It appears that there are two stages of light-dependent trafficking out of the rhabdomeres. Both stages of the TRPL translocation require rhodopsin activity; however, the mechanisms are otherwise different.
In flies expressing a constitutively active derivative of TRP [ ], TRPL is detected primarily in the basolateral membrane even in the dark [ , ]. Furthermore, the mislocalized TRPL was found in a pattern similar to that observed after the first stage of light-dependent translocation out of the rhabdomeres [ ]. Thus, shuttling of TRPL back to the rhabdomeres may also be a two step process.
However, in a second study, elimination of both arr1 and arr2 caused a defect in light-induced movement out of the rhabdomeres, rather than dark-mediated shuttling into the rhabdomeres [ ]. The basis for this difference is unclear. The basis for this latter observation is unclear but could be due to a variety of differences in the experimental protocols, which remain to be resolved.
NINAC would be predicted to be required only for movement into the rhabdomeres, since it may be a plus-end-directed motor, based on analyses of a mammalian myosin III [ — ], and the barbed ends of the actin filaments are at the distal end of the rhabdomeres [ ]. Many of the proteins that function in Drosophila phototransduction are organized into a supramolecular signaling complex the signalplex; Fig.
Each of the three target proteins that are included in the core complex is expressed at similar levels [ ] and may always be present in the complex as they depend on INAD for normal localization in the rhabdomeres and protein stability [ , ].
The signalplex. Members of the core complex depend on the signalplex for normal rhabdomere localization. This additional role may account for the observations that TRP is present in approximately stoichiometric proportions with INAD [ ], which is a level tenfold higher than is necessary for a normal light-response [ ].
The mutual requirement for TRP and INAD for localization reflects mutual roles in retention in the rhabdomeres, rather than for targeting [ , ]. In addition to the core binding proteins, several other proteins bind INAD. Noncore binding proteins do not depend on INAD for localization in the rhabdomeres [ , ]. INAD has the capacity to nucleate a large array of target proteins since it self-assembles into a polymer, and does so through different interaction interfaces in two PDZ domains than those involved in binding other target proteins [ ].
As described above, the core-binding proteins depend on INAD for localization in the rhabdomeres and for protein stability [ , ]. It also seems plausible that nucleation of an array of signaling proteins in a complex would promote rapid signaling.
Interactions of signaling proteins with INAD appear to function in rapid termination of phototransduction. An additional potential function for the signalplex is that INAD might serve to compartmentalize protein kinases and substrates into the same complex reminiscent of the complexes containing receptors for activated C-kinase RACK [ ] and A-kinase anchoring proteins AKAPs [ ].
The Drosophila visual system has proven to be a powerful model for dissecting the molecular mechanisms underlying retinal degeneration. Mutations in almost any gene that functions in phototransduction result in photoreceptor cell death and the majority of the retinal degenerations are light-dependent. The molecular bases underlying the retinal degenerations are diverse Table 3.
The first mutations linked to retinal degeneration were in ninaE , which encode the major rhodopsin, Rh1 [ 40 , 41 , , ]. This observation turned out to have relevance to human retinal dystrophies, as mutations in human rhodopsin were shown subsequently to account for a large proportion of the cases of autosomal dominant retinitis pigmentosa disease ADRP [ — ]. Since Rh1 plays a structural role in photoreceptor cells [ ], in addition to functioning as a light-receptor, most loss-of-function ninaE alleles result in light-independent retinal degeneration [ , ].
The degeneration in ninaE is not dependent on the signal transduction cascade as the severity of photoreceptor cell death mutation is not reduced by disruption of the PLC NORPA , which is required for phototransduction [ , ].
The cell death is associated with accumulation of membranes in the subrhabdomeral regions, although there is considerable variation in the severities of the phenotypes. Many of the mutations in ninaE are dominant [ , ], as is the case for human ADRP disease resulting from mutations in rhodopsin [ , , ]. The dominance may be attributable to misfolding of the rhodopsin derivatives, which in turn interferes with the posttranslational maturation of wild-type rhodopsin [ , , ].
As a consequence, the levels of mature Rh1 are dramatically reduced. The dominant ninaE mutations primarily affect the maturation and transport of Rh1 into the rhabdomeres [ , ]. In Drosophila photoreceptor cells, rhodopsin is synthesized and core-glycosylated in the endoplasmic reticulum ER , transported through the Golgi, and delivered to the rhabdomeres where it functions in phototransduction.
