Knockout of TgTEP results in trans-Golgi fragmentation. (A) The cis-Golgi marked with GRASP-RFP (in yellow) is unaffected upon knockout of TgTEP (shown in magenta). This was observed in 100% of cases. (B) TEM images demonstrate that in non-induced parasites (-Rapa), Golgi stacks are organized adjacent to the nucleus. At 48- and 72-h after treatment with 50 nM rapamycin (+Rapa), TgTEP knockout results in the appearance of large electron-lucent vesicular structures and disruption of Golgi integrity. Scale bars: 1 μm. (C) Upon knockout of TgTEP (in magenta), the trans-Golgi, labelled by endogenously tagged SortLR with Halo-tag (in yellow), was seen to fragment. (D) Quantification confirms significantly increased trans-Golgi fragmentation at 48 h post-induction (hpi) with rapamycin in TgTEP-KO compared with controls. (E) The compartment marked by DrpB (yellow) is seen to fragment upon TgTEP (in magenta) knockout. (F) Quantifications showed that in knockout parasites, the fragmentation of the post-Golgi compartment marked by DrpB was significantly higher than that in WT parasites after 48 hpi with rapamycin. (G) Syntaxin-6 distribution is similarly disrupted following TgTEP loss. (H) Knockout of TgTEP (in magenta) was not seen to affect the ELC labelled with anti-ProM2AP antibodies (in yellow). All immunofluorescence assays were done three times, with a minimum of 100 parasite vacuoles quantified per condition per replicate. Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s multiple comparison test were done, with P values being represented as follows: ns ≥ 0.05; ** = 0.001–0.01; **** ≤0.0001. All scale bars are 5 μm. KO, knockout.
Figure 4.

Knockout of TgTEP results in trans-Golgi fragmentation. (A) The cis-Golgi marked with GRASP-RFP (in yellow) is unaffected upon knockout of TgTEP (shown in magenta). This was observed in 100% of cases. (B) TEM images demonstrate that in non-induced parasites (-Rapa), Golgi stacks are organized adjacent to the nucleus. At 48- and 72-h after treatment with 50 nM rapamycin (+Rapa), TgTEP knockout results in the appearance of large electron-lucent vesicular structures and disruption of Golgi integrity. Scale bars: 1 μm. (C) Upon knockout of TgTEP (in magenta), the trans-Golgi, labelled by endogenously tagged SortLR with Halo-tag (in yellow), was seen to fragment. (D) Quantification confirms significantly increased trans-Golgi fragmentation at 48 h post-induction (hpi) with rapamycin in TgTEP-KO compared with controls. (E) The compartment marked by DrpB (yellow) is seen to fragment upon TgTEP (in magenta) knockout. (F) Quantifications showed that in knockout parasites, the fragmentation of the post-Golgi compartment marked by DrpB was significantly higher than that in WT parasites after 48 hpi with rapamycin. (G) Syntaxin-6 distribution is similarly disrupted following TgTEP loss. (H) Knockout of TgTEP (in magenta) was not seen to affect the ELC labelled with anti-ProM2AP antibodies (in yellow). All immunofluorescence assays were done three times, with a minimum of 100 parasite vacuoles quantified per condition per replicate. Data are presented as mean ± SD. One-way ANOVA followed by Tukey’s multiple comparison test were done, with P values being represented as follows: ns ≥ 0.05; ** = 0.001–0.01; **** ≤0.0001. All scale bars are 5 μm. KO, knockout.

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