Disruption of Plasmodium falciparum kinetochore proteins destabilises the nexus between the centrosome equivalent and the mitotic apparatus

Plasmodium falciparum is the causative agent of malaria and remains a pathogen of global importance. Asexual blood stage replication, via a process called schizogony, is an important target for the development of new antimalarials. Here we use ultrastructure-expansion microscopy to probe the organisation of the chromosome-capturing kinetochores in relation to the mitotic spindle, the centriolar plaque, the centromeres and the apical organelles during schizont development. Conditional disruption of the kinetochore components, PfNDC80 and PfNuf2, is associated with aberrant mitotic spindle organisation, disruption of the centromere marker, CENH3 and impaired karyokinesis. Surprisingly, kinetochore disruption also leads to disengagement of the centrosome equivalent from the nuclear envelope. Severing the connection between the nucleus and the apical complex leads to the formation of merozoites lacking nuclei. Here, we show that correct assembly of the kinetochore/spindle complex plays a previously unrecognised role in positioning the nascent apical complex in developing P. falciparum merozoites.

ndc80/nuf2 sequence, GOI is gene of interest (ndc80/nuf2) and LoxPint is loxP artificial introns (coral arrowheads).PAM is protospacer adjacent motif, which is the target site for the CRISPR-Cas9 system to sever the downstream DNA sequence.b, c PCR analysis at different time points confirms the correct integration of the plasmid into the ndc80 (b) and nuf2 (c) loci and illustrates the progress of gene deletion.The integration forward (IntF) primer was used with the glmS-R reverse primers to screen for integration and gene excision.The ndc80-glmS product is 2425 bp and the excised ndc80-glmS product is 791 bp.The nuf2-glmS product is 2288 bp and the excised nuf2-glmS product is 746 bp.d, e PfNDC80-HA (73 kDa) (d) and PfNuf2-HA (58 kDa) (e) proteins are detected by Western blotting (anti-HA) at the expected sizes at different time points, illustrating efficient cKO/KD, when compared to DMSO controls.Anti-Pfaldolase (40 kDa) is used as a loading control.Samples for PCR and Western blotting were collected at 24, 30, 36 and 41 hpi.The experiment was performed two times.f, g Immunofluorescence microscopy of samples labelled with anti-HA, anti-βtubulin (anti-β-tub) and DAPI, showing the location of PfNDC80 (f) and PfNuf2 (g) in mitotic schizonts at 36 hpi.All images are displayed as z-projections.The experiment was performed three times.All scale bars are 2 μm.used to highlight membranes (a, green) while NHS-PBS staining was used to highlight protein rich structures (b, inverse greyscale).PfNDC80-HA was labelled with anti-HA (a, greyscale or b, green, yellow arrowheads), microtubules with anti-β-tubulin (red, anti-β tub) and chromatin with DAPI (blue).Sub-pellicular microtubules are indicated by white arrows.The data relate to Fig. 1c.All images are displayed as zprojections.The experiment was performed three times.All scale bars are 5 μm, except the zoom images which are 2 μm.NHS-PBS (b, inverse greyscale).PfNuf2-HA was labelled with anti-HA (a, greyscale or b, green, yellow arrowheads), microtubules with anti-β-tubulin (red, anti-β tub) and chromatin with DAPI (blue).Subpellicular microtubules are indicated by white arrows.The data relate to Fig. 1c.All images are displayed as z-projections.The experiment was performed three times.All scale bars are 5 μm, except the zoom images which are 2 μm.