Among the mutations in rhodopsin that affect maturation is one N20I that disrupts the N-linked glycosylations site of Rh1 [ 69 ]. The Rh1 N20I protein is retained in the secretory pathway, resulting in accumulation of ER cisternae and retinal degeneration. Mutations in loci encoding molecular chaperones, which are necessary for rhodopsin maturation, also cause retinal degeneration.
One chaperone, NINAA, is a cyclophilin-related protein, which is thought to promote the proper folding of Rh1 [ 57 , 58 , 60 ].
Mutations in the gene encoding another rhodopsin chaperone, calnexin cnx , result in a phenotype bearing some similarities to ninaA mutant flies [ 70 ].
Rh1 accumulates in the ER and there is retinal degeneration; however, in contrast to ninaA , the retinal degeneration in the cnx mutant is enhanced in the presence of light. Rab6 also appears to be required for rhodopsin maturation, since expression of a dominant negative form of Rab6 causes defective rhodopsin maturation, and trafficking and triggers retinal degeneration [ 64 ].
Taken together, these findings demonstrate that disruption in rhodopsin maturation leads to retinal degeneration. There are at least two possible mechanisms through which defective rhodopsin maturation leads to photoreceptor degeneration. In null or very strong ninaE alleles, the retinal degeneration is a consequence of the structural requirement for rhodopsin during morphogenesis.
Rhodopsin may comprise the majority of total membrane protein in the rhabdomeres, and complete absence of Rh1 during development results in architectural defects that initiate during pupal development [ , ]. Production of even small amounts of Rh1 is sufficient for production of normal rhabdomeres [ , ]. However, the level of Rh1 and the degree of retinal degeneration do not always correlate among the dominant NinaE alleles [ , ].
Rather, retinal degeneration due to dominant NinaE mutations appears to result from inhibition of rhodopsin trafficking, which leads to accumulation of ER cisternae and unfolded rhodopsin in the ER. Constitutive or uncontrolled activity of rhodopsin can also lead to retinal degeneration, such as in the dominant allele, NinaE PP [ ]. Mutation of arr2 also leads to light-dependent retinal degeneration presumably due to uncontrolled activity of rhodopsin [ 95 , ].
Arrestin is necessary for deactivation of rhodopsin, and the cell death in the arr2 mutant is blocked by the dG q 1 mutation [ 95 ] suggesting that it results from excessive activation of the phototransduction cascade. Loss of arr2 also leads to decreased endocytosis of Rh1 as reviewed in the next section.
It appears that during and after light exposure, a small proportion of the rhodopsin pool is removed from the rhabdomeral membrane and degraded, possibly as a quality control mechanism to dispose of photodamaged or constitutively active rhodopsin [ 98 ]. Abnormal increases or decreases in the rhodopsin turnover pathway appear to cause retinal degeneration. In wild-type flies, removal of Rh1 is initiated by interactions with Arr1 [ 99 ] and Arr2 [ ] and subsequent endocytosis of Rh1, which may then be trafficked to lysosomes through direct interactions with a tetraspanin, Sunglasses Sun , present in the membranes of late endosomes and lysosomes [ 31 ].
Mutations in either arr1 , arr2 , or sun result in retinal degeneration because of decreased endocytosis or degradation of rhodopsin. Thus, a defect in degradation of internalized rhodopsin may be toxic. The cell death resulting from loss of arr2 is countered by overexpressing ceramidase [ , ], which cleaves ceramide to produce sphingosine. The ceramidase may decrease the toxicity resulting from absence of Arr2 by increasing endocytosis [ , ]. Unlike the light-dependent cell death in sun [ 31 ] and arr2 flies [ 95 ], the degeneration in arr1 is light-independent [ 99 ].
However, no degeneration occurs in arr1 ; arr2 double mutant flies [ 99 ]. The converse of retinal degeneration resulting from too little endocytosis of rhodopsin is the cell death that occurs from excessive endocytosis due to stable interactions between Rh1 and Arr2. Mutation of the dynamin GTPase shibire , which prevents clathrin-mediated endocytosis, suppresses the retinal degeneration in the rdgC mutant [ 97 ].
