An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites

(A) Dual-color STED nanoscopy images of different schizogony stages of 3D7 parasites expressing tagged nuclear pore protein Nup313-HA_glms, labeled with anti-centrin (green), anti-tubulin (magenta) antibodies and stained with Hoechst (blue). Single slices are shown except for the extended spindle. Quantification of percentage of hemispindles in mononucleated cells with and without centrin signal was performed in 3D7 wild-type cells (n = 52 cells, 1 replicate) imaged with confocal microscopy. (B) Confocal U-ExM images of individual schizont nuclei of the 3D7 Nup313-3xHA_glms strain in hemispindle and mitotic spindle phase, labeled as in (A), except for using three instead of one anti-tubulin antibody. Maximum intensity projections are shown. (C) Quantification of lengths (n = 217, corrected by a measured expansion factor of 4.5) and number of hemispindle branches per nucleus (n = 38) and mitotic spindle lengths (n = 38) of 3D7 Nup313-3xHA_glms expressing cells imaged with U-ExM in 2 replicates. (D) Like (A) with anti-tubulin (magenta) and anti-CenH3 (yellow) showing centromere positioning in hemispindle phases. All scale bars are 1 μm.

(A) Dual-color STED nanoscopy images of a 3D7 schizont expressing tagged nuclear pore protein Nup313-3xHA_glms, labeled with anti-HA (yellow), anti-tubulin (magenta), and overlayed with confocal images of anti-centrin (green) and Hoechst staining (blue). (B) Confocal images of a 3D7 schizont ectopically expressing 3xNLS-mCherry. Signal was enhanced with RFP-Booster-Atto594 (magenta) and cells labeled with anti-centrin (green) and with Hoechst (blue) and DRAQ5 (turquoise) to detect DNA. (C) Quantification of dimensions of the DNA-free region in 3D7wt hemispindle (n = 36) and mitotic spindle (n = 23) stages using single image slices acquired in one immunofluorescence staining. Dimensions were measured as indicated in schematic. Depth was measured from underneath the centrin signal to the deepest point of the DNA-free region. Width was measured at the widest diameter of the DNA-free region where the nuclear membrane is expected. (D) Transmission EM image of the centriolar plaque region (annotated copy on the right) in a NF54 wt schizont shows no invagination of the nuclear membrane (blue) but suggests a boundary-like structure (black) delineating an intranuclear region from which microtubules (magenta) emanate. Green arrow indicates electron-dense region likely associated with the centriolar plaque. (E) Correlative in-resin widefield fluorescence and electron tomography (CLEM) images of thick sections (300 nm) of a high-pressure frozen and embedded NF54 schizont expressing PfCentrin1-GFP (green) and stained for DNA with 5-SiR-Hoechst (blue). Same cell region containing clear PfCentrin1-GFP foci imaged by fluorescence microscopy was overlayed with an electron tomogram slice. In zoom-ins, arrows indicate a microtubule (magenta) and a boundary-like region (black) for two tomogram slices. (F) Confocal images of individual 3D7 Nup313-3xHA_glms schizont nuclei expanded with U-ExM in hemispindle and mitotic spindle phase. Proteins were labeled in bulk using an NHS-ester Atto594 dye conjugate (white). Brighter staining therefore indicates higher protein density. Cells were additionally stained with anti-tubulin (magenta) and Hoechst (blue). (G) as in (F), but cells were labeled with anti-centrin (green) instead of anti-tubulin. (H) Quantification of width and depth of the NHS conjugate-stained intranuclear region at the centriolar plaque for the full area as well as the highly protein-dense region as example image and schematic indicate. To test for significant differences, we used the Mann–Whitney U test. In total, we analyzed NHS-stained regions of 14 schizonts (one replicate).

(A) 3D electron tomograms of thick sections (200 nm) of a schizont nucleus (NF54 PfCentrin1-GFP strain) in hemispindle stage. Corresponding surface rendering of microtubules (magenta), nuclear membrane (blue), microtubule minus ends (white), and electron-dense regions in the nuclear membrane (green) associated with the potential centriolar plaque are shown. (B) as (A) for mitotic spindle stage. All scale bars are 1 μm. (C) Schematic model of centriolar plaque organization during hemispindle and mitotic spindle phase in blood-stage schizonts. Content of the DNA-free, protein-rich intranuclear region harboring microtubule nucleation sites is unknown.