FLA3 is an essential protein in the bloodstream form o<i>f T. brucei.</i>

<p>Panel A. Northern blot analysis of 15 µg of total RNA from a cloned FLA3 bloodstream RNAi cell line grown under induced or noninduced conditions for 24 h. The probe used for hybridisation was the same fragment employed for RNAi. The lower part of the panel presents the ethidium bromide staining of the RNA as a control for loading. The arrow indicates the presence of the dsRNA (∼ 600 bp) in the induced cells. Panel B. An immunoblot analysis confirmed loss of the FLA3 protein in the induced cells after 30 h induction. Each lane contained 5×10⁶ cells and a loading control was performed with antibodies against trypanosome actin. Panel C. The effect of knock down of FLA3 on the growth of a representative cloned FLA3 RNAi cell line cultured in the presence (○) or absence of tetracycline (•). Panel D. Analysis of the number of nuclei and kinetoplasts per cell in a FLA3 RNAi cell line cultured in the presence of tetracycline. For each time point individual cells were assessed for the presence of kinetoplast and nucleus and scored as 1N1K, 1N2K, 2N2K and cells with clearly more than 2N. The results were expressed as the mean ± SD of four separate surveys of 50 cells.</p

Knockdown of FLA3 leads to flagellar detachment in the bloodstream form of <i>T. brucei.</i>

<p>Panel A. FLA3 RNAi cells were grown in the presence of tetracycline. Samples were removed, fixed and processed for phase contrast and fluorescence microscopy to assess flagellar attachment (Phase) and DNA content (DAPI) of the cells. Bar = 10 µm. Panel B. At each time point the number of cells with detached flagella was assessed as a % of the total number of cells in the population. The results were expressed as the mean ± SD of four separate surveys of 50 cells.</p

The effect of the loss of FLA3 on the flagellar attachment zone.

<p>Panel A. Detail of the flagellar attachment zone in noninduced FLA3 RNAi cells showing the regularly spaced FAZ filament structure (arrow). Panel B. Detail of the flagellar attachment zone in an induced FLA3 RNAi cells at the point where the flagellum became detached reveals the loss of the regularly spaced FAZ filament structure (arrows). The panel on the left presents a close up of the region where detachment occurred. Bar = 500 nm.</p

Co-localization of FLA3 with markers for the FAZ and flagellum.

<p>Cells were probed using affinity purified α-FLA3 rabbit antibodies and a mouse monoclonal antibody against FAZ1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052846#pone.0052846-Vaughan2" target="_blank">[19]</a> or PFR2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052846#pone.0052846-Kohl4" target="_blank">[18]</a>. The cells were examined using an Olympus Fluoview 1000 confocal microscope. The panels are presented as merges of the phase and the individual immunofluorescence images plus a combined antibody fluorescence merged image. Bar = 10 µm. Panel A. The location of FLA3 (green), FAZ1 (magenta) and the nucleus/kinetoplast stained with DAPI (blue). Panel B. The location of FLA3 (green), PFR2 (magenta) and the nucleus/kinetoplast (blue). PFR2 but not FLA3 is associated with the flagellum and the free flagellum at the anterior end of the cell (black arrow).</p

Localization of FLA3 in the bloodstream form of <i>T. brucei</i>.

<p>Trypanosomes were purified from infected blood, fixed and processed for immunofluorescence using the affinity purified α-FLA3 antibodies. Upper panels present a merge of the fluorescence and phase images and reveal the location of FLA3 (green) and the nucleus/kinetoplast (blue); lower panels present the fluorescence image alone. Bar = 10 µm. Panel A. FLA3 was detected as a punctate pattern of staining along the FAZ. The black arrows in the upper panel (α-FLA3) indicate the absence of FLA3 along the free flagellum. Non specific binding was investigated by including the peptide antigen (10 µg.ml⁻¹) (α-FLA3+ pep). Panel B. FLA3 is associated with existing and new (arrows) FAZ. Panel C. FLA3 remains associated with the cell body at regions where the flagellum has become detached (black arrows in the phase/fluorescence merge). The white arrows indicate more pronounced staining of FLA3 frequently observed at the posterior and anterior end of the attachment zone (lower panels).</p

TL blotting on Tf and glycophorin.

<p>Different amounts of proteins (up to 5 μg) were loaded. The lectin blot analysis indicates that TL does not recognize Tf but reacts with the sialoglycoprotein glycophorin.</p

Inhibition of uptake of Tf by TL in epimastigote forms of <i>T</i>. <i>cruzi</i>.

<p>Trypanosomes preincubated with biotinylated TL in the presence of 20 μM FMK-024 (25 μg/ml) and in the absence (A, left panel) or presence of competing chitin hydrolysate (A, right panel), were then incubated with Tf Alexa-594 for 5 or 30 min at 27°C. Cells were then fixed and treated for fluorescence microscopy. Similar incubations wherein TL was substituted by GSLII (B) were performed to assess the specificity of the TL labeling. Furthermore, live parasites preincubated with DyLight 488-TL and 20 μM protease inhibitor (FMK-024) for 5 min and then incubated for 60 min in the presence of Alexa Fluor 594 conjugated Tf showed a lectin labeling in the cytostome/cytopharynx (arrowhead), while no Tf labeling (red signal) was observed in these conditions (C, upper panel). In presence of a molar excess of chitin hydrolysate an intense labeling of Tf exclusively concentrate into reservosomes (arrow) while no green signal corresponding to TL was observed anymore (C, lower panel). Inhibition of trypanosomes Tf uptake with TL was furthermore quantified by flow cytometry (D). The TL signal was dropping from 913 to 273 of mfi in the absence or presence of chitin hydrolysate, respectively (D, left histogram). Conversely, Tf signal was increasing from 597 to 3793 of mfi in the absence or presence of chitin hydrolysate, respectively (D, right histogram).</p

Localization of TL and GSLII binding sites in <i>T</i>. <i>cruzi</i>.

<p>Endocytosis kinetics of fluorescent Alexa Fluor 594 conjugated Tf was performed in order to follow <i>T</i>. <i>cruzi</i> endocytic pathway from the flagellar pocket/cytostome to the reservosomes. Parasites were fixed at different time points and probed with biotinylated TL (A), biotinylated ricin (B) or Alexa 488 conjugated GSLII (C). The addition of chitin hydrolysate clearly shows inhibition of TL and GSLII staining. (A) Co-localization of biotinylated-TL (green) and Tf (red). (B) Co-localization of biotinylated-ricin (green) and Tf (red). Addition of 200 mM galactose abolished the ricin staining. (C) Co-localization of Alexa 488 conjugated GSLII (green) and Tf (red). (D) Co-localization of Alexa 488 conjugated GSLII (green) and TcJ6 (red). (E) Co-localization of Alexa 488 conjugated GSLII (green) and anti-BiP (red). (F) GSLII blotting of cell extracts enriched by GSLII chromatography. GSLII blots of <i>T</i>. <i>cruzi</i> CHAPS- and Triton-soluble (CHAPS+Triton X-114) cell lysate fractions were enriched by GSLII chromatography and then treated (+) or not (-) with PNGase F. Blots were probed with biotinylated-GSLII. The GSLII blot indicates the presence of <i>N-</i>acetylglucosamine modification in both soluble and membrane fractions. Treatment of the fractions with PNGase F decreased the reactivity of GSLII confirming <i>N</i>-glycoprotein type modification.</p

Uptake of Dextran in the presence of TL in epimastigote forms of <i>T</i>. <i>cruzi</i>.

<p>Flow cytometry profiles of uptake of Dextran Alexa-647 by trypanosomes in the presence or absence of biotinylated TL. Trypanosomes preincubated (A) or not (B) with biotinylated TL in the presence of 20 μM FMK-024 (25 μg/ml) and in absence (A, left histogram) or presence of competing chitin hydrolysate (A, right histogram), were then incubated with Dextran Alexa-647 for 30 min at 27°C.</p

Subcellular localization of TL-binding sites in <i>T cruzi</i> by transmission electron microscopy (TEM).

<p>Parasites were incubated for 5 min in PSG medium in presence (F) or absence (A-E) of BSA-gold as endocytic tracer (10 nm). Cells were fixed and processed for ultrathin frozen sectioning (Tokayasu method, [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163302#pone.0163302.ref042" target="_blank">42</a>]). Cryosections were sequentially probed with biotinylated TL, rabbit anti-biotin antibodies, protein A-gold (5 nm) and finally mounted in methyl cellulose-uranyl acetate films. Representative images are shown. K: kinetoplast, M: mitochondrion, R: reservosome, N: nucleus, FP: flagellar pocket, F: flagellum, G: golgi, Cy: cytostome. Arrows and arrowhead, point to gold particles that mark the presence of TL binding sites and BSA-gold particles, respectively. Asterisk show TL-binding matrix near the opening of the cytostome. Bars = 200 nm.</p