Proc. Natl. Acad. Sci. USAVol. 93, pp. 571-576, January 1996Colloquium Paper

This paper was presented at a colloquium entitled "Vision: From Photon to Perception," organized by John Dowling,Lubert Stryer (chair), and Torsten Wiesel, held May 20-22, 1995, at the National Academy of Sciences in Irvine, CA.

The biology of vision in Drosophila(G protein/phospholipase C/ion channels/signal transduction)

CHARLES S. ZUKERHoward Hughes Medical Institute and Departments of Biology and Neurosciences, University of California at San Diego, La Jolla, CA 92093-0649

ABSTRACT Phototransduction systems in vertebratesand invertebrates share a great deal of similarity in overallstrategy but differ significantly in the underlying molecularmachinery. Both are rhodopsin-based G protein-coupled sig-naling cascades displaying exquisite sensitivity and broaddynamic range. However, light activation of vertebrate pho-toreceptors leads to activation of a cGMP-phosphodiesteraseeffector and the generation of a hyperpolarizing response. Incontrast, activation of invertebrate photoreceptors, like Dro-sophila, leads to stimulation of phospholipase C and thegeneration of a depolarizing receptor potential. The compar-ative study of these two systems of phototransduction offersthe opportunity to understand how similar biological prob-lems may be solved by different molecular mechanisms ofsignal transduction. The study of this process in Drosophila, asystem ideally suited to genetic and molecular manipulation,allows us to dissect the function and regulation of such acomplex signaling cascade in its normal cellular environment.In this manuscript I review some ofour recent findings and thestrategies used to dissect this process.

The Drosophila compound eye is made up of 800 ommatidia orunit eyes. Each ommatidium is composed of 20 cells including8 photoreceptor neurons (for review, see ref. 1). The eightphotoreceptor neurons can be divided into three main classesdepending on their spectral sensitivity: the six outer photore-ceptors are blue sensitive and contain the major rhodopsin inthe retina, Rhi (2, 3). Of the two central neurons, the distal R7cells express one of two different UV-sensitive opsins (4), andthe proximal R8 cell expresses a blue-green rhodopsin (5).Each photoreceptor cell contains a specialization of the plasmamembrane, known as a rhabdomere, composed of -60,000microvilla; these are the functional equivalent of the discs inthe rod outer segments and contain rhodopsin and the ma-chinery involved in phototransduction (the large increase insurface area provided by the rhabdomeres allows the photo-receptor neurons to pack >100 million molecules of rhodopsinper cell).

Fig. 1 shows a highly schematized, summarized view of thephototransduction cascade in Drosophila. The light receptormolecule rhodopsin (R) is composed of a protein, opsin,covalently linked to a chromophore, 3-hydroxy-11-cis-retinal.Upon absorption of a light photon the chromophore is isomer-ized from the 11-cis to the all-trans configuration. This changein the conformation of the chromophore leads to a change inthe conformation of the protein and to the activation of itscatalytic properties. Activated rhodopsin, or metarhodopsin(M), activates a heterotrimeric G protein of the Gq-family (6,7), which in turn activates a phospholipase C (PLC) encodedby the norpA gene (8). PLC catalyzes the breakdown of the

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minor membrane phospholipid phosphatidyl 4,5-bisphosphate(PIP2) into the two intracellular messengers inositol trisphos-phate (IP3) and diacylglycerol (DAG). This reaction leads tothe opening of cation-selective channels and the generation ofa depolarizing receptor potential (Drosophila photoreceptors,like most invertebrates, depolarize as opposed to hyperpolar-ize in response to light). In addition to excitation, photore-ceptor neurons have evolved sophisticated mechanisms tocontrol termination of the light response (deactivation) andlight and dark adaptation (for review, see D. Baylor in thisissue). Molecular, genetic, and physiological studies suggestthat as many as 50 different gene products are dedicated to thefunctioning and regulation of this one signaling cascade in thefruit fly Drosophila melanogaster (9-11).

