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European Journal of Cell Biology 90 (2011) 620–630

Contents lists available at ScienceDirect

European Journal of Cell Biology

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he Drosophila TRPL ion channel shares a Rab-dependent translocation pathwayith rhodopsin

laudia Oberegelsbacher, Carina Schneidler, Olaf Voolstra, Alexander Cerny, Armin Huber ∗

epartment of Biosensorics, Institute of Physiology, University of Hohenheim, 70599 Stuttgart, Germany

r t i c l e i n f o

rticle history:eceived 30 November 2010eceived in revised form 1 February 2011ccepted 7 February 2011

a b s t r a c t

The Drosophila visual transduction cascade is embedded in the rhabdomeres of photoreceptor cells andculminates in the opening of the two ion channels, TRP and TRPL. TRPL translocates from the rhabdomeresto the cell body upon illumination and vice versa when flies are kept in the dark. Here, we studiedthe mechanisms underlying the light-dependent internalization of TRPL. Co-localization of TRPL andrhodopsin in endocytic particles revealed that TRPL is internalized by a vesicular transport pathway that

eywords:rosophilandocytosison channelab protein

is also utilized, at least partially, for rhodopsin endocytosis. TRPL internalization is attenuated underlight conditions that result in a high rate of rhodopsin internalization and is highest in orange light thatresult in very little rhodopsin internalization. In line with a canonical vesicular transport pathway, wefound that rab proteins, Rab5 and RabX4, are required for the internalization of TRPL into the cell body.

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hodopsinRPLision

Our results provide insigsuperfamily.

ntroduction

Signaling proteins in the apical plasma membrane of polarizedells undergo a turnover that is mediated on the one hand by vesic-lar transport of newly synthesized or stored proteins to the plasmaembrane and on the other hand by internalization of membrane

roteins through endocytic pathways. Regulated incorporation andemoval of signaling proteins determine the number of signal-ng molecules in the plasma membrane and hence the signalingapacity of the cell. For example, many G-protein coupled recep-ors become internalized upon stimulation, resulting in reductionf the number of receptors at the cell surface and a decrease inhe sensitivity to subsequent stimuli (for reviews see Achour et al.,008; Ferguson, 2001; Marchese et al., 2003).

In the Drosophila compound eye, the proteins of the visualignal transduction cascade are incorporated into tightly packedicrovilli of the apical, light-sensitive membrane compartment,hich forms the rhabdomere of the photoreceptor cell. Activa-

ion of rhodopsin by light triggers the phototransduction cascadend also results in internalization and degradation of the activatedhodopsin molecule, referred to as metarhodopsin (Schwemer,

984; Satoh and Ready, 2005; Alloway et al., 2000; Kiselev et al.,000). The TRPL ion channel is another protein of the Drosophilahototransduction cascade that undergoes light induced internal-

zation. TRPL and the homologous ion channel TRP are activated

∗ Corresponding author. Tel.: +49 711 45923611; fax: +49 711 45923152.E-mail address: Armin.Huber@uni-hohenheim.de (A. Huber).

171-9335/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.ejcb.2011.02.003

o stimulus-dependent internalization of a prominent member of the TRP

© 2011 Elsevier GmbH. All rights reserved.

in response to light absorption by rhodopsin and mediate Na+ andCa2+ influx into the photoreceptor cell thus generating the depolar-izing receptor potential (Hardie and Minke, 1992; Niemeyer et al.,1996). While the amount of rhabdomeral TRP does not changesignificantly under different light conditions, TRPL translocatesfrom the rhabdomeres to the cell body upon illumination and viceversa when flies are kept in the dark, thereby altering the bio-physical properties of the photoreceptor membrane (Bähner et al.,2002). By investigating visual transduction mutants, we and oth-ers showed that internalization of TRPL depends on the activationof the phototransduction cascade, including the central effectorenzyme of the pathway, phospholipase C�, and the TRP ion chan-nel (Meyer et al., 2006; Cronin et al., 2006). While activation ofthe phototransduction cascade has been identified as the triggerfor TRPL internalization, the cell biological mechanism underly-ing TRPL trafficking is still elusive. Detailed immunocytologicalanalysis by Cronin et al. suggested that TRPL internalization is atwo-stage process (Cronin et al., 2006). In the first stage, the chan-nel is transported to the base of the rhabdomere of photoreceptorcells and to the adjacent stalk membrane probably by lateral mem-brane transport. In the second stage, the channel is removed fromthe rhabdomere and transported to the cell body. However, themolecular mechanism mediating the internalization of TRPL fromthe base of the rhabdomere and the stalk membrane is not known

so far.

Rab proteins are Ras-like GTPases that regulate intracellularvesicular membrane transport and fusion of vesicles with mem-brane compartments (Pfeffer, 2003). In Drosophila, 29 Rab proteinshave been identified (Pereira-Leal and Seabra, 2001). Except for a

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roup of six Rab proteins, termed RabX1 to RabX6, the Drosophilaab proteins are homologous to the corresponding Rab proteinsf humans. Trangenic flies expressing putative dominant nega-ive variants of all Drosophila Rab proteins have been constructedZhang et al., 2007). In these Rab constructs, a serine or threonineesidue in the GTP binding site was changed to asparagine pre-umably resulting in a defect in GTP binding. The use of dominantegative Rab constructs previously led to the identification of Rabroteins that are involved in the transport of Drosophila rhodopsin.xpression of dominant negative Rab1 resulted in the accumula-ion of immature, glycosylated rhodopsin in the rough endoplasmiceticulum (rER) indicating that Drosophila Rab1 is required forhe transport of rhodopsin from the rER to the Golgi appara-us (Satoh et al., 1997). The anterograde transport of rhodopsinhrough the ER-Golgi complex depends also on Rab6 (Shetty et al.,998). Photoreceptor cells expressing a dominant negative Rab6xhibit decreased rhodopsin content and accumulation of imma-ure, glycosylated rhodopsin. Post-Golgi trafficking of rhodopsin is

