Drosophila visual transductionCraig Montell

Departments of Biological Chemistry and Neuroscience, Center for Sensory Biology, The Johns Hopkins University School of

Medicine, Baltimore, MD 21205, USA

Review

Visual transduction in the Drosophila compound eyefunctions through a pathway that couples rhodopsin tophospholipase C (PLC) and the opening of transientreceptor potential (TRP) channels. This cascade differsfrom phototransduction in mammalian rods and cones,but is remarkably similar to signaling in mammalianintrinsically photosensitive retinal ganglion cells(ipRGCs). In this review, I focus on recent advancesin the fly visual system, including the discovery of avisual cycle and insights into the machinery and mech-anisms involved in generating a light response inphotoreceptor cells.

IntroductionDrosophila phototransduction has been scrutinized formore than 40 years, and provides a genetic paradigm forsignaling cascades that employ phosphoinositides and TRPchannels [1–4]. During the past decade, it has become clearthat there exists a new class of photoreceptor cells inmammals, referred to as ipRGCs [5], which functionthrough a signaling cascade akin to fly visual transduction[6–9]. The ipRGCs contribute primarily to photoentrain-ment of circadian rhythm, pupillary constriction and sleep[10–15]. In this review, I focus on recent advances inunderstanding the fly visual system. Such findings includethe discovery of a visual cycle, insights into the mechanismactivating the TRP channels, the demonstration of dynam-ic interactions with the inactivation but no afterpotential D(INAD) signaling complex and plastic changes in rhodopsinexpression. Furthermore, I discuss the finding that light-dependent shuttling of a signaling protein depends on asecond type of cascade that is coupled to rhodopsin.

Anatomy of compound eyeThe Drosophila compound eye comprises approximately750–800 reiterating hexagon-shaped ommatidia(Figure 1a), each of which contains 20 cells, including eightphotoreceptors. Six photoreceptor cells (R1–6) extend thefull depth of the retina, whereas the R7 and R8 cells aresituated in the top (distal) and bottom (proximal) halves ofthe retina, respectively. Thus, only seven of the photore-ceptor cells lie in any plane of section (Figure 1b). Photo-reception and signal transduction take place in therhabdomeres, which comprise stacks of microvilli. Approx-imately 50 000 are present in the larger R1–6 cells(Figure 1c,d). These approximately 1.2–1.5 mm-long micro-villi are only approximately 50 nm in diameter and containan F-actin filament, but are devoid of internal organelles.

Corresponding author: Montell, C. (cmontell@jhmi.edu).Keywords: TRP channels; rhodopsin; phototransduction; phospholipase C.

356 0166-2236/$ – see front matter � 2012 Elsevier Ltd. All rights res

The secondary retinal pigment cells (RPCs) are the maincells surrounding the photoreceptor cells (28 PC;Figure 1b). Tertiary RPCs and mechanosensory bristlecells occupy alternating vertices of the ommatidia (38 PCand bristle cell; Figure 1b).

In addition to the compound eye, adult flies have threesmaller and simpler eyes (ocelli) located on the top of thehead. Ocelli seem to be more important for detectingchanges in light intensities during flight than for imageformation [16].

Fly phototransduction and its relationship to thecascades in rods and conesPhototransduction cascades serve to amplify single photonresponses, and to allow cells to adapt to light with intensi-ties that differ over many orders of magnitude. The initia-tion of these signaling pathways depend on light sensorsthat comprise a seven transmembrane-containing protein(opsin) linked to a chromophore (3-hydroxy 11-cis-retinalin Drosophila, and 11-cis-retinal in rods and cones). Lightpromotes isomerization of the chromophore to the all-transconfiguration, thereby inducing a conformation change inthe protein subunit. The photoactivated visual pigmentstimulates GDP–GTP exchange in a heterotrimeric G-pro-tein. In the fly eye, the G-protein (Gq) [17] activates thePLC encoded by norpA [18], which hydrolyzes phosphati-dylinositol 4,5-bisphosphate (PIP2), leading to opening ofthe TRP channel and the related TRP-like (TRPL) cationchannel in photoreceptor cells (Figure 2) [19–23]. NORPAalso accelerates the intrinsic GTPase activity of the Gqa

[24–26]. Thus, NORPA promotes activation and negativefeedback regulation through distinct domains [24–26]. Theincrease in Ca2+ is counterbalanced by extrusion throughthe Na+–Ca2+ exchanger, CalX [27].