Genetic and Molecular Dissection of Phototransduction

We and others have used three general strategies to identifymolecules involved in phototransduction. The first relies on theexpectation that many of the proteins involved in this processwill be encoded by genes preferentially expressed in the visualsystem. This is not an unreasonable assumption because thehigh degree of specialization seen in photoreceptors was mostlikely accompanied by the evolution of dedicated components.Thus, by taking advantage of mutants lacking compound eyesand using highly sensitive subtraction hybridization protocols,it has been possible to isolate a large number ofgenes encodingeye-specific proteins. Examples include ninaA, eye-PKC andG-protein subunits. The second strategy, and perhaps the mostpowerful, relies on classical genetics and functional screens.Nearly 30 yr ago Seymour Benzer isolated the first Drosophilavisual mutants by screening for defects in visual behavior (thiswas also the birth of the field of neurogenetics) (12). Severalyears later, Bill Pak and coworkers at Purdue Universitypioneered the use of electrophysiological screens to search formutant flies with defects in visual physiology (13). Since then,several groups, including our own, have extended these screensto include a wide range of genetic, physiological, and behav-ioral assays of photoreceptor function. The molecular andphysiological analysis of many of these mutants is providingfundamental insight into the biology of this process (seebelow). The third strategy relies on enhancer trap screens, atechnique developed in Walter Gehring's laboratory severalyears ago. In essence, by using a bacterial ,3-galactosidasereporter element and assaying for flies with blue eyes, it hasbeen possible to tag and isolate genes that are preferentiallyexpressed in the visual system. Using combinations of thesethree strategies we have been able to identify a number ofgenes involved in phototransduction (9-11). Of particularinterest are those whose role could have not been predicted on

Abbreviations: IP3, inositol trisphosphate; DAG, diacylglycerol; PIP2,phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; PKC,protein kinase C; [Ca2+]i, intracellular Ca2+ concentration.

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{rhZ rh4 } opsin

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Ty-GDPGTPI* dgq (PGDP

gbeTsx-GTP

norpA

(trp-l) trp

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Intracellular

Extracellular

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FIG. 1. Phototransduction in Drosophila photoreceptors. Absorption of a photon of light causes a conformational change in the rhodopsinmolecule (R) and activates its catalytic properties. Active metarhodopsin (M*) catalyzes G protein activation. The G protein exchanges GDP forGTP and releases the inhibitory fry subunits. Active G protein catalyzes the activation of the norpA-encoded PLC. PLC hydrolyzes PIP2 into theintracellular messengers IP3 and DAG. cGMP has also been implicated as a possible intracellular messenger mediating excitation. Extracellularsodium and calcium enter the cell through the light-activated conductance and cause the depolarization of the photoreceptor cells. Thelight-activated conductance appears to be composed of at least two types of channels. The trp gene is required for a class of channels with highcalcium permeability. DAG is thought to modulate a photoreceptor cell-specific PKC (encoded by inaC) that regulates deactivation anddesensitization of the light response. Metarhodopsin is inactivated via phosphorylation by rhodopsin kinase (RoK) and arrestin binding (encodedby the arrl and arr2 genes). Inactive metarhodopsin is photoconverted back to rhodopsin and then presumably dephosphorylated by therdgC-encoded phosphatase. The box in the upper right indicates a pathway likely to be required for synthesis of PIP2. rdgA encodes DAG-kinaseand rdgB encodes a protein with significant sequence homology to phosphatidylinositol-transfer protein. cds refers to CDP-DAG synthase. dgq andgbe are the genes encoding the photoreceptor cell-specific isoforms of Ga and G,9 subunits, respectively. ninaB and ninaD are genes required forretinal biogenesis, and ninaA is a cyclophilin homolog required for rhodopsin biogenesis. rhl, rh2, rh3, and rh4 are the structural genes for the fourknown rhodopsins. IP3R and PIPase refer to the IP3 receptor and inositol polyphosphate phosphatase (an enzyme required to break down IP3).Mutations in all gene products highlighted in red are now available (refs. 11 and 22, and unpublished work from C.S.Z. laboratory).

biochemical grounds but in which a genetic approach providedfundamental insight as to their functional requirement. Ex-amples are the cyclophilin homologue ninaA and its role inrhodopsin biogenesis (14-17), an eye-specific protein kinase C(PKC) required for deactivation and calcium feedback regu-lation (18, 19), the role of Gp in the termination of the lightresponse (20), and a number of enzymes involved in inositolphospholipid metabolism and shown to be required for pho-toreceptor cell excitation (10, 21).

