Localisation of PABP-interacting proteins to starvation stress granules.

<p>PABP interacting proteins were expressed as eYFP fusion proteins in a cell line expressing the starvation stress granule marker PABP2 as a C-terminal mChFP fusion. One representative Z-stack projection image is shown for untreated and starved parasites for proteins that localise to granules <b>(A)</b> and proteins that are (largely) absent from granules <b>(B)</b>. For all PBS treated cells, the average stress granule enrichment ratio with standard deviation is shown on top of each image: Granules were defined on the mChFP image (PABP2) by threshold settings using the maximal entropy method of ImageJ (<a href="https://imagej.nih.gov/ij/" target="_blank">https://imagej.nih.gov/ij/</a>) and the background corrected fluorescence within the granule divided by the background corrected fluorescence next to the granule was calculated for the three largest granules of ten cells for all five eYFP fusion proteins. Broad-field images of all cell lines are available in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006679#pntd.0006679.s002" target="_blank">S1 Fig</a>.</p

Overview of eukaryotic phylogeny emphasising the supergroup affiliation of organisms discussed here.

<p>Each of five recognised eukaryotic supergroups is shown as a coloured triangle to indicate that it contains a great many lineages, which are under continual diversification; groups not discussed are in gray, whilst Excavata (teal), stramenopiles, alveolates, and Rhizaria (SAR, red), and Opisthokonta (purple) are shown with icons for representative organisms. All of these groups radiated rapidly following the origin of eukaryotes and evolution of the LECA. Relationships are based on recent views of the branching order but should not be considered definitive.</p

Model integrating suramin-sensitivity pathways, trafficking and ISG turnover.

<p>A simplified schematic of the trypanosome endomembrane system is shown, with the flagellar pocket at top left. Teal arrows indicate ISG degradative and recycling trafficking routes, red putative AP-1-mediated transport from the Golgi complex to the lysosome [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005236#ppat.1005236.ref014" target="_blank">14</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005236#ppat.1005236.ref027" target="_blank">27</a>] and gray exocytic/biosynthetic pathways. The predominant locations of ISG75, ESAG6/7 (the transferrin receptor) and p67 (the major lysosomal protein) are indicated by icons. Evidence suggests that ISG75 is ubiquitylated at, or close to the surface (magenta) and deubiquitylation by TbUsp7 and/or TbVdu1 is proposed to take place prior to the sorting step at the early endosome that selects for the recycling or degradative arm of the endocytic system. TbVdu1 is known to associate with structures in this region, whilst TbUsp7 is likely cytosolic. Approximate percent changes in the proportion of ISG75 transiting the different arms of the endocytic system are calculated from differences in half-life derived for observed ISG75 turnover changes upon TbUsp7 knockdown (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005236#ppat.1005236.g005" target="_blank">Fig 5</a>) and assuming a recycling cycle time of ~10 min. AP-1 is proposed to mediate pathways required to deliver components to the lysosome required for suramin to translocate to the cytosol.</p

Assessment of ISG65 and ISG75 transcription and biosynthesis under TbUsp7 and TbVdu1 knockdown.

<p>(A) ISG65 and ISG75 transcript levels were determined in the presence (+, open bars) or absence (-, closed bars) of TbUsp7 and TbVdu1 knockdown by qRT-PCR normalised to β-tubulin. Error bars denote standard error of the mean. (B) Biosynthesis of ISG65 and ISG75 were monitored by immunoprecipitation using specific polyclonal antibodies, in the presence (+) or absence (-) of TbUsp7 and TbVdu1 knockdown. Bar graphs represent the mean of three independent knockdown experiments (open bars) normalised to uninduced (closed bars) cells, with the standard error indicated. Statistical analysis: Student’s t-test; *p<0.05, **p<0.01.</p

Immunofluorescence of cells ectopically expressing the TbLAP1-HA protein, double labeled with polyclonal anti-HA (green) and anti-TAC102 antibodies (red).

<p>The latter antibody visualizes the dynamics of the tripartite attachment complex (TAC). DAPI (blue) shows the location of the nucleus and kDNA; arrowheads and asterisks denote aberrant kDNA. Squares denote area displayed as zoom. B) After 2 hrs (A) and 6 hrs of ectopic expression of TbLAP1-HA (B), the apparition of 1K2Ndiv cells (not yet segregated cells with kDNA in division) became evident as depicted by two cells failing to segregate their already divided kDNAs; note the structure of the nabelschnur holding the kDNAs and TAC together at the posterior end of the cell. Division of TAC and nucleus occurred, as shown by two TAC102 signals and two nuclei, without prior segregation of the TAC structures and the kDNAs, which remained at the posterior end of the cell (C). Representative cell displaying accumulation of TbLAP1-HA around aberrantly sized kDNAs (D). Scale bars: 1 μm.</p

Volcano plots of protein abundance changes.

<p>Normalised SILAC ratios, averaged from duplicates (TbUsp7) or triplicates (TbVdu1), are plotted against the respective -log10 transformed standard deviation. Data points representing protein groups significantly shifted after 48 hours are labeled (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005236#ppat.1005236.t001" target="_blank">Table 1</a>). ISG65 and ISG75 paralogs are highlighted in green and orange, respectively.</p