Expression of a human variant of CHMP2B linked to neurodegeneration in Drosophila external sensory organs leads to cell fate transformations associated with increased Notch activity.

Proper function of cell signaling pathways is dependent upon regulated membrane trafficking events that lead to the endocytosis, recycling, and degradation of cell surface receptors. The endosomal complexes required for transport (ESCRT) genes play a critical role in the sorting of ubiquitinated cell surface proteins. CHMP2BIntron5 , a truncated form of a human ESCRT-III protein, was discovered in a Danish family afflicted by a hereditary form of frontotemporal dementia (FTD). Although the mechanism by which the CHMP2B mutation in this family causes FTD is unknown, the resulting protein has been shown to disrupt normal endosomal-lysosomal pathway function and leads to aberrant regulation of signaling pathways. Here we have misexpressed CHMP2BIntron5 in the developing Drosophila external sensory (ES) organ lineage and demonstrate that it is capable of altering cell fates. Each of the cell fate transformations seen is compatible with an increase in Notch signaling. Furthermore, this interpretation is supported by evidence that expression of CHMP2BIntron5 in the notum environment is capable of raising the levels of Notch signaling. As such, these results add to a growing body of evidence that CHMP2BIntron5 can act rapidly to disrupt normal cellular function via the misregulation of critical cell surface receptor function.

The Drosophila rhodopsin cytoplasmic tail domain is required for maintenance of rhabdomere structure.

The ninaE-encoded Rh1 rhodopsin is the major light-sensitive pigment expressed in Drosophila R1-6 photoreceptor cells. Rh1 rhodopsin localizes to and is essential for the development and maintenance of the rhabdomere, the specialized membrane-rich organelle that serves as the site of phototransduction. We showed previously that the vertebrate bovine rhodopsin (Rho) is expressed and properly localized in Drosophila photoreceptor cells. Drosophila photoreceptors expressing only Rho have normal rhabdomere structure at young ages, but the rhabdomeres are not maintained and show extensive disorganization by 7-10 days of age. A series of Rho-Rh1 opsin chimeric rhodopsins were used to identify Rh1 domains required for maintenance of rhabdomeric structure. The results show that the Rh1 rhodopsin cytoplasmic tail domain, positioned to interact with cytoplasmic structural components, plays a major role in promoting rhabdomeric organization.

Probing rhodopsin-transducin interaction using Drosophila Rh1-bovine rhodopsin chimeras.

Invertebrate and vertebrate rhodopsins share a low degree of homology and are coupled to G-proteins from different families. Here we explore the utility of fly-expressed chimeras between Drosophila rhodopsin Rh1 and bovine rhodopsin (Rho) to probe the interactions between the invertebrate and vertebrate visual pigments and their cognate G-proteins. Chimeric Rh1 pigments carrying individual substitutions of the cytoplasmic loops C2 and C3 and the C-terminus with the corresponding regions of Rho retained the ability to stimulate phototranduction in Drosophila, but failed to activate transducin. Surprisingly, chimeric Rho containing the Rh1 C-terminus was fully capable of transducin activation, indicating that the C-terminal domain of vertebrate rhodopsins is not essential for the functional coupling to transducin.

Heterologous expression of bovine rhodopsin in Drosophila photoreceptor cells.

PURPOSE: Vertebrate and invertebrate visual pigments are similar in amino acid sequence, structural organization, spectral properties, and mechanism of action, but possess different chromophores and trigger phototransduction through distinct biochemical pathways. The bovine opsin gene (Rho) was expressed in Drosophila, to examine the properties of a vertebrate opsin within invertebrate photoreceptor cells. METHODS: Transgenic Drosophila expressing the bovine opsin gene (Rho) in photoreceptors were created. Protein expression and cellular location of bovine rhodopsin was assessed by protein blots and immunofluorescence. The glycosylation state was determined by mobility profiles in SDS-PAGE before and after treatment with endoglycosidase. The rhodopsin chromophore was determined by HPLC-mass spectroscopy (MS) and the spectral properties by spectroscopy. The ability of the bovine rhodopsin to couple to Drosophila phototransduction components was assessed by electroretinography and to couple to vertebrate transducin by light-mediated GTPgammaS-binding assays. RESULTS: Rho showed stable expression even in the absence of endogenous Rh1 opsin and chromophore. It was correctly targeted to the rhabdomeric membranes. Rho remained glycosylated during the maturation process and possessed a distinct glycosylation pattern from that of native Rho. The Drosophila-expressed Rho associated with the 3-hydroxyretinal chromophore but failed to evoke an electroretinogram response from fly photoreceptors. However, the Drosophila-expressed Rho activated transducin in a light-dependent manner. CONCLUSIONS: Drosophila photoreceptors express a vertebrate rhodopsin as a functional visual pigment, but the expression does not activate the Drosophila phototransduction pathway. The system allows the characterization and comparison of vertebrate and invertebrate visual pigment properties in a common cell type.

The role of Drosophila ninaG oxidoreductase in visual pigment chromophore biogenesis.

We previously reported (Sarfare, S., Ahmad, S. T., Joyce, M. V., Boggess, B., and O'Tousa, J. E. (2005) J. Biol. Chem. 280, 11895-11901) that the Drosophila ninaG gene encodes an oxidoreductase involved in the biosynthesis of the (3S)-3-hydroxyretinal serving as chromophore for Rh1 rhodopsin and that ninaG mutant flies expressing Rh4 as the major opsin accumulate large amounts of a different retinoid. Here, we show that this unknown retinoid is 11-cis-3-hydroxyretinol. Reversed phase high performance liquid chromatography coupled with a photodiode array UV-visible absorbance detector and mass spectrometer revealed a major product eluting at a retention time, t(r), of 3.5 min with a lambda(max) of approximately 324 nm and with a base peak in the mass spectrum at m/z 285. These observations are identical with those of the 3-hydroxyretinol standard. The base peak in the electrospray ionization mass spectrum arises from the loss of a water molecule from the protonated molecule at m/z 303 because of fragmentation in the ion source. These results suggest that 11-cis-3-hydroxyretinol is an intermediate required for chromophore biogenesis in Drosophila. We further show that ninaG mutants fed on retinal as the sole source of vitamin A are able to synthesize 3-hydroxyretinoids. Thus, the NinaG oxidoreductase is not responsible for the initial hydroxylation of the retinal ring but rather acts in a subsequent step in chromophore production. These data are used to review chromophore biosynthesis and propose that NinaG acts in the conversion of (3R)-3-hydroxyretinol to the 3S enantiomer.

The Drosophila ninaG oxidoreductase acts in visual pigment chromophore production.

The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaG(P330) mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaG(P330). These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal.