Actin Filaments in Plasmodium Development Figure 1. An apicomplexan parasite-specific anti-actin antibody. A) Sequence comparison between human non-muscle actin amino acids 237–251 (the basis of anti-Gly245 [39]) and apicomplexan actin I orthologues over the amino acids 239–253 (the basis for anti-Act239–253). B) Surface representation of the structures of rabbit G-actin (PDB:1J6Z; A) and a protomer in rabbit F-actin (PDB:3G37; B) showing anti-Gly245 epitope. Residues in yellow indicate polymorphisms between mammalian and P. falciparum actin. C) Representative immunoblot showing reactivity of rabbitH anti- Act239–253 serum with human erythrocytes (hRBC), asexual P. falciparum (3D7), mouse erythrocytes (mRBC), asexual P. berghei (ANKA), human foreskin fibroblasts (HFF) and T. gondii tachyzoites (RH). Lower panel shows same hRBC and 3D7 sample probed with mouse (m) anti-Act239–253 serum. D) As C but using generic anti-actin monoclonal C4. doi:10.1371/journal.pone.0032188.g001 concentrate broadly at the pellicular regions of free merozoites, ookinetes and salivary gland sporozoites (Fig. 2A–C, upper panels). Pellicular labelling in merozoites was further supported by serial section immunoelectron microscopy of free P. falciparum merozo- ites (Fig. S1A). When treated with the cyclodepsipeptide Jasplakinolide (JAS), which arrests actin filament turnover [25], labelling became even more pronounced in pellicular and apical regions highlighting these areas as foci for actin turnover (Fig. 2A– C, lower panels). Prominent structures, reminiscent of an acrosomal process described in JAS treated T. gondii tachyozites [26,28] and P. falciparum merozoites [24], could be seen in all three motile or invasive stages (Fig. 2A–C, lower panels). In sporozoites, labelling was generally greatest in apical and posterior regions, known sites of substrate attachment in these cells [15] (Fig. 1B, Movie S1). In ookinetes, labelling along the flanks post-JAS treatment remained associated with the pellicular region and was frequently strongest at the flexed portion of the parasite cell (Fig. 2C), which may represent a key point of traction in line with recent observations using ookinetes that express constitutive GFP- actin [21]. In ookinetes where apical or basal concentrations of labelling were strongest, three-dimensional reconstruction of fluorescent images demonstrated clear capping of the ookinete poles (Fig. 2D, Movie S2). We explored this further by three- dimensional structured illumination microscopy (3D-SIM), a technique able to resolve structures beyond the normal resolution limits of conventional fluorescence microscopy [43]. Untreated ookinetes demonstrated broad cytosolic localisation of actin with variable pellicular concentrations under 3D SIM conditions (Fig. 2E, Movie S3). Following JAS treatment, actin labelling again redistributed to polar regions with capping structures resolved into branched rod-like fibres, frequently with three or more arms (Fig. 2F, Movie S4) [21,27]. Why these actin-rich structures should be so uniform is unclear, but they are similar to bundles of actin seen following JAS treatment by electron microscopy in Toxoplasma tachyzoites [26]. Electron microscopy of untreated versus JAS-treated ookinetes was unable to resolve directly the nature of these structures (Fig. S2A–B). Pellicular actin is concentrated in the supra-alveolar space and associates with membranes Broad cortical localisation of actin in each lifecycle stage is suggestive of an association of actin with membranes in the parasite pellicle. Entirely consistent with this, carbonate extraction of P. falciparum schizont lysate (to separate membrane associated from cytosolic proteins) indicated a substantial portion of the actin pool does associate strongly with membranes when compared to cytosolic controls (Fig. S3A). Immunoelectron microscopy of ookinetes, labelled with anti-Act239–253, was consistent with a pellicular association, demonstrating strong associations of gold labelling in both apical and supra-alveolar compartments (Fig. 3A). Attempts to define conclusively the pellicular membrane associ- ated with actin using Clostridium septicum alpha toxin, which has been used extensively to separate the plasma membrane away from IMC in T. gondii tachyzoites [9], were unsuccessful with all malaria parasite zoite forms (data not shown). Given the conservation of the reactive actin 239–253 epitope in T. gondii (Fig. 1A, C, Fig. S3B), we undertook immunofluorescence assays with extracellular tachyzoites treated with the pore forming toxin. Tachyzoites labelled with anti-Act239–253 demonstrated broad pellicular association of actin with marked redistribution following JAS treatment to apical and cortical regions (Fig. 3B–C) [28]. Using markers for the plasma membrane (SAG1) and IMC (IMC4ty, Bradin and Tonkin, unpublished data), alpha-toxin treatment defined labelling to this compartment as within the supra-alveolar space (Fig. 3D). Combining toxin treatment with JAS, labelling shifted from within the supra-alveolar space to the PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e32188