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. 2013 May 21;32(12):1702–1716. doi: 10.1038/emboj.2013.113

Figure 6.

Figure 6

Overexpression of TgMyoF tail acts as a dominant negative mutant. (A) Schematic representation of the FKBP destabilization domain (DD) constructs and dominant negative effect of DDMyoF-tail caused by the formation of an inactive heterodimer that poisons the endogenous TgMyoF. (B) Western blot analysis using anti-Myc showed the stabilization of DDMyoF-tail at the predicted size (118 kDa) after Shld-1 treatment for 48 h. Catalase serves as loading control. (C) IFA (anti-Myc) detected DDMyoF-tail in the cytosol and in enlarged residual bodies (arrowhead). (D) The 24h stabilization of DDMyoF-tail led to a defect in apicoplast (anti-ATrx1) inheritance with the majority found outside the parasites (arrowhead). (E) Golgi (*) was not affected by stabilization of DDMyoF-tail. Dividing parasite were visualized with anti-ISP1. Arrow, mother cell; arrowhead, nascent daughter cell. In the presence of Shld-1, few mitochondrial fragments stained with anti-F1-ATPase (5F4) were observed in residual bodies (arrowhead). Microneme (anti-MIC3) and rhoptry contents (anti-ROP7) were found accumulating in residual bodies (arrowhead) and some rhoptries were dispersed in the cytosol (arrow). (F, left) Intracellular growth assay was performed on parental (RH) and DDMyoF-tail parasites grown for 30 h±Shld-1 before fixation and no alteration of growth was observed. (F, right) RH and DDMyoF-tail parasites were pretreated±Shld-1 during 20 h. Host cells were inoculated and the pretreated parasites were grown for 30 h±Shld-1 before fixation. Stabilization of DDMyoF-tail led to a severe arrest at 2–4 parasites per vacuole. Data are mean values±s.d. from three independent experiments. (G) The 48h stabilization of DDMyoF-tail led to the accumulation of Cpn60 precursor and decrease of the processed Cpn60 by western blot. TgACT1 was used as a loading control. The percentage of Cpn60 precursor was quantified. Data are mean values±s.d. from three independent experiments. pCpn60, Cpn60 precursor; mCpn60, Cpn60 mature; *nonspecific binding. (H) DDGFPMyoF-tail was stably expressed in MyoF-3Ty parasites. Immunoprecipitation (IP) was performed with anti-GFP coupled beads. Bound fractions were analysed by western blot and revealed the presence of MyoF-3Ty only when DDGFPMyoF-tail is stabilized (+Shld-1). Scale bar, 2 μm.