Figure 8.
TgTEP knockout alters MyoF dynamics but has no effect on FRM2. (A) In WT parasites, MyoF is seen to localize around the periphery of the cells and near the actin nucleation center proximal to the Golgi body. Upon knockout of TgTEP (in magenta; + 72 h Rapa), MyoF (in yellow) was seen to form aggregates within the parasites. This was observed in both live as well as fixed parasites. MyoF and TgTEP images were recorded with STED, while the nuclei (labelled with Hoechst) were taken with the confocal setting. Scale bars are 3 μm. (B) The number of parasitophorous vacuoles with altered MyoF localization was seen to be significantly higher compared with WT parasites starting at 48 h post-induction (hpi) with 50 nM rapamycin. The assay was done thrice, with a minimum of 100 vacuoles quantified per condition per replicate. Data are plotted as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was applied, with P values being represented as follows: ns ≥ 0.05; * = 0.01–0.05; **** ≤0.0001. (C) FRM2, typical localizing at the Golgi region, seemed unaffected upon knockout of TgTEP. (D) Knockout of FRM2 has no influence on the location of TgTEP at the TGN. (E) KO of TgTEP (72 hpi) hampered transport kinetics of MyoF. Estimated displacement (total and net displacement) of MyoF in the PV was significantly affected. (F) Inducible FRM2 KO cell line showed that TgTEP-dependent traffic was affected 72 hpi with rapamycin, suggesting a role of actin regulating the distribution of TgTEP. (G) Proposed model: TgTEP vesicles depend on actin polymerization for directional trafficking, mediated by MyoF. Upon TgTEP deletion, MyoF accumulates in immobile aggregates, whereas FRM2 positioning remains unchanged. All scale bars except in A are 5 μm. KO, knockout.

TgTEP knockout alters MyoF dynamics but has no effect on FRM2. (A) In WT parasites, MyoF is seen to localize around the periphery of the cells and near the actin nucleation center proximal to the Golgi body. Upon knockout of TgTEP (in magenta; + 72 h Rapa), MyoF (in yellow) was seen to form aggregates within the parasites. This was observed in both live as well as fixed parasites. MyoF and TgTEP images were recorded with STED, while the nuclei (labelled with Hoechst) were taken with the confocal setting. Scale bars are 3 μm. (B) The number of parasitophorous vacuoles with altered MyoF localization was seen to be significantly higher compared with WT parasites starting at 48 h post-induction (hpi) with 50 nM rapamycin. The assay was done thrice, with a minimum of 100 vacuoles quantified per condition per replicate. Data are plotted as mean ± SD. One-way ANOVA with Tukey’s multiple comparison test was applied, with P values being represented as follows: ns ≥ 0.05; * = 0.01–0.05; **** ≤0.0001. (C) FRM2, typical localizing at the Golgi region, seemed unaffected upon knockout of TgTEP. (D) Knockout of FRM2 has no influence on the location of TgTEP at the TGN. (E) KO of TgTEP (72 hpi) hampered transport kinetics of MyoF. Estimated displacement (total and net displacement) of MyoF in the PV was significantly affected. (F) Inducible FRM2 KO cell line showed that TgTEP-dependent traffic was affected 72 hpi with rapamycin, suggesting a role of actin regulating the distribution of TgTEP. (G) Proposed model: TgTEP vesicles depend on actin polymerization for directional trafficking, mediated by MyoF. Upon TgTEP deletion, MyoF accumulates in immobile aggregates, whereas FRM2 positioning remains unchanged. All scale bars except in A are 5 μm. KO, knockout.

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