Moreover, mutations in arr2 that disrupt the interaction between Arr2 and AP-2 prevent the retinal degeneration in norpA photoreceptor cells [ ]. The severity of the retinal degeneration is greatly suppressed in the calx , trp double mutant [ ]. Trp P flies, which express a constitutively active TRP channel, undergo retinal degeneration. Both the trp 14 and trp P phenotypes are suppressed by loss-of-function mutation in calx and by mutations in arr2 [ ].
Overexpression of the eye-enriched PA phosphatase, Lazaro Laza , which catalyzes dephosphorylation of PA and generation of DAG, enhances both the increased activation of the photoresponse and the retinal degeneration in rdgA mutant, whereas mutation of laza suppresses both aspects of the rdgA phenotype [ 32 , 33 ]. Therefore, the increased DAG appears to be the major reason for the retinal degeneration in rdgA. Consistent with the concept that DAG contributes to excitation during the photoresponse, the retinal degeneration in the rdgA mutant is suppressed by mutations in trp or norpA [ , ].
Operating in opposition to RDGA is Laza, and mutations that eliminate this PA phosphatase result in light-dependent retinal degeneration [ 32 , 33 ].
Overexpression of the PLD results in light-dependent retinal degeneration [ ], which is suppressed by the laza mutation [ 33 ]. Therefore, the retinal degeneration in both the rdgB and cds mutants is likely to be due to decreased PIP 2 levels. However, this possibility is counter to the observation that the retinal degeneration in rdgB is suppressed by the trp mutation but not by mutation of the eye-enriched PKC INAC [ ], which results in slower TRP channel inactivation [ ].
Moreover, chemical inhibition of the channels also inhibits the retinal degeneration phenotype of rdgB [ ]. In addition to the retinal degeneration resulting from mutations in genes that function in phototransduction, many studies in mammals demonstrate that continuous long-term exposure to light of modest intensity can result in photoreceptor cell death and diminish the photoresponse [ ].
The underlying basis of this phototoxicity is poorly understood and it has been presumed that the decrease in the visual response is a secondary consequence of the retinal degeneration.
In flies, constant light also causes loss of the photoresponse and retinal degeneration, which is paralleled by a loss of Rh1 [ ]. The visual impairment and retinal degeneration resulting from continuous light occur through distinct mechanisms [ ].
Conversely, mutations known to suppress most retinal degenerations do not protect against light-induced visual impairment. Despite the diversity in mechanisms underlying photoreceptor cell death in Drosophila , many of the retinal degenerations show features of apoptosis, which is reminiscent of human retinal dystrophies [ 31 , 98 , , ]. Although a lot of progress has been made over the past few years, there remain many unanswered questions regarding the mechanisms of retinal degeneration in Drosophila.
Mutations in Drosophila crumbs and its human homolog lead to light-induced retinal degeneration, but the mechanisms underlying these retinal degenerations are poorly understood [ , ]. The Drosophila retinal pigment cells appear to function similarly to human RPE cells in the generation of the chromophore [ 81 ], and many genetic defects in human RPE cells are known to cause photoreceptor cell degeneration.
Thus, further characterization of the Drosophila retinal pigment cells is warranted. Finally, the recent indications that the ipRGCs operate through a visual cascade remarkably similar to that in Drosophila [ 11 ] have resulted in renewed interest in the mechanism of Drosophila phototransduction. Nature — Pak WL Study of photoreceptor function using Drosophila mutants. In: Breakfield XO ed Genetic approaches to the nervous system.
Elsevier, New York, pp 67— Google Scholar. Hardie RC Whole-cell recordings of the light induced current in dissociated Drosophila photoreceptors: evidence for feedback by calcium permeating the light-sensitive channels. Proc R Soc Lond B — Cell — Montell C, Rubin GM Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron — Pflugers Arch in press.
Berson DM Phototransduction in ganglion-cell photoreceptors. J Neurosci — Science — Brain Res Mol Brain Res — Hannibal J, Hindersson P, Knudsen SM, Georg B, Fahrenkrug J The photopigment melanopsin is exclusively present in pituitary adenylate cyclase-activating polypeptide-containing retinal ganglion cells of the retinohypothalamic tract. J Neurosci RC PubMed Google Scholar. Montell C, Rubin GM The Drosophila ninaC locus encodes two photoreceptor cell specific proteins with domains homologous to protein kinases and the myosin heavy chain head.
Biophys J — J Physiol Lond — Curr Top Dev Biol — Harris WA, Stark WS Heriditary retinal degeneration in Drosophila melanogaster: a mutant defect associated with the phototransduction process. J Gen Physiol — EMBO J — Kwon Y, Montell C Dependence on the Lazaro phosphatidic acid phosphatase for the maximum light response.
Curr Biol — The isolation of Drosophila mutant fly arrestin2 arr2 , enabled demonstrating the physiological effect, in vivo, of ARR2 on the light response Dolph et al. Accordingly, these flies showed a slow response termination at the macroscopic level Figure 5 B. Further investigations have shown that single photon absorption in these flies results in a train of quantum bumps while in wild-type flies it elicits a single bump Figure 5 B.
Moreover, under the assumption that each bump is produced in a single microvillus, the train of bumps separated by intervals suggests a possible inactivation process of the microvilli Hardie and Raghu, Only upon photoregeneration of Mpp to Rpp, is ARR2 released and the Rpp is exposed to phosphatase activity by rhodopsin phosphatase, encoded by the rdgC gene Steele et al. These combined actions are crucial for preventing reinitiating of phototransduction in the dark, as the dissociation of ARR2 is coupled to conversion of Mpp to Rpp, thereby directing the protein phosphatase only towards the inactive Rpp Byk et al.
They furthermore showed that the phosphorylation of ARR2 is required for its dissociation from Mpp upon photoconversion and that ARR2 phosphorylation prevents endocytotic internalization of the ARR2-Mpp complex by a clathrin-mediated mechanism Alloway and Dolph, ; Alloway et al. Upon illumination, ARR2 translocates from the cell body to the rhabdomere, thereby elevating its concentration in the signaling compartment Byk et al. This process enables the ARR2-dependent inactivation of M, operating in massive photoconversion of R to M in bright daylight, thus preventing response saturation and ensures sufficient time resolution of the light response.
Interestingly, the electrophysiological phenotype of the ninaC mutant is similar to that of arr2 mutant Figures 5 B,C and may be the consequence of reduced ARR2 concentration in the rhabdomere caused by the ninaC mutation. It has been well established in photoreceptors of several invertebrate species that photoexcited R activates a heterotrimeric G-protein Fein, The first experiments, conducted on fly photoreceptors, showed that when pharmacological agents, known to activate G-proteins, are applied to Musca photoreceptors in the dark, they mimic the light-dependent activation of the photoreceptor cells Minke and Stephenson, Later studies using genetic screens isolated two genes encoding visual specific G-protein subunits.
These genes, dgq Lee et al. Figure 7. B Number of mean gold particles in cross-sections of 20 different single rhabdomeres. Error bars are SEM. C Whole-cell voltage clamp recordings of spontaneous bumps observed in complete darkness of various mutants as indicated. D Histogram plotting the mean bump frequency of the various mutants. Heterotrimeric G-proteins relay signals between membrane-bound receptors and downstream effectors. A long exposure to light followed by minutes of darkness resulted in reduction in the efficiency with which each absorbed photon elicited single photon responses, while the size and shape of each single photon response did not change.
The key evidence for the participation of PLC in visual excitation of the fly was achieved by the isolation and analysis of Drosophila PLC gene, designated no receptor potential A norpA. Mutant flies in the norpA gene show a drastically reduced receptor potential. Transgenic Drosophila , carrying the norpA gene on a null norpA background, rescued the transformant flies from all the physiological, biochemical and morphological defects, which are associated with the norpA mutants Bloomquist et al.
The norpA mutant thus provides essential evidence for the critical role of inositol-lipid signaling in phototransduction, by showing that no excitation takes place in the absence of functional PLC Bloomquist et al. However, the events required for light excitation downstream of PLC activation remain unresolved. A reduction in the levels of PLC in mutant flies affects the amplitude and activation kinetics of the light response Pearn et al. Biochemical and physiological studies conducted in Drosophila have revealed the requirement for PLC in the induction of GAP activity in vivo.
The virtually complete dependence of GAP activity on PLC provides an efficient mechanism for ensuring the one photon, one bump relationship Yeandle and Spiegler, , which is critical for the fidelity of phototransduction in dim light.
The apparent inability to hydrolyze GTP without PLC ensures that every activated G-protein eventually encounters a PLC molecule and thereby produces a response by the downstream mechanisms. The instantaneous inactivation of the G-protein by its target, the PLC, guarantees that every G-protein produces no more than one bump Cook et al. Accordingly, genetic elimination of regulators of G-protein signaling RGS proteins reduces and slows down GAP activity and leads to slow response termination to light Chen et al.
Figure 8. Slow response termination composed of bumps characterizes norpA mutants. A,B Upper panels: Whole-cell voltage clamp recordings of quantum bumps in response to continues dim light in wild-type and the weak allele of norpA , norpA P57 mutant flies. A,B Lower panels: Whole-cell voltage clamp recordings of normalized macroscopic responses of wild-type and the corresponding mutants in response to ms light pulses. In contrast to the fast response termination of wild-type, slow termination of the light response of norpA P57 mutant flies is revealed.
This slow response termination can be resolved into continuous production of bumps in the dark at a later time inset, at higher magnification. C Electroretinogram ERG responses showing superimposed traces recorded from wild-type and norpA P76 a weak norpA allele to a brief flash red arrow and continuous light.
The graph plots the relative steady state amplitude of the ERG to prolonged lights as a function of relative light intensity. The dual action of PLC as an activator and a negative regulator nicely accounts for all features of the PLC-deficient mutants. A striking demonstration of the poor temporal resolution of mutants with reduced PLC levels relative to wild-type flies is shown in Figure 8 C, which compares the ability of wild type and the PLC-deficient mutant norpA P76 to discriminate between intense lights of different durations flash, red arrow, pulse, blue line.
In contrast to the wild-type fly, where there is a pronounced difference between the responses to a flash compared with a long stimulus, no such difference is observed in the norpA mutants, where the two responses overlap Figure 8 C. This result indicates that the PLC-deficient mutants cannot discriminate between long and short light stimuli.
The continuous functionality of the photoreceptors during illumination is maintained by rapid regeneration of PIP 2 in a cyclic enzymatic pathway the PI pathway, Figure 9.
Figure 9. The phosphoinositide cycle. PA can also be converted back to DAG by lipid phosphate phosphohydrolase. Mutations in most proteins of the PI pathway result in retinal degeneration. Although it is possible to partially rescue the degeneration phenotypes by reducing the level of TRP Raghu et al. A spontaneously occurring Drosophila mutant, showing a decline in the receptor potential to baseline during prolonged illumination Cosens and Manning, , was designated transient receptor potential trp by Minke et al.
The cloning of the trp locus by Montell and Rubin revealed a novel membrane protein. However, the significance of the trp sequence, as a gene encoding a putative channel protein, was only first appreciated after a trp homolog, the trp-like trpl gene was cloned.
This was done by a screen for calmodulin-binding proteins which identified a TM protein. The first direct physiological evidence for the notion that TRP is the major light-activated channel came from a comparative patch clamp study of isolated ommatidia of wild type and the trp mutant Hardie and Minke, The final evidence showing that TRP and TRPL are the light-activated channels came from the isolation of a null mutant of the trpl gene and the construction of the double mutant, trpl;trp , which is blind Niemeyer et al.
Heterologous expression in HEK cells has revealed a functional channel Jors et al. However, in Drosophila photoreceptors this channel cannot generate any light-activated conductance in isolation as revealed in the trpl;trp double null mutant and therefore its role in phototransduction, if any, is not clear. Figure The electrophysiological properties of WT, trp and trpl mutants. A Whole-cell voltage clamp recordings of quantum bumps in response to continuous dim light in wild-type, trpl and trp P null mutant flies.
Highly reduced amplitude of trp P bumps is observed. B Whole-cell voltage clamp recordings in response to a 3-s light pulse of WT and the corresponding mutants.
The transient response of the trp P mutant is observed. C A family of light-induced currents to ms light pulse at voltage steps of 3 mV measured around E rev. The block mainly influenced the TRP channel and affected its voltage dependence. These studies indicated that the voltage dependence of the TRPL channel is not an intrinsic property, as is thought for some other members of the TRP family, but arises from divalent cations open channel block that can be removed by depolarization.
The open channel block by divalent cations is thought to play a role in improving the signal to noise ratio of the response to intense light and may function in light adaptation and response termination Parnas et al.
Since null trp and trpl mutants both respond to light, each can clearly function without the other. However, heterologous co-expression studies and co-immunoprecipitation, led to the suggestion that the TRP and TRPL channels can assemble into heteromultimers Xu et al. Detailed measurements of biophysical properties, questioned this conclusion since they found that the wild-type conductance could be quantitatively accounted for by the sum of the conductances determined in the trp and trpl mutants Reuss et al.
