunits of the replication machinery, although further experiments are needed to explore these interactions. PPT PowerPoint slide

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TIFF original image Download: Fig 4. NEK1 co-immunoprecipitates with proteins involved in the axoneme assembly. Fold enrichment (Log 2 transformed ratio) of normalized iBAQ values of proteins found in co-immunoprecipitates of NEK1-GFP versus control GFP. Multiple testing correction was performed using the Hochberg method, setting the FDR at 5% meaning the initial p-value was set at 0.001 to conclude significant enrichment (red dotted lines). Additional proteins involved in the axoneme (CFAPs) that are enriched (2.25 times), but do not meet the significance criteria, are specifically indicated. The data underlying this figure can be found in S1 Table. https://doi.org/10.1371/journal.pbio.3002802.g004

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