Actin Filaments in Plasmodium Development Figure 2. Spatial distribution of actin in free malaria parasite zoites. A) Widefield IFA of P. berghei merozoites with and without 1 mM JAS, labelled with rabbit anti-Act239–253 (Green) and the nuclear marker DAPI. Scale bar = 2 mm. B) Widefield IFA of P. berghei sporozoites with and without 1 mM JAS, labelled with rabbit anti-Act239–253 (Green), surface marker PbCSP (Red) and DAPI (Blue). Scale bar = 5 mm. See also Movie S1 C) Widefield IFA of P. berghei ookinetes with and without 1 mM JAS, labelled with rabbit anti-Act239–253 (Green), surface marker Pbs28 (Red) and DAPI (Blue). Scale bar = 5 mm. D) 3D reconstruction of widefield IFA with deconvolution of 1 mM JAS treated P. berghei ookinete, labelled with rabbit anti-Act239–253 (Green), Pbs28 (Red), and DAPI (Blue). Scale bar = 5 mm. See also Movie S2. E–F) 3D structured illumination microscopy (3D SIM) of P. berghei ookinetes labelled with rabbit anti-Act239–253 (Green), surface marker Pbs28 (Red) and DAPI (Blue) in the absence (E) and presence of 1 mM JAS (F). Scale bar = 2 mm. See also Movie S3, S4. Gamma settings were altered in 3D reconstructions. doi:10.1371/journal.pone.0032188.g002 bounding membranes of the IMC (Fig. 3E). In conjunction with differential solubilisation (Fig. S3A) this distribution supports an affinity of the dynamic actin filament pool for the pellicular membranes that bound the supra-alveolar space (Fig. 3F). Combined, the intensity and restriction of fluorescence labelling (following JAS treatment in particular) and lack of cross reactivity with a variety of host cells used corroborates the specificity of the anti-Act239–253 serum for parasite actin. Furthermore, the data provide clear support that microfilament dynamics are focussed at the apex and pellicle of motile or invasive parasite stages, likely associated with membranes of the supra-alveolar space. These observations are entirely in line with current models for actin’s proposed function in apicomplexan parasites and its dominant role in driving motility through the IMC immobilised gliding motor [12]. PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32188