Several of the key aspects of Drosophila phototrans-duction are distinct from the cascades in mammalian rodsand cones. In flies, rhodopsin is bistable (i.e. the chromo-phore usually stays bound to the opsin following exposureto light) [28]. A second photon of light is necessary toconvert the 3-OH-all-trans retinal back to the 3-OH-11-cis-retinal. If the flies are exposed to blue (480 nm) light,the major rhodopsin (Rh1) remains active in the dark. Inrods and cones, the light-activated chromophore, all-trans-retinal, is released from the opsin and must berecycled through an enzymatic pathway [29,30]. The het-erotrimeric G-protein that is activated by the mammalianvisual pigments (transducin; Gt) stimulates a phospho-sphodiesterase, causing a decline in cGMP levels andsubsequent closure of the cGMP-gated channels [31].Thus, light induces opposite affects on the state of the

erved. doi:10.1016/j.tins.2012.03.004 Trends in Neurosciences, June 2012, Vol. 35, No. 6

(d)

Microvilli inrhabdomere

Cellbody

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(a)

4

(b)

12

36

7

53° PC

2° PC

rhab.

Bristlecell

500 nm

(c)

rhab.

Cellbody

Cell body

Figure 1. Anatomy of the Drosophila compound eye and photoreceptor cells. (a) Scanning electron microscope (SEM) image of an adult compound eye. The eye contains

approximately 750–800 ommatidia. The red box indicates one ommatidium. (b) Cartoon illustrating the various cell types in a cross-sectional view of an ommatidium (distal

region). Seven photoreceptor cells, each containing a rhabdomere, are shown: bristle cell, mechanosensory bristle cell; cell body, photoreceptor cell body; rhab.,

rhabdomere; 28 PC, secondary retinal pigment cells; and 38 PC, tertiary retinal pigment cells. The broken blue line indicates a single photoreceptor cell. (c) Transmission EM

cross-section through one photoreceptor cell. (d) Cartoon showing a longitudinal view of one photoreceptor cell. Approximately 50 000 microvilli are present in the

rhabdomeres of each R1–6 cell. The microvilli are not drawn to scale. Normally, there are 30–35 (50-nm wide) microvilli per cross-section.

Review Trends in Neurosciences June 2012, Vol. 35, No. 6

cation channels in the rhabdomeres, and in the outersegments of rods and cones.

Mammals sensing light like fliesDirect light activation of the ipRGCs is mediated by thevisual pigment melanopsin, which bears greater sequence

2+

Gqα

PIP2 IP3 + H+ + DAG

Rhodopsin

DAGlipase

1+

2-

3 Na+ Cal1

2

34

6

GTP

RAL

PLCNORPA

βγ

Figure 2. Model of the Drosophila phototransduction cascade. Events in phototransduct

heterotrimer Gq protein. The activated Gqa subunit associates with GTP; (3) stimulatio

lipase encoded by inaE hydrolyzes DAG to produce 2-MAG and FA. Minor products are 1

lipase; (5) TRP and TRPL are activated following PLC stimulation, although the mechani

exchanger (CalX) extrudes Ca2+ out of the photoreceptor cell. Abbreviations: DAG,

monoacylglycerol; PIP2, phosphatidylinositol 4,5-bisphosphate; P, pore loop indicat

chromophore (3-OH-11-cis-retinal); TRP, transient receptor potential (channel); TRPL, T

homology to fly rhodopsin than to the mammalian rod andcone visual pigments [5,8,10]. Similar to the major Drosoph-ila rhodopsin (Rh1), melanopsin is maximally activated byblue light, and appears to be a bistable photopigment [7,32].

A variety of pharmacological, electrophysiological andexpression studies in heterologous systems and in native

TRP and TRPL

MAGlipase?

-MAG PUFA

MAG + FA

PUFA

Ca2+

X

5

Ca2+

Na+?