ediated by Rab11. Besides rhodopsin, Rab11 is needed for traffick-ng of the TRP channel to the rhabdomere. Attenuation of Rab11ctivity via expression of Rab11 RNAi or of a dominant negativeab11 protein in developing photoreceptor cells lead to prolifera-ion of rhodopsin-bearing vesicles in the cytosol and to abnormallyhaped rhabdomeres. Likewise, TRP accumulated in intracellularesicles (Satoh et al., 2005). Rab5 has been implicated to func-ion in rhodopsin internalization. Rab5 localizes to invaginationsf the plasma membrane and to endosomes referred to as multi-esicular bodies (MVBs) of Drosophila photoreceptor cells (Shimizut al., 2003). These MVBs represent a part of the endocytic pathwayf rhodopsin and can be stained with anti-rhodopsin antibod-es (Satoh et al., 2005). Expression of a dominant negative Rab5esulted in a marked decrease in the number of rhodopsin-bearingVBs suggesting that rhodopsin internalization is affected in thisutant (Satoh et al., 2005; Shimizu et al., 2003).In the present study, we investigated the light-triggered inter-

alization of the TRPL ion channel. We provide evidence that thenternalization of TRPL employs the same vesicular pathway thats utilized for rhodopsin endocytosis, although the light-triggerednternalization of rhodopsin and TRPL internalization depend onhe function of different phototransduction proteins. We also showhat both TRPL and rhodopsin internalization strongly depend onhe light quality albeit in a different way: Rhodopsin internalizationeaks at blue light whereas the maximum of TRPL internalization isbserved under orange light illumination. Internalization of TRPLepends on the function of at least two Rab proteins, RabX4 andab5. The latter has also been implicated in rhodopsin internal-

zation. We suggest a model in which both rhodopsin and TRPLre internalized from the base of the rhabdomeral microvilli androm the adjacent stalk membrane by the same endocytic pathwayhereas the fate of these proteins after internalization may be dif-

erent: TRPL is transported to a storage compartment in the cellody while internalized rhodopsin becomes degraded.

aterials and methods

ly stocks and screen of dominant negative Rab mutants

The following strains and mutants of Drosophila melanogasterere used: wild type Oregon R, w Oregon R, yw;trpP343 (Yang

t al., 1998), w,norpAP24 (Bloomquist et al., 1988), yw,P[y+,pRh1-rpl-eGFP] (Meyer et al., 2006). yw;;P[pRh1–GAL4]3,ry506 flies were

btained from the Bloomington Drosophila Stock Center at Indiananiversity, yw,P[y+,pRh1-TRPL-eGFP];;P[pRh1–GAL4]3,ry506 (gen-rated using standard Drosophila genetics). Flies were raised on atandard corn meal diet at 25 ◦C. 1–3-day-old flies were used in allxperiments.

l of Cell Biology 90 (2011) 620–630 621

For the Rab-screen, males of the driver line (yw,P[y+,pRh1-trpl-eGFP];;P[pRh1–GAL4]3,ry506) were crossed to virgins expressingdominant negative YFP-tagged Rab proteins under the control ofthe UAS promoter (Zhang et al., 2007). The female offspring of thesecrosses was screened for an altered TRPL localization by analyz-ing the TRPL-eGFP signal in the deep pseudopupil as described byMeyer et al. (2008). Flies were analyzed after incubation in the darkfor 16 h and after illumination with orange light for 6 h. Amongthe dominant negative Rab stocks described by Zhang et al. (2007),all stocks except the ones expressing putative dominant negativeforms of CG9807 and CG32673 were analyzed. These stocks con-tained the dominant negative Rab constructs either on the secondor third chromosome. For each dominant negative Rab allele, bothinsertions were tested in the screen.

Light conditions, water immersion microscopy, andimmunocytochemistry

Prior to immunocytochemistry and water immersionmicroscopy, flies were kept in the dark overnight and werethen illuminated for the time period indicated in the figure leg-ends. The following light qualities were used: White light (18 Wfluorescent lamp, 2400 lx), blue light (acrylic glass wide-band filtertransmitting light between 310 and 490 nm, 30 lx), green light(acrylic glass wide-band filter transmitting light between 460 and610 nm, 140 lx), orange light (acrylic glass cut-off filter transmit-ting light >560 nm, 1300 lx) and red light (acrylic glass cut-off filtertransmitting light > 630 nm, 270 lx). Dark-adapted flies were keptin the dark for 16 h and dissected under dim red light (Schott RG630, cold light source KL1500, Schott, Germany), whereas flieskept in white or colored light were prepared under the respectivelight condition. Detection of TRPL-eGFP fluorescence in intact eyesby water immersion microscopy and quantification of the relativeamount of TRPL in the rhabdomere was performed as alreadydescribed (Meyer et al., 2006). Briefly, the eGFP-fluorescencesignals of images obtained by water immersion microscopy weremeasured inside and outside of the rhabdomeres. The relativeamount of TRPL-eGFP present in the rhabdomeres (R) was calcu-lated using the formula R = (Ir − Ib)/[(Ir − Ib) + (Ic − Ib)] where Ir, Ib,and Ic are the fluorescence intensities in the rhabdomeres, in thebackground, and in the cell body, respectively. For each image, fiveommatidia were analyzed by measuring the fluorescence in therhabdomeres and the cell bodies of photoreceptor cells R1-6. Atleast three individual flies were analyzed per data point.