In addition, a study demonstrated that the TRPL, but not the TRP channel reversibly translocates from the rhabdomere to the cell body upon illumination Bahner et al. In neurons the expression pattern of ion channels determines the physiological properties of the cell.
Besides regulation at the level of gene expression that determines which channels are present in a given neuron, trafficking of ion channels into and out of the plasma membrane is an important mechanism for manipulating the number of channels at a specific cellular site for reviews see Lai and Jan, ; Sheng and Lee, The TRPL translocation process occurs in two stages, a fast translocation 5 min to the neighboring stalk membrane and a slow translocation over 6 h to the basolateral membrane Cronin et al.
Thus, the TRPL translocation timescale conforms to day night cycle and act in light adaptation Bahner et al. Signal dependent translocation of mammalian TRP channels was found to be a widespread phenomenon Bezzerides et al.
Nevertheless, many of these researches are conducted on TRP channels expressed in tissue culture cells. This makes the Drosophila photoreceptors a unique system in which TRPL channels translocation can be studied in vivo. However, the mechanism by which PLC activity results in channels opening is still under debate. Several hypotheses have been presented through the years. This mechanism of activation has also been suggested for a number of mammalian TRPC channels Putney, ; Yuan et al.
In addition, direct activation of the channels as in the Limulus ventral photoreceptors Payne et al. Furthermore, genetic elimination of the only InsP 3 R in Drosophila had no effect on the light response Acharya et al. Therefore, the InsP 3 hypothesis was abundant.
However, mutations in the ePKC, encoded by the inaC gene lead to defects in response termination with no apparent effects on activation Hardie et al. In addition, a detailed analysis of the rdgA mutant encoding DAG kinase has established the importance of the DAG branch in channel activation. This mutant shows light-independent retinal degeneration and constitutive activity of the light-activated channels, while a partial rescue of the degeneration is achieved by eliminating the TRP channel in the double mutant rdgA;trp P Raghu et al.
Furthermore, it has been shown that the double mutant norpA P24 , rdgA partially rescues the light response in the almost null norpA P24 mutant. Recently, the inaE gene was identified as encoding a homologue of mammalian sn-1 type DAG lipase and was shown to be expressed predominantly in the cell body of Drosophila photoreceptors Figure 9.
Mutant flies, expressing low levels of the inaE gene product, have an abnormal light response, while the activation of the light-sensitive channels was not prevented Leung et al. The discovery of the inaE gene is a first step in an endeavor to elucidate lipids regulation of the channels see review, Raghu and Hardie, However, Hardie et al.
Accordingly, disruption of this interaction by membrane lipid modification through PLC activation causes the opening of the channels Parnas et al. It is important to realize that PLC activation, which converts PIP 2 , a charged molecule, containing a large hydrophilic head-group, into DAG, devoid of the hydrophilic head-group, is known to cause major changes in lipid packing and lipid—channel interactions Janmey and Kinnunen, This may in turn act as a possible mechanism of channel activation Parnas et al.
This hypothesis suffers from insufficient direct demonstration both in cell expression systems and in vivo. The inaC P and inaD P mutants reveal slow response termination of the macroscopic response to light and of the single bumps. A—C Upper panels: Whole-cell voltage clamp quantum bump responses to continues dim light in wild-type, inaC P and inaD P mutant flies. A slow termination of the bumps is observed in inaC P and inaD P mutant flies. A—C Lower panels: Whole-cell voltage clamp recordings of normalized responses to a ms light pulse of the above mutants.
An important step towards understanding Drosophila phototransduction has been achieved by the finding that some of the key elements of the phototransduction cascade are incorporated into supramolecular signaling complexes via a scaffold protein, INAD Figure 4.
The first discovered inaD mutant, the inaD P , was isolated by Pak and was subsequently cloned and sequenced by Shieh and Niemeyer These domains are recognized as protein modules which bind to a diversity of signaling, cell adhesion and cytoskeletal proteins Dimitratos et al. This binding pattern is still under debate due to several contradictory reports.
Such binding, however, must be dynamic. Accordingly, the association of TRP into transduction complexes may be related to increasing speed and efficiency of transduction events as reflected by the immediate vicinity of TRP to its upstream activator, PLC, and its possible regulator, ePKC Huber et al.
Indeed, genetic elimination of INAC affected the shape of the quantum bump of the inaC null mutant, by inducing slow termination of the bump, composed of dumped oscillating current noise of an unclear underlying mechanism Hardie et al. This conclusion was derived from the use of Drosophila mutants in which the signaling proteins, which constitute the INAD complex, were removed genetically, and also by deletions of the specific binding domains, which bind TRP to INAD.
To demonstrate that a specific interaction of INAD with TRP is required for rhabdomeric localization of the complex, the binding site at the C-terminal of TRP was removed or three conserved residues in PDZ3, which are expected to disrupt the interaction between PDZ domains and their targets were modified. One important function is to preassemble the proteins of the signaling complex.
Another important function, at least in the case of PLC, is to prevent degradation of the unbound signaling protein. A recent study by Ranganathan and colleagues has suggested that the binding of signaling proteins to INAD may be a dynamic process that allows an additional level of phototransduction regulation. This conformational change has light-dependent dynamics that was demonstrated by the use of transgenic Drosophila flies expressing INAD with a point mutation disrupting the formation of the disulfide bond.
They proposed a model in which, ePKC phosphorylation at a still unknown site promotes the light-dependent conformational change of PDZ5, distorting its ligand-binding groove to PLC and thus regulating phototransduction Mishra et al. The study of fly photoreceptors has opened new avenues in biological research, mainly through the exploitation of the power of Drosophila molecular genetics.
Processes and proteins that were discovered in Drosophila have been found to be highly conserved through evolution and thus paved the way for the discovery of important proteins and mechanisms in development and cell signaling in mammals. A striking example is the discovery of the TRP channel protein in the Drosophila photoreceptors, which led to the discovery of the widespread TRP superfamily, which plays crucial roles in sensory signaling of insects and mammals. The activation and regulation of Drosophila TRPs by the inositol-lipid signaling pathway and the major role of PLC in the activation of these channels has wide implications for understanding the activation and regulations of mammalian TRPs.
Even today Drosophila photoreceptors are one of the few systems in which TRP channels are studied in vivo. Another novel molecule that was discovered in Drosophila photoreceptors is the INAD scaffold protein which forms a supramolecular signaling complex. This protein has introduced new concepts in cell signaling dynamics which are still under investigations. An additional advantage of using the fly for research on cellular signaling is that frequently the fly system is less evolutionary evolved relative to mammals, making it simpler to study, while maintaining its core function.
It is therefore anticipated that research using the Drosophila sensory and motor systems will continue to identify new proteins and mechanisms of high biological importance. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Acharya, J. InsP 3 receptor is essential for growth and differentiation but not for vision in Drosophila. Neuron 18, — This reaction leads to the opening of cation-selective channels trp and trpl genes and causes the depolarization of the photoreceptor cells.
Drosophila melanogaster fruit fly [GN: dme ]. PMID: Drosophila photoreceptors and signaling mechanisms. Cell DOI: Primary processes in sensory cells: current advances. Phototransduction and retinal degeneration in Drosophila. Pflugers Arch DOI: TRP channels in Drosophila photoreceptors: the lipid connection. Drosophila phototransduction is one of the best model systems for the study of G protein-coupled PLC signaling 10 , 22 , Not only is the system amenable to molecular genetic analysis but also it can report activity with exquisite sensitivity and specificity: photoreceptor cells are sensitive to single photons, and the signaling pathway can be turned on and off with millisecond kinetics phototransduction in Drosophila is the fastest known G protein cascade, taking just a few tens of milliseconds to go from light activation of rhodopsin to the generation of a receptor potential.
IP3 mobilizes calcium from internal stores, which affects and modulates many cellular processes, and DAG activates members of the PKC family of proteins. Given the central role of PIP2 in signaling, its levels may be expected to be tightly regulated in the cell. Using enhancer trap technology, we identified an eye-specific form of CDP-DAG and isolated mutations in this gene eyecds To determine whether eye-cds mutants have a defect in their signaling properties, wild-type and mutant animals were assayed for their ability to maintain a continuously activated state of the photoreceptor cells because such a state would require the continuous availability of the second messenger PIP 2.
Our results demonstrated that light activation depletes a pool of PIP2 necessary for excitation that cannot be replenished in eye-cds mutants Fig. This phenotype is due exclusively to a defect in eye-cds, because introduction of the wild-type eye-CDS cDNA into mutant hosts fully restores wild-type physiology Fig. Furthermore, inclusion of PIP 2 in the patch pipette is sufficient to restore signaling in the depleted eye-cds mutants Fig. On the basis of these findings, we reasoned that it should be possible to modulate the output of this cascade by experimentally manipulating the levels of eye-CDS.
