Phospholipase D and retromer promote recycling of TRPL ion channel via the endoplasmic reticulum.

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 PLD3.1 mutants results in enhanced degradation of TRPL. In the retromer mutant vps35MH20 , TRPL is trapped in a Rab5-positive compartment. Evidenced by epistatic analysis in the double mutant PLD3.1 vps35MH20 , 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.

Classification of solutions for guided waves in fluid-loaded viscoelastic composites with large numbers of layers.

Guided ultrasonic waves are used for the inspection of multilayered composite aerospace structures. Calculating the corresponding dispersion diagrams is challenging for thick-walled composites with more than 100 layers, such as in modern rocket booster pressure vessels. The Dispersion Calculator (DC) is an open source software for calculating such dispersion diagrams and mode shapes of guided waves. Attenuation caused by viscoelasticity and fluid-loading makes the dispersion curve tracing much more difficult than in the nonattenuated case because the modal solutions are sought in the complex wavenumber plane. The tracing problem is mastered by a reliable algorithm. Whereas leaky Lamb and Scholte waves in coupled and decoupled cases are modeled using the stiffness matrix method, shear horizontal (SH) waves are traced using the transfer matrix method without facing the numerical instability. Through implementation of mode family specific dispersion equations in both matrix techniques for nonattenuated and attenuated cases, symmetric, antisymmetric, and nonsymmetric leaky Lamb, Scholte, and SH waves can be traced separately with better efficiency and robustness. The capabilities of DC are demonstrated by calculating dispersion diagrams and mode shapes for a viscoelastic composite with 400 layers immersed in water. Results are compared against DISPERSE (Imperial College London, London, UK) for selected cases.

Multilevel regulation of the glass locus during Drosophila eye development.

Development of eye tissue is initiated by a conserved set of transcription factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control identity of photoreceptor as well as non-photoreceptor cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintenance of differentiated tissues. The molecular mechanisms that control expression of glass, however, remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alternative small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible Glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but is able to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is largely unaffected by targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye.

The Role of Reversible Phosphorylation of Drosophila Rhodopsin.

Vertebrate and fly rhodopsins are prototypical GPCRs that have served for a long time as model systems for understanding GPCR signaling. Although all rhodopsins seem to become phosphorylated at their C-terminal region following activation by light, the role of this phosphorylation is not uniform. Two major functions of rhodopsin phosphorylation have been described: (1) inactivation of the activated rhodopsin either directly or by facilitating binding of arrestins in order to shut down the visual signaling cascade and thus eventually enabling a high-temporal resolution of the visual system. (2) Facilitating endocytosis of activated receptors via arrestin binding that in turn recruits clathrin to the membrane for clathrin-mediated endocytosis. In vertebrate rhodopsins the shutdown of the signaling cascade may be the main function of rhodopsin phosphorylation, as phosphorylation alone already quenches transducin activation and, in addition, strongly enhances arrestin binding. In the Drosophila visual system rhodopsin phosphorylation is not needed for receptor inactivation. Its role here may rather lie in the recruitment of arrestin 1 and subsequent endocytosis of the activated receptor. In this review, we summarize investigations of fly rhodopsin phosphorylation spanning four decades and contextualize them with regard to the most recent insights from vertebrate phosphorylation barcode theory.

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System.

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.

Ca2+ Signaling in Drosophila Photoreceptor Cells.

In Drosophila photoreceptor cells, Ca2+ exerts regulatory functions that control the shape, duration, and amplitude of the light response. Ca2+ also orchestrates light adaptation allowing Drosophila to see in light intensity regimes that span several orders of magnitude ranging from single photons to bright sunlight. The prime source for Ca2+ elevation in the cytosol is Ca2+ influx from the extracellular space through light-activated TRP channels. This Ca2+ influx is counterbalanced by constitutive Ca2+ extrusion via the Na+/Ca2+ exchanger, CalX. The light-triggered rise in intracellular Ca2+ exerts its regulatory functions through interaction with about a dozen well-characterized Ca2+ and Ca2+/CaM binding proteins. In this review we will discuss the dynamic changes in Ca2+ concentration upon illumination of photoreceptor cells. We will present the proteins that are known to interact with Ca2+ (/CaM) and elucidate the physiological functions of these interactions.

The Phosphorylation State of the Drosophila TRP Channel Modulates the Frequency Response to Oscillating Light In Vivo.

