Fig. 3: TgLIPIN depletion results in gross membrane anomalies early in the process of ATc downregulation.

Transmission electron micrographs showing wild-type (a–c) and TgLIPIN-ikD parasites at 1% (d–f) or 10% FBS (g–i), after 12, 24, and 48 h of ATc treatment. After 12 h of growth, wild-type parasites showed vacuoles containing (a) 2–4 parasites, (b) 8–16 parasites at 24 h, and (c) after 48 h, the first round of egress/reinvasion has occurred showing cells with multiple small vacuoles showing regular nuclei and organelles. d–f TgLIPIN-ikD parasites show membrane anomalies as early as d–d¹ 12 h of induction. Vacuoles showed large inclusions in the parasitophorous vacuole, red asterisks. d¹ Nuclei showed a multilobed shape even in the absence of early signs of mitosis. e–e¹ At 24 h, nuclear envelope showed pronounced anomalies with elongations connected with rough ER tubules, red arrows. e¹ The ER showed exaggerated proliferation generating big membrane whorls. Incomplete cytokinesis was observed, black arrow. f Forty-eight hours after induction, parasites were full of ER-derived membranes and there was the fusion of the parasitophorous membrane (PVM) and parasite plasma membrane (PM), red arrow. f¹ Endodyogeny defect: a new round of mitosis started, shown by the presence of IMC buds (black arrows) and centrocone (red arrowhead), while two sister cells from the previous generation have not separated. g–i All membrane defects were present and enhanced in 10% FBS. g–g¹ At 12 h, nucleus elongation and cytoplasmic evaginations were observed. g² Cytokinesis failure was observed. h–h² At 24 h, ER proliferation and cytokinesis defect were observed. Vacuoles showed signs of necrosis with PM rupture and cytoplasmic leakage in the vacuolar space. i At 48 h, TgLIPIN-ikD did not reinvade, the remaining vacuoles showed profoundly abnormal parasites, i¹ PVM or i² PM rupture/cytokinesis failure. Nu nucleus. Scale bar: 2 µm, n = 3.