P

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ion: (1) light activates rhodopsin; (2) coupling of light-activated rhodopsin with the

n of PLC leads to hydrolysis of PIP2 and production of IP3, DAG and H+; (4) a DAG

-MAF and PUFA. The 2-MAG might be metabolized into PUFA by an unknown MAG

sm remains controversial; and (6) following activation of the channels, a Na+–Ca2+

diacylglycerol; FA, saturated fatty acid; IP3, inositol 1,4,5-trisphosphate; MAG,

ed in TRP; PLC, phospholipase C; PUFA, polyunsaturated fatty acid; RAL, the

RP-like.

357

Rh1

Rh1

Rh1Rh1

Rh1

Rh1Rh1

Rh1

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Rh1 Rh1

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Rh1

Wild type (c)(b)

Yellow ommatidia

rh6 mutant

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Rh3R7

R8

def

Yellow ommatidia~70%

Rh6

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R8

Ss

def

Rh5

Rh4Rh4

Rh5 Rh5

Rhodopsin Expression pattern Peak sensitivityrhodopsin (nm)

Peak sensitivitymetarhodopsin (nm)

~566~506~468~470~494~468~515R8 (~70%)

R7 (~70%)R8 (~30%)

R7 (~30%)

~442~355~331~418~486R1–R6

Ocelli

???

Rh1Rh2Rh3Rh4Rh5Rh6Rh7

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Figure 3. Expression of Drosophila rhodopsins. (a) Five of the six characterized

rhodopsins (Rh1 and Rh3–6) are expressed in photoreceptor cells in the compound

eye. Rh2 is expressed in the ocelli. The peak light sensitivities of the non-activated

(rhodopsin) and light-activated pigments (metarhodopsin) are indicated. (b)

Shown are the spatial distribution of Rh1 and Rh3–6 in the eight photoreceptor

cells within an ommatidium (wild-type fly reared under a normal light–dark cycle).

Rh1 is expressed in the R1–6 cells of all ommatidia. Rh3–6 are expressed in non-

overlapping subsets of R7 and R8 cells, as indicated. Yellow ommatidia normally

express Rh4 and Rh6 in the R7 and R8 cells, respectively. Pale ommatidia express

Rh3 and Rh5 in the R7 and R8 cells, respectively. Expression of Rh4 is induced by

the Spineless (Ss) transcription factor [36]. If Ss is not expressed in a given Rh7

cell, then Rh3 is turned on as the default (def) state. Rh5 is induced in R8 cells that

are below Rh3-expressing R7 cells. As a default, Rh6 is expressed in an R8 cell if it

is not below an Rh7 cell that expresses Rh3. Expression of Rh6 in R8 cells inhibits

expression of Rh5 in these cells. (c) Rh5 is turned on in most yellow ommatidia of

rh6 mutant flies [37]. If wild-type flies are maintained constantly in the dark, some

yellow ommatidia express low levels of Rh5 in addition to Rh6 [37].

Review Trends in Neurosciences June 2012, Vol. 35, No. 6

cells indicate that melanopsin engages a Gq–PLC–TRPCsignaling cascade [6,7,33,34]. The requirement for PLCsignaling has been solidified in a recent study demonstrat-ing that loss of the mouse homolog of Drosophila NORPA(i.e. PLCb4) dramatically reduces the light response of theM1-ipRGCs, which are the most light-responsive ipRGCs[9]. Phototransduction in these cells depends on the TRPcation (TRPC) channels TRPC6 and TRPC7, as a doublemutant eliminated photosensitivity [9]. Given that theDrosophila TRP and TRPL channels are the classic TRPCchannels, these findings further underscore the commonfeatures of phototransduction in ipRGCs and fly photore-ceptor cells.

The sphincter muscle cells in the iris of certain noctur-nal and crepuscular (i.e. most active at dawn and dusk)mammals are also photosensitive and contribute to a localpupillary light response. It is proposed that the iris fromnocturnal and/or crepuscular animals is directly photosen-sitive because their eyes comprise mostly rods and, underbright light, the pupil may be constricted to such an extentthat insufficient light reaches retinal photoreceptor cells todirect a pupillary response [9]. The phototransductioncascade in these photosensitive muscle cells is dependenton melanopsin and PLCb4, as is the case for the ipRGCs[9]. However, their light response is not dependent on aTRPC channel, or on the TRP vanilloid (TRPV) channel,TRPV4, which functions in smooth muscle cells [9]. Nev-ertheless, the phototransduction cascade in the iris may bedependent on another TRP channel. If so, light signaling inthese sphincter muscle cells would represent another Dro-sophila-like phototransduction cascade.

