Fig 1.

Conservation of the fission machinery in T. gondii: TgFis1 is not essential for mitochondrial fission A) Illustration of the main events that lead to mitochondrial fission in higher eukaryotes. ER and actin enwrap the mitochondrial membrane at future sites of fission, and an “adaptor complex” recruits Drp1/Dnm1, which forms spirals around the MOM and constricts it. (B) Scheme of the promoter replacement strategy adopted for the conditional KD of TgFis1; red and blue arrows indicate oligonucleotides designed to verify promoter exchange via genomic PCR. (C) Genomic PCR confirms the replacement of the endogenous promoter of TgFis1 with the inducible promoter Tet07-Sag1. A clonal parasite line was used for genomic PCR; primer positions and amplicon length are specified. (D) Confirmation of protein depletion upon addition of ATc for 48h in Fis1KD. In absence of ATc, western blot analysis shows the expected protein size of 17 kDa, tagged with c-MYC; the induction of TgFis1 knockdown with ATc shows that at 48 hours the protein is no longer detectable by western blot. (E) Plaque assay on parasites grown on HFF cells for 7 days in presence and absence of ATc shows that depletion of TgFis1 is not deleterious for parasite fitness. The images shown are representative of three experiments. (F) Immunofluorescence analysis shows that TgFis1 is evenly distributed in the mitochondrial outer membrane. In presence of ATc, the signal of Fis1 is no longer detectable, but mitochondria morphology is not affected. The experiment was performed in triplicate; representative images are shown. Scale bar: 2 μm.