OSI21 partially localizes on the endo-lysosomal system.

Newly eclosed flies were exposed to bright light (2900 lux) for 90 min. A single ommatidium prepared from fly retina was visualized by confocal microscopy. (A–B) The OSI21 protein was marked with GFP. Each subcellular structure was marked with Lysotracker -Red (lysosomes, A) and Rhodopsin-RFP (endosomes, B). OSI21 partially localizes on lysosomes (Pearson's correlation coefficient: 0.617, A). OSI21 partially localizes on the rhodopsin positive vesicles (Pearson's correlation coefficient: 0.635, B). (A) w; Rhi1::Gal4, UAS::Osi21-GFP/+ (B) w; Rhi1::Gal4, UAS::Osi21-GFP/+; UAS::Rh1-RFP/+. (arrowheads) lysosomes or endosomes colocalized with OSI21-GFP. Flies were kept in the 25°C fly culture room, 18 h light/8 h dark cycles. (C–D) Localization of endocytosed rhodopsin with lysosomes. Newly eclosed flies were exposed to bright light (2900 lux) for 90 min. A single ommatidium was prepared from fly retina and examined using confocal microscopy. GFP-labeled rhodopsin and Lysotracker -red were used for visualizing rhodopsin endocytosis and lysosomes. Lysosomes (red) in norpA^p24 photoreceptor cells are smaller in number and are not overlapped with endocytosed rhodopsin (green) (Pearson's correlation coefficient: 0.342, C). (Arrowhead in C) Endocytosed rhodopsin escaped Osi21 blockage, reflecting regular lysosomal rhodopsin-turnover. On the other hand, lysosomes are greatly proliferated and are colocalized with endocytosed rhodopsin in the norpA^p24 mutant photoreceptor with a Osi21-RNAi transgene (Pearson's correlation coefficient: 0.604, D). (Arrowheads) Lysosomes collocalized with endocytosed rhodopsin. (C) norpA^p24; Rh1::Gal4, UAS::Rh1-GFP/+ (D) norpA^p24; Rh1::Gal4, UAS::Rh1-GFP/UAS::Osi21-RNAi. (E) Relative number of green-labeled vesicles among red-labeled population was calculated from three representative photoreceptor cells of each genotype in triplicate. Error bars indicate SEM. ***p<0.01, **p<0.05.