Additional file 1: of Multiple paralogues of α-SNAP in Giardia lamblia exhibit independent subcellular localization and redistribution during encystation and stress

Figure S1. Circular dichroism spectrum of giardial SNAPs. Far-UV circular dichroism spectra of the three giardial SNAPs, in 20 mM sodium phosphate buffer at 16°C. Predicted helicity percentage, calculated by using the BeStSel server (bestsel.elte.hu/), is indicated in each spectrum. Percent helicity of Sec17, calculated on the basis of the crystal structure of Sec17 (1QQE), is 73%. Figure S2. Expression of the putative SNAPs during the life cycle of Giardia. The expression of the three putative SNAP orthologues of Giardia were determined by reverse transcriptase PCR, using the cDNA prepared from trophozoites, encysting cells and cysts. The primers used for this analysis are given in Additional file 2: Table S4. PCR products were visualized on 1.2% agarose gel. The length of all the PCR products for each SNAP correspond to the expected size. Figure S3. Specificities of the antibodies for giardial α-SNAPs and NSF. a Western blot with trophozoite extract and each of the three purified giardial α-SNAPs. Expression and purification of the proteins have been described in the Experimental Procedures section. The 6xHis-tag was removed from all three proteins prior to western blotting. The upper blot was incubated with anti-α-SNAP17224 antibody while the middle and lower blots were incubated with anti-α-SNAP16521 and anti-α-SNAP10856 antibodies, respectively. All antibodies were diluted 1000× prior to use. The presence of a ~34 kDa band in both the giardial extract and the corresponding overexpressed protein fraction in each blot indicates the specificity of that particular antibody. b Western blot with anti-GlNSF antibody using extracts of Giardia trophozoites and E. coli overexpressing GlNSF. Instead of the full-length protein, a stretch of 200-amino acids from the N-terminal segment of GlNSF was tagged with the 6xHis tag and expressed in E. coli. The expected size of this overexpressed protein is 39 kDa, while the size of the full-length GlNSF is 91 kDa. Figure S4. Localization of α-SNAP10856 to peripheral vesicles. a Colocalization of α-SNAP10856 with Lucifer yellow, a fluorescent dye that specifically stains acidic compartments, and, b a giardial PX domain-containing protein encoded by ORF GL50803_16548 and known to localize to the peripheral vesicles indicates that α-SNAP10856 localizes to the peripheral vesicles of trophozoites. The scattergram in each row indicates the analysis of colocalization between the two fluorophores, over the entire z-stack, by considering all the pixels within the whole area occupied by that cell. The values for Pearson correlation coefficient (Pr) and overlap coefficient (OC) are indicated within the respective scattergrams. The intensity plot at the right in the lower panel indicates changes in fluorescence intensity of the red and green signals across the dotted white line drawn across the ROI. Scale-bars: a, b, 5 μm. Figure S5. Immunolocalization of α-SNAP17224 and α-SNAP16521 to various subcellular structures. a Localization of α-SNAP17224 at the perinuclear region persisted after 30 h of encystation. The caret marks the perinuclear region. b α-SNAP16521 is located both at the peripheral vesicles (PV) and the paraflagellar dense rods (PDRs) of different flagella at 1.5 h post induction of encystation. However, at 4 h post induction, α-SNAP16521 is exclusively present at the PDRs, with no signal at the PVs. The triangle and arrow heads indicate PVs and the PDRs of different flagella, respectively. c Colocalization of α-tubulin (red) with α-SNAP16521 (green) in 16 h encysting trophozoites. The red and green signals are juxtaposed to each other at the anterior flagella, without any significant overlap. Cells were visualized with Alexa Fluor 488 and Cy5-conjugated secondary antibodies that bind to rabbit anti-α-SNAP16521 and mouse anti α-tubulin, respectively. d Two individual z-sections showing localization to the ventral disc periphery (left panel) and the PDRs (right panel). Scale-bars: a, b, c, d, 5 μm. Figure S6. Colocalization of α-SNAP10856 and a PX-domain containing protein in cysts. Both α-SNAP10856 and a PX-domain containing protein (GL50803_16548), a known lipid-interactor that localizes to vesicles, colocalize to cytoplasmic puncta in cysts. Carets mark some of the regions where there is considerable overlap of the red and green signals. The lower panels show regions of colocalization across the entire z-stack, with the left panel showing colocalization of the green signal (α-SNAP10856) on red (GL50803_16548) and the right panel shows the opposite, i.e. red on green. The intensity plot at the extreme right indicates changes in fluorescence intensity of the red and green signals across the dotted white line in the ROI. The values for Pearson (Pr) and overlap (OC) coefficient are given in the scatter plot. Scale-bar: 5 μm. Figure S7. Localization of GlNSF and α-SNAP16521. Localization of GlNSF (green) at the brush border (BB) and α-SNAP16521 (red) at the paraflagellar dense rods (PDRs) in 48 h encysting trophozoites. Each of the two fluorescent images has been individually merged with the DIC image to show the distribution of the fluorescent signals w.r.t. the flagella. Scale-bar: 5 μm. Figure S8. Detection of intracellular reactive oxygen species (ROS) by H2DCFDA. Giardia trophozoites were treated with either 150 μM H2O2 or 1μg/ml metronidazole for 1 h and intracellular ROS generation was monitored using H2DCFDA. The top row shows untreated cells with no fluorescence signal of DCF. Green fluorescence signal was observed in cells exposed to oxidative stress, with either H2O2 (middle row) or metronidazole (bottom row) due to the formation of reactive oxygen species. Scale-bar: 5 μm. (PPTX 3310 kb