ABSTRACT Phototransduction in Drosophila is mediated by phospholipase C-dependent hydrolysis of PIP 2­ , and is an important model for phosphoinositide signalling. Although generally assumed to operate by generic machinery conserved from yeast to mammals, some key elements of the phosphoinositide cycle have yet to be identified in Drosophila photoreceptors. Here, we used transgenic flies expressing fluorescently tagged probes (P4M and Tb R332H ), which allow in vivo quantitative measurements of PI4P and PIP 2 dynamics in photoreceptors of intact living flies. Using mutants and RNA interference for candidate genes potentially involved in phosphoinositide turnover, we identified Drosophila PI4KIIIα (CG10260) as the PI4-kinase responsible for PI4P synthesis in the photoreceptor membrane. Our results also indicate that PI4KIIIα activity requires rbo (the Drosophila orthologue of Efr3) and CG8325 (orthologue of YPP1), both of which are implicated as scaffolding proteins necessary for PI4KIIIα activity in yeast and mammals. However, our evidence indicates that the recently reported central role of dPIP5K59B (CG3682) in PIP 2 synthesis in the rhabdomeres should be re-evaluated; although PIP 2 resynthesis was suppressed by RNAi directed against dPIP5K59B , little or no defect was detected in a reportedly null mutant ( dPIP5K 18 ).

ABSTRACT Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila melanogaster , the main light-sensing rhodopsin (Rh1; encoded by ninaE ) and the downstream ion channel, transient receptor potential like (TRPL), are endocytosed in response to light, but the mechanism is unclear. By using an RNA-Sequencing (RNA-Seq) approach, we discovered a protein we named CULD, a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein, that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1-positive or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1, but not of TRPL, suppressed retinal degeneration in culd -null mutant flies. Our data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity.

ABSTRACT In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, Tb R332H (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5) P 2 ], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4 P ). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5) P 2 and very little PtdIns4 P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca 2+ -permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4 P ( t ½ ∼12 s) was faster than PtdIns(4,5) P 2 ( t ½ ∼40 s), but both were greatly slowed in mutants of DAG kinase ( rdgA ) or PtdIns transfer protein ( rdgB ). The results indicate that Ca 2+ - and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4 P to PtdIns(4,5) P 2 is the rate-limiting step of the cycle.

ABSTRACT Retinal photoreceptor cells contain a specialized outer segment (OS) compartment that functions in the capture of light and its conversion into electrical signals in a process known as phototransduction. In rods, photoisomerization of 11- cis to all- trans retinal within rhodopsin triggers a biochemical cascade culminating in the closure of cGMP-gated channels and hyperpolarization of the cell. Biochemical reactions return the cell to its ‘dark state’ and the visual cycle converts all- trans retinal back to 11- cis retinal for rhodopsin regeneration. OS are continuously renewed, with aged membrane removed at the distal end by phagocytosis and new membrane added at the proximal end through OS disk morphogenesis linked to protein trafficking. The molecular basis for disk morphogenesis remains to be defined in detail although several models have been proposed, and molecular mechanisms underlying protein trafficking are under active investigation. The aim of this Cell Science at a Glance article and the accompanying poster is to highlight our current understanding of photoreceptor structure, phototransduction, the visual cycle, OS renewal, protein trafficking and retinal degenerative diseases.

Summary Family members of the cationic transient receptor potential (TRP) channels serve as sensors and transducers of environmental stimuli. The ability of different TRP channel isoforms of specific subfamilies to form heteromultimers and the structural requirements for channel assembly are still unresolved. Although heteromultimerization of different mammalian TRP channels within single subfamilies has been described, even within a subfamily (such as TRPC) not all members co-assemble with each other. In Drosophila photoreceptors two TRPC channels, TRP and TRP-like protein (TRPL) are expressed together in photoreceptors where they generate the light-induced current. The formation of functional TRP–TRPL heteromultimers in cell culture and in vitro has been reported. However, functional in vivo assays have shown that each channel functions independently of the other. Therefore, the issue of whether TRP and TRPL form heteromultimers in vivo is still unclear. In the present study we investigated the ability of TRP and TRPL to form heteromultimers, and the structural requirements for channel assembly, by studying assembly of GFP-tagged TRP and TRPL channels and chimeric TRP and TRPL channels, in vivo . Interaction studies of tagged and native channels as well as native and chimeric TRP–TRPL channels using co-immunoprecipitation, immunocytochemistry and electrophysiology, critically tested the ability of TRP and TRPL to interact. We found that TRP and TRPL assemble exclusively as homomultimeric channels in their native environment. The above analyses revealed that the transmembrane regions of TRP and TRPL do not determine assemble specificity of these channels. However, the C-terminal regions of both TRP and TRPL predominantly specify the assembly of homomeric TRP and TRPL channels.

Transient receptor potential (TRP) channels across species are expressed in sensory receptor cells, and often localized to specialized subcellular sites. In Drosophila photoreceptors, TRP-like (TRPL) channels are localized to the signaling compartment, the rhabdomere, in the dark, and undergo light-induced translocation into the cell body as a mechanism for long-term light-adaptation. We show that translocation of TRPL channels occurs in two distinct stages, first to the neighboring stalk membrane then to the basolateral membrane. In the first stage, light-induced translocation occurs within 5 minutes, whereas the second stage takes over 6 hours. The exclusive apical localization of TRPL channels in the first stage of translocation suggests that channels are released from the rhabdomere and diffuse laterally through the membrane into the adjoining stalk membrane. In the second stage, TRPL channels are localized in the basolateral membrane, implicating a different transport mechanism. Genetic analyses suggest that activation of the other light-activated TRP channel and eye-protein-kinase C (eye-PKC) are both required for the second stage of TRPL translocation in R1 to R6 photoreceptor cells, whereas only phospholipase C (PLC) is required for the first stage. Finally, we show that arrestin2 is required for the rhabdomeric localization and stability of TRPL channels.

Signal-mediated translocation of transient receptor potential (TRP) channels is a novel mechanism to fine tune a variety of signaling pathways including neuronal path finding and Drosophila photoreception. In Drosophila phototransduction the cation channels TRP and TRP-like (TRPL) are the targets of a prototypical G protein-coupled signaling pathway. We have recently found that the TRPL channel translocates between the rhabdomere and the cell body in a light-dependent manner. This translocation modifies the ion channel composition of the signaling membrane and induces long-term adaptation. However, the molecular mechanism underlying TRPL translocation remains unclear. Here we report that eGFP-tagged TRPL expressed in the photoreceptor cells formed functional ion channels with properties of the native channels, whereas TRPL-eGFP translocation could be directly visualized in intact eyes. TRPL-eGFP failed to translocate to the cell body in flies carrying severe mutations in essential phototransduction proteins, including rhodopsin, Gαq, phospholipase Cβ and the TRP ion channel, or in proteins required for TRP function. Our data, furthermore, show that the activation of a small fraction of rhodopsin and of residual amounts of the Gq protein is sufficient to trigger TRPL-eGFP internalization. In addition, we found that endocytosis of TRPL-eGFP occurs independently of dynamin, whereas a mutation of the unconventional myosin III, NINAC, hinders complete translocation of TRPL-eGFP to the cell body. Altogether, this study revealed that activation of the phototransduction cascade is mandatory for TRPL internalization, suggesting a critical role for the light induced conductance increase and the ensuing Ca 2+ -influx in the translocation process. The critical role of Ca 2+ influx was directly demonstrated when the light-induced TRPL-eGFP translocation was blocked by removing extracellular Ca 2+ .