A genetic screen to uncover mechanisms underlying lipid transfer protein function at membrane contact sites

(A) Cartoon depicting classes of VAP interactors used in the present genetic screen. Three classes of genetic interactors of rdgB are shown based on the likely molecular mechanism: loss of A, a direct physical interactor of VAP-A; loss of B, a direct interactor of VAP-A that also interacts with C, a protein required for RDGB function; and loss of C, a protein required for rdgB function but only interacts with VAP-A via B. Depletion of a specific VAP interactor is depicted with a dotted line. Fly homologs were filtered using DIOPT in FlyBase (http://flybase.org/). (B) Genetic scheme used to find either enhancers or suppressors of the retinal degeneration phenotype of rdgB⁹. (C) Pseudopupil imaging: (i) rdgB⁹ showed retinal degeneration by day 4 in dark when checked via deep pseudopupil imaging (depicted by *). (ii) The degeneration was partially suppressed when levels of G_αq were down-regulated in rdgB⁹ on day 4. (iii) Selected hits that showed suppression of retinal degeneration in rdgB⁹ on day 4 (scale bar 225 μm). (D) Table showing the full list of genes used in the screen and the number of suppressor genes identified. (E) Positive hits (suppressor genes) are divided into different categories depending on their cellular functions. n = 5 flies/RNAi line.

(A) Scheme used to test for genetic interaction of each of the 52 su(rdgB) with norpA^p24 under illumination conditions (constant light 2000 Lux). (B) norpA^p24 flies degenerate by day 3 under light conditions, and examples of su(RDGB) candidates that suppressed the norpA^p24 retinal degeneration phenotype. n = 5 flies/RNAi line. (C) Complete list of 13 genes with their cellular functions that suppressed the norpA^p24 phenotype. ERG screen. (D) Of 52 candidates, five su(RDGB) showed reduced (CG9205, Yeti, Apc7, Set, and dCert) and one (CG3071) showed higher ERG phenotype (traces and quantification shown) when down-regulated in an otherwise WT background. The number of flies used for the experimental set is mentioned along with the quantification. Scatter plots with the mean ± SEM are shown. Statistical tests: unpaired t test.

(A) Suppression of retinal degeneration when dCert RNAi line (35579/TRiP, BDRC) was expressed using Rh1 promoter. After eclosion, flies were kept in the dark and assayed on either day 1 or 3: (i) on day 1, there was no appreciable difference in two genotypes and rhabdomeres were intact; and (ii) on day 3, down-regulation of dCert in rdgB⁹ suppressed the retinal degeneration observed in rdgB⁹ control. (B) When subjected to ERG analysis, down-regulation of dCert using Rh1-GAL4 in the background of rdgB⁹ did not suppress the ERG phenotype: (i) ERG trace and (ii) quantification. n = 6 flies. Scatter plots with the mean ± SEM are shown. Statistical tests: unpaired t test. (C) Double mutant of rdgB⁹;dcert¹ showed enhancement of retinal degeneration: (i, ii) by day 1 alone, double mutant has severely enhanced retinal degeneration phenotype when compared to rdgB⁹. (D) Enhancement of retinal degeneration when dCert (35579/TRiP, BDRC) was down-regulated with a whole-body Actin-Gal4 promoter in the rdgB⁹ background: (i) on day 1, rhabdomere loss is significant in the experimental files compared with control that worsens by day 3 and phenocopies the retinal degeneration present in the double mutant. For optical neutralization experiments, scoring was done by quantifying 10 ommatidia/fly head, n = 5 fly heads.

(A) Suppression of retinal degeneration when CG9205 RNAi line (29079/GD, VDRC) was expressed using Rh1 promoter. After eclosion, flies were kept in the dark and assayed on either day 1 or 3: (i) on day 1, there was no appreciable difference in two genotypes and rhabdomeres were intact; and (ii) on day 3, down-regulation of CG9205 in rdgB⁹ suppressed the retinal degeneration observed in rdgB⁹ control. (B) When subjected to ERG analysis, down-regulation of CG9205 using Rh1-GAL4 in the background of rdgB⁹ did not suppress the ERG phenotype, whereas down-regulation of CG9205 using Rh1-GAL4 in an otherwise WT background shows reduced ERG amplitude: (i) ERG trace and (ii) quantification. n = 8 flies. scatter plots with the mean ± SEM are shown. Statistical tests: unpaired t test. (C) Double mutant of rdgB⁹;CG9205^KO showed enhancement of retinal degeneration: (i, ii) by day 1 alone, double mutant has severely enhanced retinal degeneration phenotype when compared to rdgB⁹. (D) Enhancement of retinal degeneration when CG9205 (29079/GD, VDRC) was down-regulated with a whole-body Actin-Gal4 promoter in the rdgB⁹ background: (i) on day 1, rhabdomere loss is significant in the experimental files compared with control that remains the same on day 3 and phenocopies the retinal degeneration present in the double mutant. For optical neutralization experiments, scoring was done by quantifying 10 ommatidia/fly head, n = 5 fly heads.