Actin Filaments in Plasmodium Development Figure 3. Location of anti-PfAct239–253 labelling to supra-alveolar membranes. A) Transmission electron micrographs with anti-Act239–253 (rabbit) immunogold labelling (arrowheads) of P. berghei ookinetes (including inset and independent ookinete apical end). B–E) Widefield IFA of free T. gondii tachyzoites under various treatments probed with anti-PfAct239–253 (Green) versus anti-TgSAG1 (a plasma membrane marker) or anti-Ty (IMC4, and IMC marker) (Red). DAPI (Blue) and Scale bar = 5 mm. B) Untreated. C) Following treatment with 5 mM JAS. D) Following treatment with C. septicum a toxin 1/100. E) Following treatment with 5 mM JAS and a-toxin. F) Schematic for labelling seen following treatment with a toxin alone or in combination with 5 mM JAS localising actin filaments. doi:10.1371/journal.pone.0032188.g003 Actin filament dynamic also localise to the nuclear periphery in multiple lifecycle stages In several instances, immunofluorescence assay of ookinetes and sporozoites revealed a sizable proportion of actin localised at the nucleus or around the nuclear periphery (Fig. 4A, B, Movie S5). This may suggest that actin dynamics function in nuclear architecture or gene regulation, as is seen in other eukaryotes [35]. To explore the generality of this observation in other lifecycle stages (in particular those in which nuclear activity is high) early ring stage asexual parasites were labelled with anti-actin and the nuclear marker DAPI. Very early rings demonstrated a consistent punctate labelling of actin within DAPI staining of the nucleus (Fig. 4C). Treatment with JAS transformed this punctate pattern into a clear ring surrounding the nucleus PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32188