Spatial localisation of different rhoptry proteins before and during merozoite invasion.

<p>(<b>A</b>) IEM of free PfRON2-HA merozoites (pre-invasion) dual labeled with immunogold anti-HA (18 nm) and rhoptry bulb marker RAP1 (12 nm). Scale bar = 0.2 µm. (<b>B</b>) IEM of free PFF0645c-HA merozoites (pre-invasion) dual labeled with immunogold anti-HA (18 nm) and rhoptry bulb marker RAP1 (12 nm). Scale bar = 0.2 µm. (<b>C</b>) Widefield IFA of E64-treated schizonts (to prevent egress – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#s4" target="_blank">Materials and Methods</a>) labeled with anti-PfRh2, anti-PfRON4 and DAPI. Scale bar = 5 µm. (<b>D–G</b>) Independent replicate imaging of merozoites from (<b>D</b>) PfRON2-HA, (<b>E</b>) PfASP-HA (two classes of distribution seen), (<b>F</b>) RAP1 and (<b>G</b>) PFF0645c-HA mid-way through invasion colabeled with anti-PfRON4 and DAPI.</p

<i>In silico</i> integrative genomic search strategy to identify <i>P. falciparum</i> invasins.

<p>(<b>A</b>) To compile a list of proteins that include invasins, <i>P. falciparum</i> genes with homologues in the tight junction forming <i>T. gondii</i>, <i>P. berghei, P. chabaudi, P. vivax, P. yoelii</i> and <i>P. knowlesi</i> were selected (blue). Orthologues found in the non-tight junction forming <i>C. parvum</i> and <i>C. hominus</i> (pink) were removed from the dataset. Transcriptomic and proteomic data from <i>P. berghei</i> and <i>P. gallinaceum</i> ookinetes (brown) was used to remove proteins involved in motility but not invasion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#pone.0046160.s003" target="_blank">Table S1</a> and Supplemental Experimental Procedures for data sources). (<b>B</b>) The top 50 candidate invasins ranked according to <i>P. falciparum</i> asexual cycle maximum fold change in transcript expression and relative protein abundance in <i>P. falciparum</i> merozoite and sporozoite proteomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#pone.0046160.s004" target="_blank">Table S2</a>). Accession numbers are from PlasmoDB version 8.2. Number of transmembrane domains (TM), presence of a signal peptide (SP) and expression maximum during intra-erythrocytic cycle are listed. Heat diagram demonstrate intra-erythrocytic expression levels, given across 48 hr lifecycle with red representing high relative and green low relative expression. Proteins tagged in this study with an HA epitope are highlighted in yellow, and proteins where tagging was attempted but unsuccessful are highlighted blue.</p

Cellular localisation of invasins in <i>P. falciparum</i> schizonts and <i>T. gondii</i> tachyzoites.

<p>(<b>A–E</b>) IFA of schizonts from HA tagged <i>P. falciparum</i> parasite lines labeled with anti-PfRON4, to mark the rhoptry neck, DAPI, to mark nuclei and anti-HA. (<b>A</b>) PfRON2 (<b>B</b>) PFF0645c (<b>C</b>) PfASP and (<b>E</b>) PF14_0578. (<b>D</b>) PF14_0375 was also colabeled with MitoTracker® Deep Red, labeling mitochondria, and anti-PfACP, labeling apicoplasts. (<b>F</b>) IFA of intracellular TGME49_115220-YFP tachyzoites (PFF0645c orthologue in <i>T. gondii</i>), colabeled with TgGAP45 (IMC) and TgMIC2 (micronemes). (<b>G</b>) Early intracellular development and extracellular TGME_116540-mCherryHA tachyzoites (PF14_0578 orthologue in <i>T. gondii</i>) colabeled with anti-TgGAP45 (IMC) and anti-TgSAG1 (surface). <i>C. septicum</i> alpha-toxin treatment swells the plasma membrane away from IMC. Scale bar = 5 µm throughout.</p

Subcompartmentalisation of rhoptries and invasin dynamics during apicomplexan host-cell invasion.

<p>A schematic for how the role that spatial distribution of rhoptry proteins pre-invasion facilitates different stages of the merozoite invasion of the erythrocyte and development post-invasion as a model for apicomplexan host-cell entry.</p

RON2 follows the tight junction during <i>P. falciparum</i> and <i>P. berghei</i> merozoite invasion.

