Hairless (H) is the major antagonist within the Notch signalling pathway of Drosophila melanogaster. By binding to Suppressor of Hairless [Su(H)] and two co-repressors, H induces silencing of Notch target genes in the absence of Notch signals. We have applied genomic engineering to create several new H alleles. To this end the endogenous H locus was replaced with an attP site by homologous recombination, serving as a landing platform for subsequent site directed integration of different H constructs. This way we generated a complete H knock out allele H attP, reintroduced a wild type H genomic and a cDNA-construct (H gwt, H cwt) as well as two constructs encoding H proteins defective of Su(H) binding (H LD, H iD). Phenotypes regarding viability, bristle and wing development were recorded, and the expression of Notch target genes wingless and cut was analysed in mutant wing discs or in mutant cell clones. Moreover, genetic interactions with Notch (N 5419) and Delta (Dl B2) mutants were addressed. Overall, phenotypes were largely as expected: both H LD and H iD were similar to the H attP null allele, indicating that most of H activity requires the binding of Su(H). Both rescue constructs H gwt and H cwt were homozygous viable without phenotype. Unexpectedly, the hemizygous condition uncovered that they were not identical to the wild type allele: notably H cwt showed a markedly reduced activity, suggesting the presence of as yet unidentified regulatory or stabilizing elements in untranslated regions of the H gene. Interestingly, H gwt homozygous cells expressed higher levels of H protein, perhaps unravelling gene-by-environment interactions.

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Drosophila photoreceptor cells are employed as a model system for studying membrane protein transport. Phototransduction proteins like rhodopsin and the light-activated TRPL ion channel are transported within the photoreceptor cell, and they change their subcellular distribution in a light-dependent way. Investigating the transport mechanisms for rhodopsin and ion channels requires accurate histochemical methods for protein localization. By using immunocytochemistry the light-triggered translocation of TRPL has been described as a two-stage process. In stage 1, TRPL accumulates at the rhabdomere base and the adjacent stalk membrane a few minutes after onset of illumination and is internalized in stage 2 by endocytosis after prolonged light exposure. Here, we show that a commonly observed crescent shaped antibody labeling pattern suggesting a fast translocation of rhodopsin, TRP, and TRPL to the rhabdomere base is a light-dependent antibody staining artifact. This artifact is most probably caused by the profound structural changes in the microvillar membranes of rhabdomeres that result from activation of the signaling cascade. By using alternative labeling methods, either eGFP-tags or the self-labeling SNAP-tag, we show that light activation of TRPL transport indeed results in fast changes of the TRPL distribution in the rhabdomere but not in the way described previously.

Cell communication in metazoans requires the highly conserved Notch signaling pathway, which is subjected to strict regulation of both activation and silencing. In Drosophila melanogaster, silencing involves the assembly of a repressor complex by Hairless (H) on Notch target gene promoters. We previously found an in-frame internal ribosome entry site in the full length H transcript resulting in two H protein isoforms (Hp120 and Hp150). Hence, H may repress Notch signalling activity in situations where cap-dependent translation is inhibited. Here we demonstrate the in vivo importance of both H isoforms for proper fly development. To this end, we replaced the endogenous H locus by constructs specifically affecting translation of either Hp150 or Hp120 isoforms using genome engineering. Our findings indicate the functional relevance of both H proteins. Based on bristle phenotypes, the predominant isoform Hp150 appears to be of particular importance. In contrast, growth regulation and venation of the wing require the concomitant activity of both isoforms. Finally, the IRES dependent production of Hp120 during mitosis was verified in vivo. Together our data confirm IRES mediated translation of H protein in vivo, supporting strict regulation of Notch in different cellular settings.

Plasma membrane protein trafficking is of fundamental importance for cell function and cell integrity of neurons and includes regulated protein recycling. In this work, we report a novel role of the endoplasmic reticulum (ER) for protein recycling as discovered in trafficking studies of the ion channel TRPL in photoreceptor cells of Drosophila. TRPL is located within the rhabdomeric membrane from where it is endocytosed upon light stimulation and stored in the cell body. Conventional immunohistochemistry as well as stimulated emission depletion super-resolution microscopy revealed TRPL storage at the ER after illumination, suggesting an unusual recycling route of TRPL. Our results also imply that both phospholipase D (PLD) and retromer complex are required for correct recycling of TRPL to the rhabdomeric membrane. Loss of PLD activity in PLD 3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35 MH20 , TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD 3.1 vps35 MH20 , PLD activity precedes retromer function. We propose a model in which PLD and retromer function play key roles in the transport of TRPL to an ER enriched compartment.

The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.

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