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First published online 8 March 2005
doi: 10.1242/jcs.01712

Journal of Cell Science 118, 1373-1384 (2005)
Published by The Company of Biologists 2005
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Functional INAD complexes are required to mediate degeneration in photoreceptors of the Drosophila rdgA mutant

Plamen Georgiev1, Isaac Garcia-Murillas1, Danny Ulahannan2, Roger C. Hardie2 and Padinjat Raghu1,*

1 The Inositide Laboratory, Babraham Institute, Babraham Research Campus, Cambridge, CB2 4AT, UK
2 Department of Anatomy, University of Cambridge, Downing St, Cambridge, CB2 3DY, UK



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  		Fig. 1. Assessment of retinal degeneration in live flies using the FDPP. Images of w,rdgA1, P[rh1-Rh1EGFP] and w,rdgA1, P[rh1-Rh1EGFP];trp343 viewed on a stereomicroscope and recorded digitally. Rescue of retinal degeneration by trp343 results in a crisp pseudopupil in which the rhabdomeres of photoreceptors R1-R6 can be distinctly seen and counted.

 


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  		Fig. 2. Genetic scheme showing crosses that were used in the screen to isolate suppressers of rdgA using the whole-eye mosaic strategy. The stocks shown were generated from flies obtained.

 


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  		Fig. 3. (A) Visualization of the fluorescent deep pseudopupil (FDPP) in live flies. In the starting stock used for the screen (w,rdgA1, P[rh1-Rh1EGFP];parent), no FDPP can be seen. A distinct FDPP can be seen in the three suppressers isolated [rdgA1;su(1), rdgA1;su(40) and rdgA1;su(100)]. (B) TEM image of a single ommatidium from wild-type and rdgA1 mutants compared with those from rdgA1;su(1) and rdgA1;su(40) confirming the rescue of retinal degeneration as assessed by the FDPP. Similar results were obtained with su(100) (data not shown).

 


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  		Fig. 4. Rescue of rhabdomere degeneration in w;Trp365. In comparison to wild-type flies, w;Trp365 homozygous flies show severe rhabdomere degeneration (A). This can be rescued by su(40);Trp365 (C) but not in su(100);Trp365 (B) double mutants. The effect of su(1) on Trp365 was equivalent to that of su(100) with minimal, if any, rescue of rhabdomere structure (D). Arrows indicate rhabdomeres.

 


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  		Fig. 5. (A) Cytogenetic representation of the 59B region showing the three overlapping deficiencies relevant to this study. The extent of the deficiencies (coloured boxes) and their breakpoints have been taken from curated data on the Drosophila database FlyBase. (B) FDPP images of rdgA1;su(100) showing rescue of degeneration. Comparable images showing rescue of degeneration by Df(2R)59AD/su(100) but not Df(2R)59AB/su(100) and Df(2R)3_70/su(100).

 


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  		Fig. 6. (A) Rhabdomere structure in rdgA1 is rescued to equivalent levels by su(1) and InaD1. (B) Western blot using an antiserum against INAD showing wild-type INAD protein missing from head extracts of su(1) and su(100) but not su(40). The established protein-null mutant InaD1 is used as a negative control to identify the INAD band. Control panel shows the same blot detecting syntaxin as a loading control to show equivalent levels of protein in all samples.

 


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  		Fig. 7. (A) Rescue of INAD protein levels in su(1) and su(100). Western-blot analysis of head extracts with an INAD antiserum. Levels of INAD protein in su(1), su(100) and InaD1 are compared to that in the same mutants also carrying the P[GMR-InaD] transgene. Levels of INAD in head extracts from the parent strain used for mutagenesis (2120) are also shown. A control blot shows equivalent levels of protein loading between the various samples. (B) The rescue of degeneration (assayed by FDPP) seen in rdgA1;su(100)/InaD1 and rdgA1;su(100) mutants can be reversed by introducing the transgene P[GMR-InaD] in rdgA1;su(100);P[GMR-InaD]. The original rdgA1;parent that shows degeneration is shown for comparison.

 


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  		Fig. 8. Schematic representation of the multiple PDZ domains of INAD. The principal binding protein partners that are known to interact with INAD and the PDZ domains involved are shown; TRP-TRP channel; PKC eye-enriched protein kinase C and PLCß-NORPA. Only key interactions about which there is general agreement among workers in the field are shown. The positions of point mutations in several INAD alleles including InaD1, InaD2, InaDP215, su(1) and su(100) are shown with respect to the PDZ domains.

 


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  		Fig. 9. Effect of specifically disrupting the TRP-INAD interaction on rdgA1. (A) TEM of rdgA1;InaDP215 double mutants showing no rescue of degeneration. (B) Rescue of rdgA1 by trpCM assayed by optical neutralization. When grown at 29°C, the restrictive temperature for trpCM, rdgA1;trpCM show rescue of degeneration compared with that at the permissive temperature of 20°C. The images of rhabdomeres R1-R7 can be seen. Expression of TRP{Delta}1272 channels in rdgA1;trpCM mutants at 29°C results in severe retinal degeneration.

 


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  		Fig. 10. (A) Western blot showing reduced levels of NORPA in head extracts from su(1) and su(100) but not su(40). Levels of dSyx on the same blot were used as a loading control. (B) TEM of a single ommatidium showing equivalent levels of rhabdomere rescue by InaD1 and InaD2 in rdgA1. (C) Western blot showing that the reduced levels of NORPA in InaD1 and su(100) can be restored by complementation with P[GMR-InaD]. An irrelevant band from the same blot is used as a control. Extracts from norpAP24, a null allele were used to identify the NORPA band.

 


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  		Fig. 11. Effects of modulating phototransduction on retinal degeneration in rdgA3. (A) Time course of retinal degeneration in rdgA3 compared with wild-type (Oregon-R ROR) flies. Flies were grown in a normal laboratory incubator and experienced brief pulses of light when the incubator doors were opened. ROR flies show no degeneration under these conditions. On eclosion, rdgA3 flies show wild-type rhabdomere structure by optical neutralization but then progressively degenerate over 72 hours. (B) Rates of degeneration compared in rdgA3 flies grown in normal incubator (NI), constant darkness in black photographic bags (24h D) and in a illuminated cooled incubator on light/dark cycles (12h L/12h D). (C) Rates of degeneration of rdgA3 compared with rdgA3;Gq1. In this experiment, flies were grown in the normal incubator. (D) Rates of degeneration in ROR, rdgA3 and norpAP24,rdgA3. There is virtually complete suppression of degeneration in norpAP24,rdgA3. In this experiment, flies were grown in a normal incubator.

 

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© The Company of Biologists Ltd 2005