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. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Nat Microbiol. 2021 Jan 25;6(4):425–434. doi: 10.1038/s41564-020-00854-z

Fig. 1 |. Rhoptry secretion is dependent on rosette formation.

Fig. 1 |

a, Immunofluorescence assay (IFA) of control untagged line and endogenously HA3-tagged TgNd6 and HA3-tagged TgNd9 tachyzoites. The green arrow points to TgNd6-HA3 apical puncta. ARO is a marker for rhoptries. b, Super-resolution microscopy of the apical dot of TgNd6. Schematic of apical end of a tachyzoite. Maximum intensity projections of z-stacks of TgNd6-HA3 parasites transiently expressing RNG1_GFP (top) and of TgNd6-HA3 parasites expressing centrin 2-Ty3 (bottom). The protein RNG1_GFP marks the apical polar ring (APR) and CEN2_GFP marks pre-conoidal rings (PCR). Higher magnifications show that TgNd6-HA3 (red arrow) localizes above the apical polar ring (green arrow) and co-localizes partially with CEN2. DIC: differential interference contrast. c, Immunogold labelling of TgNd6-HA3. Right panel shows TgNd6-HA3 on the apical vesicle. Insert panel: higher magnification of the apical vesicle. Micronemes (m) and rhoptries (Rh) are visible in transverse section of the conoid (Co). Bar is 200 nm. d, Quantification of invasion after depletion of TgNd6 and TgNd9. Mean ± SD of n=3 independent experiments. e, Immunoblot showing microneme secretion (arrow=processed/secreted TgAMA1) in Tgnd6-iKD ± IAA 24h (top) and Tgnd9-iKD ± ATC 72h (bottom). P = pellet, Sup: supernatant, Ind: propanolol-induced secretion. GRA3, loading control. f, Rhoptry secretion assay by Secreted Cre, epitope-tagged (SeCrEt). Rhoptry secretion quantification of Tgnd6i-KD (left) ± IAA and Tgnd9-iKD (right) ± ATC. Mean ± SD of n=3 independent experiments. f, Left: Freeze-fracture electron microscopy of a T. gondii tachyzoite (P face) showing a rosette of intramembranous particles (white arrow). Middle: Higher magnification of the right panel. The white arrows point to the eight IMPs of the rosette. Right: Quantification of rosettes of IMPs in Tgnd9-iKD ± ATc 72h and Tgnd6-iKD ± IAA 24h using freeze fracture. (d, f) Unpaired two tail student’s t test: **** p-value < 0.0001, *** p-value < 0.001, ** p-value < 0.01, * p-value < 0.05.