Evidence that Egfr signaling is negatively regulated by endocytosis in the AS.

<p>(A–D) <i>zip</i> FISH on embryos late in germband retraction. (A) Wild-type embryo showing <i>zip</i> expression in AS. (B) Expression of Ack in the AS using the <i>Gal4^c381</i> driver causes an increase in <i>zip</i> levels in this tissue relative to wild-type. (C) Ack fails to elevate <i>zip</i> levels when co-expressed with Egfr–EGFP. (D) <i>zip</i> levels are elevated when Ack is co-expressed with control <i>lacZ</i> gene. (E–É ´´) AS in which Ack had been over-expressed in <i>prd</i> stripes, triple-stained with anti-phosphotyrosine (anti-PY) (E), anti-Egfr (É) and anti-Rab5 (É ´). (E) Cells over-expressing Ack are marked by high levels of anti-PY (outlined with dotted lines). (É) Egfr shows strong cortical localization in wild-type AS cells but a more cytoplasmic distribution in Ack-over-expressing cells. (É ´) There is an increase in Rab5-positive early endosomes in Ack-over-expressing cells. (É ´´) Merge of panels É and É ´. Arrowheads and arrows mark Egfr-positive early endosomes in wild-type cells and Ack-over-expressing cells, respectively. (F) <i>Egfr-EGFP</i> expressed in the AS using the <i>Gal4^NP3312</i> driver shows vesicular accumulation in addition to being at the plasma membrane. (G) AS cells in embryo in which Apoliner has been expressed with <i>LP1-Gal4</i> driver showing localization of Apoliner-RFP signal to membranes. (H) AS cells in embryo in which Apoliner and kinase-dead Ack have been co-expressed with <i>LP1-Gal4</i> driver showing punctate localization of Apoliner-RFP signal. Scale bars: 50 µm in A-D; 5 µm in E-H.