Drosophila photoreceptors respond to oscillating light of high frequency (∼100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. Here, we report that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca2+-dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC). Electroretinogram measurements of the frequency response to oscillating lights in vivo revealed that dark-reared flies expressing wild-type TRP exhibited a detection limit of oscillating light at relatively low frequencies, which was shifted to higher frequencies upon light adaptation. Strikingly, preventing phosphorylation of the S936-TRP site by alanine substitution in transgenic Drosophila (trpS936A ) abolished the difference in frequency response between dark-adapted and light-adapted flies, resulting in high-frequency response also in dark-adapted flies. In contrast, inserting a phosphomimetic mutation by substituting the S936-TRP site to aspartic acid (trpS936D ) set the frequency response of light-adapted flies to low frequencies typical of dark-adapted flies. Light-adapted rdgC mutant flies showed relatively high S936-TRP phosphorylation levels and light-dark phosphorylation dynamics. These findings suggest that RDGC is one but not the only phosphatase involved in pS936-TRP dephosphorylation. Together, this study indicates that TRP channel dephosphorylation is a regulatory process that affects the detection limit of oscillating light according to the light rearing condition, thus adjusting dynamic processing of visual information under varying light conditions.SIGNIFICANCE STATEMENTDrosophila photoreceptors exhibit high temporal resolution as manifested in frequency response to oscillating light of high frequency (≤∼100 Hz). Light rearing conditions modulate the maximal frequency detected by photoreceptors, thus enabling them to maintain high sensitivity to light and high temporal resolution. However, the precise mechanisms for this process are not fully understood. Here, we show by combination of biochemistry and in vivo electrophysiology that transient receptor potential (TRP) channel dephosphorylation at a specific site is a fast, light-activated and Ca2+-dependent regulatory process. TRP dephosphorylation affects the detection limit of oscillating light according to the adaptation state of the photoreceptor cells by shifting the detection limit to higher frequencies upon light adaptation. This novel mechanism thus adjusts dynamic processing of visual information under varying light conditions.

Ectopic Expression of Mouse Melanopsin in Drosophila Photoreceptors Reveals Fast Response Kinetics and Persistent Dark Excitation.

The intrinsically photosensitive M1 retinal ganglion cells (ipRGC) initiate non-image-forming light-dependent activities and express the melanopsin (OPN4) photopigment. Several features of ipRGC photosensitivity are characteristic of fly photoreceptors. However, the light response kinetics of ipRGC is much slower due to unknown reasons. Here we used transgenic Drosophila, in which the mouse OPN4 replaced the native Rh1 photopigment of Drosophila R1-6 photoreceptors, resulting in deformed rhabdomeric structure. Immunocytochemistry revealed OPN4 expression at the base of the rhabdomeres, mainly at the rhabdomeral stalk. Measurements of the early receptor current, a linear manifestation of photopigment activation, indicated large expression of OPN4 in the plasma membrane. Comparing the early receptor current amplitude and action spectra between WT and the Opn4-expressing Drosophila further indicated that large quantities of a blue absorbing photopigment were expressed, having a dark stable blue intermediate state. Strikingly, the light-induced current of the Opn4-expressing fly photoreceptors was ∼40-fold faster than that of ipRGC. Furthermore, an intense white flash induced a small amplitude prolonged dark current composed of discrete unitary currents similar to the Drosophila single photon responses. The induction of prolonged dark currents by intense blue light could be suppressed by a following intense green light, suggesting induction and suppression of prolonged depolarizing afterpotential. This is the first demonstration of heterologous functional expression of mammalian OPN4 in the genetically emendable Drosophila photoreceptors. Moreover, the fast OPN4-activated ionic current of Drosophila photoreceptors relative to that of mouse ipRGC, indicates that the slow light response of ipRGC does not arise from an intrinsic property of melanopsin.

The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells.

Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cß-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14P75L mutant. The ttd14P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane.

Classification of solutions for guided waves in anisotropic composites with large numbers of layers.

Guided waves are used for the non-destructive evaluation in automotive and aerospace industries. There is a trend leaning away from isotropic materials to the manufacturing based on composites. However, the elastic wave dynamics in such materials is considerably more complicated. Much effort has been committed to the calculation of guided waves' dispersion curves in composites. Lots of methods and tools are available, but it becomes difficult when there are more than one hundred layers. In this paper the calculation of dispersion diagrams and mode shapes using the stiffness matrix method is demonstrated. Boundary conditions are implemented into the stiffness matrix method that allow for the separate tracing of the various mode families. Shear horizontal modes are modeled with the transfer matrix method without facing any numerical instability. It is elucidated just how the occurrence of the mode families depends on the system's symmetry and wave propagation direction. As a result, the robustness and reliability of guided wave modeling by using the stiffness method is improved, and more information about the modes is yielded. This is demonstrated on exemplary layups of the fiber reinforced polymer T800/913, with up to 400 layers. Referencing is made against results from DISPERSE® (Imperial College London, London, UK) for selected cases.