Drosophila rhodopsins and plasticity in the adult eyeSeven opsin genes (Rh1–Rh7) are encoded in Drosophila,six of which (Rh1–6) fully account for the photoresponses ofthe different classes of photoreceptor cell in the adultcompound eyes and ocelli (Figure 3a). The major rhodop-sin, Rh1, is expressed in the six outer photoreceptor cells(R1–6 cells), which respond to dim light (Figure 3a,b). TheR7/R8 photoreceptor cells each express one of four differentrhodopsins, and are involved in color vision, including thedetection of ultraviolet light by the R7 cells (Figure 3a andb) [35]. A random set of approximately 30% of the omma-tidia coordinately express Rh3 in the R7 cells and Rh5 inthe R8 cells (pale ommatidia; Rh3/Rh5; Figure 3b). Most ofthe remaining approximately 70% of ommatidia expressRh4 and Rh6 in the R7/R8 photoreceptor cell pairs (yellowommatidia; Rh4/Rh6). In addition, there are two minorclasses of ommatidium located near the dorsal rim thatdeviate from this pattern.

The control underlying the limitation of one opsin per R7or R8 neuron in the pale and yellow ommatidia begins withstochastic expression of the Spineless (Ss) transcriptionfactor in 70% of R7 cells, and induction of Rh4 expressionin these cells [36]. As a default, R7 cells that do not expressSs turn on Rh3 (Figure 3b). The Rh7 cells that express Rh3then induce expression of Rh5 in the underlying R8 cells inpale ommatidia (Figure 3b). Expression of Rh6 in the R8cells is the default if Rh3 is not expressed (Figure 3a).

The continued exclusion of Rh5 from R8 cells inyellow ommatidia occurs through a mechanism involving

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a negative feedback signal induced by Rh6, which inhibitstranscription of rh5 [37]. The signal requires activity ofRh6 and the Gqa, but not NORPA or the TRP and TRPLchannels. In rh6 mutant flies, expression of Rh5 graduallyexpands with age to include nearly all R8 cells, includingthose in yellow ommatidia (Figure 3c) [37]. If wild-type fliesare maintained in the dark, a low level of Rh5 expression isturned on in some yellow R8 cells that also express Rh6[37]. The mechanism that links Rh6 activity with tran-scriptional repression of rh5 remains to be determined.

The finding that Rh6 prevents expression of Rh5 illus-trates a role for a rhodopsin that is distinct from its classicfunction in vision. The major rhodopsin, Rh1, has two light-independent roles, one of which includes a requirement forthis rhodopsin for rhabdomere morphogenesis [38]. Inaddition, Rh1 initiates a thermosensory signaling cascade,which permits larvae to select their preferred temperaturein the comfortable range [39]. Whether Drosophila rhodop-sins have other light-independent or non-visual roles hasnot been reported, but are intriguing possibilities.

Bistable photopigments and an enzymatic visual cycleThe Drosophila visual pigments are bistable pigments;therefore, they require absorption of one photon to activate

Review Trends in Neurosciences June 2012, Vol. 35, No. 6

rhodopsin, and a second photon to convert the light-acti-vated metarhodopsin back to the non-active rhodopsin.Because the regeneration of the chromophore is normallya light-driven process, it has been assumed that Drosophi-la rhodopsins do not rely on an enzymatic cycle to regen-erate the chromophore. However, recent findingsdemonstrate that flies use a visual cycle after all.

During light stimulation, a fraction of the rhodopsinpool is internalized and the opsin is degraded, therebyreleasing the chromophore. The free 3-OH-all-trans-retinalis then recycled through an enzymatic pathway thatincludes the retinal dehydrogenases, pigment cell dehy-drogenase (PDH) [40] and retinal dehydrogenase B(RDHB) [41], and an isomerase that remains to be identi-fied (Figure 4). These enzymatic steps take place in theRPCs that surround the photoreceptor cells. The essentialrole for the RPCs is reminiscent of the requirements forcells in the retinal pigment epithelium and Mu ller cells forregeneration of the chromophore used in rods and cones,respectively [29,30].