HA and PfNuf2-HA parasite lines.b Western blotting of PfNDC80-HA vs PfNDC80-HA/GFP-PfCENH3 and PfNuf2-HA vs PfNuf2-HA/GFP-PfCENH3 parasite lines, detected using anti-HA (PfNDC80-HA, 73 kDa; and PfNuf2-HA, 58 kDa) and anti-GFP (GFP-PfCENH3, 47 kDa).Anti-Pfaldolase was used as a loading control (40 kDa).c, d Genomic DNA PCR and Western blotting reveal efficient excision of ndc80 (c) and reduction of PfNDC80-HA (d) protein expression.Sample of PfNDC80-HA/GFP-PfCENH3 conditional knockout/knockdown (cKO/KD) schizonts were collected at 37 hpi.e, f The nuf2 gene (e) was successfully excised and PfNuf2-HA protein (f) expression was significant reduced in PfNuf2-HA/GFP-PfCENH3 cKO/KD schizonts (38 hpi).Western blotting and genomic DNA PCR experiments were performed three times.The integration forward (IntF) primer was used with the glmS-R reverse primers to screen for integration and gene excision.For primer locations used for PCR refer to Supplementary Fig. 1a.PBS (inverse greyscale).PfNuf2-HA or PfNDC80-HA was labelled with anti-HA (green, yellow arrowheads) and PfCENH3 with anti-GFP (red, yellow arrowheads).The signals overlap with the anti-HA signal shown, appearing yellow in the merged images.The chromatin is labelled with DAPI (blue).The outer centriolar plaque structures are indicated by aqua arrowheads.The data relate to Fig. 1d.The experiment was performed three times.All images are displayed as z-projections.All scale bars are 5 μm, except the zoom images which are 2 μm.appear to be closely associated with the outer centriolar plaque.The experiment was performed three times.(d) Locations of the kinetochore marker, PfNDC80-HA (green, aqua arrowheads), relative to centrin (red, yellow arrowheads) and NHS-PBS-labelled rhoptries (purple arrowheads).The experiment was performed twice.(e, f) U-ExM of NHS-BSA-labelled parasites (green) showing different microtubule populations (anti--tubulin, anti--tub, red) in late mitotic schizonts.The minus-end of the subpellicular microtubules (aqua arrowheads) is marked with anti-polyE (greyscale, orange arrowheads) (e), while the inner membrane complex is marked with anti-anti-PfGAP45 (anti-GAP45, greyscale, magenta arrowheads) (f).The spindle remnants are indicated with yellow arrowheads.DAPI stains the chromatin (blue).The images are displayed as zprojections.Supplementary Fig. 6e experiment was performed one time and 6f was performed two times.Full image/zoom scale bars are 5 μm and 2 μm.Additional images are presented in Supplementary Fig. 7-9.greyscale).The outer centriolar plaque is labelled with anti-centrin (red, yellow arrowheads).The inner centriolar plaque structures are evident as NHS-PBS-labelled puncta (aqua arrowheads).The nascent subpellicular microtubules (white arrowheads) and rhoptries (purple arrowheads) are evident in a later round of nuclear division (b) and in an early segmented schizont (c).The data extend Supplementary Fig. 6a-c.All images are displayed as z-projections.The experiment was performed three times.All scale bars are 5 μm, except the zoom images which are 2 μm.

Supplementary
anti-centrin (red, orange arrowheads), chromatin marked with DAPI (blue), and NHS-PBS (inverse greyscale).The nascent rhoptries are NHS-PBS-labelled (purple arrowheads).A physical nexus is evident between the kinetochore-containing compartment, at the nuclear periphery, through to the outer centriolar plaque and the apical organelles.These data extend the data in Supplementary Fig. 6d.All images are displayed as zprojections.The experiment was performed twice.All scale bars are 5 μm, except the zoom images which are 2 μm.knockout/knockdown on nuclear division.U-ExM images of control (DMSO) and PfNDC80-cKO/KD (conditional knockout/knockdown, treated with rapamycin and glucosamine) in early stage mitotic schizonts (a), late mitotic schizonts (b) and early segmented schizonts (c, d).NHS-PBS (inverse greyscale) or NHS-BSA (green) are the general protein labels and DAPI (blue) stains the chromatin.Anti-α-tubulin (green or greyscale, anti-α-tub) or anti-β-tubulin (red, anti-β-tub) are used to label microtubules.Spindle microtubules (a) and remnant spindle microtubules (b, c, d) are indicated with yellow arrowheads.Sub-pellicular microtubules are indicated with aqua arrowheads.a, b.Anti-centrin (red, orange arrowheads) labels the outer centriolar plaque.The data relate to Fig. 3c.The experiment was performed twice.c.Anti-polyE (greyscale) indicates sub-pellicular microtubules (aqua arrowheads), which are also labelled with anti-β-tubulin (red).The data relate to Fig. 3e.The experiment was performed one time.d.Anti-PfGAP45 (greyscale) labels the inner membrane complex (magenta arrowheads).The data relate to Fig. 3f.The experiment was performed twice.All images are displayed as z-projections.All scale bars are 5 μm, except the zoom images, which are 2 μm.
