Figure 8. A refined model for the apicomplexan host-cell invasion. An apicomplexan zoite during host-cell invasion. Abbrevia- tions: ama1, apical membrane antigen 1; F-actin, actin filaments (turning over); hpm, host plasma membrane; imc, inner membrane complex; myoA, myosin A; ppm, parasite plasma membrane; ron- complex, rhoptry neck protein complex; trap, thrombospondin related anonymous protein. Question mark indicates unknown linkage between motor complex (actin and myosin) and tight junction complex (ama1-ron complex). Red arrow indicates movement of actin filaments rearwards by myoA driving motility. Movement of actin drives trap-like adhesin (Blue arrow). Green arrow marks movement of traction point complexes (light green) during gliding or invasion. doi:10.1371/journal.pone.0032188.g008 merozoite invasion but not sporozoites invasion (Fig. 5) (nor that of tachyzoites, data not shown) may reconcile these conflicting observations. Recently, we have demonstrated that P. falciparum actin depolymerising factor 1 (ADF1) has a broad cytosolic localisation but is excluded from the tight junction during merozoite invasion [42]. As such it may be a combination of nucleation, stabilisation and depolymerisation factors that ulti- mately dictates the restricted localisation of actin to the tight junction zone. A second question is what links the actin (and possibly aldolase) raft along with its associated surface bound adhesins to the tight junction RON-AMA1 complex? Proteins responsible for this linkage would be of great interest. Ultimately, precise imaging of each individual component of the motility and invasion machinery by fixed and live imaging approaches is required to resolve current models for the molecular basis of gliding. Live imaging of dynamic actin in particular, and the development of tools for this purpose, would be a major step towards this goal. Furthermore, increased efforts in understanding the regulation of actin’s spatiotemporal localisation, filament turnover, and association with the plasma membrane may help explain how motility is regulated. More broadly, efforts to understand the repertoire of functions played by actin in malaria parasite development and movement should reveal potent chemo- therapeutic targets that transcend stage specificity and may work as the basis for treatment and also transmission blocking drugs. Actin Filaments in Plasmodium Development Materials and Methods Ethics statement The culture of P. falciparum parasites using donated blood and serum from the Australian Red Cross Society and use of mice for growing P. berghei have been approved by The Walter and Eliza Hall Institute Human Ethics (HEC 86/17) and Animal Ethics Committees (AEC Project 2009-023). Antibody production A synthetic peptide spanning amino acids 239–253 of PfActin (PlasmoDB ID: PFL2215w) was used to raise polyclonal rabbit antisera (Genscript, USA) and mouse antisera against malaria actin (anti-Act239–253). Parasite culture and maintenance and immunoblot analysis P. falciparum (3D7 and D10 strains), P. berghei (ANKA strain) and T. gondii (RH strain) parasites were each maintained using standard procedures. P. falciparum cultures were grown in human O+eryth- rocytes at 4% hematocrit with 0.5% Albumax II (Invitrogen). 3D7 is a cloned line derived from NF54, obtained from the late David Walliker at Edinburgh University, UK. P. berghei lines were maintained in Balb/c mice as described previously [66]. In vitro conversion to ookinetes followed Moon et al. [67]. T. gondii was propagated in human foreskin fibroblasts (HFF) grown in Dulbecco’s modified Eagle’s medium (DMEM) with 1% fetal calf serum (GIBCOBRL). For a-toxin treatment, needle passaged IMC4-ty transfectant tachyzoites (C. H. Bradin and C. J. Tonkin, unpub- lished) were incubated in 50 ml of Clostridium septicum culture supernatant. Lysates from saponin-treated schizont stage in vitro cultures of both P. falciparum and P. berghei along with tachyzoites were harvested by centrifugation, resuspended in reduced sample buffer and analysed by Western blot probing with rabbit or mouse anti-Act239–253 antisera at 1:1000 and 1:200, with anti-Actin (clone C4, Millipore) at 1:1000. Signal was detected by anti-rabbit or mouse IgG horseradish peroxidase conjugate (HRP) (Millipore), and visualised via enhanced chemiluminescence (ECL, Amersham Biosciences). For solubility analysis, isolated 3D7 strain P. falciparum merozoites resuspended in water (Complete Protease Inhibitors, Roche) were snap frozen and incubated on ice for 10 min to release the cell content. Water soluble and insoluble proteins were separated by ultracentrifugation at 100,0006 g for 30 min at 4uC (TLA100.2 rotor, Beckman Optima TL Ultracentrifuge, Beckman Coulter). Water insoluble fractions were further treated with Na2CO3 pH 11.5 for 1 hour at 4uC. Carbonate soluble and insoluble fractions were isolated by further ultracentrifugation. Samples were subject to SDS-PAGE and immunoblot analysis. Membranes were incubated with antisera (rabbit anti-Act239–253 [1:500], rabbit anti- PfADF1 [1:1000] [42] and rabbit anti-MTRAP [1:200] [13]). Zoite invasion preparation Blood stage P. falciparum parasites and P. berghei (ANKA) were cultured through to schizogony and prepared for merozoite invasion following Boyle et al [68]. For sporozoite invasion P. berghei sporozoites were dissected from infected Anopheles stephensi salivary glands and kept on ice in HepG2 culture media (Advanced MEM (GibcoH) supplemented with 10% foetal calf serum (Bovogen), 1% L-Glutamin (Thermo Scientific), 1% Penicillin/Streptomycin (Thermo Scientific), 0.1% Amphotericin B (Thermo Scientific) until dissection was completed. Sporozoites were added to 16105 HepG2 cells per well in a 24 well plate. The plate was spun at 800 rpm at 4uC for 4 min to facilitate sporozoite – HepG2 cell interaction. The plate was put at 37uC and 5% CO2 for 5 or 10 min, carefully washed once with PBS and invasion PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e32188