Actin Filaments in Plasmodium Development Figure 7. The tight junction is composed of dynamic actin filaments that localise posterior to the junction during invasion. A) Widefield IFA with deconvolution and B) 3D reconstruction of P. berghei merozoites incubated with and without 1 mM JAS and labelled with anti- Act239–253 (Green) and DAPI (Blue). Scale bar = 2 mm. Arrows show direction of invasion. C) Graphic representation of actin labelling in P. berghei merozoites with and without the addition of JAS. n = 124 merozoites for each of three replicates, mean is shown. D) 3D structured illumination microscopy (3D SIM) of three separate invading P. falciparum merozoites labelled with rabbit (upper row) and mouse (lower row) anti-Act239–253. Labelling shows actin (Red), RON4 (Green) and DAPI (Blue). See also Movie S6. Gamma settings were altered in 3D reconstructions. doi:10.1371/journal.pone.0032188.g007 restricted to the junction or to regions of anti-Act239–353 labelling (Fig. 6D, E). Combined, this evidence would support anti-Act239–253 having a strong affinity and potential preference for actin in a filamentous over monomeric state and the existence of actin filaments at the tight junction. By extension, this also implies that pellicular and apical actin labelling in free motile and invasive zoite forms (Fig. 2–3) and peripheral nuclear actin labelling across development stages (Fig. 4) is likely of a filamentous nature. Imaging of actin filament-like structures at the tight junction It has previously been shown that treatment of merozoites with actin inhibitors arrests invasion subsequent to tight junction formation [23,41]. To further explore the nature of actin at the tight junction, we compared untreated merozoites with those treated with high concentrations of JAS (post-attachment) to prevent complete invasion (see Materials and Methods). When compared to untreated controls (Fig. 7A, upper panel), P. berghei merozoites incubated with erythrocytes but treated with JAS following attachment showed a breakdown of anti-Act239–253 labelling (Fig. 7A, lower panel). Instead of a clear ring of actin, labelling appeared in elongated furrows surrounding the invading parasite (Fig. 7B). Similar results were seen with P. falciparum merozoites (data not shown). Given the greater numbers of invading merozoites seen with P. berghei, we attempted quantification of actin labelling under different treatments. Whilst circumferential (i.e. pellicular) actin labelling of merozoites associated with PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e32188