Fig. 7.

Endosomal acidification is necessary for Wg–DFz2 interaction and signaling. (A–C) Confocal images of wing discs, surface labeled on ice with A568 anti-Wg and A647 anti-DFz2 (A), or surface labeled and pulsed for 3–5 min followed by an 8-min chase either without (B) or with Baf (C) followed by acid wash to strip off the surface-bound antibodies and visualize endosomes (as detailed in Fig. 1A, step 3). Images were obtained from subapical and medial planes where both Wg and DFz2 are found. The Insets (magnified ∼2–3×) show the region taken for FRET measurements by donor (A568) dequenching after acceptor photobleaching method. Within the outlined boxes, the acceptor (A647 anti-DFz2) was bleached, and both donor and acceptor intensities, pre- and postbleaching is shown in the panels below. See LUT bar in E to compare differences in donor intensities. Endosomes with both Wg and DFz2 (described in Fig. 1 E and F and step 4 of Fig. 1A) were only used for the assay. (D) Graph shows FRET efficiencies obtained from outlined images, as in A–C, and calculated as described in SI Materials and Methods. Note FRET efficiency is much higher in endosomes compared with that at the cell surface (P < 10⁻¹²), and decreases upon neutralization of the pH of endosomes with Baf (P < 10⁻¹⁰). Control used here are punctae labeled with only the donor fluorophore. (E and F) Confocal images (E) of surface-labeled wing discs maintained on ice for 2 h at pH 6.0, and corresponding graph (F) shows FRET efficiencies between Wg and DFz2 on surface at pH 6.0 or 7.2. The data indicate that the efficiency is enhanced upon incubation in acidic environment (P < 10⁻¹⁵). Data represented is average (±SEM) of FRET efficiencies taken from 60 to 100 structures (endosomes or surface clusters) from four to five discs from two separate experiments. (G–I) Confocal images of wing discs depicting the distribution of Wg signal transduction cascade downstream player, Dsh-GFP (expressed under its native promoter), upon incubating wing discs at different pH on ice. Z projection of subapical planes is shown in G and graphs show the distribution (H) and the average intensities from planes enriched in the Dsh-GFP spots (I) in response to the incubation at different pH. Significantly more Dsh-GFP is recruited to enriched clusters at lower pH (P < 10⁻³), and their distribution is uniform at pH 6.3 (D, dorsal; V, ventral). All images are background-subtracted and intensities are appropriately scaled for representation. (Scale bars, 10 μm in A–C and E and 50 μm in G.)