Immunocytochemistry

Immunocytochemistry was performed essentially as describedby Chorna-Ornan et al. (2005). Dissected eyes were fixed in 2% PFAin PBS (175 mM NaCl, 8 mM Na2HPO4, and 1.8 mM NaH2PO4, pH7.2) for 1 h at RT, and then washed twice in 0.1 M phosphate buffer(0.1 M Na2HPO4 and 0.1 M NaH2PO4). This was followed by threewashes in 10% sucrose and two washes in 25% sucrose for 15 mineach. Eyes were then infiltrated with 50% sucrose overnight at 4 ◦C,cryofixed in melting pentane, and sectioned at 10 �m thickness ina Leica CM3050S cryostat (Leica, Germany) at −25 ◦C. Cryosectionswere incubated in 2% PFA in PBS for 10 min. Sections of white eyedflies were washed twice in PBS, and then blocked in 1% BSA and 0.3%Triton X-100 in PBS (PBS-T) for 2 h at RT. Sections from flies express-ing a functional white gene were washed in PBS (175 mM NaCl,8 mM Na2HPO4, 1.8 mM NaH2PO4, pH 7.2), 0.5% Triton X-100 at 4 ◦C

overnight prior to the blocking step in order to remove the red pig-ments. The sections were incubated with �-DmTRPL (Meyer et al.,2008), �-GFP (Roche, Germany), and �-Rh1 rhodopsin (4C5, Devel-opmental Studies Hybridoma Bank) in PBS-T overnight at 4 ◦C. Thesections were subsequently washed three times in PBS and were

622 C. Oberegelsbacher et al. / European Journal of Cell Biology 90 (2011) 620–630

F ongituw cted ac ), co-T le bar

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ig. 1. Co-localization of TRPL and rhodopsin inendocytic particles (EPs). (A–C) A lhite light was probed with an anti-TRPL antibody (green) and an antibody dire

orresponds to the area indicated by the white box. In the merged images (C and FRPL and rhodopsin in EPs. Arrowheads depict EPs that contain rhodopsin only. Sca

ncubated with �-rabbit-AlexaFluor 680, �-mouse-AlexaFluor 488,nd phalloidin-AlexaFluor 546 (Invitrogen, Germany) in 0.5% fishelatine and 0.1% ovalbumin in PBS for at least 4 h at RT. The sec-ions were finally washed three times in PBS, mounted in Mowiol.88 (Polyscience), and examined with an AxioImager.Z1m micro-cope (objective: EC Plan-Neofluar 40×/1.3 Oil, Zeiss, Germany)ith an ApoTome module (Zeiss, Germany) at room temperature.

mages were imported into Adobe Photoshop CS2 for cropping andontrast adjustment before being assembled into figures using theame software. For a quantitative analysis of rhodopsin and TRPLnternalization, the number of rhodopsin- and TRPL-containing EPser ommatidium was counted for each genotype and light quality

n cross sections obtained from three to five flies. Per section, eightmmatidia were analyzed.

lectroretinogram recordings

For electroretinogram recordings, flies were anesthetized once and mounted with a mixture of colophonium and bee’s wax.o analyze RabDN-expressing flies, light stimuli of 5 s durationere delivered using an orange light-emitting diode (LED, Roith-er, Austria) in a setup of two collimating lenses (Linos, Germany)ithin the light path. The light intensity at the position of the fly

ye was 2.15 mW/cm2. To investigate the influence of differentight qualities on the electrophysiological response, a white LEDRoithner, Austria) was used and the above-mentioned acrylic glasside-band filters were put in the light path. Light intensities were

ttenuated using neutral density filters (Linos, Germany) to match

dinal section through the eye of a wild type fly that was illuminated for 2 h withgainst the major rhodopsin, Rh1 (red). (D–F) show a magnified view of A–C thatlabeling of TRPL and rhodopsin appears yellow. Arrows indicate co-localization ofs, 10 �m.

those applied in the other experiments. All ERG recordings wereperformed following 3 min of dark adaptation at room temperature.Signals were amplified using a DPA-2FS amplifier (NPI electronic,Germany) with a low pass filter (700 Hz) applied. Analog to digi-tal conversion was accomplished with a BNC-2090A rack-mountedterminal block (National Instruments, Germany) and a PCI-6221 PCcard (National Instruments, Germany). Data was recorded and visu-alized using the Whole Cell Analysis Program software (Universityof Strathclyde). The recording electrode glass capillary was filledwith Davenport solution (100 mM NaCl, 2 mM KCl, 1 mM CaCl2,1.8 mM NaHCO3, pH 7.2).

Results

Internalization of TRPL and rhodopsin is mediated by a commonvesicular pathway

The major rhodopsin Rh1 of Drosophila photoreceptor cells R1-6 undergoes light-triggered internalization by vesicular transportfrom the rhabdomere to the endosome. In contrast, the light-dependent internalization of TRPL has been suggested to occuralong the plasma membrane by a translocation of TRPL fromthe apical membrane, containing the rhabdomeral and the stalk

membrane, to the basolateral portion of the plasma membrane(Cronin et al., 2006). In order to test whether TRPL internalizationoccurs via a similar vesicular pathway as rhodopsin internaliza-tion or via a fundamentally different mechanism, we performedimmunohistochemical stainings of sections through the eyes of

C. Oberegelsbacher et al. / European Journa

Fig. 2. Time course of TRPL and rhodopsin internalization. Cross-sections throughthe eyes of dark-kept wild type flies (A–A”’) and of flies illuminated with white lightfor the indicated periods of time (B–G”’). The sections were probed with anti-TRPL(green), anti-Rh1 (red) antibodies, and phalloidin labeling the rhabdomeres (white).Note that the anti-Rh1 antibody does not label the whole rhabdomere probably dueto restricted accessibility. Overlay of green and red in the merged panels appearsyellow. Arrows indicate co-localization of TRPL and rhodopsin in endocytic particles(EPs). Arrowheads point to EPs that contain rhodopsin only. In dark-kept flies, TRPLand rhodopsin were located in the rhabdomere and almost no EPs were observed.

l of Cell Biology 90 (2011) 620–630 623

illuminated Drosophila with antibodies directed against TRPL andRh1 rhodopsin. After 2 h of white light illumination, numerousTRPL-containing vesicular structures were observed in the vicin-ity of the rhabdomeres in longitudinal sections (Fig. 1A and D).The TRPL-positive structures appeared similar in size and shapeto rhodopsin-containing vesicular structures, termed ERPs (endo-cytic Rh1 particles) (Han et al., 2007) or RLVs (Rh1-containing largevesicles) (Satoh et al., 2005), respectively. These structures repre-sent a collection of small endocytic vesicles that were suggestedto correspond to multivesicular bodies (MVBs) detected on theelectron microscopic level (Satoh et al., 2005). Indeed, co-labelingwith an antibody directed against Rh1 was observed in many ofthe TRPL-positive vesicular structures (Fig. 1C and F), in the fol-lowing called endocytic particles (EPs). Besides EPs that containedboth rhodopsin and TRPL, we observed vesicular structures thatwere stained by anti-rhodopsin antibody alone (Fig. 1D–F). Theseresults show that light-triggered TRPL internalization is mediatedby a vesicular transport pathway that at least partially correspondsto the internalization pathway of rhodopsin.

For a quantitative analysis of TRPL and rhodopsin internaliza-tion, we counted the number of TRPL- and rhodopsin-containingEPs in cross sections of eyes derived from flies that wereswitched from darkness to white light for different periods oftime (Fig. 2). In dark-kept flies, both TRPL and rhodopsin werelocalized to the rhabdomeres and EPs that contained eitherTRPL or rhodopsin were observed at a very low frequency (<0.5EPs/ommatidium) (Fig. 2A–A”, H). After 5 min of light exposure,numerous rhodopsin-containing EPs (3.3 ± 1.8 EPs/ommatidium)were observed (Fig. 2B’, H). The maximum of rhodopsin-containingEPs (4.6 ± 1.8 EPs/ommatidium) was observed after 30 min of illu-mination (Fig. 2C’ and H) and stayed at about this level for the restof the period analyzed (10 h) (Fig. 2D’, E’, F’, G’, H). After 10 h of illu-mination, neither a depletion of rhodopsin in the rhabdomere noran accumulation of rhodopsin in an intracellular compartment wasobserved (Fig. 2G’).

After 5 min of illumination, the number of TRPL-containing EPswas still small (0.5 ± 0.8 EPs/ommatidium) (Fig. 2B and H). Thisdelay in the formation of TRPL-containing EPs may reflect thetime required for the first step of TRPL internalization, namely itstransport to the base of the rhabdomere and to the adjacent stalkmembrane. Formation of TRPL-containing EPs lagged behind theformation of rhodopsin-containing EPs and reached a maximumafter 2 h of light exposure (3.5 ± 0.9 EPs/ommatidium) (Fig. 2C, D,H). Contrary to the number of rhodopsin-containing EPs, the num-ber of TRPL-containing EPs declined when the flies were exposedto white light for more than 2 h and went down to zero after 10 hof illumination (Fig. 2E–H). After 6–10 h of illumination, the TRPLstaining in the rhabdomeres diminished and an increasing diffusestaining in the cell body was detected instead (Fig. 2F and G). Asshown before in longitudinal sections (Fig. 1), the cross sectionsanalyzed in the time course revealed that many TRPL-containing

EPs co-localized with rhodopsin (Fig. 2C”, D”, E”, F”, H).

Albeit sharing common EPs, the internalization of rhodopsin andTRPL is probably triggered by different mechanisms. It was reportedthat rhodopsin internalization does not require phospholipase C

TRPL-containing EPs were observed from 5 min to 6 h of illumination (B–F). Notethat the TRPL staining disappeared from the rhabdomere and appeared in the cellbody after prolonged illumination (6 and 10 h; F and G). Rhodopsin-containing EPswere observed at any time point after switching the flies from the dark to the light(B’, C’, D’, E’, F’, G’). Most TRPL-containing EPs also contained rhodopsin (B”, C”, D”,E”, F”). (H) Results of a quantitative analysis of the number of TRPL-containing EPs(green curve), Rh1-containing EPs (red curve) and EPs containing both Rh1 andTRPL (yellow curve). Mean values of the number of EPs per ommatidium derivedby counting vesicles of 30–40 ommatidia from sections of at least four fly eyes perdata point are displayed. Error bars show S.E.M. Scale bar, 5 �m.

624 C. Oberegelsbacher et al. / European Journal of Cell Biology 90 (2011) 620–630

Fig. 3. Formation of TRPL- and rhodopsin- (Rh1) containing EPs and receptor responses under different light qualities. Shown are representative cross sections through theeyes of wild type flies kept in darkness (A–D) or illuminated for 2 h with either blue (E–H), green (I–L), orange (M–P) or white (Q–T) light. The sections were probed withanti-TRPL (green), anti-Rh1 (red) antibodies, and phalloidin labeling the rhabdomeres (white). In the merged panels, co-labeling of TRPL and rhodopsin appears yellow (C,G, K, O, S). (U) Results of a quantitative analysis of the number of TRPL-containing EPs (green bars), rhodopsin-containing EPs (red bars) and EPs containing both TRPL andrhodopsin (yellow bars). Mean values of the number of EPs per ommatidium derived by counting vesicles of 20–40 ommatidia from sections of at least three fly eyes per datap cted ai (J andS lightt

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oint are displayed. Error bars show S.D. Numerous TRPL-containing EPs were detellumination (E and U). Rhodopsin-containing EPs formed after blue (F and U), greencale bar, 5 �m. (V) Electroretinogram recordings of flies illuminated with differento similar responses.

ctivity (Han et al., 2007; Alloway et al., 2000; Kiselev et al., 2000)hile TRPL translocation strongly depends on a functional pho-

otransduction cascade (Cronin et al., 2006; Meyer et al., 2006).n order to provide evidence for the aforementioned differenceetween the triggering of TRPL and rhodopsin internalization withespect to activation of the phototransduction cascade, we ana-yzed rhodopsin- and TRPL-containing EPs in trpP343 and norpAP24

utants after 2 h of white and orange light illumination (Fig. S1).s expected, in both mutants, we observed a large number ofhodopsin-containing EPs (>7 EPs/ommatidium) and almost noRPL-containing EPs (<0.4 EPs/ommatidium).

nhanced rhodopsin internalization reduces TRPL internalization

Since rhodopsin and TRPL share, at least partially, the samenternalization pathway, we wondered whether changing themount of rhodopsin endocytosis might affect TRPL internaliza-ion. The amount of rhodopsin endocytosis depends on the amountf rhodopsin converted to metarhodopsin (the light-activatedorm of rhodopsin) (Schwemer, 1984). Therefore, the rhodopsinnternalization rate can be altered by adjusting the fraction of

etarhodopsin present in the rhabdomeres. The ratio between

hodopsin and metarhodopsin depends on the light quality used forllumination. Orange light leads to the generation of a small amount<2%) of metarhodopsin, whereas green light converts about 50% ofhodopsin to metarhodopsin and blue light shifts the rhodopsin-etarhodopsin photoequilibrium to 70% metarhodopsin (Meyer

fter green (I and U), orange (M and U) and white (Q and U), but not after blue lightU), white (R and U), but only very rarely after orange light illumination (N and U).

qualities. ERG recordings show that illumination with different light qualities lead

et al., 2006). Despite the different amounts of metarhodopsingenerated by different light qualities, the electrophysiologicalresponses to these stimuli were similar and within the physiolog-ical range (Fig. 3V). To determine the TRPL internalization rate inthe presence of different amounts of metarhodopsin, we exposedflies to blue, green, orange and white light for 2 h before subjectingthem to immunocytochemistry (Fig. 3). As expected, the number ofrhodopsin-containing EPs was highest after illumination with bluelight (6.6 ± 1.3 EPs/ommatidium) (Fig. 3F and U). Green or whitelight illumination generated an intermediate number of rhodopsin-containing EPs (5.5 ± 1.5 and 4.5 ± 1.2 EPs/ommatidium) (Fig. 3J, R,U) while very few rhodopsin-containing EPs were found after illu-mination with orange light (<0.3 ± 0.4/ommatidium) (Fig. 3N andU). The maximal number of TRPL-containing EPs was observed afterorange light illumination (5.8 ± 1.4 EPs/ommatidium) (Fig. 3M andU), the light quality under which very few rhodopsin-containingEPs were observed. White and green light resulted in a signifi-cantly lower number of TRPL-containing EPs (3.5 ± 0.9 and 4.9 ± 1.1EPs/ommatidium, respectively) (Fig. 3I, Q, U). After illuminationwith blue light, almost no TRPL-containing EPs were observed (<0.4EPs/ommatidium) (Fig. 3E and U). Thus, the light condition thatresults in the highest number of rhodopsin EPs causes the lowest

number of TRPL EPs and vice versa.

We assume that TRPL-containing EPs are a major part of theinternalization route of TRPL during light adaptation. Therefore, thetime courses of TRPL-eGFP translocation from the rhabdomere tothe cell body were expected to differ between blue, green, orange

C. Oberegelsbacher et al. / European Journal of Cell Biology 90 (2011) 620–630 625

Fig. 4. Time course of TRPL-eGFP translocation from the rhabdomere to the cell body under different light conditions. (A) Water immersion microscopy images of TRPL-eGFPfluorescence in intact eyes. Flies were illuminated with blue (1st row), green (2nd row), orange (3rd row), or white light (4th row) for the time periods indicated. (B) Resultso rhabdv d from( w S.E.

amatp

f a quantitative analysis of the relative amount of TRPL-eGFP fluorescence in thealues of the relative amount of TRPL in the rhabdomere from 15 ommatidia derivegreen curve), orange (orange curve), and white light (dotted curve). Error bars sho

nd white light exposure. To test this hypothesis, water immersionicroscopy of intact fly eyes was used to determine the relative

mount of TRPL-eGFP remaining in the rhabdomeres accordingo Meyer et al. (2006) (Fig. 4). TRPL-eGFP internalization indeedroceeded fastest when flies were illuminated with orange light

omere (see Materials and methods) calculated from images as shown in (A). Meanthree flies per data point are shown for illumination with blue (blue curve), green

M. Scale bar, 10 �m.

(Fig. 4A and B). Illumination with green or white light resultedin a slightly delayed TRPL-eGFP internalization while blue lightexposure lead to the slowest TRPL-eGFP internalization (Fig. 4Aand B). Therefore, the speed of TRPL-eGFP internalization nega-tively correlates with the amount of metarhodopsin suggesting

626 C. Oberegelsbacher et al. / European Journal of Cell Biology 90 (2011) 620–630

Fig. 5. TRPL-eGFP translocation in flies expressing dominant negative Rab5 (3rd chromosome) and RabX4 (2nd chromosome). TRPL-eGFP fluorescence in the deep pseudopupil(dpp) (A, B, E, F, I, J) and in water immersion microscopy images (C, D, G, H, K, L). In dark-adapted flies, TRPL-eGFP was located in the rhabdomeres (A, C, E, G, I, K). After 6 h ofi abdoo 00 �m

tm

Rb

prbttcpepteGcmpnbflgptun

llumination with orange light, TRPL-eGFP fluorescence was detected outside the rhverexpressing dominant negative Rab5 (F and H) and RabX4 (J and L). Scale bars, 1

hat TRPL internalization might be attenuated by an excess ofetarhodopsin.

