Fig 1.
Rab6 and syntaxin6 co-localize in a post-Golgi compartment.
(A) Schematic diagram of the endomembrane system in T. gondii. Created with BioRender.com. (B) RH parasites expressing Grasp55-mCherry (Golgi marker; yellow) and NeonFP-Rab5 (magenta; upper panel) or EmGFP-Rab6 (magenta; lower panel) were fixed and stained with DAPI (cyan). (C) RH parasites expressing EmGFP-Rab6 (magenta; panel i) or AppleFP-Rab6 (magenta; panels ii-vii) and markers of the endomembrane system as indicated (yellow). Nuclei were stained with DAPI (cyan). Panels i-iv are deconvolved images of a single focal plane from fixed intracellular parasites. Panels v-vi are a single focal plane from live intracellular parasites expressing AppleFP-Rab6 and SAG1ΔGPI-GFP (marker for the dense granules) or Syn6-GFP respectively. Panel vi is a single extracellular parasite expressing AppleFP-Rab6 that fixed and stained with an anti-CPL antibody and DAPI. Scale bar = 5μm. Inset scale bar in panel v is 1 μm.
Fig 2.
The Rab6 and syntaxin6 PGC is dynamic.
(A) Live imaging of RH parasites expressing AppleFP-Rab6 and Syn6-GFP. Rab6 vesicles indicated with the arrow head. Areas in the white box were used to make inset images in panels (B) and (C). Scale bar = 5μm. (B) Upper panel. Inset 1 from panel (A). Dashed line was used to make line scan in lower panel. Bracket indicates Rab6 positive and Syn6 negative tubule. Magenta arrow indicates vesicular compartment containing both Rab6 and Syn6. Lower panel. Line scan of Rab6 and Syn6. Shift in peaks between Rab6 and Syn6 likely due to sequential image acquisition between the two imaging channels. Scale bar is 1μm. (C) Left panel. Image from Inset 2 from panel (A), taken at times indicated. Bracket indicates Rab6 positive and Syn6 negative tubule. Magenta arrow indicates vesicular compartment containing both Rab6 and Syn6. Dashed lines were used to make line scan in right panel. Right panel. Line scans of Rab6 and Syn6 at times indicated. Scale bar is 1μm.
Fig 3.
Rab6 vesicle transport is actin dependent.
(A, D &G) RH parasites expressing EmGFP-Rab6 treated with DMSO (A) or cytochalasin D (CD) (D) or oryzalin (G). Dashed oval indicates the PV surrounding a 2-parasite vacuole. Location of the nucleus is indicated by the blue circles. Area in the yellow box was used to make inset (panels B, E and H). Arrow indicates parasites apical end. (B, E and H) Rab6 compartment dynamics. Images taken at 5 second intervals. (C, F and I)Time lapse images (left) and kymograph (right) depicting Rab6 directed vesicle motion. No directed motion was observed after cytochalasin D treatment indicated by the vertical line in kymograph. (J) Immunofluorescence image of parasites expressing EmGFP-Rab6 (magenta) treated with DMSO or oryzalin for 15 hours. Parasites were fixed and stained with an anti-IMC1 antibody (yellow) and DAPI (cyan). (K) Number of Rab6 vesicles per parasite was quantified in DMSO and CD treated parasites. (L) Run frequency (defined as the number of directed runs/parasite/minute) observed in DMSO and CD treated parasites. (M) Number of Rab6 vesicles per vacuole was quantified in DMSO and oryzalin treated parasites. (N) Run frequency observed in DMSO and oryzalin treated vacuoles. In (K-N) results are from three independent experiments. Mean from each independent experiment is indicated with large circles. These values were used to calculate the average (horizontal bar), standard error of the mean (error bars), and P value. Raw data is shown with smaller colored circles. Experiment 1 in magenta, experiment 2 in cyan, experiment 3 in grey.
Fig 4.