In Drosophila, the visual cycle allows the flies to maintainchromophore levels and a normal visual response underconditions in which dietary limitations prevent them fromsynthesizing new chromophore [40]. Melanopsin alsoappears to be a bistable pigment [7,32], although it is notknown whether it depends on a visual cycle. If so, the Mu llerglia are candidate cells for participating in this processbecause they are situated near the ipRGCs and functionin the visual cycle necessary for the cone pigments [15].

Retinal pigment cells

Isomerase?

(Light-dependent)

3-OH-11- cis-retinol

RDHB

3-OH-11- cis-retinal

3-OH-11- cis-retinal

3-OH-all-trans-retinol

PDH

HO HO HO

HO O HO

OH

O

Photoreceptor cells

3-OH-11- cis-retinol

Opsinsynthesis

Opsindegradation

3-OH-all-trans-retinal

HO N K

K N

HO

3-OH-all-trans-retinal

3-OH-all-trans-retinal

Light

Light

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Figure 4. Model of the visual cycle. Following light stimulation of rhodopsin, a

proportion of the rhodopsin pool is internalized and degraded, thereby liberating the

3-OH-all-trans-retinal. The 3-OH-all-trans-retinal is then transported to primary and

secondary retinal pigment cells, where it is converted into 3-OH-11-cis-retinal

through several enzymatic steps that depend on at least two retinal dehydrogenases,

pigment cell dehydrogenase (PDH) [40] and retinal dehydrogenase B (RDHB) [41],

and a putative isomerase that has not been identified. The 3-OH-11-cis-retinal is

then transported into the photoreceptor cells.

Mechanism of activation of TRPTRP is the classic member of the TRP superfamily[19,20,42], and activation of this Ca2+-permeable cationchannel is strictly dependent on stimulation of the PLCb

encoded by norpA [18]. However, the mechanism linkingPLC with the opening of TRP, and the related TRPLchannels, remains controversial. Stimulation of PLCresults in hydrolysis of PIP2 and production of inositol1,4,5-trisphosphate (IP3) and diacylglycerol (DAG;Figure 2). Although largely ignored, a single H+ is alsogenerated [43] (Figure 2). Thus, TRP and TRPL could beactivated either by a reduction in inhibitory PIP2, or anincrease in H+, IP3, DAG, or by metabolites that areproduced by these products, such as polyunsaturated fattyacids (PUFAs). IP3 does not appear to contribute to channelgating because release of caged IP3 does not mimic the lightresponse, and mutation of the sole IP3 receptor encoded bythe fly genome does not impair activation [44–46].

There are currently two prevailing models. The first isthat PUFAs activate the channels. This concept is sup-ported by whole-cell and single-channel recordings of iso-lated ommatidia indicating that TRPL is activated byPUFAs [47,48]. DAG lipases metabolize DAG, and muta-tion of a gene (inaE) encoding a putative DAG lipase(Figure 2), severely impairs the light response [49]. Asecond model, supported by a recent study, is that adecrease in inhibitory PIP2, in combination with a localacidification, activates TRP and TRPL [43]. This work alsoprovides in vivo evidence that light stimulation leads toPLC-dependent acidification. However, acidification withthe protonophore 2,4-dinitrophenol is most effective atactivating the channels, and this chemical is a mitochon-drial uncoupler. Given that metabolic stress can also acti-vate the channels [50], it cannot be excluded that this is thebasis for activation by 2,4-dinitrophenol. Another possibil-ity is that full activation of the channel involves a decline inPIP2 in combination with a rise in DAG [51], PUFA, H+ andpossibly other metabolites.

Light-dependent movements of signaling proteinsMultiple signaling proteins in mammalian and fly photo-receptor cells undergo light-dependent changes in spatialdistribution [52]. In Drosophila photoreceptor cells, TRPL,Gq and Arrestin1 and Arrestin2 (Arr1 and Arr2) shuttle inand out of the rhabdomeres dynamically [53–57]. Theselatter two proteins bind to rhodopsin, and contribute totermination of signaling by blocking the rhodopsin–Gqinteraction.

The light-induced movements of some of these proteinsare opposite in direction, and correlate with their rolesduring phototransduction. TRPL and Gq function in acti-vation and are concentrated in the rhabdomeres of dark-adapted flies [56–59]. Upon light stimulation, these twoproteins translocate to the cell bodies over the course ofminutes. By contrast, Arr1 and Arr2 participate in termi-nation of phototransduction and shuttle from the cellbodies into the rhabdomeres in response to light [54,55],with a time constant of under 10 sec [60]. The oppositevector of these movements makes sense, given that thelight-dependent translocations participate in light adapta-tion [55,56]. A decline in TRPL and Gq in the rhabdomeres,

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Review Trends in Neurosciences June 2012, Vol. 35, No. 6

or an increase in Arr1 and Arr2, all serve to attenuatesignaling.

Translocation of TRPL, Gq and Arr1/2 between the twomajor compartments of the photoreceptor cells depends onrhodopsin, which is expected given that the trafficking islight dependent [59,61,62]. However, there are some sur-prising findings concerning the requirements for otherphototransduction proteins. Two studies indicate thatthe phototransduction cascade comprising PLC, TRPand TRPL is not required for movement of Gqa [57,58].Similarly, Arr2 can translocate into the rhabdomeres inthe absence of any of classic proteins that function down-stream of Rh1, such as PLC and TRP [61]. However, thekinetics of the movements is reduced at least ten times inthe absence of these proteins owing to a contribution ofCa2+ influx for the translocation [60]. Nevertheless, thereis a Gq–PLC–TRP-independent mechanism that contrib-utes to light-induced movement of Arr2. This second path-way includes the small GTPase, Rac2, which associateseither directly or with Rh1 in vivo [61]. Thus, in addition tothe classic phototransduction cascade, there is a secondRac2-dependent cascade in Drosophila photoreceptor cells.

Several studies focusing on vertebrate rhodopsin indi-cate that a similar non-classic phototransduction pathwayexists [63]. Mammalian Rac1, which is the homolog of flyRac2, associates with mammalian rhodopsin and is acti-vated by light. These findings raise the possibility thatsuch as a cascade also participates in Arr2 translocation inrod photoreceptor cells.

The effector proteins that couple with Rac2 in fly pho-toreceptor cells are not known. However, one candidate isphospholipase D (PLD), an enzyme that can be controlledby small G proteins [64]. G protein-coupled receptors of therhodopsin family can activate PLD. Furthermore, in bovineretinas, the small GTPase RhoA has been reported toregulate PLD activity in a light-dependent manner [65],and the Drosophila genome encodes a PLD that appears tocontribute to phototransduction [66].

The light-dependent movement of TRPL out of therhabdomeres occurs in a two-step process. During the firststage, which occurs over the course of a few minutes, TRPLtranslocates to the apical region of the plasma membranejust outside the rhabdomeres (stalk membrane) [59]. Thesecond stage proceeds for several hours and results in aredistribution of TRPL over the basolateral membrane ofthe photoreceptor cells. NORPA is required for the firststage, whereas the entire phototransduction cascade, in-cluding activation of the TRP channel, participates in thesecond stage [59]. In addition, two small GTPases, Rab5and RabX4 [67], as well as both the N- and C-terminalregions of TRPL, appear to contribute to internalization ofTRPL [68]. The proteins that bind to these domains andpromote the translocation of TRPL are not known.

A controversial issue is whether the neither inactivationnor afterpotential C (NINAC) myosin III participates indynamic movements of signaling proteins. Three indepen-dent studies have found that NINAC participates in thetranslocation of Gq, TRPL and Arr2 [58,62,69]. Anotherstudy has challenged this conclusion, at least with respectto the contribution of NINAC to shuttling of Arr2 [53].NINAC has been reported to promote the light-dependent

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translocation of TRPL from the rhabdomeres to the cellbodies [62]. In the case of Gqa (Ga49B) [58] and Arr2 [69],NINAC may function in movements from the cell bodies tothe rhabdomeres, although this occurs under opposite lightconditions (dark and light, respectively). The interactionbetween NINAC and Arr2 is indirect, and may be mediatedby interactions with the same vesicles, given that bothproteins bind phosphoinositides [55,69].