stains the chromatin.The data relate to Fig. 4a, b.Supplementary Fig. 12a experiments were performed twice and 12b one time.All images are displayed as z-projections.All scale bars are 5 μm, except the zoom-in images which are 2 μm.Quantification of nuclei (c) and merozoite (d) numbers in control (DMSO) and PfNDC80-deficient segmented schizonts (cKO/KD, rapamycin and glucosamine) based on expansion microscopy data (n = 24 cells).Statistical differences were determined using an unpaired Welch's t-test (c, ns = 0.1758 and d, ***p = 0.0002).e Ratio of the number of merozoites to the number of nuclei per cell in control and PfNDC80-deficient segmented schizonts (n = 24 cells).Differences were determined using an unpaired Welch's t-test, ****p < 0.0001.f Quantification of the volumes of the nuclei in control (n = 452 nuclei in 20 cells) and cKO/KD (n = 545 nuclei in 20 cells) segmented schizonts.Volumes were assessed using Imaris software.The difference was determined by an unpaired t-test with Welch's correction, ****p < 0.0001.The confidence intervals of all the quantitative estimates are 95%.Supplementary Fig. 12 c, d, e, f images for analysis were acquired from three different experiments.

Figure 6 .
Reorganisation of the mitotic machinery and apical organelles during mitotic division.a-c Different stages of nuclear division and schizogony imaged by U-ExM in samples labelled with NHS-PBS (inverse greyscale), anti--tubulin (green, anti--tub), chromatin (blue) and the outer centriolar plaque marker, anti-centrin (red).An illustration for each stage shown.Microtubules are evident as a hemispindle (a), a mitotic spindle (b), and a post-mitotic spindle remnant (c) and sub-pellicular microtubules (c, white arrowheads) as the PfNDC80-HA parasite matures.Centrin is associated with an NHS-PBS-labelled structure just outside the nucleus (yellow arrowheads).NHS-PBS-labelled structures (aqua arrowheads; inner centriolar plaque) are evident at the site of origin of the hemispindles (a), associated with the mitotic spindle (b) and at the site of the post-mitotic spindle remnant (c).NHS-PBS-labelled rhoptries (purple arrowheads) 3 times, and the mean and standard deviation values are shown.b-e U-ExM images of control and PfNuf2-cKO/KD parasites during early (b), mid (c) and later (d) rounds of nuclear division and in a segmented schizont (e).NHS-BSA (c, green) or NHS-PBS (b, d, e inverse greyscale) labels general proteins and DAPI (blue) labels chromatins.Anti-HA (b, d, e green or c, greyscale) labels the PfNuf2-HA.Anti-β-tubulin (antiβ-tub, red) marks both spindle microtubules (yellow arrowheads) and sub-pellicular microtubules (aqua arrowheads).The rhoptries are evident as NHS-PBS-labelled areas (purple arrowheads; c and d).The data relate to Fig.5a, b.The experiment was performed three times.All images are displayed as z-projections.All scale bars are 5 μm, except the zoom images, which are 2 μm.