Decreasing Rh1 levels suppresses PR degeneration in <i>Vps26</i> mutants.

<p>(A) Decreasing endocytosis with a temperature-sensitive dynamin mutation (<i>shi^ts</i>) suppresses ERG defects associated with the loss of Vps26. Animals were grown at 18°C in the dark. Newly eclosed flies were selected and kept in the dark for an additional 12 h at 24°C to reduce dynamin function in <i>shi^ts1</i> or <i>Vps26¹,shi^ts1</i>. Subsequently, flies are shifted to LD or kept in DD at 24°C. ERG is performed at the end of the fourth LD cycle. <i>shi^ts1</i> flies exhibit a normal ERG response. ERG defects in <i>Vps26¹</i> mutant PRs are suppressed when dynamin function is partially reduced, as the <i>Vps26¹,shi^ts1</i> double mutants show a stronger depolarization than that in <i>Vps26¹</i>. (B) Quantification of ERG amplitude of the flies tested in (A). Ten flies were measured for each genotype after completing the fourth light cycle. ERG amplitudes are normalized to control (iso). Student's <i>t</i> test; error bars represent SEM; * <i>p</i><0.05; ** <i>p</i><0.01; ns, no significance. (C) Reducing Rh1 suppresses ERG defects in <i>Vps26¹</i> mutants. Heterozygous <i>Rh1¹⁷</i> mutants exhibit a normal ERG response. After 4 d in LD, loss of one copy of the <i>Rh1</i> gene strongly suppresses the reduction of ERG amplitude in <i>Vps26¹</i> mutants. (D) Quantification of ERG amplitude of the flies tested in (C). Ten flies were recorded for each genotype at day 4. ERG amplitudes are normalized to control. Student's <i>t</i> test; error bars represent SEM; * <i>p</i><0.05; ** <i>p</i><0.01; ns, no significance. (E) Reducing dietary vitamin A (vitA) strongly suppresses PR degeneration in <i>Vps26¹</i> mutants. Decreasing vitA lowers the levels of retinal, which is required for Rh1 maturation <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001847#pbio.1001847-Ozaki1" target="_blank">[39]</a>. Loss of retinal therefore leads to loss of Rh1. Bright field images show that PR degeneration is strongly suppressed in <i>Vps26¹</i> mutants raised on the low vitA–containing food when flies are kept in LD for 10 d. As vitA is a key component for Rh1 maturation and Rh1 is a key structural component of rhabdomeres, the rhabdomere size is much reduced when the flies were raised on low vitA food. Regular, regular fly food; low vitA, low vitamin A fly food. Scale bar, 2 µm. (F) Quantification of intact rhabdomere numbers in flies described in (E). Thirty ommatidia from three animals are examined for each genotype. Student's <i>t</i> test; error bars represent SEM. ** <i>p</i><0.01; ns, no significance. (G) Quantification of ERG amplitudes of control and <i>Vps26¹</i> raised on regular or low vitA fly food in LD after 10 d. Ten ERG traces were measured for corresponding genotypes and conditions. The amplitudes are normalized to control flies that are raised on regular food. Student's <i>t</i> test; error bars represent SEM; ** <i>p</i><0.01; ns, no significance.