If NINAC participates in the movements of signalingproteins, the question arises as to whether this activity ismediated by the motor activity of this myosin III. Humanmyosin III is a plus-ended motor [70]. However, NINACmotor activity has not been demonstrated, despite con-siderable effort (e.g. Porter, Sellers and Montell, unpub-lished observations). The plus end of F-actin orientstowards the distal tips of the rhabdomeres [71], whichis consistent with a function for NINAC in the transloca-tion of Gq and Arr2 into the rhabdomeres. However, anactivity as a plus-ended motor is inconsistent with a rolein the shuttling of TRPL from the rhabdomeres to the cellbodies. Thus, NINAC may not retain motor activity.Rather, it is possible that the cell body and rhabdo-mere-specific isoforms of NINAC (i.e. p132 and p174,respectively) [72,73] function in dynamic spatial redis-tributions of signaling proteins by passive association,and so act as a sink in one compartment or the other. If so,this would support the recent contention that the shut-tling of signaling proteins is driven by diffusion, ratherthan by an active motor [60].

The INAD signalplex and redox modulation of signalingMany of the proteins that function in Drosophila photo-transduction are grouped in the rhabdomeres into a largemacromolecular assembly referred to as the ‘signalplex’[74–78]. The central scaffold in the signalplex is INAD, aprotein consisting of five tandem, approximately 90 aminoacid protein interaction modules referred to as PDZdomains. The core complex includes INAD and three targetproteins: TRP, PLC (NORPA) and a protein kinase C (PKC)encoded by the inaC gene [79]. Loss of INAD results ininstability of these three proteins and disrupts their locali-zation in the rhabdomeres [74,80]. The stability and spa-tial distribution of INAD is reciprocally dependent on TRP[79,81]. Thus, TRP is required both as a cation channel andas a molecular anchor. The concentration of INAD alsodeclines if it not bound to the NINAC myosin III, or if thereare mutations in a membrane occupation and recognitionnexus (MORN)-domain containing protein, tetinophilin,which is required for stability of NINAC [82].

INAD also binds to itself and forms a homo-oligomerthrough a PDZ–PDZ interaction interface distinct from thesurface groove that functions in target binding [77]. Thus,INAD may couple an extensive array of TRP channels withthe other proteins required for phototransduction. Severalother proteins that might bind to INAD include calmodu-lin, TRPL and a fraction of the rhodopsin (Rh1) pool[74,77]. These signaling proteins are not dependent on thisinteraction for stability or localization, and their interac-tions with INAD might be dynamic. In contrast to themembers of the core complex, there is not a consensusthat these latter proteins bind INAD [83].

Review Trends in Neurosciences June 2012, Vol. 35, No. 6

Two studies demonstrate that interactions of at leastsome of the signaling proteins that bind to INAD aredynamic, and are regulated by light-dependent conforma-tional changes in PDZ5 [84,85]. PDZ domains are b-barrelstructures that contain a surface groove into which targetproteins form hydrogen bonds [86]. PDZ5 in INAD isunusual in that two cysteines juxtaposed on either sideof the surface groove form disulfide bonds, but only uponlight stimulation (Figure 5a) [84]. Once generated, thisprecludes binding of target proteins. Under dark condi-tions, the disulfide bond is reduced, thereby creating atypical binding pocket [84].

The dynamic oxidation and reduction of the disulfidebonds in PDZ5 depend on interactions with the adjacentPDZ4 domain [85]. In the dark, the two PDZ domainsinteract stably (Figure 5b), thereby promoting the reducedstate by increasing the redox potential of the disulfide bondby 330 mV. Upon exposure to light, PDZ4–PDZ5 couplingis disrupted, resulting in disulfide formation. It is proposedthat light dissociates PDZ4 from PDZ5 owing to the acidi-fication generated by hydrolysis of PIP2 [85].

The light-induced oxidation of PDZ has physiologicalconsequences, because it prevents target binding. One such

SHHS

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Ligand binding disruptedLigand binding

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Oxidized statePDZ5

Reduced statePDZ5

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3

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(a)

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Dark?

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Figure 5. Dynamic binding of TRP with INAD. (a) The two cysteines lining the

surface groove in INAD PDZ5 are reduced in the dark [84,98]. As a consequence,

PDZ5 can bind to binding proteins, including the C-terminus of TRP [84]. Light

results in oxidation of the two key cysteines in PDZ5, which precludes target

binding. (b) In the dark, PDZ4 and PDZ5 interact, thereby promoting the reduced

state in PDZ5 [85]. Under these conditions TRP binds to PDZ5 through the C

terminus, and to PDZ3 via a separate binding site near the C terminus. Following

light stimulation, the PDZ4–PDZ5 interaction is disrupted, leading to oxidation of

PDZ5 [85]. This prevents binding of the TRP C terminus to PDZ5. Given that the

affinity of the internal binding site in TRP to PDZ3 is weak, binding to PDZ3 may

dissociate as a secondary consequence of the oxidation of PDZ5. However, the

light-induced impairment of the TRP–PDZ3 interaction is speculative, as indicated

by a question mark. Abbreviations: INAD, inactivation but no afterpotential D; TRP,

transient receptor potential (channel).