3D non-contrast-enhanced ECG-gated MR angiography of the lower extremities with dual-source radiofrequency transmission at 3.0 T: Intraindividual comparison with contrast-enhanced MR angiography in PAOD patients.

OBJECTIVE: To compare prospectively image quality and diagnostic confidence of flow-sensitive 3D turbo spin echo (TSE)-based non-contrast-enhanced MR angiography (NE-MRA) at 3.0 T using dual-source radiofrequency (RF) transmission with contrast-enhanced MRA (CE-MRA) in patients with peripheral arterial occlusive disease (PAOD). METHODS: After consent was obtained, 35 patients (mean age 69.1 ± 10.6 years) with PAOD stage II-IV underwent NE-MRA followed by CE-MRA. Signal-to-noise ratio and contrast-to-noise ratio were calculated. Subjective image quality was independently assessed by two radiologists and stenosis scoring was performed in 875 arterial segments. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for stenosis classification were calculated using CE-MRA as a reference method. Diagnostic agreement with CE-MRA was evaluated with Cohen's kappa statistics. RESULTS: NE-MRA provided high objective and subjective image quality at all levels of the arterial tree. Sensitivity and specificity for the detection of relevant stenosis was 91 % and 89 %, respectively; the NPV was 96 % and the PPV 78 %. There was good concordance between CE-MRA and NE-MRA in stenosis scoring. CONCLUSIONS: 3D electrocardiography (ECG)-gated TSE NE-MRA with patient-adaptive dual-source RF transmission at 3.0 T is a promising alternative for PAOD patients with contraindications for gadolinium-based contrast agents. It offers high sensitivity and NPV values in the detection of clinically relevant arterial stenosis. KEY POINTS: • Flow-sensitive TSE NE-MRA is a promising technique for PAOD evaluation. • Diagnostic accuracy is comparable to contrast-enhanced MRA. • NE-MRA eliminates the risk of NSF in patients with renal insufficiency. • Costs arising from the use of contrast agents can be avoided.

Drosophila TRP and TRPL are assembled as homomultimeric channels in vivo.

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.

Mutation of light-dependent phosphorylation sites of the Drosophila transient receptor potential-like (TRPL) ion channel affects its subcellular localization and stability.

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.

Dose response of the intravascular contrast agent gadofosveset trisodium in MR perfusion imaging of the myocardium using semiquantitative evaluation.

PURPOSE: To evaluate appropriate injection protocols for gadofosveset at 1.5 and 3 T magnetic resonance imaging (MRI) for semiquantitative myocardial perfusion analysis. MATERIALS AND METHODS: Eighteen young healthy volunteers were subjected to first-pass perfusion cardiac scans at 1.5 and 3 T MRI using three different injection protocols for gadofosveset (0.00375, 0.0075, and 0.0150 mmol/kg bodyweight) and two perfusions. At both field strengths a T1-weighted saturation recovery turboFLASH sequence with parallel imaging was employed. Peak signal-to-noise ratio (SNR), maximum contrast enhancement ratio (CER), peak-baseline difference, and upslope values were assessed. Moreover, sectors with dark banding artifacts were evaluated. RESULTS: Significant differences of the upslope values for first compared to second perfusion could be observed for the medium- and high-dose groups at 1.5 T (P < 0.01), but not at 3 T. Sectors with dark banding artifacts during first perfusion occurred significantly more often at the highest dose of gadofosveset compared to the lowest dose at 1.5 T (P = 0.04) and 3 T (P < 0.01). CONCLUSION: The best injection protocol for semiquantitative perfusion analysis at 1.5 T is 0.00375 mmol/kg, as higher doses lead to lower upslope values during the second perfusion. At 3 T 0.0075 mmol/kg should be used to avoid dark banding artifacts.

In vivo identification of Drosophila rhodopsin interaction partners by biotin proximity labeling.

Proteins exert their function through protein-protein interactions. In Drosophila, G protein-coupled receptors like rhodopsin (Rh1) interact with a G protein to activate visual signal transduction and with arrestins to terminate activation. Also, membrane proteins like Rh1 engage in protein-protein interactions during folding within the endoplasmic reticulum, during their vesicular transport and upon removal from the cell surface and degradation. Here, we expressed a Rh1-TurboID fusion protein (Rh1::TbID) in Drosophila photoreceptors to identify in vivo Rh1 interaction partners by biotin proximity labeling. We show that Rh1::TbID forms a functional rhodopsin that mediates biotinylation of arrestin 2 in conditions where arrestin 2 interacts with rhodopsin. We also observed biotinylation of Rh1::TbID and native Rh1 as well as of most visual signal transduction proteins. These findings indicate that the signaling components in the rhabdomere approach rhodopsin closely, within a range of ca. 10 nm. Furthermore, we have detected proteins engaged in the maturation of rhodopsin and elements responsible for the trafficking of membrane proteins, resembling potential interaction partners of Rh1. Among these are chaperons of the endoplasmic reticulum, proteins involved in Clathrin-mediated endocytosis as well as previously unnoticed contributors to rhodopsin transportation, such as Rab32, Vap33, or PIP82.

