TgTEP interacts with the actomyosin system. (A) Immunofluorescence images suggested that knockout of TgTEP (in magenta) resulted in a change in actin filament formation (chromobody-emerald in yellow). In WT parasites, the filaments primarily localize at the actin polymerization center near the Golgi body and connect the parasites within the parasitophorous vacuole at the basal end. Upon knockout of TgTEP, less actin filaments were observed at the actin nucleation center around the Golgi, and the filaments connecting the parasites appeared more prominent. (B and C) In live movies (see Video 1), TgTEP (in magenta) was seen colocalizing and moving along actin filaments (marked with the Cb-emerald in yellow). (B) Still images taken from the live movies wherein the white arrow indicates vesicles that are moving along actin filaments close to the actin nucleation center. (C) The yellow arrow points toward vesicles, which are moving along actin filaments along the periphery of the parasites. (D and E) Representative kymographs and analyzed tracks of inducible TgTEP KO in actin-chromobody emerald-LoxP-TgTEP cell lines. Top panels show the ROI path (green) for kymograph analysis. Bottom panels show kymograph with tracks (green and red) chosen for extracting quantitative data. (D and E) The left panel shows an untreated parasitophorous vacuole, while (E) shows an example of a vacuole 72 h after induction with rapamycin. (F) Actin kinetics estimated as a measurement of actin chromobody displacement support no changes in actin kinetics as a result of abrogation of TgTEP. KO, knockout.