ab5 and RabX4 are required for translocation of TRPL to the cellody

Rab proteins are important mediators of vesicular trans-ort pathways (Pfeffer, 2003). Having established that TRPL, likehodopsin, is transported from the rhabdomere to the cell bodyy a vesicular transport pathway, we wondered whether Rab pro-eins are involved in TRPL translocation as is the case for rhodopsinranslocation. In order to screen for possible defects in TRPL translo-ation, we utilized a collection of transgenic flies that expressutative dominant negative YFP-tagged Rab variants (RabDN) ofach of the 29 Drosophila Rab proteins under the control of a UASromoter (Zhang et al., 2007). In order to drive the expression ofhe dominant negative Rab variants in the photoreceptor cells R1-6,ach of these fly stocks was crossed with a driver stock expressingal4 under the control of the Rh1 promoter. The driver stock alsoontained a TRPL-eGFP transgene under the control of the Rh1 pro-oter which allowed for direct monitoring of the TRPL-eGFP fusion

rotein in intact eyes. At first, Drosophila expressing the dominantegative Rab proteins were screened for TRPL translocation defectsy observing the deep pseudopupil of both dark- and light-adaptedies. Wild type flies expressing TRPL-eGFP displayed an intensely

reen fluorescing pseudopupil when kept in the dark but a darkseudopupil when kept in orange light (Fig. 5A and B). This reflectshe translocation of TRPL from the rhabdomeres to the cell bodypon illumination (Meyer et al., 2006, 2008). None of the dominantegative Rab proteins affected the localization of TRPL-eGFP in the

meres of wild type flies (B and D) but largely remained in the rhabdomeres of fliesfor dpp and 10 �m for water immersion.

rhabdomere of dark-adapted flies. However, six dominant nega-tive Rab proteins (Rab1DN, Rab4DN, Rab5DN, Rab10DN, Rab19DN,and RabX4DN) gave rise to an abnormal pseudopupil in the light ascompared to the wild type (Fig. 5 and Fig. S3). In a second step,these flies were analyzed by water immersion microscopy pro-viding a higher resolution. This rescreen revealed that expressionof Rab5DN and RabX4DN lead to a strong and robust phenotype(Fig. 5E–L). In flies expressing Rab5DN and RabX4DN, TRPL-eGFPfluorescence was observed in the rhabdomeres irrespective of thelight condition. In the case of Rab5DN, the strength of the phenotypediffered between the two lines available and was stronger in the lineharboring the insertion on the 3rd chromosome (Fig. 5E–H) than inthe line harboring the insertion on the 2nd chromosome (data notshown). Western blot analysis demonstrated that the amount ofRab5DN protein was higher using the insertion on the 3rd chro-mosome compared to the insertion on the 2nd chromosome (datanot shown). Therefore, the insertion on the 3rd chromosome wasused for further analysis. In contrast to the YFP-Rab5DN lines, bothYFP-RabX4DN insertions gave rise to the same phenotypic strength(data not shown).

To study the localization of TRPL in flies expressing Rab5DN orRabX4DN in more detail, we carried out immunocytochemistry. Forthis purpose, a driver line containing Rh1-Gal4 but not TRPL-eGFPwas used, and the subcellular localization of endogenous TRPL wasdetermined using an anti-TRPL antibody (Fig. 6). After 10 h of illu-

mination with white light, TRPL was distributed throughout thecell body of the photoreceptor cells of wild type flies (Fig. 6A andB), whereas in flies expressing Rab5DN or RabX4DN, TRPL labelingwas observed at the base of the rhabdomere and in the adjacentstalk membrane (Fig. 6C–F). This result indicates that in the pres-

C. Oberegelsbacher et al. / European Journa

Fig. 6. Localization of endogenous TRPL in flies expressing dominant negative Rab5and RabX4. Cross sections through the eyes of flies that were illuminated with whitelight for 10 h were probed with an anti-TRPL antibody (green) or phalloidin (red).Phalloidin labels the actin cytoskeleton of the rhabdomeres. In illuminated photore-ceptor cells of wild type flies, TRPL labeling was detected in the cell body (A and B)wTm

eot

tmtcRgca

(ERTia(

ber of TRPL-containing EPs during the time course differs markedly

hile upon expression of dominant negative Rab5 (C and D) and RabX4 (E and F),RPL was located predominantly at the base of the rhabdomeres and at the stalkembrane. Scale bar, 10 �m.

nce of Rab5DN or RabX4DN, light exposure resulted in transportf TRPL to the base of the rhabdomere but not in its translocationo the cell body.

To exclude an indirect effect of Rab5DN and RabX4DN on TRPLranslocation due to a failure in the phototransduction cascade, we

easured electroretinograms which reflect the depolarization ofhe photoreceptor cells after activation of the phototransductionascade. We found that the electroretinograms of flies expressingab5DN and RabX4DN were indistinguishable from electroretino-rams of wild type flies, indicating that the phototransductionascade operates normally despite impairment of Rab5 or RabX4ctivity (Fig. S2).

Rab5 and RabX4 were reported to be located in endosomesWucherpfennig et al., 2003; Zhang et al., 2007). As TRPL sharesPs with rhodopsin and rhodopsin was shown to co-localize withab5 (Han et al., 2007), we also addressed the question whether

RPL is co-localized with Rab5 and RabX4. After 2 h of white lightllumination, TRPL was identified in numerous EPs and most but notll of these TRPL-containing EPs also stained for Rab5 and RabX4Fig. 7). Together, these data strongly suggest that Rab5 and RabX4

l of Cell Biology 90 (2011) 620–630 627

are needed for the transport and/or sorting of TRPL in the processof light-induced internalization of this ion channel.