Creation of TgMyoF-KD parasites using the auxin-inducible degradation system.
(A) Cartoon of TgMyoF knockdown strategy. (B) Western blot analysis of TgMyoF-AID-HA depletion as a function of IAA treatment time. Tubulin was used as the loading control. (C) Deconvolved epifluorescence images of TgMyoF-AID parasites treated with EtOH or IAA for 15 hours before fixation. Anti-HA immunofluorescence (magenta) was used to assess TgMyoF depletion. Anti-IMC1 antibody (green) and DAPI (cyan) staining used to visualize the parasite periphery and nuclei respectively.
Fig 5.
TgMyoF knockdown results in fragmentation and dispersion of post-Golgi compartments.
(A) TgMyoF-AID parasites expressing Neon-Rab5, EmGFP-Rab6, Neon-Rab7, GFP-Syn6, GFP-DrpB (magenta), or untransfected parasites, were treated with EtOH (left panels) or IAA (right panels) for 18 hours before fixation. Parasites were stained with anti-IMC1 to highlight the parasite periphery (yellow) and DAPI to stain DNA (cyan). Untransfected parasites were stained with anti-SORTLR antibody (magenta; lower panel). Scale bar is 5μm. (B) For each post-Golgi compartment, the number of objects was quantified in control and TgMyoF knockdown parasites. Combined results from two or three independent experiments. Mean from each independent experiment is indicated with large circles. These values were used to calculate the average (horizontal bar), standard error of the mean (error bars), and P value. Raw data is shown with smaller colored circles. Experiment 1 in magenta, experiment 2 in cyan, experiment 3 in grey.
Fig 6.
TgMyoF is required for Rab6 vesicle transport.
(A-B) Left panel: TgMyoF-AID parasites expressing EmGFP-Rab6 treated with EtOH (A) or IAA (B) for 18 hours before imaging. Dashed oval indicates the PV surrounding a 2-parasite vacuole. Location of the nucleus is indicated by the blue circles. Area in the yellow box was used to make inset (middle panel). Middle panel: Images of the Rab6 compartment taken at 5 second intervals. Right panel: Kymograph depicting Rab6 vesicle motion. (C) Run frequency (# of directed runs/parasite/minute) of Rab6 vesicles in control and TgMyoF depleted parasites. Combined results from two independent experiments. Mean from each independent experiment is indicated with large circles. These values were used to calculate the average (horizontal bar), standard error of the mean (error bars), and P value. Raw data is shown with smaller colored circles. Experiment 1 in magenta, experiment 2 in cyan. (D) Frequency distribution of Rab6 vesicle velocities in RH parasites (orange) and in TgMyoF-AID parasites treated with EtOH (blue) or IAA (green) for 18 hours.
Fig 7.
TgMyoF is required for ER tubule movement.
(A-C) Left panel: RH parasites treated with DMSO, oryzalin for 18 hours or CD for 30 minutes. Center panel: magnification of area in the white box. First image of each movie (grey) was overlaid with images taken after 5s (magenta), 10s (yellow) and 15s (cyan) of imaging. Right panel: Line scan analysis of insets at 0s (grey), 5s (magenta), 10s (yellow) and 15s (cyan) time points. (D & E) Left panel: TgMyoF-AID parasites treated with EtOH (D) or IAA (E) for 15 hours where the ER is fluorescently labeled with eGFP. Center panel: magnification of area in the white box. First image of each movie (grey) was overlaid with images taken after 5s (magenta), 10s (yellow) and 15s (cyan) of imaging. Right panel: Line scan analysis of insets at 0s (grey), 5s (magenta), 10s (yellow) and 15s (cyan) time points.
Fig 8.
TgMyoF and actin control Golgi morphology.
