Figure 6. | Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

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Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation

Figure 6.

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University of Glasgow, United Kingdom; Cancer Research United Kingdom Beatson Institute, United Kingdom; University of Vermont, United States
Figure 6.
Download figureOpen in new tabFigure 6. Chromobodies specifically stain parasite F-Actin.

(A) 3D-SIM imaging of parasites transiently expressing Cb-Halo (red) and stained using actin antibody (green) (Angrisano et al., 2012b). Treatment with 100 nM Jas for 1 hr results in formation of an elaborate network, whereas treatment with 2 μM Cyt-D, the filamentous network collapses. Note that in the bottom images a field of view was selected were a transiently transfected PV expressing Cb-Halo is next to a non-transfected PV. Both show identical staining. Scale bars 2 μm. Right: Quantification of the apparent filament size in five representative PVs growth for 24 hr, as measured using default settings in the Ridge Detection Plug In (ImageJ, see Material and Methods), n is the number of filaments, max size is the longest filament found in each condition expressed in µm. Number below indicates average pixels +/- SEM (10 pixels correspond to 1 µm). (B) Expression of Cb-Halo in a conditional act1 cKO. A filamentous network is observed prior to excision of act1. As soon as 24 hr after excision of act1 the network diminishes. Right: The percentage of vacuoles with >8 parasites containing actin filaments in RH Δhxpgrt and act1 cKO were quantified at 24, 48, 72 and 96 hr after induction. Mean values of three experiments in triplicate are shown. One way ANOVA followed by Dunnett’s multiple comparisons test was used to compare means between groups. ***p<0.0001. (C) Host cell actin is not involved in formation of the filamentous network. Parasites were allowed to replicate for 24 hr, before being treated for 3 hr with indicated concentration of Latrunculin A. Host cell F-actin was visualised with Phalloidin488 (green). Scale bars; 10 μm. (D) Analysis of actin dynamics in a conditional mutant for adf. Left: Immunoblot using indicated antibodies. ADF-HA is depleted upon treatment of parasites with 1 μM ATc. Parasites were grown for 96 hr in HFF cells in the presence and absence of inducer, before being artificially released. Equal amounts of parasites were loaded. Right: Representative still image of Video 6 and 7. Parasites were grown for 96 hr in the presence or absence of ATc. Note that upon depletion of ADF no actin filaments can be detected in the cytosol of the parasites (arrows) and F-actin accumulates at the posterior and (to a lesser extent) apical pole of the parasite. Scale bar: 5 μm. For source data (A,B) see supplementary information (Figure 6—source data 1).