Figure 4.

Depletion of TgNFS2 or TgSUFC leads to membrane defects during cell division.A, TgNFS2-HA and TgSUFC-HA conditional knockdown parasites as well as a TATi ΔKu80 control were grown in the presence of ATc for up to 2 days and were stained with an anti-TgIMC3 antibody (in red, to outline parasites and internal buds - top). In a normal situation, internal budding of parasites is generally synchronous within the same vacuole. Upon depletion of TgNFS2 or TgSUFC, dividing and nondividing parasites are occasionally present together within the same vacuole (arrowheads). The scale bar represents 5 μm. DNA was labeled with DAPI (blue). B, the percentage of vacuoles presenting asynchronous division as illustrated in (A) has been quantified and is represented as a means of n = 3 experiments ±SD (bottom). Two-tailed Student’s t test p-values are indicated. C, electron microscopy analysis of TgNFS2-HA and TgSUFC-HA conditional mutants grown for 4 days in the presence of ATc shows default in plasma membrane separation during parasite division, as displayed on insets representing magnifications of selected parts of the respective left image. D, cKD TgSUFC-HA parasites that were grown in the presence of ATc for 5 days were costained with anti-TgIMC3 to outline the inner membrane complex and anti-TgCPN60 (an apicoplast marker), which highlighted abnormal membrane structures and organelle segregation problems. The scale bar represents 5 μm. DNA was labeled with DAPI. ATc, anhydrotetracycline; cKD, conditional knockdown; DAPI, 4,6-diamidino-2-phenylindole; HA, hemagglutinin; IMC, inner membrane complex; TATi, tetracycline-inducible transactivator.