INAD-binding protein is TRP, which interacts with bothPDZ3 and PDZ5 through distinct sites in the channel. TRPbinds to PDZ5 through a classic C-terminal PDZ-bindingmotif, and to PDZ3 via a site near the C terminus [79,85]. Itis proposed that, in the dark, when TRP is bound to bothPDZ domains, TRP is maximally sensitive to activation[85]. Upon light stimulation, the TRP–PDZ5 interaction isdisrupted, which is proposed to lead to full dissociation ofthe TRP–INAD interaction since TRP binding to PDZ3 istoo weak to maintain the association between the twoproteins (Figure 5b). As a consequence, the sensitivity ofTRP to activation declines. Thus, production of H+ by PIP2

might contribute to both activation and negative feedbackregulation.

Concluding remarks and future perspectivesThe discovery that light-dependent changes in redox po-tential affect dynamic interactions of TRP with PDZ5raises several questions. Is there a disulfide isomerasethat promotes the conformational switch? A protein withthis predicted activity is enriched in the fly eye [87]. Giventhat the kinetics of the oxidation of PDZ5 appears to be tooslow to account for the rapid kinetics of signaling, themechanisms that work in concert with this reaction topromote the speed of phototransduction remain to bedetermined. If light causes full dissociation of TRP fromINAD, how is TRP maintained in the rhabdomeres duringlight stimulation? This is an issue because TRP depends onits interaction with INAD for localization in the rhabdo-meres [74], and remains in the rhabdomeres during pro-longed light stimulation [56]. Furthermore, mutation ofthe C-terminal PDZ5-binding site in TRP causes misloca-lization of TRP [79]. Perhaps wild-type TRP remains boundto PDZ3 after dissociation from PDZ5, or the C terminus ofTRP binds to an alternative PDZ domain in INAD after itsinteraction with PDZ5 is occluded by the light-inducedoxidation of PDZ5.

The nexus between PIP2 hydrolysis and activation ofTRP and TRPL remains controversial. Although it is anintriguing concept that a decline in inhibitory PIP2 com-bined with a rise in H+ might be the mechanism [43], arecent study argues that PUFAs gate TRPL [88], as pro-posed previously [47]. Although there are many reportsdescribing the biophysical properties of TRPL in heterolo-gous expression system [89–94], until now there have beenonly three groups that have reported functional expressionof TRP in vitro [90,95,96]. Apparently, this is the result ofdifficulties in obtaining surface expression of TRP in ex-pression systems. This obstacle may be reduced by therecent demonstration that a protein called XPORT aug-ments the transport of TRP from the endoplasmic reticu-lum to the plasma membrane in tissue culture cells [97].

The recent insights concerning the machinery involved inthe light response in Drosophila photoreceptor cells havepotential implications for the ipRGCs, and for the light-sensitive cells in the iris of nocturnal and crepuscular mam-mals [9]. Are mammalian TRPC6 and/or TRPC7 activated byPUFAs or a combination of a decline in PIP2 and a rise in H+?The existence of a Drosophila visual cycle [40] despite thebistability of the fly visual pigments suggests that the mel-anopsin in ipRGCs also depends on an enzymatic pathway

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for regenerating the chromophore. The melanopsin andPLCb-dependent light response in the iris sphincter musclecells would appear to function through a TRP channel,although TRPC channels and TRPV4 have been excluded[9]. Nevertheless, the discovery of melanopsin-initiated sig-naling in mammalian eyes indicates that the mechanism oflight detection in flies and mammals has a common andancient origin.

AcknowledgmentsI thank the National Eye Institute for supporting the research in mylaboratory on Drosophila phototransduction, Wendy Novak for assistancewith preparation of the Figures and Jinfei Ni for providing the EM imagein Figure 1c.

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