Fig. 4: PfARK1 controls karyokinesis, spindle formation and cytokinesis during asexual division. | Nature Communications

Fig. 4: PfARK1 controls karyokinesis, spindle formation and cytokinesis during asexual division.

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

Fig. 4: PfARK1 controls karyokinesis, spindle formation and cytokinesis during asexual division.The alternative text for this image may have been generated using AI.

a Growth assays of synchronised HA-PfARK1-loxP ring stage parasites treated with DMSO or RAP. Inset: fold change in parasitemia of RAP-treated parasites relative to control (DMSO) (SEM  ±  SE, two-sided paired t-test, three independent biological replicates, *P  = 0.0345, **P = 0.0035). b Giemsa stains of HA-PfARK1-loxP parasites treated with either DMSO or RAP as described in panel (a). c HA-PfARK1-loxP parasites were treated with DMSO or RAP and cycle 1 schizonts with E64. Parasites ( ~ 40–44 hpi, cycle 1) stained with DAPI (blue). Scale bar = 2 μm. n > 70 parasites, three independent biological replicates (see Supplementary Fig. 5d). d U-ExM of HA-PfARK1-loxP schizonts ( ~ 40–44 hpi, cycle 1) treated with DMSO or RAP and stained with anti-tubulin (magenta), anti-centrin (green), SYTOX (blue) and NHS-ester (grey). Hemispindles (yellow arrow), mitotic spindles (cyan arrow), interpolar spindles (white arrow). Speckles of tubulin (grey arrows) in RAP-treated parasites are also shown as zoom. Unsegregated nuclei and clusters of centrin (squares) are also shown in zoom. Scale bar = 2 μm. Upper Right Panel: parasites with normal tubulin staining of spindles (SEM  ±  SE, two-sided unpaired t test, ****P < 0.0001, n > 55 parasites, three independent biological replicates). Lower Right Panel: % parasites with clustered and/or detached centrin foci (SEM  ±  SE, two-sided unpaired t test, ****P < 0.0001, n > 55 parasites, three independent biological replicates). HA-PfARK1-loxP parasites were treated with DMSO or RAP and cycle 1 schizonts were treated with E64. IFA (e) or U-ExM (f) was performed on unexpanded (e) or expanded (f) parasites using anti-MSP1 (e, green) or anti-GAP45 (f, magenta) and anti-tubulin (f, green). RAP-treated parasites either lacked MSP1 and GAP45 staining or contained unsegregated nuclei (f, cyan arrow, square, zoom) circumscribed by a large MSP1 contour (e, cyan arrow). Middle Panel: Quantification of parasites with normal GAP45 (bottom for panel f) or MSP1 staining (top for panel e) (SEM  ±  SE, two-sided unpaired t test, **P = 0.0016, ***P = 0.0006, n > 70 parasites for MSP1 (e) and 40 parasites for GAP45 (f), three independent biological replicates). Scale bar = 2 μm. U-ExM images are displayed as maximum intensity projection of multiple z slices (d and f). g Time lapse microscopy of HA-PfARK1-loxP cycle 1 schizonts cultured in the presence of DMSO or RAP. Selected still images from videos (Supplementary Movies 6-7) showing normal egress. RAP treated parasites that egressed from erythrocytes but continued to clump-together even after prolonged duration are indicated (magenta arrow). Scale bar = 2 μm. Right Panel: parasites undergoing normal egress (SEM  ±  SE, two-sided unpaired t test, ***P = 0.0002, n > 100 parasites, three independent biological replicates). Source data are provided as a Source Data file.

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