Awd is required for Rab5 function. YFP-tagged constitutively active Rab5 mutant Q88L (Rab5CA) were expressed in (A) wild-type or (B)awd mutant clones, using the genetic combinations en2.4-Gal4e22c, UAS-flp/UAS-YFP-RAB5Q88L; +/FRT82B or en2.4-Gal4e22c, UAS-flp/UAS-YFP-RAB5Q88L; awdj2A4, FRT82B/FRT82B, respectively. Egg chambers were processed for staining for NICD (red) and YFP (green) as indicated. awd mutants were verified by lack of Awd staining (cyan in b’). (A) YFP-Rab5CA expressed in wild-type follicle cells. In Rab5CA-expressing wild-type follicle cells, NICD is reduced and is present in either Rab5-positive (insets 2 and 3) or Rab5-negative (likely late endosomes; insets 1 and 4). Note that NICD is in the lumen of these vesicles. (B) YFP-Rab5CA expressed in awd mutant follicle cells. In Rab5CA-expressing awd mutant cells, abundant NICD is present in enlarged vesicles that are mostly Rab5-positive. NICD is enriched on the surface of these vesicles (insets 1–3). (C) A stage 8 egg chamber from hs-flp/GbeSu(H)m8-lacZ; UAS-YFP-Rab5CA/+; tub-Gal4, FRT82B, tub-Gal80/FRT82B, awdj2A4 was stained for Hrs (cyan), YFP (green) and NICD (red). There is only partial co-localization of accumulated Notch with Hrs. (D) A stage 8 egg chamber from hs-flp/GbeSu(H)m8-lacZ; UAS-YFP-Rab5CA/+; tub-Gal4, FRT82B, tub-Gal80/FRT82B, awdj2A4 was stained for Awd (cyan), YFP (green) and Hnt (red). Expression of Rab5CA in Awd-negative cells (bracket) cannot rescue the loss of Hnt expression. Note that the Awd positive signal apical to the awd mutant clone is the expression sometimes detectable in the germ cell abutting the awd mutant clones. Bars are 10 μm. Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; NICD, Notch intracellular domain; YFP, yellow fluorescent protein.
Ignesti et al. BMC Biology 2014 12:12 doi:10.1186/1741-7007-12-12