Fig. 2.

CPH1 is essential for parasite motility, invasion, and egress but not for microneme secretion. a Western blot analysis of CPH1 endogenously tagged with AID-3HA (α-HA) in the parental TIR1 line (shown for comparison). Aldolase (α-ALD) loading control. b Degradation of CPH1-AID triggered by addition of 500 µM auxin (+IAA) for different times (h), vs. mock, 0.1% ethanol (−IAA). Western blot performed as in a (Supplementary Fig. 3A). c Immunofluorescence microscopy of CPH1-AID parasites grown ± IAA for 24 h stained using mouse anti-HA (green) and rabbit anti-GAP45 (red). Scale bar = 2 µm. d Plaque formation with TIR1 and CPH1-AID lines grown ± IAA for 7 days. Scale bar = 0.5 cm. a–d Representative of three or more experiments with similar outcomes. e Microneme secretion following stimulation with bovine serum albumin (BSA) plus 1% ethanol for 10 min. f Parasite motility was recorded by video microscopy, manually scored, and plotted as percentage of total. ***P < 0.0001. g Parasite invasion into HFF cells was examined after 20 min challenge. ***P < 0.0001. e–g Parasite lines were grown in ±IAA for 40–44 h and extracellular parasites were collected and immediately used for assays. Mean ± SEM (n = 3 experiments, each with 3 technical replicates, n = 9). One-way ANOVA with Kruskal–Wallis test (e) or with Tukey’s multiple comparison (g) or two-way ANOVA (f) with Tukey’s multiple comparison. h–j Parasite lines were grown in ±IAA for 36 h and video microscopy was performed after addition of 2 μm A23187. h Representative example of images from three independent experiments showing time lapse recordings of DsRED diffusion (time stamp min:s). Scale bar = 20 µm. i Mean of replicates (n = 8 vacuoles) from each experiment plotted as separate lines (n = 3 experiments). j Mean of replicates (n = 44 −IAA, 53 +IAA in total) from each experiment plotted as separate lines (n = 3 experiments). ***P < 0.0001. Two-way ANOVA with Tukey’s multiple comparison. RLU relative luminescence units, ns not significant