Fig. 4.

Conventional actin disrupting drugs are not exclusively specific for Toxoplasma actin. Evaluation of the off-target effect of cytochalasin D (CD). a Generation of an inducible CD resistant actin strain (act1 cKO^CDr). Schematic of the inducible CD resistant act1 conditional knockout (cKO) geneswap vector, encoding the act1 resistance mutation for CD (A136G), flanked by LoxP sites with a reporter cassette of YFP. Analytical PCR checked integration with primer sets highlighted in the schematic. b Trail deposition assay for RH, CytD^r, act1 cKO and act1 cKO^CDr treated with increasing concentrations of CD (0–4 μM). No significant difference in gliding rates was observed between the act1 cKO or act1 cKO^CDr. The CytD^r parasites are inhibited in gliding motility at high concentrations of CD (>1 μM). Error bars represent ± standard error of the mean (SEM). c Trail deposition assay of RH parasites and act1 cKO parasites in 1 μM Jas. Forcing polymerisation blocks motility in wildtype cells but has no additional effect on act1 cKO. Error bars represent ± SEM. d, e Latrunculins have no effect on parasite motility. Parasites treated with increasing concentrations of either latrunculin A (d) or latrunculin B (e). Error bars represent ± SEM. All experiments were performed in biological triplicate and the datasets were compared with a two-tailed Student’s t-test, **** P < 0.0001, non-significance (ns) P >0.05