Actin Filaments in Plasmodium Development Figure 5. The spatial distribution of actin in invading merozoites and sporozoites. A) Transmission electron micrograph with anti-Act239– 253 (rabbit) immunogold labelling (arrowheads) of invading P. falciparum merozoite. Arrows show direction of invasion. B) Widefield IFA with deconvolution of invading P. falciparum merozoites labelled with mouse anti-Act239–253 (Red) or rabbit PfRON4 (Green) and DAPI (Blue). Scale bar = 2 mm. C) Widefield IFA with deconvolution of invading P. berghei merozoites labelled with rabbit anti-Act 239–253 (Green) and DAPI (Blue). Scale bar = 2 mm. Gamma settings were altered in 3D reconstruction. D) Widefield IFA with deconvolution of invading P. berghei sporozoites labelled with rabbit anti-Act239–253 (Green), anti-PbCSP (Red, exterior only) and DAPI (Blue). Scale bar = 5 mm, arrowhead shows presumed site of tight junction. doi:10.1371/journal.pone.0032188.g005 (Fig. 5D). However, exclusive labelling showing a ring of actin at the sporozoite junction was not seen. Whilst these data suggest that concentration of actin in a ring at the host-parasite tight junction is a clear feature of merozoite invasion across species, its conserva- tion in other zoite forms is still uncertain, likely confounded by the presence of traversal and general gliding in addition to invasion in these forms. Anti-Act239–253 shows preferential labelling for actin filaments Given the unusual reactivity of the parent anti-Gly245 antibody, we sought to explore the filament labelling preferences of the anti- Act239–253 antibody using quantitative imaging of sporozoites treated with either JAS (to stabilise filaments), cytochalasin D (in which filaments are capped and will be less prevalent) or left PLoS ONE | www.plosone.org 7 February 2012 | Volume 7 | Issue 2 | e32188