Discussion

In the present study, we provide evidence for co-internalizationof TRPL and rhodopsin by a vesicular transport pathway. It iswell established that the internalization of the major rhodopsinin Drosophila photoreceptors, Rh1, occurs by a vesicular transportpathway in which rhodopsin-containing particles are formed thatare composed of numerous small vesicles (Satoh and Ready, 2005;Han et al., 2007). However, the mechanisms underlying rhodopsininternalization are not entirely clear. When activated, rhodopsinbinds two arrestin proteins, arrestin1 and arrestin2, and becomesphosphorylated at its C-terminal domain by G protein coupledreceptor kinase 1 (Dolph et al., 1993; Bentrop et al., 1993; Leeand Montell, 2004). While arrestin2 is required for inactivationof the photoresponse by uncoupling rhodopsin from the visualG-protein (Dolph et al., 1993), arrestin1 was shown to mediaterhodopsin endocytosis in wild type photoreceptor cells (Satoh andReady, 2005). Arrestin2, however, may also participate in rhodopsinendocytosis in a situation when arrestin2 forms abnormally sta-ble complexes with phosphorylated rhodopsin (Alloway et al.,2000; Kiselev et al., 2000). The arrestin1- or arrestin2-dependentinternalization of activated rhodopsin resembles internalization ofthe vertebrate �2-adrenergic receptor that is mediated by bind-ing of �-arrestin to the activated receptor (Luttrell and Lefkowitz,2002). However, in adult Drosophila photoreceptors, an arrestin-independent pathway of rhodopsin endocytosis seems to exist aswell (Han et al., 2007). Han and colleagues investigated tes (ter-mination slow) mutants that have a defect in the transcriptionactivator dCAMTA and exhibit prolonged rhodopsin activation foryet unknown reasons. In tes mutants, rhodopsin becomes mas-sively internalized upon light exposure, resulting in reduction ofthe total rhodopsin level. This massive internalization is indepen-dent of arrestin1 but requires the visual G-protein, suggesting thatprolonged activation of the visual G-protein results in enhanced,arrestin1-independent rhodopsin internalization. Our analysis ofthe number of rhodopsin-containing EPs after white light illumi-nation for different periods of time revealed that after an initialphase of about 30 min, the number of rhodopsin-containing EPsreached a constant level that remained essentially unchangedfor 10 h. Despite the constant removal of rhodopsin from therhabdomere during this period, neither a detectable decrease ofrhodopsin in the rhabdomeres nor an increase of rhodopsin inthe cell body was observed. These findings suggest that the inter-nalized rhodopsin is targeted for degradation rather than storedin an intracellular compartment and that the loss of rhodopsinin the rhabdomeres is probably replaced by de novo synthesizedrhodopsin.

The translocation of TRPL from the rhabdomere to the cell bodyhas been described as a two-stage process, in which the channelis first transported to the base of the rhabdomere and to the adja-cent stalk membrane and is then removed from this apical plasmamembrane compartment (Cronin et al., 2006). The cell biologicalmechanisms underlying the internalization of TRPL and its trans-port route after removal from the plasma membrane, however, arepoorly understood. Here, we show that after activation of the pho-totransduction cascade by light, internalized TRPL is detected in thesame vesicular structures (EPs) as rhodopsin. However, the num-

from the number of rhodopsin EPs. The internalization rate of TRPLpeaked after 2 h of illumination while after 10 h, almost no TRPL-containing EPs were detected anymore. At that stage, TRPL wasfound to be completely outside the rhabdomeres. These results are

628 C. Oberegelsbacher et al. / European Journal of Cell Biology 90 (2011) 620–630

F roughw FP anto in whm

itaTss

iMfmtirTtapeltiRatmKoa(trqlmwifad

D

ig. 7. Co-localization of TRPL and Rab5-YFP or RabX4-YFP. Longitudinal sections thhite light for 2 h. The sections were probed with anti-TRPL (A and D) and an anti-G

f TRPL and Rab5-YFP or RabX4-YFP appears yellow (C and F). Arrows indicate EParked by arrowheads. Scale bar, 10 �m.

n line with previous findings, showing that TRPL translocates fromhe rhabdomeres to an intracellular storage compartment. Thus,fter a common internalization route through EPs, rhodopsin andRPL have a different fate, as most of the internalized rhodopsineems to be degraded while most of the internalized TRPL becomestored.

An interesting aspect of rhodopsin and TRPL co-internalizations the dependence of TRPL internalization on the light quality.

easuring the internalization of a TRPL-eGFP reporter protein, weound that TRPL internalization was lowest for blue light, inter-

ediate for green light and maximal for orange light. One wayo explain this phenomenon is that the mechanism for trigger-ng the light-dependent internalization is different for TRPL andhodopsin and that massive rhodopsin internalization might limitRPL internalization. We and others have shown previously thatranslocation of TRPL from the rhabdomere to the cell body requiresctivation of the phototransduction cascade including a functionalhospholipase C� and TRP ion channel (Cronin et al., 2006; Meyert al., 2006). The phototransduction cascade is activated by anyight quality capable to convert rhodopsin into metarhodopsin andherefore, within the physiological range, TRPL should becomenternalized upon illumination irrespective of the light-quality.hodopsin internalization on the other hand does not depend onctivation of the phototransduction cascade as it also occurs inhe phospholipase C� null mutant norpAP24 and in the TRP null

utant trpP343 (Fig. S1) and Han et al. (2007), Alloway et al. (2000),iselev et al. (2000). Instead, rhodopsin internalization dependsn the amount of metarhodopsin generated, to which arrestin1nd arrestin2 bind, and maybe on the amount of active G proteinHan et al., 2007) (see above). The amount of metarhodopsin inurn is determined by the photoequilibrium established betweenhodopsin and metarhodopsin and therefore depends on the lightuality in a way that blue light causes a maximal (70%), green

ight an intermediate (50%) and orange light a minimal (<2%)etarhodopsin content (Meyer et al., 2006). This readily explainshy the rhodopsin internalization rate is higher under blue light

llumination than under orange light. But why is the reverse true

or TRPL internalization? We suggest that the generation of highmounts of metarhodopsin inhibits TRPL internalization maybeue to sequestration of a factor required for TRPL internalization.