(A) Diagram of centrosome and Golgi division. (B) TgMyoF-AID parasites expressing Grasp55-mCherry (a marker of the cis-Golgi; magenta) were fixed and stained with anti-IMC1 (green) and DAPI (cyan) after 15 hours of EtOH or IAA treatment. (C) TgMyoF-AID parasites expressing mCherry-Grasp55 were treated with DMSO or CD for 60 minutes before fixation and DAPI staining. White arrows indicate Golgi fragments. (D) Quantification of the number of Golgi/parasite in TgMyoF-AID parasites treated with EtOH for 15 hours (blue), IAA for 15 hours (light green) or CD for 60 minutes (orange). Results are combined data from at least 2 independent experiments. N>50 parasites/experiment. (E) Quantification of Golgi localization in apical only, basal only or both the apical and basal ends of the parasite. Results are combined data from 2 independent experiments. N>50 parasites/experiment. (F) In TgMyoF-AID parasites, the number of Golgi per parasite was quantified as a function of time after IAA addition (time 0). (G) In TgMyoF-AID parasites, the number of parasites with a fragmented and distributed Rab6 compartment was quantified as a function of time after IAA addition (time 0). (H) TgMyoF-AID parasites expressing EmGFP-Rab6 (yellow) and Grasp55-mCherry (magenta) treated with IAA for 0, 1.5, 3 and 6 hours.
Fig 9.
TgMyoF knockdown does not affect centrosome number.
(A) TgMyoF-AID parasites expressing Grasp55-mCherry (magenta) and centrin 1-GFP (as a marker of the centrosome; yellow) were treated with EtOH and IAA for 15 hours then fixed and stained with DAPI (cyan). Arrowhead indicates the centrosome. Arrows indicate the Golgi. (B) Quantification of centrosome and Golgi number in control and TgMyoF depleted parasites. Combined results from 2 independent experiments. N = 77 parasites for control, N = 93 parasites for IAA treated. (C) Centrosome number per TgMyoF-AID parasite treated with EtOH or IAA. Combined results from 2 independent experiments. N = 77 parasites for control, N = 93 parasites for IAA treated.
Fig 10.
TgMyoF depletion affects apical positioning of the rhoptries and Rop1 vesicle movement.
(A) TgMyoF-AID parasites expressing Rop1-NeonFP (magenta) were treated with EtOH and IAA for 15 hours before fixation and staining with an anti-AMA1 (green) and anti-GAP45 (yellow) antibodies and DAPI (cyan). Magenta arrow indicates Rop1 vesicles in the cytosol. Yellow arrow indicates the residual body (RB). (B) Quantification of TgMyoF-AID parasites with micronemes (visualized with anti-AMA1 antibody) and rhoptries (visualized by expression of Rop1-NeonFP) in the residual body. N = 57 vacuoles for AMA-1 quantitation, N = 77 vacuoles for Rop-1 quantitation from two independent experiments. (C&D) TgMyoF-AID parasites expressing Rop1-NeonFP were treated with EtOH or IAA for 15 hours before live cell imaging. Right panel: Rop1 localization. Magenta arrow indicates Rop1 mislocalization to the parasites basal end. White arrow indicates parasite apical end. Dashed oval indicates the PV in a 2-parasite vacuole. Area in the white box was used to make inset in the left panel. Left panel: Dynamics of rhoptries in the first 15 seconds of imaging. (E) Run frequency (# of directed runs/parasite/minute) of Rop1 vesicles in control and TgMyoF depleted parasites. See S1 Table for more details. (F) Number of Rop1 vesicles per parasite. N = 63 and 76 parasites in control and TgMyoF-KD parasites respectively in 3 independent experiments. (G) Ratio of Rop1 fluorescence in the apical and basal ends of the parasite. N = 63 and 76 parasites in control and TgMyoF-KD parasites respectively in 3 independent experiments. (E-G) Mean from each independent experiment is indicated with large circles. These values were used to calculate the average (horizontal bar), standard error of the mean (error bars), and P value. Raw data is shown with smaller circles; experiment 1 in magenta, experiment 2 in cyan, experiment 3 in grey.