Actin Filaments in Plasmodium Development Figure 4. Concentration of actin labelling in the nucleus and around the nuclear periphery. Widefield IFA of representative P. berghei A) ookinetes and B) sporozoites that show pronounced nuclear labelling using rabbit anti-Act239–253 (Green) surface markers Pbs28 or PbCSP (Red) and DAPI (Blue). Scale bar = 5 mm. See also Movie S5. C) Widefield IFA of P. falciparum rings labelled with rabbit anti-Act239–253 (Red) and DAPI (Blue). D) As C but following 6 hour JAS treatment. E) Two colour widefield IFA using rabbit anti-Act239–253 (Red), rat anti-ERD2 (Green) and DAPI (Blue) in absence or presence of 1 mM JAS. All scale bars = 5 mm. doi:10.1371/journal.pone.0032188.g004 (Fig. 4D). The concentration of stabilised actin filaments at the nuclear periphery was confirmed using ERD2, a cis-Golgi marker that localises to defined sites adjacent to the nucleus [44] (Fig. 4E). Demonstration of actin in the nucleus, specifically its dynamic nature at the nuclear periphery, is consistent with emerging understanding of the roles of actin and myosin in movement of chromosome ends to the nuclear periphery [45,46]. Indeed, two recent studies have corroborated just such a role for actin in gene regulation during P. falciparum ring stage develop- ment [47,48]. The association of actin with the tight junction during merozoite and sporozoite invasion of host cells We have recently shown that rabbit serum against Act239–253 labels a ring of actin at the merozoite-erythrocyte tight junction during P. falciparum merozoite invasion [41,42]. We confirmed the presence of a concentration of actin at the electron dense junction by immunoelectron microscopy using rabbit antiserum (Fig. 5A) and by immunofluorescence with new antiserum raised in mice (Fig. 5B). To explore the generality of this observation in other species, we undertook immunofluorescence imaging with mero- zoites and salivary gland sporozoites from P. berghei. Invading P. berghei merozoites showed a consistent ring of actin at the junction during invasion (Fig. 5C). Sporozoite invasion of hepatocytes is notoriously hard to capture [49]. Furthermore, sporozoites are actively involved with gliding as well as invasion (for example traversing cells in the absence of true invasion [50]) and differentiating between the two can be challenging. Sporozoites dissected from mosquito salivary glands and applied to cultured HepG2 liver cells showed a consistent, seemingly structured, concentration of actin in the internalised portion of the entering sporozoite (as marked by restriction of CSP to the exterior) (Fig. 5D, inset). In some instances patterns of labelling included a distinct band of actin associated with a tight junction constriction PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32188