Journal: PLoS Pathogens

Article Title: Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

doi: 10.1371/journal.ppat.1008317

Figure Lengend Snippet: (A) Membrane-supported lipid arrays spotted with gradients of different phosphorylated variants of phosphatidylinositol (PtdIns), from 100pmol (A) to 1.56pmol/spot (G), were probed with fixed amounts (2.5 μg) of clathrin assemblies-associated epitope-tagged PIP-binding domains from Gl PXD1-6, Gl NECAP1 and Gl FYVE, followed by immunodetection of the epitope tag. The protein fusion partner MBP (MBP alone) and for antibodies raised against PI(4,5)P 2 (anti-PI(4,5)P 2 ) were included as negative and positive controls for binding, respectively. No signal using arrays was obtained for MBP- Gl PXD4 and MBP- Gl PXD5 however, binding preferences for these fusions were determined using lipid strips . (B) Plots of densitometric analyses using FIJI for each MBP-fused PIP-binding domain and each spotted PI/PIP residue based on array data presented in (A). (C) Testing of the binding affinity of the MBP-fused PIP-binding domain from Gl NECAP1 on a wider range of lipid residues detects cardiolipin as the preferred substrate.

Article Snippet: Specific PIP residues were detected using anti-PI(3)P (1:100); Purified anti-PI(3)P IgG, Z-P003 Echelon Biosciencies), anti-PI(4,5)P 2 (1:100; Purified anti-PI(4,5)P 2 IgM, Z-P003 Echelon Biosciencies) and anti-PI(3,4,5)P 3 (1:100; Purified anti-PI(3,4,5)P 3 IgM, Z-P045 Echelon Biosciencies) followed by an anti-mouse antibody coupled to fluorochrome in all three cases (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 594, Cat. Nr. A-11005, Thermo Fischer Scientific or Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 488, Cat. Nr. A-11017, Thermo Fischer Scientific).

Techniques: Binding Assay, Immunodetection

Journal: PLoS Pathogens

Article Title: Phosphoinositide-binding proteins mark, shape and functionally modulate highly-diverged endocytic compartments in the parasitic protist Giardia lamblia

doi: 10.1371/journal.ppat.1008317

Figure Lengend Snippet: (A-D) Light microscopy-based immunofluorescence analysis of representative transgenic trophozoites expressing Legionella -derived PIP-binding constructs. (A-B) Compared to low 2xFYVE-GFP-expressing cells from the same population, reduction of PI(3)P binding sites in cells highly expressing a regulated encystation-dependent epitope-tagged construct 2xFYVE-GFP (GFP) inhibits uptake of fluid-phase marker Dextran-R. Scale bars: 1 μm. (C-D) Expression levels of PI(4)P-binding epitope-tagged construct GFP-P4C expression (GFP) have no visible impact on Dextran-R signal at PVs of transfected cells. Scale bars: 1 μm. (E) Dextran-R uptake in non-transgenic wild-type cells as negative controls for construct-induced uptake phenotypes. Scale bars: 1 μm (F) Box-plot representing the distribution of cell width (in μm) across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) increase in median cell width with respect to non-transgenic cells is detected for 2xFYVE-GFP- but not GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (G) Box-plot representing the distribution of measured Dextran-R signal intensity across at least 100 wild-type, 2xFYVE-GFP- and GFP-P4C- expressing cells selected in an unbiased fashion. A statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal intensity, normalized to wild-type cells (100%), is detected for 2xFYVE-GFP- but not for GFP-P4C- expressing cells. Asterisks indicate statistical significance. n.s.: not significant. (H) A statistically significant (p<0.5) linear correlation exists between Dextran-R signal ( x -axis, intensity_red channel [%]) and 2xFYVE-GFP expression ( y -axis, intensity_green channel [%]) measured across 100 cells. (I) The apparent linear correlation between GFP-P4C expression ( y -axis, intensity_green channel [%]) and Dextran-R signal ( x -axis, intensity_red channel [%]) is not statistically significant (p<0.5). (J) Wide-field microscopy-based immunofluorescence analysis of the impact of neomycin treatment on Dextran-R uptake to deplete PI(4,5)P 2 binding sites in non-transgenic wild-type cells. With respect to non-treated cells (WT; left panel), Dextran-R signal at PVs is visibly impacted in non-transgenic neomycin-treated cells (WT_Neo; right panel). Scale bars: 10 μm for full wide-field image, 1 μm for a single cell. (K) Box-plot representing the distribution of measured Dextran-R signal intensity across 100 wild-type cells, either untreated (WT) or treated with neomycin (WT_Neo). Neomycin treatment causes a statistically significant (two-sided t-test assuming unequal variances, p<0.05) decrease in Dextran-R signal. Scale bars: wide-field: 10 μm; single cells: 1 μm. For all images, nuclei are labelled with DAPI (blue). Insets: DIC images.

Article Snippet: Specific PIP residues were detected using anti-PI(3)P (1:100); Purified anti-PI(3)P IgG, Z-P003 Echelon Biosciencies), anti-PI(4,5)P 2 (1:100; Purified anti-PI(4,5)P 2 IgM, Z-P003 Echelon Biosciencies) and anti-PI(3,4,5)P 3 (1:100; Purified anti-PI(3,4,5)P 3 IgM, Z-P045 Echelon Biosciencies) followed by an anti-mouse antibody coupled to fluorochrome in all three cases (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 594, Cat. Nr. A-11005, Thermo Fischer Scientific or Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa flour 488, Cat. Nr. A-11017, Thermo Fischer Scientific).

Techniques: Light Microscopy, Immunofluorescence, Transgenic Assay, Expressing, Derivative Assay, Binding Assay, Construct, Marker, Transfection, Microscopy