MTX treatment of cells expressing mitosome-targeted DHFR affects processing of a mitosomal matrix reporter.

<p>Subcellular distribution of a matrix-targeted reporter (<i>Gl</i>17030-HA) without MTX (A) or after addition of 1μM MTX for 24 h (B) in transgenic cells expressing mitosome-targeted DHFR. Note the accumulation of HA-signal in the cytoplasm. Nuclear DNA is stained with DAPI (blue). Insets: DIC images. (C) Immunoblot analysis detects accumulation of unprocessed <i>Gl</i>17030-HA in the presence of MTX.</p

MTX treatment of cells expressing mitosome-targeted DHFR affects processing of a mitosomal matrix reporter.

<p>Subcellular distribution of a matrix-targeted reporter (<i>Gl</i>17030-HA) without MTX (A) or after addition of 1μM MTX for 24 h (B) in transgenic cells expressing mitosome-targeted DHFR. Note the accumulation of HA-signal in the cytoplasm. Nuclear DNA is stained with DAPI (blue). Insets: DIC images. (C) Immunoblot analysis detects accumulation of unprocessed <i>Gl</i>17030-HA in the presence of MTX.</p

An integrated model for mitosome interactome networks.

<p>Schematic representation of all proteins identified via protein-protein interaction data through serial coIP assays using 6 different mitosomal bait proteins are shown in a model. Previously identified/ known mitosomal proteins are depicted in yellow. Newly identified mitosome localized hypothetical proteins are shown in blue. Proteins with dual localization (mitosomes and ER) are shown in blue/green. As yet un-identified pore (translocase) in the inner membrane is shown in red. Positioning of these identified proteins on the model is based on <i>in silico</i> data (presence/ absence of (1) mitochondrial targeting sequence, (2) transmembrane domain), localization data and socio- affinity interaction of these proteins with their respective and other bait proteins.</p

Conditional expression of GlDRP-K43E elicits a mitosome morphogenesis phenotype.

<p>(A-C) Subcellular localization of a C-myc tagged <i>Gl</i>Tom40 (red) by IFA in cells induced to express a wild type <i>Gl</i>DRP (green) or GTP-locked variant <i>Gl</i>DRP-K43E (D-F). Note the altered size and distribution of organelles labeled with Tom40-myc in <i>Gl</i>DRP-K43E expressing lines. Nuclear DNA is stained with DAPI (blue). Insets: DIC images. Scale bar: 1 μM (G) -Cell fractionation experiments confirm fixed membrane localization of <i>Gl</i>DRP-K43E. (H) TEM: normal morphology of mitosomes (black arrows) in the CMC in cells expressing wild type <i>Gl</i>DRP whilst cells expressing <i>Gl</i>DRP-K43E show enlarged dumbbell-shaped mitosomes (black arrows in I, J) indicative of defective organelle division. Nu: nucleus. Scale bars: 100 nm.</p

Co-IP with HA_tagged GlTom40 yields numerous candidate interacting proteins.

<p>(A) Immunofluorescence microscopy: C-terminally HA-tagged <i>Gl</i>Tom40 (<i>Gl</i>Tom40-HA) is an exclusive marker for mitosomes (green). Nuclear DNA is stained with DAPI (blue). Inset: DIC image. (B) Venn diagram indicating 46 <i>Gl</i>Tom40 specific hits. (C) Parsing of 46 <i>Gl</i>Tom40-specific and 6 enriched proteins in metabolic categories based on available annotations in <a href="http://www.giardiaDB.org" target="_blank">www.giardiaDB.org</a>.</p

Subcellular localization of co-precipitated GlTom40 interaction partners.

<p>(A-N) Immunofluorescence microscopy: subcellular localization of C-terminally HA-tagged <i>Gl</i>Tom40 and 13 putative interaction partners (green) falls into 3 categories: Typical mitosome localization (A-E); dual localization to mitosomes and ER (F-I); no or ambiguous mitosome localization (J-N). Nuclear DNA is stained with DAPI (blue). Insets: DIC image. Scale bars: 1μm. (O) Partially validated <i>Gl</i>Tom40 interactome showing the bait protein (orange sphere), matrix proteins (purple), previously identified and mitosome-localized proteins (black), and mitosome-localized hypothetical proteins (blue). The stringency parameters used for detection (high, medium, and relaxed) are represented by bold, dashed, and dotted arrows, respectively.</p

Detected and validated mitosomal proteins in this report and in [33,49,72].

<p>Detected and validated mitosomal proteins in this report and in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006036#ppat.1006036.ref033" target="_blank">33</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006036#ppat.1006036.ref049" target="_blank">49</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006036#ppat.1006036.ref072" target="_blank">72</a>].</p

Selected MOMTiP proteins for validation by light microscopy.

<p>Selected MOMTiP proteins for validation by light microscopy.</p

G. lamblia mitosomes are immobilized and do not form dynamic networks.

<p>(A-B) Detection of typical organelle distribution of GFP-tagged <i>Gl</i>Tom40 (green) in time-lapse microscopy. (B) Overlay showing the CMC between the two nuclei and PMs which show a canonical dispersed localization throughout the cell. (C) Tracking of organelles during a period of 30 min shows no significant movement of mitosomes in the cytosol. (D-G) FRAP experiments performed on cells conditionally expressing GFP-tagged <i>Gl</i>Tom40 suggest that outer membrane proteins are not able to move amongst/in between organelles. (E) Photobleaching of a single mitosome (region of interest 1 (ROI 1)) in a living cell is shown. (F-G) Fluorescence in a bleached organelle (green line in the graph) does not recover even after several minutes (>20 min). Purple and brown lines in the graph represent fluorescence in unbleached areas (ROIs 2 and 3). (G) Fluorescence micrographs from the image series at the start (0 sec) of the experiment, during bleaching, and at the beginning of the recovery phase (20 sec). Arbitrary units of fluorescence are indicated [I]. Broken lines connect pre- and post-bleaching values in the graph. Scale bar: 1 μM.</p

Expansion and validation of the GlTom40 interactome by reverse co-IP with MOMTiP-1.

<p>(A) The “string” mitosome phenotype observed upon constitutive expression of GFP-tagged MOMTiP-1 (MOMTiP-1-GFP). Left panel: MOMTiP-1-GFP localized exclusively to mitosomes and in some cases virtually all of the peripheral organelles have been replaced by a single long tubular mitosome spanning both daughter cells length-wise. Middle panel: DIC image. Right panel: Overlay of the two channels. (B) Venn diagram showing MOMTiP-1-specific proteins identified after filtering the dataset. (C-F) Subcellular localization of selected C-terminally HA-tagged novel hypothetical proteins by IFA (green). Nuclear DNA is stained with DAPI (blue). Insets: DIC images. (G) Preliminary interactome of <i>Gl</i>Tom40 and MOMTiP-1 showing validated hits. Bait proteins (orange spheres), matrix proteins (purple), previously identified and localized proteins (black), and localized hypothetical proteins (blue). The stringency parameters used for detection (high, medium, and relaxed) are represented by bold, dashed, and dotted arrows, respectively. (H) Venn diagram showing the intersection of <i>Gl</i>Tom40 and MOMTiP-1datasets.</p