Myocardium: dynamic versus single-shot CT perfusion imaging.

PURPOSE: To determine the diagnostic accuracy of dynamic computed tomographic (CT) perfusion imaging of the myocardium for the detection of hemodynamically relevant coronary artery stenosis compared with the accuracy of coronary angiography and fractional flow reserve (FFR) measurement. MATERIALS AND METHODS: This study was approved by the institutional review board and the Federal Radiation Safety Council (Bundesamt für Strahlenschutz). All patients provided written informed consent. Thirty-two consecutive patients in adenosine stress conditions underwent dynamic CT perfusion imaging (14 consecutive data sets) performed by using a 256-section scanner with an 8-cm detector and without table movement. Time to peak, area under the curve, upslope, and peak enhancement were determined after calculation of time-attenuation curves. In addition, myocardial blood flow (MBF) was determined quantitatively. Results were compared with those of coronary angiography and FFR measurement by using a receiver operating characteristic (ROC) analysis. In addition, threshold values based on the Youden index and sensitivity and specificity were calculated. RESULTS: Area under the ROC curve, sensitivity, and specificity, respectively, were 0.67, 41.4% (95% confidence interval [CI]: 23.5%, 61.1%), and 86.6% (95% CI: 76.0%, 93.7%) for time to peak; 0.74, 58.6% (95% CI: 38.9%, 76.5%), and 83.6% (95% CI: 72.5%, 91.5%) for area under the curve; 0.87, 82.8% (95% CI: 64.2%, 94.1%), and 88.1% (95% CI: 77.8%, 94.7%) for upslope; 0.83, 82.8% (95% CI: 64.2%, 94.1%), and 89.6% (95% CI: 79.6%, 95.7%) for peak enhancement; and 0.86, 75.9% (95% CI: 56.5%, 89.7%), and 100% (95% CI: 94.6%, 100%) for MBF. The thresholds determined by using the Youden index were 148.5 HU · sec for area under the curve, 12 seconds for time to peak, 2.5 HU/sec for upslope, 34 HU for peak enhancement, and 1.64 mL/g/min for MBF. CONCLUSION: The semiquantitative parameters upslope and peak enhancement and the quantitative parameter MBF showed similar high diagnostic accuracy. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.13121441/-/DC1.

Characterization of the transmitted near-infrared wavefront error for the GRAVITY/VLTI Coudé Infrared Adaptive Optics System.

The adaptive optics system for the second-generation VLT-interferometer (VLTI) instrument GRAVITY consists of a novel cryogenic near-infrared wavefront sensor to be installed at each of the four unit telescopes of the Very Large Telescope (VLT). Feeding the GRAVITY wavefront sensor with light in the 1.4 to 2.4 micrometer band, while suppressing laser light originating from the GRAVITY metrology system, custom-built optical components are required. In this paper, we present the development of a quantitative near-infrared point diffraction interferometric characterization technique, which allows measuring the transmitted wavefront error of the silicon entrance windows of the wavefront sensor cryostat. The technique can be readily applied to quantitative phase measurements in the near-infrared regime. Moreover, by employing a slightly off-axis optical setup, the proposed method can optimize the required spatial resolution and enable real time measurement capabilities. The feasibility of the proposed setup is demonstrated, followed by theoretical analysis and experimental results. Our experimental results show that the phase error repeatability in the nanometer regime can be achieved.

Translocation of the Drosophila transient receptor potential-like (TRPL) channel requires both the N- and C-terminal regions together with sustained Ca2+ entry.

In Drosophila photoreceptors the transient receptor potential-like (TRPL), but not the TRP channels undergo light-dependent translocation between the rhabdomere and cell body. Here we studied which of the TRPL channel segments are essential for translocation and why the TRP channels are required for inducing TRPL translocation. We generated transgenic flies expressing chimeric TRP and TRPL proteins that formed functional light-activated channels. Translocation was induced only in chimera containing both the N- and C-terminal segments of TRPL. Using an inactive trp mutation and overexpressing the Na(+)/Ca(2+) exchanger revealed that the essential function of the TRP channels in TRPL translocation is to enhance Ca(2+)-influx. These results indicate that motifs present at both the N and C termini as well as sustained Ca(2+) entry are required for proper channel translocation.