Figure 5. | 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 5.

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University of Glasgow, United Kingdom; Cancer Research United Kingdom Beatson Institute, United Kingdom; University of Vermont, United States
Figure 5.
Download figureOpen in new tabFigure 5. Chromobodies are specific for parasite F-actin and do not influence total amount of F-actin in the parasite.

(A) Coomassie stained gel showing recombinant Cb purified from bacteria. (B) Left. rCb affinity assays. Coomassie stained gels showing in vitro binding of purified Cb (4 μM) to variable range of skeletal chicken F-Actin concentration (80 μM down to 0 μM). Supernatant (S) and pellet (P) were separated by ultracentrifugation. Right. Quantitative analysis. Ratio between purified Cb in the supernatant and pellet was determined. Solid line is a fit of the binding equation to the data (Kd = 5 ± 1.2 mM). Results obtained from two independent experiments. (C) Interaction between Cb-Halo and actin in T. gondii Cb-Halo expressing strain. Western blot comparison of input lysate (I) and elution (E) obtained from co-immunoprecipitation using beads against the halo-tag with the Cb-Halo strain and RH. Actin pull-down was only detected in the Cb-Halo expressing strain. (D) Sedimentation assays. Actin sedimentation, with and without Jasplakinolide (1 μM) was evaluated for Cb-Halo strain and RH. GRA7 was used as loading control and signal intensity normalisation between conditions. Increased amount of F-actin was found in the pellet of parasites incubated in the presence of Jasplakinolide. However, no difference between RH and parasite expressing Cb-Halo could be detected in both control and Jas treated condition (n = 6).

DOI: http://dx.doi.org/10.7554/eLife.24119.014