Inositol Phospholipids and Phototransduction

Drosophila phototransduction is one of the best model systemsfor the study of G protein-coupled PLC signaling (10, 22, 23).Not only is the system amenable to molecular genetic analysisbut also it can report activity with exquisite sensitivity andspecificity: photoreceptor cells are sensitive to single photons,and the signaling pathway can be turned on and off withmillisecond kinetics (phototransduction in Drosophila is thefastest known G protein cascade, taking just a few tens ofmilliseconds to go from light activation of rhodopsin to thegeneration of a receptor potential). As described above, activePLC catalyzes the hydrolysis of the minor membrane phos-pholipid PIP2 into the second messengers inositol IP3 andDAG. IP3 mobilizes calcium from internal stores, which affectsand modulates many cellular processes, and DAG activates

members of the PKC family of proteins. Given the central roleof PIP2 in signaling, its levels may be expected to be tightlyregulated in the cell.

Fig. 2 shows an expanded view of the PIP2 cycle. CDP-DAGsynthase (CDS) is an enzyme required to convert phosphatidicacid into CDP-DAG, the acceptor for the inositol head group.

Using enhancer trap technology, we identified an eye-specificform of CDP-DAG and isolated mutations in this gene (eye-cds) (21). To determine whether eye-cds mutants have a defectin their signaling properties, wild-type and mutant animalswere assayed for their ability to maintain a continuouslyactivated state of the photoreceptor cells because such a statewould require the continuous availability of the second mes-

senger PIP2. Our results demonstrated that light activationdepletes a pool of PIP2 necessary for excitation that cannot bereplenished in eye-cds mutants (Fig. 3 a-d). This phenotype isdue exclusively to a defect in eye-cds, because introduction ofthe wild-type eye-CDS cDNA into mutant hosts fully restoreswild-type physiology (Fig. 3 e-f). Furthermore, inclusion ofPIP2 in the patch pipette is sufficient to restore signaling in thedepleted eye-cds mutants (Fig. 3 g-h). These results suggest,contrary to expectations, that the pool of PIP2 required forsignaling is quite small and likely synthesized "on demand". Onthe basis of these findings, we reasoned that it should bepossible to modulate the output of this cascade by experimen-tally manipulating the levels of eye-CDS. Indeed, we made

rdgB

1 ' >~~~~~~~~~'pIP2 CDP) DAG

/ cdsphosphatcdic Acid

/ rdgA

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a00.

CMP

C_-) roGctaolipin

,ol (norpA) N4 _A

lIntracellular signaling

Intermediatemetabolism

FIG. 2. PIP2 regeneration cycle. Upon activation, PLC hydrolyzesPIP2 to yield IP3 and DAG. To regenerate the PIP2, DAG is phos-phorylated by DAG kinase (dgk) to produce phosphatidic acid (PA).CDP-DAG synthase (CDS*) adds on CMP to phosphatidic acid. Thisproduct, CDP-DAG, is the activated donor of the phosphatidyl groupto inositol. The phosphatidylinositol (PI) is phosphorylated by PIkinase and PIP kinase to yield PIP2. Listed in parentheses are the sitesof action of known photoreceptor cell-specific proteins in Drosophila(adapted from ref. 21).

transgenic animals that overexpressed the enzyme and gener-ated photoreceptor neurons that now display responses thatare 300-400% larger than wild-type cells (21). These resultsopen up the possibility of genetically and pharmacologicallymanipulating PIP2 signaling in vivo and highlight three unex-pected aspects of PLC signaling. (i) PIP2 pools required forsignaling are distinct from the general pool. This is demon-strated by the observation that eye-cds mutants are onlydefective in signaling and only in response to light activation.(ii) PIP2 levels involved in signaling are low and likely gener-ated "on-line". (iii) PIP2 availability is rate limiting andregulates the output of this signaling pathway. This is furtherdemonstrated by the observation that eye-cds mutants displaya reduction in the amplitude of their response as a function ofthe number of light flashes (and thus their state of depletion).A search for second-site mutations that enhance or suppress

the eye-CDS phenotype should produce mutations in othercomponents of this cycle and make it possible to carry out acomprehensive genetic dissection of the various players re-quired for the functioning and regulation of PIP2 and itsmetabolites.

