Actin Filaments in Plasmodium Development Figure 6. Anti-Act239–253 shows preferential labelling of actin filaments. A) Widefield IFA with deconvolution of sporozoites treated with DMSO control, 1 mM cytochalasin D or 1 mM JAS labelled with rabbit anti-Act239–253 (Green), PbCSP (Red) and DAPI (Blue). Scale bar = 2 mm. Mask determined by anti-PbCSP labelling (see Materials and Methods). B) Maximum and total fluorescence levels of sporozoites treated with 1 mM JAS, 1 mM cytochalasin D and DMSO control. Significance as shown, unpaired t-test. C) Widefield IFA with deconvolution of invading P. falciparum merozoite labelled with rabbit anti-Act239–253 (Red) mouse anti-PfRON4 (Green) and DAPI (Blue). Scale bar = 2 mm. Arrows show direction of invasion. D–E) Widefield IFA with deconvolution of invading P. falciparum merozoites labelled with mouse anti-Actin (C4, Red) co-labelled with rabbit anti- PfRON4 (Green) (D) or rabbit anti-Act239–253 (Green) (E). DAPI (Blue) and Scale bar = 2 mm. doi:10.1371/journal.pone.0032188.g006 untreated (Fig. 6A). We reasoned that if the antibody recognises filament ends, fluorescence intensity should increase in cells that have more filaments. In agreement with this, maximum fluores- cence intensity for JAS treated sporozoites (where the number of normally transient filament ends is stabilised and concentrates in areas of dynamic actin) was significantly higher (p,0.005, unpaired t-test), whereas that for cytochalasin D treated parasites (having reduced F-actin) was significantly lower (p,0.05, unpaired t-test) than untreated controls (Fig. 6B). Total fluorescence decreased for both treatments (Fig. 6B). This decrease in maximal fluorescence intensity would be expected for cytochalasin D. Following JAS- treatment, the reduction likely results from two contributing factors. First, since JAS treatment elongates filaments, the total number of filament ends may be reduced. Second, whilst sporozoites that were relatively flat in the plane of view were selected for observation, 3D reconstructions demonstrated that a significant proportion have fluorescence that frequently lies outside the plane of imaging therefore removing a major proportion of actin labelling from quantification (Fig. S4). As further evidence, we re-visited P. falciparum merozoite invasion, reasoning that since actin filaments are required for invasion, the tight junction is likely filamentous in nature whilst non-polymerised actin (recognised by a generic anti- Actin antibody) would be cytosolic. Fluorescent labelling with anti- Act239–353 was again concentrated to the junction (co-labelling with RON4) (Fig. 6C), whereas that seen when using a generic anti-Actin monoclonal (C4) showed a broad cytosolic distribution not PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e32188