Localization of dEsyt to the ER–PM contact sites requires calcium binding to dEsyt. (A) Schematic showing the site-directed mutations introduced into the nine conserved calcium-binding residues spread across the three C2 domains of dEsyt (Image generated using IBS, Illustrator for Biological sciences Version 1.0 software http://ibs.biocuckoo.org/) (Liu et al., 2015). (B) Confocal images showing the localization of exogenously expressed dEsyt::GFP and dEsyt^CaBM::GFP driven using eye-specific Rh1-Gal4 in 1-day-old dark-reared flies. Rh1-Gal4 is shown as a control. Transverse sections of an individual ommatidium are shown. Phalloidin marks F-actin staining and highlights rhabdomeres R1–R7 (Magenta), red indicates the localization of exogenously expressed dEsyt and dEsyt^CaBM protein tagged to GFP, and cyan represents RDGB localization at the base of the rhabdomere (marker for SMC). Scale bar: 2 µm. White arrows indicate the colocalization of GFP-tagged dEsyt and endogenous RDGB at the base of the rhabdomere. (C) Confocal images showing the localization of endogenous dEsyt (Cyan) in 1-day-old dark-reared flies of wild type and norpA^P24. dEsyt^KO is shown as a control. A single ommatidium is shown. Scale bar: 2 µm. Phalloidin marks F-actin staining (Magenta) and highlights rhabdomeres R1–R7.