Ultrastructure of <i>Toxoplasma gondii</i>.

<p>(<b>A</b>) Intracellular <i>T. gondii</i> tachyzoite showing the MICs (Mn), ROPs (Rh), micropore (Mp), Golgi (G), nucleus (N), endoplasmic reticulum (ER), and dense granules (DG). (<b>B</b>) A schematic picture of <i>T. gondii</i> entering into a nucleated mammalian host cell. The apical exocytosis of MICs deploys onto the parasite surface MIC proteins required for parasite motility and the formation of moving junction. ROP secretion provides the ROP proteins that are involved in host cell invasion and modulation of immune responses. The constitutive secretion of dense granules (DG) is involved in the modification of the parasitophorous vacuole (PV). (<b>C</b>) Higher magnification of the single Golgi apparatus of <i>T. gondii</i>. (<b>D</b>) Higher magnification of the <i>T. gondii</i> ER. Scale bars, 1 µm.</p

A model for TgSORTLR functions in protein sorting and the biogenesis of apical secretory organelles.

<p>We propose that TgSORTLR has a distinct role as a type I transmembrane cargo-protein receptor for ROPs and MICs of apicomplexan parasites. We observed TgSORTLR-positive structures that could be transport vesicles destined for the endolysosomal system or they might be integral to the endolysosomal system, i.e., early (EE) and late (LE) endosomes. The model further proposes that the cytoplasmic tail of TgSORTLR binds to AP-1, Sec23/24, clathrin, clathrin-associated adaptor protein, and VPS9, and this defines it as a key receptor involved in the anterograde transport of cargo ROP and MIC proteins. The binding of TgSORTLR to the retromer VPS26/VPS35 also indicates that this receptor is also involved in the retrograde transport of components. <i>T. gondii</i> lysosome-like, acidic vacuolar compartment (VAC), also termed the Plant-Like Vacuole (PLV), contains cathepsin proteases implicated in the proteolytic maturation of proproteins targeted to MICs. Proteolytic maturation likely occurs in the LE where conditions are thought to be more conducive for limited proteolysis.</p

TgSORTLR co-localizes with TgVsp26 and Tgμ1-adpatin.

<p>(<b>A</b>) Confocal images of tachyzoites expressing endogenously tagged TgVps26-HA (green) that co-localizes with TgSORTLR (red). (<b>B</b>) Confocal images of tachyzoites expressing endogenously tagged Tgμ1adaptin-HA (red) and TgSORTLR (green). White circles indicate the zoomed areas showing co-distribution between TgSORTLR and Tgμ1adaptin-HA or TgVPS26-HA in the Golgi and post-Golgi. Scale bars, 5 µm.</p

Comparative bioinformatics analysis of genes coding components of vesicle-mediated trafficking and endosomal sorting in apicomplexan parasites, <i>Saccharomyces cerevisiae</i>, and <i>Homo sapiens</i>.

<p>Most of these genes and their corresponding accession numbers were collected from Eupathdb.org (for apicomplexan parasites) and uniprot.org (yeast and human cells). The data from apicomplexan parasites <i>Toxoplasma gondii</i> (<i>T. gondii</i>), <i>Plasmodium falciparum</i> (<i>P. falciparum</i>), <i>Theileria parva</i> (<i>T. parva</i>), and <i>Cryptosporidium parvum</i> (<i>C. parvum</i>) were compared with human (<i>H. sapiens</i>) and the yeast <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>). AP, adaptor protein; GGAs, Golgi-localized, γ-ear–containing, ADP-ribosylation factor binding protein; COPI, Coatomer complex I (retrograde transport from trans-Golgi apparatus to cis-Golgi and endoplasmic reticulum); COPII, Coatomer complex II (anterograde transport from ER to the cis-Golgi); ESCRT, Endosomal Sorting Complex Required for Transport.</p

TgChromo1 participates to the nuclear organisation of the nucleus.

<p>TgChromo1 participates in the functional organisation of the nucleus. The schematic represents the chromosomes in the nucleus with the centromere and telomere clusters occupied by TgChromo1 and their position at the periphery of the nucleus.</p

TgChromo1 binds to peri-centromeric heterochromatin.

<p>ChIP on chip was performed with the TgChromo1 antibody (anti-CHD1, red) or the anti-HA antibody (HA, black) and hybridized on a genome-wide tiling microarray. The regions of enrichment for H3K9me3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032671#pone.0032671-Brooks1" target="_blank">[12]</a> are represented in blue. A snapshot of the 12 chromosomes where a centromere was identified <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032671#pone.0032671-Brooks1" target="_blank">[12]</a> is presented. ChIP on chip signals are represented as a log2 ratio of the signal given by the immunoprecipitated DNA over the input and plotted according to the genomic position of the oligonucleotide.</p

Subtelomeric repeats occupy the same nucleus territory as TgChromo1.

<p>FISH/IFA of TgChromo1-HA (green) and chromosome IX telomeric repeats (red). Parasite nuclei are labelled with DAPI (blue). Colocalising signals from FISH and IFA are arrowed.</p

TgChromo1 is maintained near the centrosome and the centrocone throughout the cell cycle.

<p><b>A:</b> IFA of TgChromo1-HA (green) and centrin1 (red), a marker of the centrosome. Parasite nuclei are labelled with DAPI (blue) at the interphase (G1), mitosis and the beginning of budding. <b>B:</b> IFA of TgChromo1-HA (green) and MORN1 (red), a marker of the centrocone. Parasite nuclei are labelled with DAPI (blue) at the interphase (G1), mitosis and the beginning of budding.</p

Colocalisation of TgChromo1 and peri-centromeric sequences and identification of a missing centromere on chromosome IV.

<p><b>A:</b> Genomic localisation of the FISH probes. Fish probes (red rectangles) approximate locations are plotted against the signal of the TgChromo1-HA ChIP on chip. <b>B:</b> FISH/IFA of TgChromo1-HA (green) and chromosome IX peri-centromeric repeats (red). Parasite nuclei are labelled with DAPI (blue). <b>C:</b> FISH/IFA of TgChromo1-HA (green) and chromosome IV putative peri-centromeric repeats (red). Parasites nuclei are labelled with DAPI (blue). <b>D:</b> Quantification of the overlap coefficient between the signals of FISH and IFA. The coefficient is expressed as a ratio of the number of pixel overlapping in IFA over FISH in a given area.</p

TgChromo1 expression is cell cycle regulated.

<p><b>A:</b> IFA of TgChromo1-HA (green) and IMC1 (red), a marker of the inner membrane complex, throughout the cell cycle. Parasites representative of interphase (G1) and mitosis are presented. Parasite nuclei are labelled with DAPI. <b>B:</b> Comparison of the intensity of the signal produced by IFA of TgChromo1-HA in interphase (G1) and during budding (Budding, B). IMC1, a marker of the inner membrane complex, is used to identify emerging daughter cells during the budding. Parasites during budding (B) are arrowed. Parasite nuclei are labelled with DAPI. <b>C:</b> TgChromo1 is concentrated in foci of different intensity. IFA was performed using an anti-HA antibody and the IMC1 antibody. Parasites nuclei are labelled with DAPI. Foci of lesser intensity are arrowed.</p