Calcium in Drosophila Photoreceptors

C

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FIG. 3. Defects shown by eye-cds mutants in photoreceptor cellfunction. To determine whether eye-cds mutants have a defect in theirsignaling properties, we assayed wild-type and mutant animals for theirability to maintain a continuous supply of the second-messenger PIP2.Control and mutant cells were dissected and transferred to a bathsolution with nominally zero calcium. The excitation mechanisms werethen depleted before patching by subjecting the cells to 40 min of alight pulse protocol, consisting of 3 sec of intense light pulses followedby 3 min in the dark. If wild-type cells are patched after the depletionprotocol with 700 nM [Ca2+]i in the patch pipette, the light responsereliably recovers (a, b). If the same depletion protocol is applied to cdsmutant cells, the light response does not recover (c, d). This phenotypeis due exclusively to a defect in eye-cds because introduction of thewild-type eye-CDS cDNA into mutant hosts fully restores wild-typephysiology (e, f). Depleted cds mutants can be rescued by supplyingPIP2 through the patch pipette (g). (a, c, and e) Responses ofwild-type,eye-cds and P[cds+] photoreceptors before depletion, respectively.Arrows indicate the position of the stimulating light flash. See ref. 21for further details.

An important and unresolved issue in the study of invertebratephototransduction has been the identification of the intracel-lular messenger(s) that mediate the opening of the light-activated ion channels. IP3, calcium, and cGMP have beenimplicated in this process (23, 24). Although the messenger(s)that actually gates the plasma membrane ion channels remainselusive, patch clamp studies have provided strong evidenceimplicating calcium in the regulation of the light response (18,25, 26). For example, extracellular calcium influx is bothsufficient and necessary to regulate activation and deactivationkinetics of the light-activated conductance. In the absence ofexternal calcium, photoreceptors display slow activation anddeactivation kinetics. Conversely, high extracellular calciumsolutions, or release of caged-intracellular Ca2+ ([Ca2+]1)during a light response (27), cause a transient acceleration inactivation kinetics followed by rapid deactivation.

Despite the role of calcium in regulation, all available dataindicate that calcium release from internal stores is neither thesignal nor is it required for the opening of the light-activatedchannels (28, 29). However, internal calcium stores are re-quired both for the developmental maturation of the light-activated currents (30) and for maintaining a responsive state(31). For instance, mature currents (e.g., adult-like) can beinduced in immature pupae by artificially raising [Ca2+]i, andphotoreceptors depleted for their internal stores show a dra-matic loss of sensitivity that can be rescued by raising [Ca2+]1.

In efforts to directly image calcium changes in intact pho-toreceptors, Rama Ranganathan, Brian Backsai, Roger Tsien,and myself (29) developed a preparation suitable for simulta-neous recording of light-activated currents and the dynamics of[Ca2+], using fluorescent calcium indicators. Past attempts tomeasure simultaneously light-induced current and fluores-

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cence signals in vertebrate (32, 33) and invertebrate (28, 34)photoreceptors have been compromised by the inability tofunctionally separate the stimulating light from that requiredfor fluorescence excitation. Two possible solutions to thisproblem are as follows: (i) to use a fluorescent calciumindicator whose excitation spectrum is well separated from theaction spectra of the cell (not currently available) (35, 36), or(ii) to retune the cell's response to become spectrally separatedfrom available fluorescent calcium indicators. Indeed, wegenetically engineered flies that express a UV-specific rho-dopsin in place of the normal rhodopsin (4), so that longwavelength light can be used to image [Ca2+]i while minimallyexciting the photoreceptor cell. To achieve high temporal andspatial resolution in recording [Ca2+]i, the cells were imagedon a high-speed laser scanning confocal microscope acquiringimages at 30 frames per sec (37). Using this preparation, weshowed that influx of external calcium is responsible for allmeasured light-dependent changes in [Ca2+], (29).The first response to the UV stimulus is seen as a highly

