Fig. 6: APR2 is required for integrity and apical anchorage of APR-SPMT.

a Representative images from scanning electron microscopy (SEM) of 17XNL and Δapr2 ookinetes. Ookinete apical (white dashed line in the left panel) was zoomed in and shown in the right panel. Scale bars: 200 nm. Three independently performed experiments with similar results. b Representative images from transmission electron microscopy (TEM) of 17XNL and Δapr2 ookinetes. Ookinete apical (black dashed line in the left panel) was zoomed in and shown in the right panel. APR, SPMT, PM and IMC are indicated. A gap (blue arrow) between apical IMC and APR was observed in the mutant parasites. Micronemes were labeled with asterisk. Scale bars: 200 nm. Three independently performed experiments with similar results. c Representative images from the U-ExM of 17XNL and Δapr2 ookinetes stained with the NHS-ester dyes. Ookinete apical (white dashed line in the left panel) was zoomed in and shown in the right panel. APR and IMC are indicated. A gap (blue arrow) between apical IMC and APR was observed in the mutant parasites. Scale bars: 200 nm. Three independently performed experiments with similar results. d Representative images from negative staining TEM (NS-TEM) of 17XNL and Δapr2 ookinetes. A gap (blue arrow) between SPMT and apical membrane-like layer was emerged in the Δapr2 ookinetes, but not in 17XNL ookinetes. The distance (d) between SPMT and the apical membrane-like layer was quantified and shown in the low right panel. Scale bars: 200 nm. n is the number of ookinetes in each group. Boxes show means (red line) with interquartile ranges, whiskers: min to max show all points. ****P = 6e−29, two-sided Mann–Whitney test. Three independently performed experiments with similar results. e Flow-chart of Proximity Labeling coupled with U-ExM (PL-U-ExM) for detecting a high-resolution 3D structure of APR within ookinete. Two modified strains ara1::TurboID (Tb-ARA1) and ara1::TurboID;∆apr2 (Tb-ARA1/∆apr2) with endogenous ARA1 tagged with a TurboID::HA motif were generated. APR was labeled via the biotin ligase TurboID-based PL in living ookinetes after incubation with 50 µM biotin. The ookinetes were expanded and co-stained with the fluorescent-conjugated streptavidin (SA-488) and the NHS-ester dye for APR and pellicle imaging. f Representative maximum intensity projection (MIP) images from PL-U-ExM of the Tb-ARA1 and Tb-ARA1/∆apr2 ookinetes. APR (magenta) and pellicle (black) in the ookinete apical were shown. Ookinetes were expanded by 4×fold. Scale bars: 200 nm. Three independently performed experiments with similar results. g Quantification of APR surface area and volume in f, n is the number of cells analyzed. Boxes show means (red line) with interquartile ranges, whiskers: min to max show all points. From top to buttom: ****P = 9e−20 and ****P = 4e−08, by two-sided Mann–Whitney test.