Indeed, we made. PI P 2 regeneration cycle. Listed in parentheses are the sites of action of known photoreceptor cell-specific proteins in Drosophila adapted from ref.
These results open up the possibility of genetically and pharmacologically manipulating PI P 2 signaling in vivo and highlight three unexpected aspects of PLC signaling. This is demonstrated by the observation that eye-cds mutants are only defective in signaling and only in response to light activation.
This is further demonstrated by the observation that eye-cds mutants display a reduction in the amplitude of their response as a function of the 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 other components of this cycle and make it possible to carry out a comprehensive genetic dissection of the various players required for the functioning and regulation of PI P 2 and its metabolites.
An important and unresolved issue in the study of invertebrate phototransduction has been the identification of the intracellular messenger s that mediate the opening of the lightactivated ion channels. I P 3 , calcium, and cGMP have been implicated in this process 23 , Although the messenger s that actually gates the plasma membrane ion channels remains elusive, patch clamp studies have provided strong evidence implicating calcium in the regulation of the light response 18 , 25 , For example, extracellular calcium influx is both sufficient and necessary to regulate activation and deactivation kinetics of the light-activated conductance.
In the absence of external calcium, photoreceptors display slow activation and deactivation kinetics. Defects shown by eye-cds mutants in photoreceptor cell function. To determine whether eye-cds mutants have a defect in their signaling properties, we assayed wild-type and mutant animals for their ability to maintain a continuous supply of the second-messenger PI P 2.
Control and mutant cells were dissected and transferred to a bath solution with nominally zero calcium. The excitation mechanisms were then depleted before patching by subjecting the cells to 40 min of a light pulse protocol, consisting of 3 sec of intense light pulses followed by 3 min in the dark.
If the same depletion protocol is applied to cds mutant cells, the light response does not recover c , d. This phenotype is due exclusively to a defect in eye-cds because introduction of the wild-type eye-CDS cDNA into mutant hosts fully restores wild-type physiology e , f. Depleted cds mutants can be rescued by supplying PIP2 through the patch pipette g. Arrows indicate the position of the stimulating light flash.
See ref. Despite the role of calcium in regulation, all available data indicate that calcium release from internal stores is neither the signal nor is it required for the opening of the light-activated channels 28 , However, internal calcium stores are required both for the developmental maturation of the lightactivated currents 30 and for maintaining a responsive state For instance, mature currents e.
Past attempts to measure simultaneously light-induced current and fluores. Two possible solutions to this problem are as follows: i to use a fluorescent calcium indicator whose excitation spectrum is well separated from the action spectra of the cell not currently available 35 , 36 , or ii to retune the cell's response to become spectrally separated from available fluorescent calcium indicators.
Such ultramicro domains would, of course, be too small to be resolved by present optical techniques. This provides an elegant avenue to prevent signal cross-talk in intracellular signaling pathways. What are the targets of calcium in mediating negative regulation? An electrophysiological screen for Drosophila phototransduction mutants with defects in deactivation kinetics demonstrated that photoreceptors from inaC mutants 39 are specifically defective in the calcium-dependent negative regulatory mechanisms These results suggest a model in which the light-dependent generation of DAG from the breakdown of PI P 2 together with the influx of external calcium activates eye-PKC.
Thus, this is a wonderful regulatory loop in which activation of regulatory mechanisms is intimately tied to the productive activation of the signaling cascade. Active PKC could then phosphorylate specific target s and mediate termination of the light response by catalyzing the inactivation of active intermediates.
The target s of eye-PKC have not yet been identified. The Drosophila light-activated conductance is nonselective for cations and is primarily permeable to calcium ions 18 , Unexpectedly, the light-activated conductance is composed of at least two biophysically distinct channels.
One of these is encoded by the trp gene product and is responsible for the majority of the calcium permeability Although the molecular identity of the non- trp -dependent light-activated channels is not known, Phillips et al. Spatial localization of light-induced calcium transients in transgenic animals expressing a UV rhodopsin in the blue photoreceptor cells. Calcium changes relative to preflash levels are displayed using a pseudocolor scale.
Images were acquired at a video frame rate of 30 Hz.
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