localized elevation in [Ca2+]i at the junction of the rhabdomereand cell body (Fig. 4); this is consistent with localization oflight-sensitive channels at the base of the rhabdomeres. Thisinitial response is rapidly followed by a general increase in[Ca2+]i at the rhabdomere, which generates a dramatic gradi-ent of calcium levels within the cell. The existence of func-tionally relevant, ultramicro domains of high [Ca2+]i has beenpostulated in a number of signaling processes (for review, seeref. 38). Theoretical calculations indicate that [Ca2+], canreach tens to hundreds of micromolarity within 10 to 100 nmfrom each conducting channel. Such ultramicro domainswould, of course, be too small to be resolved by present opticaltechniques. Using a combination of calcium chelators, elec-trophysiology, and calcium imaging, we showed that the Ca2+influx initially generates large-amplitude submicroscopicallylocalized [Ca2+]i transients that are >6 ,uM and are crucial fornegative regulation. These studies provided experimental sup-port for a functional requirement of spatially localized [Ca2+],

and suggest that the transducing machinery may be organizedand compartmentalized as a "transducisome" responding tohighly localized signals. This provides an elegant avenue toprevent signal cross-talk in intracellular signaling pathways.What are the targets of calcium in mediating negative

regulation? An electrophysiological screen for Drosophila pho-totransduction mutants with defects in deactivation kineticsdemonstrated that photoreceptors from inaC mutants (39) arespecifically defective in the calcium-dependent negative reg-ulatory mechanisms (40). When compared to wild-type con-trols, inaC cells have deactivation kinetics that are >20 timesslower. Molecular cloning of the inaC locus showed that itencodes an eye-specific isoform of PKC (eye-PKC) (19).eye-PKC is found only in photoreceptors, and within photo-receptors, the protein localizes to the light-sensitive microvillarmembranes (19). These results suggest a model in which thelight-dependent generation of DAG (from the breakdown ofPIP2) together with the influx of external calcium activateseye-PKC. Interestingly, eye-PKC is also required for normalkinetics of the return of [Ca2+], to resting levels (29). Thus, thisis a wonderful regulatory loop in which activation of regulatorymechanisms is intimately tied to the productive activation ofthe signaling cascade. Active PKC could then phosphorylatespecific target(s) and mediate termination of the light responseby catalyzing the inactivation of active intermediates. Thetarget(s) of eye-PKC have not yet been identified.

Light-Activated Ion Channels

The Drosophila light-activated conductance is nonselective forcations and is primarily permeable to calcium ions (18, 25).Unexpectedly, the light-activated conductance is composed ofat least two biophysically distinct channels. One of these isencoded by the trp gene product and is responsible for themajority of the calcium permeability (31). Although the mo-lecular identity of the non-trp-dependent light-activated chan-nels is not known, Phillips et al. (41) identified a protein with

FIG. 4. Spatial localization of light-induced calcium transients in transgenic animals expressing a UV rhodopsin in the blue photoreceptor cells.Sequential images of a cell in response to a 33-msec UV stimulus at t = 0 are shown. Calcium changes relative to preflash levels are displayed usinga pseudocolor scale. Images were acquired at a video frame rate of 30 Hz. The rhabdomere of this cell (rhabdo) is distinctly resolved from thecell body (nucl. = nucleus). By the third image (t = 67 msec), the first response in [Ca2+]i is noted as a highly localized increase at the rhabdomerebase. This time window (between 33 and 67 msec) is consistent with the time latency of the light response (11). The subrhabdomeric calcium burstis rapidly followed by a wave of elevated [Ca2+]i largely localized to the rhabdomere. The [Ca2+]i in the cell body also rises, but to a lesser degree.To enhance signals above noise, we time averaged images from five identical flash trials, and each resulting image was subjected to spatial averaging.The bottom, right-hand panel shows a black and white photograph of the same cell at low contrast levels to emphasize the difference between cellbody and rhabdomere. [Reproduced with permission from ref. 29 (copyright Cell Press).]

