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Fig. 5. Evidence for a Lqf-dependent pathway of Dl endocytosis. (A) Clone of lqf- cells (marked by the absence of ß-Gal, green) in a wing disc overexpressing Myc-tagged Dl, stained for extracellular (red) and total (blue) Dl accumulation. Plane of focus is at the apical cell surface. Surface accumulation of Dl is not impaired by the absence of Lqf. (A') Same clone as in A. Four adjacent planes of focus located around 10-15 µm below the cell surface; elements in focus in each plane are shown, summed, by Auto-montage software (Syncroscopy). Punctate accumulation is detected only by staining for total Dl protein (blue); no accumulation is detected using the extracellular staining protocol (red), as expected. The punctate accumulation of Dl is not impaired by the absence of Lqf. (B) Clone of lqf- cells (marked by the absence of ß-Gal, green) in a wing disc overexpressing both Myc-tagged Dl and Neur, stained for Myc (red) and Hrs (blue). Plane of focus is at the apical cell surface. Surface depletion of Dl is impaired in the lqf- clone. (B') Same clone as in B, shown at a deeper plane of section (around 10 µm beneath the surface). Note that the accumulation of Dl in cytosolic puncta is not affected by the absence of Lqf. Although the number of Dl staining puncta appears similar to that seen in in A', where Neur was not overexpressed, only one focal plane has been sampled, as opposed to the four planes in A'. (B'') High magnification view of the boxed region in B', showing Hrs and Dl staining. Note that some Dl staining puncta co-stain with Hrs (arrows). (C) Clone of lqf- cells overexpressing Myc-tagged Dl (red) and Neur (marked by CD8-GFP, green) fail to induce Cut (blue), indicating that the absence of Lqf blocks the signaling activity of Dl, even when co-overexpressed with Neur. (C',C'') High magnification of the boxed region in C, showing Dl staining (red) at apical and sub-apical planes of focus. As in B', Dl accumulates at both the surface and in cytosolic puncta; nevertheless, signaling is blocked, owing to the absence of Lqf.