Fig. 7: Cortical arrangement of SPMT is impaired in the APR2-null ookinetes.

a Full section projections of fluorescent signal in living 17XNL and Δapr2 ookinetes stained with SiR-Tubulin (MT fluorescent probe). Representative maximum intensity projection (MIP) images were shown. a, apical end; b, basal end. Scale bars: 5 μm. Three independently performed experiments with similar results. b Quantification of fluorescent signal in a along pellicle from apical to basal. The maximum intensity was set as 1.0 and all signals were normalized. n is the number of ookinetes analyzed from two independent experiments. Data are shown means ± SEM. c IFA of SPMTs by staining the glutamylated tubulin (PolyE) in the expanded 17XNL and Δapr2 ookinetes. Representative maximum intensity projection (MIP) image was shown. Scale bars: 5 μm. Two independently experiments performed. d PL-U-ExM of the Tb-ARA1 and Tb-ARA1/∆apr2 ookinetes with APR stained with SA-488 (red) and SPMT stained with α- and β-Tubulin antibodies (green) respectively. Representative maximum intensity projection (MIP) images showing SPMT (green) and APR (red) in left panel, and enlarged views of the apical in right panels. Scale bars: 5 μm. Three independently performed experiments with similar results. e TEM of ookinete cross sections showing the arrangement of SPMTs underneath IMC in 17XNL and Δapr2 parasites. Approximately 60 hollow SPMTs are associated with IMC, distributing evenly around pellicle in 17XNL ookinetes. Approximately half of SPMTs lost association with IMC in the Δapr2 ookinetes. One representative cell in each parasite is shown with two selected areas zoomed in. Scale bars: 100 nm. Three independently performed experiments with similar results. f Quantification of distance (d1) between SPMT and PM and distance (d2) between the adjacent IMC-associated SPMTs in ookinetes in e. Values are means ± SD for n = 647 for d1 and 108 for d2 (17XNL) and n = 365 for d1 and 108 for d2 (∆apr2) measurements in 20 cells each group from two independent experiments; From top to buttom: ****P = 2e−21 and ****P = 3e−07, by Kolmogorov–Smirnov test. g Protein solubility assay detecting membrane association of α- and β-Tubulin in 17XNL and Δapr2 ookinetes 2.0 × 10⁶ ookinetes were lysed in each sample. Cytosolic soluble proteins are in hypotonic buffer (Hypo), peripheral membrane proteins in carbonate buffer (Carb), integral membrane proteins in Triton X-100 buffer (Trx), and insoluble proteins in pellet (Pel). P28 is an integral plasma membrane protein, and GAPDH is a cytosolic soluble protein. The numbers are the relative intensities of band in the immunoblot. Two independent experiments were repeated.