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significant sequence similarity to the Trp protein that is alsoexpressed in photoreceptors. This gene, called Trp-like (trpl),encodes a protein that displays 39% amino acid identity withTrp.

trp mutants were isolated over 25 yr ago (42) and shown tobe defective in maintenance of the light response (thus thename transient-receptor-potential). We now know that inter-nal calcium is required to maintain a receptor potential andthat trp mutants have lost the major light-activated calciumentry pathway (28, 31, 34). On the basis of these findings,Hardie and Minke (23) have suggested that the Trp phenotyperesults from a failure in the refilling of the internal stores. trpmutant photoreceptors are also inactivated after a strong lightstimulus. This is likely due to the emptying of the stores anda subsequent decrease in the efficiency of the excitationprocess (43, 44).

Analysis of the Trp sequence showed regions of weaksimilarity with neuronal voltage-gated Ca2+ channels (45, 46),consistent with the notion that trp encodes a plasma membranechannel with high calcium permeability. Interestingly, a num-

ber of studies suggest that Trp may be related to the elusivevertebrate Icrac ion channel (calcium-release-activated-channel)(11, 23), and thus Trp homologs may be critically important inthe regulation of intracellular calcium. In efforts to determinewhether Trpl also encodes a component of the light-activatedconductance, we set up to isolate mutations in this gene. Adifficulty in setting up a screen for mutations in trpl is the lackof a reliable, predictable phenotype that defines its loss offunction and the possibility that trp and trpl may serve redun-dant functions. Because of these concerns, we used a screeningstrategy that was based on the loss of Trpl antigen on immu-noblots (20, 47). The advantage of this screen is that it does notrely on a hypothetical physiological or behavioral defect butonly on the presence or absence of Trpl protein. After screen-

ing several thousand chromosomes, we isolated a knock-outmutation in trpl. These mutants are now being subjected todetailed genetic, biochemical, and physiological characteriza-tion. Interestingly, Trp and Trpl are not subunits of the same

channel (B. Niemeyer and C.S.Z., in preparation). Recently,vertebrate homologs of Trp and Trpl have been cloned. Theanalysis of these channels and the trp and trpl mutants is likelyto provide important insight into the biology of this novel classof ion channels and their role in calcium homeostasis.

Future Challenges

Phototransduction has proven to be an ideal model system forthe study of G protein-coupled signaling cascades. Basiccellular phenomena like signal amplification and integration,response deactivation, and adaptation have been first ad-dressed in this signaling pathway. The study of this process alsoresulted in the molecular cloning of the first seven transmem-brane domain receptor (48), the first cyclic nucleotide-gatedion channel (49), and the crystal structure of the first Gaprotein subunit (50). Furthermore, the genetic dissection ofthis pathway in humans and flies has provided fundamentalinsight into the molecular and cellular basis of inherited retinaldisorders (51, 52). However, despite these great advances,many important questions still remain. For example, what are

the determinants of the kinetics of activation? What are thedetailed molecular mechanisms of light and dark adaptation?How do the different signaling molecules interact with eachother and regulate their output? How is response deactivationcontrolled? What are the intracellular messengers in inverte-brate phototransduction? How is signal cross-talk prevented?A complete understanding of the phototransduction process

will have to wait until all the gene products that have a role inthis process are identified and studied by the physiologicaleffect of their loss or dysfunction. It is here where the study ofphototransduction in Drosophila offers unprecedented versa-

tility. The study of this signaling cascade in the fruit flyDrosophila melanogaster makes it possible to use powerfulmolecular genetic techniques to identify novel transductionmolecules and then to examine the function of these moleculesin vivo, in their normal cellular and organismal environment.Recent advances in mouse knockout technology also offer anexciting opportunity for a genetic dissection of this process invertebrates. The combination of these two systems of studymay provide the answer to the biggest challenge for the future:how is the entire response of a photoreceptor cell orchestratedin vivo?

I deeply thank past and present members of my laboratory for theircontributions. This research was funded by grants from the NationalEye Institute, the Pew Foundation, the McKnight foundation, and theMarch of Dimes. C.S.Z. is an investigator of the Howard HughesMedical Institute.

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