<p>(<b>A</b>) Widefield 3D imaging of PfRON2-HA merozoites labeled with anti-HA, anti-PfRON4 (tight junction) and DAPI showing early, mid and late invasion events as well as parasites captured within 10 min post-invasion (<10 min p.i.). Scale bar = 5 µm. (<b>B</b>) IEM of free, invading and post-invasion (<10 min p.i.) PfRON2-HA merozoites labeled with anti-HA (white arrows). Scale bar = 0.2 µm. (<b>C</b>) Widefield 3D imaging of PbRON2-myc merozoites labeled with anti-myc, anti-parasite actin and DAPI captured mid invasion. IFA Scale bars = 5 µm. 3D reconstruction with 0.2 µm grid intervals. (<b>D</b>) 3D SIM of a PfRON2-HA merozoite captured mid way through invasion and labeled with anti-HA, anti-PfRON4 and DAPI. 3D reconstruction with 0.2 µm grid intervals.</p

PFF0645c is released from the rhoptries only after completion of merozoite invasion.

<p>(<b>A</b>) IEM of PFF0645c-HA merozoites pre- and post-erythrocyte invasion labeled with immunogold anti-HA (white arrows). Scale bar = 0.2 µm. (<b>B</b>) Widefield 3D imaging of PFF0645c-HA merozoites labeled with anti-HA, anti-PfRON4 and DAPI showing early, mid and late invasion events. Scale bar = 5 µm. 3D reconstruction with 0.2 µm grid intervals. (<b>C</b>) Widefield 3D imaging of PFF0645c-HA early rings (<10 min post-invasion) labeled with anti-HA, anti-PfRON4 (∼PVM) anti-RAP1 (PV) or anti-MSP1 (plasma membrane) and DAPI. Scale bar = 5 µm. 3D reconstruction with 0.2 µm grid intervals.</p

PfASP shows a dual localisation to both the tight junction and merozoite apex during erythrocyte invasion.

<p>Widefield 3D imaging of ASP-HA merozoites labeled with anti-HA, anti-PfRON4 and DAPI showing early invasion events (<b>A</b>) and two classes of mid/late invasion (<b>B, C</b>) distributions seen during invasion. Scale bar = 5 µm. 3D reconstruction with 0.2 µm grid intervals.</p

Concentration of actin labelling in the nucleus and around the nuclear periphery.

<p>Widefield IFA of representative <i>P. berghei </i><b>A</b>) ookinetes and <b>B</b>) sporozoites that show pronounced nuclear labelling using rabbit anti-Act_239–253 (Green) surface markers Pbs28 or PbCSP (Red) and DAPI (Blue). Scale bar = 5 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188.s009" target="_blank">Movie S5</a>. <b>C</b>) Widefield IFA of <i>P. falciparum</i> rings labelled with rabbit anti-Act_239–253 (Red) and DAPI (Blue). <b>D</b>) As <b>C</b> but following 6 hour JAS treatment. <b>E</b>) Two colour widefield IFA using rabbit anti-Act_239–253 (Red), rat anti-ERD2 (Green) and DAPI (Blue) in absence or presence of 1 µM JAS. All scale bars = 5 µm.</p

The spatial distribution of actin in invading merozoites and sporozoites.

<p><b>A</b>) Transmission electron micrograph with anti-Act_239–253 (rabbit) immunogold labelling (arrowheads) of invading <i>P. falciparum</i> merozoite. Arrows show direction of invasion. <b>B</b>) Widefield IFA with deconvolution of invading <i>P. falciparum</i> merozoites labelled with mouse anti-Act_239–253 (Red) or rabbit PfRON4 (Green) and DAPI (Blue). Scale bar = 2 µm. <b>C</b>) Widefield IFA with deconvolution of invading <i>P. berghei</i> merozoites labelled with rabbit anti-Act _239–253 (Green) and DAPI (Blue). Scale bar = 2 µm. Gamma settings were altered in 3D reconstruction. <b>D</b>) Widefield IFA with deconvolution of invading <i>P. berghei</i> sporozoites labelled with rabbit anti-Act_239–253 (Green), anti-PbCSP (Red, exterior only) and DAPI (Blue). Scale bar = 5 µm, arrowhead shows presumed site of tight junction.</p

The tight junction is composed of dynamic actin filaments that localise posterior to the junction during invasion.

<p><b>A</b>) Widefield IFA with deconvolution and <b>B</b>) 3D reconstruction of <i>P. berghei</i> merozoites incubated with and without 1 µM JAS and labelled with anti-Act_239–253 (Green) and DAPI (Blue). Scale bar = 2 µm. Arrows show direction of invasion. <b>C</b>) Graphic representation of actin labelling in <i>P. berghei</i> merozoites with and without the addition of JAS. <i>n</i> = 124 merozoites for each of three replicates, mean is shown. <b>D</b>) 3D structured illumination microscopy (3D SIM) of three separate invading <i>P. falciparum</i> merozoites labelled with rabbit (upper row) and mouse (lower row) anti-Act_239–253. Labelling shows actin (Red), RON4 (Green) and DAPI (Blue). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032188#pone.0032188.s010" target="_blank">Movie S6</a>. Gamma settings were altered in 3D reconstructions.</p