Figure 6. Evidence that Egfr signaling is negatively regulated by endocytosis in the AS.

(A–D) zip FISH on embryos late in germband retraction. (A) Wild-type embryo showing zip expression in AS. (B) Expression of Ack in the AS using the Gal4^c381 driver causes an increase in zip levels in this tissue relative to wild-type. (C) Ack fails to elevate zip levels when co-expressed with Egfr–EGFP. (D) zip levels are elevated when Ack is co-expressed with control lacZ gene. (E–É ´´) AS in which Ack had been over-expressed in prd 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) Egfr-EGFP expressed in the AS using the Gal4^NP3312 driver shows vesicular accumulation in addition to being at the plasma membrane. (G) AS cells in embryo in which Apoliner has been expressed with LP1-Gal4 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 LP1-Gal4 driver showing punctate localization of Apoliner-RFP signal. Scale bars: 50 µm in A-D; 5 µm in E-H.