PI3K/Akt/Rheb signalling promotes shuttling of the PATs to endosomal compartments.
(A, C) CG1139-GFP expressed in the larval fat body using Lsp2-GAL4 is localised to both the plasma membrane (white arrow) and also to intracellular LELs throughout the cytoplasm (yellow arrow; A). Overexpression of Rheb leads to an increase in the relative proportion of intracellular, including perinuclear, protein compared to cell surface CG1139-GFP (C). (B, D) When CG1139-GFP is co-expressed with a dominant negative version of Shibire, ShiK44A, which blocks endocytosis, in either the presence (D) or absence (B) of Rheb, the transporter is mostly located at the plasma membrane (white arrow), strongly suggesting that PATs are normally shuttled to LELs from the cell surface. (E, F) The ratio of the GFP signal intensity in a 2.25 µm perinuclear region and a 2.25 µm region at the plasma membrane (see E) was measured for genotypes in A–D (grey bars; error bars = s.d. in F). Average cell size for each genotype is also shown in F (blue bars; error bars = s.d.). n = 25; *P<0.001 (increased) and ■ P<0.001 (decreased) relative to non-Rheb/non-ShiK44A-expressing control; ■ ■ P<0.001, decreased relative to Rheb-overexpressing control. Rheb-induced changes in intracellular PATs and PAT-induced growth are entirely dependent on endocytosis. (G, H) In the larval eye imaginal disc, CG1139-GFP, expressed under GMR-GAL4 control (G), is mostly located at or near the plasma membrane, for example at the surfaces of flattened non-photoreceptor cells that surround each ommatidium (white arrows). Co-overexpression of Rheb (H) results in much larger ommatidia, with reduced staining around the ommatidial border (white arrow) and more CG1139-GFP cytoplasmic expression, including intense intracellular punctae of staining (yellow arrows), consistent with Rheb promoting endocytosis of PATs in this tissue. An outline of an individual ommatidium in G and H is marked with a dashed line. Scale bar in A is 20 µm and applies to panels A–D, scale bar in G is 5 µm and applies to panels G and H.