Analysis of egress, invasion, and motility in parental and mutant lines.

<p><b>A</b>. Parasites grown for 30 hr ± IAA (500 μM vs 0.1% ethanol) were stimulated with 3 μM A23187 to simulate egress. Rabbit anti-GRA7 (red) and mouse anti-IMC1 (green) antibodies were used to distinguish intact vs. egressed vacuoles. *** <i>P ≤ 0</i>.<i>0001</i>, significant for the time points of 2, 5, 10 and 15 min, but not significant for 0 and 20 min. Scale bar, 5 μM. <b>B</b>. Quantitative analysis of invasion by parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) and used to challenge fresh HFF monolayers on coverslips for 20 min. Extracellular parasites (invaded) were distinguished from those that remained extracellular (attached) by differential IFA staining (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006379#sec002" target="_blank">methods</a>). *** <i>P</i> ≤ 0.0001. <b>C.</b> Evaluation of cell entry past the moving junction. Parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) were used to challenge fresh HFF monolayers on coverslips for 3 min, fixed and stained with rabbit anti-RON4 (green) and mouse anti-SAG1 (red) without permeabilization. Parasites with RON4 dots were considered to be apically attached (red column), and parasites with RON4 positive rings were classified as partially invaded (green column). *** <i>P ≤ 0</i>.<i>0001</i>. Scale bar, 2 μM. <b>D</b>. Parasite motility as monitored by video microscopy. Parasites grown for 2 days ± IAA (500 μM vs 0.1% ethanol) were allowed to glide on serum-coated coverslips. Time-lapse video microscopy was used to score different motile behaviors. *** <i>P</i> ≤ 0.0001, the cam2^KOCaM1-AID line showed significant decrease in twirling and increase non-productive movement when grown in +IAA <i>vs</i>. -IAA or the TIR1 parental line, **, <i>P</i> ≤ 0.0001, the CaM3-AID line showed a significant decrease in twirling and increase in circling when grown in +IAA <i>vs</i>. -IAA or the TIR1 parental line. Panels <b>A</b>, <b>B</b>, <b>C</b>, <b>D</b> represent means ± S.D. from three independent experiments with triplicates for each (n = 9). Two-way ANOVA with Tukey’s multiple comparison test for <b>A</b>, <b>C</b> and <b>D</b>, and one-way ANOVA with Tukey’s multiple comparison test for <b>B</b>.