Fluorescent tagging and absence of introns can impact profilin function.

<p>(A) Quantification of profilin-mCherry fluorescence per volume in ring stages, trophozoites and sporozoites (red shading). Each dot represents the fluorescence intensity normalized to parasite volume for one parasite. Horizontal bar: median. Significances were tested with Mann-Whitney tests; * denotes p<0.05; *** p<0.001.(B) Quantification of total profilin-mCherry fluorescence intensity of ookinetes. Note that fluorescence was much stronger in the nucleus of ookinetes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006412#ppat.1006412.s003" target="_blank">S3B Fig</a>) and thus ookinetes were not directly comparable with other stages. Significance was tested with a Mann-Whitney test. (C, D) Average speed with median (line) and mean (arrowheads) of ookinetes (C) and sporozoites (D) from the different parasite lines; Wt: wild type; Pfn⁺ⁱ mCh: Profilin-mCherry with introns; Pfn^-i mCh: Profilin-mCherry without introns; Pfn^-i // mCh: Profilin-mCherry without introns and long linker, which is cleaved (see panel E). At least 50 parasites per line were tracked over at least half of the acquisition time (sporozoites: 5 min, ookinetes: 15 min); significance was tested with a Kruskal-Wallis test; *** denotes p<0.001. (E) Western blot showing a weak band for the profilin-mCherry fusion protein (arrowhead) and a major band for a cleaved mCherry of the parasite line expressing profilin-mCherry with a long linker (Pfn^-i // mCh) detected with an anti-mCherry antibody in blood stage schizont lysates (lane 1). Note that at least 75% of profilin-mCherry is cleaved. Lane 2: control line expressing only mCherry. Loading control: anti-HSP70.