Data Availability StatementAll data are contained in the paper or the associated supplemental materials. proteins, that the exocyst complex and the microtubule-based motor proteins dynein and kinesin promote the secretion of Eys and Rh1, and that Syntaxin 7/Avalanche controls the endocytosis of Rh1, Eys, and Crb. photoreceptor cells (PRCs) are an important model for the epithelial differentiation of a sensory cell and to study vesicle trafficking and neuro-degeneration (for reviews see Tepass and Harris 2007; Shieh 2011; Xiong and Bellen 2013; Schopf and Huber 2017). PRCs have specialized apical and basolateral membranes that are segregated by an epithelial adherens junction, the zonula adherens. While the basolateral membrane extends an axon, the apical membrane differentiates a light sensing organelle, the rhabdomere. In addition to the rhabdomere, the apical membrane of PRCs contains the stalk membrane domain that connects the rhabdomere to the zonula adherens. Here, we have identified factors that contribute to the trafficking of three proteins – Rhodopsin 1 (Rh1), Crumbs (Crb), and Eyes shut (Eys) to the apical membrane of PRCs to further our understanding of how the vesicle trafficking machinery contributes to the maintenance and function of a complex epithelial sensory cell. Rhodopsin photopigments Rabbit Polyclonal to CDH23 GLPG0974 are seven-pass mutant PRCs show light-induced degeneration (Johnson PRCs (see Figure 1). Examples include Rab1 and Syntaxin 5 (Syx5) that are essential in ER to Golgi trafficking (Satoh PRCs. (A) Schematic of PRC showing major known trafficking pathways. Arrows highlight basolateral (gray) and apical (red, Crb; blue, Eys; green, Rh1) trafficking pathways. Abbreviations: EN, endosome; ER, endoplasmatic reticulum; GO, Golgi apparatus; IRS, interrhabdomeral space; LY, lysosome; RB, rhabdomere; SM, stalk membrane; TW, terminal web; ZA, zonula adherens. (B) Schematic of PRC showing site of action of major known vesicle trafficking factors. See text for description. It appears that the apical and basolateral trafficking routes diverge somewhere along the Golgi prior to the action of Rab6, whereas the rhabdomeral stalk membrane route diverges downstream of Rab6 following the exit from the Golgi. Crb and Eys are thought to be targeted GLPG0974 to the stalk through a pathway distinct from the secretory pathway GLPG0974 used by rhabdomeral proteins (Beronja Stock Center (BDSC) and Vienna Resource Center (VDRC). See Table S1 for a list of lines used. UAS-RNAi constructs were expressed in the developing eye with UAS-Dicer-2 GMR-Gal4. GMR-Gal4 activity starts in the developing retina posterior to the morphogenetic furrow at third larval instar and continues to adulthood (Freeman 1996). UAS-Dicer-2 was used to amplify the effects of RNAi (Ketting stocks are available from public repositories. All other reagents are commercially available or can be sent upon request. Supplemental material available at figshare: https://doi.org/10.25387/g3.10010408. Results and Discussion Identification of genes involved in PRC vesicle trafficking PRCs are organized as elongated cylinders surrounding a lumen, the IRS, bound by PRC apical membranes (Figure 2A). PRCs deficient in Rh1, Eys, or Crb have prominent developmental defects. Rh1 is essential for rhabdomeral maintenance (Kumar and Ready 1995) and disruption of factors, such as the exocyt component Sec6 that affects the exocytosis of Rh1 and other rhabdomeral proteins show rhabdomeral deterioration (deficient PRCs appear enlarged and rectangular in cross-sections as a consequence of a distal to proximal extension defect (Pellikka mutant ommatidia lack an IRS (Husain PRCs. GMR-GAL4 was used to drive the expression of UAS-dicer-2 and UAS-RNAi constructs. UAS-dicer-2/+; pGMR-Gal4/+ was used as control. Scale bars, GLPG0974 5 m. (A) Schematic of cross-section of.