Fig. 5: PfNuf2 disruption physically separates the two domains of the centriolar plaque, but microtubule polymerising capacity is retained.

a, b Representative U-ExM images of control and PfNuf2-cKO/KD (conditional knockout/knockdown) parasites labelled with the general protein dye NHS-PBS (a inverse greyscale) or NHS-BSA (b green), at early (30 hpi) and mid (36 hpi) schizont stages. The microtubules were labelled with anti-β-tubulin (yellow arrowheads, anti-β-tub, red), chromatin is marked by DAPI (blue), and the PfNuf2-HA is labelled with anti-HA (a green or b greyscale; evident only in control samples). Aqua arrowheads indicate the NHS-PBS-labelled outer centriolar plaque structures. The experiment was performed three times. c U-ExM images of PfNuf2-HA/GFP-PfCENH3 control and cKO/KD in early (30 hpi), middle (36 hpi), and late (38 hpi) stage schizonts labelled with NHS-PBS (inverse greyscale). The kinetochore protein, PfNuf2, is labelled with anti-HA (red) and the centromere protein, PfCENH3, is labelled with anti-GFP (green, GFP- PfCENH3). The anti-GFP signal overlaps with the anti-HA signal (white arrowheads) in control cells. Both signals are lost in cKO/KD parasites. The experiments were performed three times. Early (30 hpi), mid (36 hpi) and segmented schizonts (41 hpi) were labelled with NHS-PBS (d) or NHS-BSA (e). The outer centriolar plaque is labelled with anti-centrin (red, orange arrowheads). Both spindle microtubules (d green, white arrowheads) and subpellicular microtubules (e greyscale, aqua arrowheads) are labelled with anti-α-tubulin (anti-α-tub). The experiment was repeated three times. f Control and PfNuf2-disrupted segmented schizonts are labelled with NHS-PBS (inverse greyscale), highlighting the protein-dense rhoptries (purple arrowheads). The subpellicular microtubules are labelled with anti-β-tubulin (red), the chromatin with DAPI (blue) and the inner membrane complex with anti-PfGAP45 (anti-GAP45, green, magenta arrowheads). An NHS-PBS-labelled region in control cells likely represents the spindle remnant/ inner centriolar plaque (yellow arrowheads). In PfNuf2-cKO/KO parasites, a green arrowhead indicates an unusual NHS-PBS-labelled area inside the nucleus. The experiment was performed two times. g Anti-polyE (green) is concentrated at the minus end of subpellicular microtubules (white arrowheads) close to the apically located rhoptries. The experiment was performed two times. All images are presented as z-projections. All scale bars are 5 μm, except the zoom images, which are 2 μm. Additional images of PfNuf2-cKO/KO parasites are presented in Supplementary Figs. 13, 14 and 15.