L., 2007). In both dPob4 and dPobeSatoh et al. eLife 2015;4:e06306. DOI: 10.7554/eLife.11 ofResearch Protease K Purity articleCell biologyFigure 9. Unfolded protein response (UPR) induced in dPob4 photoreceptor. (A) Projection image in the Z-series section with a 1 m interval of dPob4 mosaic retina expressing RFP (magenta) as a wild-type cell marker and Xbp1: GFP as a UPR sensor. The Xbp1:GFP signal (green) is enhanced by immunostaining making use of anti-GFP antibody. Asterisks show dPob4 homozygous photoreceptors. (B) Immunostaining of a dPob4 mosaic retina expressing RFP (magenta) as a wild-type cell marker. Phosphorylated Bifenthrin Epigenetic Reader Domain eukaryotic translation Initiation Element 2 is shown in green. Asterisks show dPob4 homozygous photoreceptors. DOI: ten.7554/eLife.06306.mutant mosaic retinas expressed Xbp1:GFP sensor in all R1-6 photoreceptors, and Xbp1:GFP fusion proteins were detected in the dPob mutant photoreceptors but not within the wild-type (Figure 9A and data not shown). Subsequent, we examined the level of eukaryotic translation Initiation Factor two (eIF2) phosphorylation due to the fact UPR is well known to induce eIF2 phosphorylation to attenuate protein translation on the ER membrane within a transduction pathway independent from IreI/Xbp1 (Ron and Walter, 2007; Cao and Kaufman, 2012). Anti-phospho-eIF2 signals have been stronger in each dPob4 and dPobe02662 photoreceptors than in wild-type photoreceptors (Figure 9B and data not shown). These results indicate that UPR is induced in the dPob-deficient photoreceptors, similar to EMC mutant.Rhabdomere development and degeneration in dPob null mutantBecause the synthesis of lots of membrane proteins was affected in dPob mutant cells, we observed the phenotype of dPob mutant throughout the developmental processes of photoreceptors. Regardless of the lack of many membrane proteins, ommatidial formation was not affected in dPob4 photoreceptors in mosaic retina; adherence junctions formed typically (Figure 6E) and the apical membrane was well differentiated into stalks and rhabdomeres (identified with Crb and phosphorylated moesin, respectively) (Figure 6B and data not shown) (Karagiosis and Prepared, 2004). The IRS was formed normally and rhabdomeres have been nonetheless separated by IRSs (Figure 8A ). We observed dPob4 mosaic retinas at 58 and 75 pupal development (pd) by electron microscopy (Figure 10A,B). The wild-type photoreceptors at 58 pd had already begun to amplify the rhabdomere membranes. The rhabdomeres had been shorter in dPob4 photoreceptors than in wild-Satoh et al. eLife 2015;4:e06306. DOI: 10.7554/eLife.12 ofResearch articleCell biologyFigure 10. Improvement and degeneration of dPob4 photoreceptor rhabdomeres. Electron microscopy of pupal and adult dPob4 mosaic retinas. Asterisks show dPob4 homozygous photoreceptors. Scale bar: 1 m. (A, B) dPob4 mosaic ommatidia from 58 pupal development (A) and 73 pupal development (B) under constant light (L) situation. (C ) dPob4 mosaic ommatidia from flies reared in full darkness (D) (C, E) or beneath 12 hr light/12 hr dark circumstances (D, F). Ommatidia from 3-day-old (C, D) and 17-day-old (E, F) flies. (D, inset) dPob4 R5 photoreceptor rhabdomere at larger magnification. DOI: 10.7554/eLife.06306.Satoh et al. eLife 2015;four:e06306. DOI: 10.7554/eLife.13 ofResearch articleCell biologytype photoreceptors, however the difference in their appearance was subtle at this stage. Until 75 pd, the microvilli of wild-type rhabdomeres had been 0.5 m extended and packed tightly. Even so, the microvilli of dPob4 rhabdomeres at 73 pd re.
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