cAMP signaling downstream of coronin relocalization modulates motility.

<p>(A) Sporozoites were incubated in RPMI + 3% BSA with the indicated kinase inhibitors at the indicated concentrations, imaged and their movement pattern quantified as gliding, waving or attached. The inhibitors stopped sporozoite motility in a dose-dependent fashion. Over 150 sporozoites were examined for each experiment. Significant differences (Fisher’s exact test) from the controls show both concentrations of H89 tested (p<0.0001) as well as 500 μM SQ22536 (SQ) (p = 0.01; p = 0.46 for 200 μM SQ22536). (B) Forskolin partially stimulates sporozoite motility when added to sporozoites in RPMI. Over 300 sporozoites were examined and their motility pattern quantified as in A. The shown difference is significant (p<0.0001; Fisher’s exact test). (C) Coronin-mCherry expressing sporozoites were investigated under various kinase inhibitors (each at 0,1 mM) with a fluorescence microscope to determine coronin-mCherry localization. The fluorescence stays at the rear in activated sporozoites arrested with PKA inhibitors. Coronin-mCherry relocalizes to the periphery when additional cytochalasin D is applied. Addition of forskolin to non-activated sporozoites leads to the localization of coronin-mCherry to the rear. Scale bar: 5 μm. (D) Table showing percentages of motile and non-motile sporozoites and the associated localization patterns (rear versus non-rear) of coronin-mCherry under the indicated conditions; low and high concentrations of SQ22536 (SQ) were 0,1 and 0,5 mM, respectively. Between 105 and 120 sporozoites were examined per condition. Statistical differences determined by Fisher’s exact test was p<0.0001 from the respective controls listed in the table of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005710#ppat.1005710.g004" target="_blank">Fig 4A</a>. (E) Examples of FRAP of motile sporozoites with coronin-mCherry localized to the rear. Scale bar: 5 μm. Circle indicates location of the bleach spot. (F) Quantitative analysis of FRAP. Coronin-mCherry recovers as fast in motile (>0.25 μm/s) as in non-motile (<0.25 μm/s) sporozoites if pooled across all conditions. There is also no difference in recovery time depending on the localization of coronin-mCherry [top graphs]. Quantitative analysis of FRAP data over a range of conditions [bottom graph]. Average values (+/- S.D.) are indicated above the graph. Bars show significant differences (* p<0.05; ** p<0.01), non-significance (ns) or p-values (Students t-test). Coronin-mCherry recovers significantly faster in non-motile sporozoites incubated in RPMI than in any other condition. With all other conditions there is no difference from each other with the exception of H89, where coronin-mCherry recovers significantly slower when compared to controls, 1 μM Cytochalasin D and 100 nM Jasplakinolide [bottom].