Our screen of flies expressing dominant negative forms ofrosophila Rab proteins revealed that Rab5DN and RabX4DN atten-

the eyes of flies expressing YFP-tagged Rab5 or RabX4 that were illuminated withibody detecting Rab5-YFP (B) and RabX4-YFP (E). In the merged images, co-labelingich TRPL and Rab-YFP colocalize. EPs containing either TRPL or Rab-YFP alone are

uated the light-triggered internalization of TRPL (Figs. 5 and 6).After light exposure of these mutants, TRPL was mainly localizedat the base of the rhabdomere and in the stalk membrane, andcompared to wild type, very little TRPL labeling was observed inthe cell body (Fig. 6). Rab5 is conserved between Drosophila andvertebrates and has been described as a regulatory factor in earlysteps of the endocytic pathway that controls protein transport fromthe plasma membrane to the early endosome and protein sort-ing within the endosome (Bucci et al., 1992, 1994; Chavrier et al.,1990). Rab5 has been implicated, for example, in the internaliza-tion of the �2-adrenergic receptor and the D2 dopamine receptor(Seachrist et al., 2000; Iwata et al., 1999). The reports of Seachristet al. (2000) and Iwata et al. (1999) provide evidence that Rab5 isnot only involved in the fusion of endocytic vesicles with the earlyendosome and in protein sorting within the endosome, but also inthe formation of endocytic vesicles at the plasma membrane. Thisnotion would be in line with our data, showing that TRPL is locatedin or at the plasma membrane in illuminated Rab5DN-expressingflies. As shown by ERG recordings, the phototransduction cascadefunctions normally in Rab5DN-expressing flies, ruling out the pos-sibility that the defect in TRPL translocation results from the lack ofa central phototransduction protein in the rhabdomere. Moreover,abolition of the phototransduction cascade should result in a distri-bution of TRPL throughout the rhabdomere as seen in norpAP24 andtrpP343 mutants (Fig. S1) and not in localization at the base of therhabdomere as was observed in Rab5DN-expressing flies (Fig. 6).This finding and the observed co-localization of TRPL and YFP-tagged Rab5 and RabX4 in endocytic particles strongly suggest thatRab5 and RabX4 directly mediate vesicular internalization of TRPLbut have no influence on the lateral membrane transport from therhabdomere to its base and to the adjacent stalk membrane (Fig. 7).Flies expressing RabX4DN display a phenotype indistinguishablefrom that of Rab5DN-expressing flies. RabX4 is a Drosophila-specificRab protein that has no close homolog in vertebrates. Among theDrosophila Rab proteins, RabX4 is most closely related to Rab8 andRab10 and rather distantly to Rab5 (Zhang et al., 2007). In DrosophilaS2 cells, YFP-tagged RabX4 was shown to co-localize with Rab5 but

not with Rab7, Rab9, or Rab11 (Zhang et al., 2007), suggesting thatRabX4 acts in the same compartment as Rab5. The identical phe-notype of Rab5DN- and RabX4DN-expressing flies with respect toTRPL internalization argues for a similar but not redundant functionof Rab5 and RabX4 in fly photoreceptors.

C. Oberegelsbacher et al. / European Journal of Cell Biology 90 (2011) 620–630 629

Fig. 8. A model of TRPL and rhodopsin internalization in photoreceptor cells. In dark-adapted photoreceptors, TRPL and rhodopsin are located in the microvilli of the apicalr merica the enT e rhabs

traralvEcvTeiaR

oTspr

A

vBrmDpDw(

habdomeric membrane. Upon illumination, TRPL moves to the base of the rhabdore formed in a Rab5- and RabX4-dependent manner and transport their cargo toRPL to a storage compartment. Dotted arrows indicate redistribution of TRPL to thtorage compartment.

In conclusion, we propose the following scheme for the light-riggered internalization of TRPL (Fig. 8). In the dark, TRPL andhodopsin are distributed throughout the rhabdomere. Withinbout 5 min of illumination, TRPL is transported to the base of thehabdomere and to the adjacent stalk membrane while rhodopsinlready becomes internalized and is detected in EPs. Upon pro-onged illumination, TRPL as well as rhodopsin are recruited toesicles and become co-localized in endocytic particles (EPs). SincePs are composed of several vesicles, we are not able to dis-riminate if TRPL and rhodopsin are incorporated into the sameesicles at the plasma membrane or if separate rhodopsin- andRPL-containing vesicles fuse and form EPs. EPs then fuse withndosomes where rhodopsin is sorted for degradation and TRPLs sorted to a storage compartment. The internalization of TRPLnd probably also of rhodopsin requires the function of Rab5 andabX4.

The identification of Rab-protein dependent vesicular transportf TRPL is a first step in solving the puzzle of stimulus dependentRPL internalization in the model organism Drosophila. Under-tanding the internalization of Drosophila TRPL might ultimatelyrovide insights into the modulation of vertebrate TRP signaling byegulated trafficking of vertebrate TRP channels.

cknowledgements

The authors thank the Bloomington Stock Center at Indiana Uni-ersity for providing Drosophila stocks. We are grateful to Gregorelusic for his invaluable help with the construction of an ERGecording setup and Jens Pfannstiel for critical comments on theanuscript. The monoclonal antibody 4C5 was obtained from the

evelopmental Studies Hybridoma Bank developed under the aus-ices of the NICHD and maintained by the University of Iowa,epartment of Biological Sciences, Iowa City, IA 52242. This workas supported by a grant of the Deutsche Forschungsgemeinschaft

Hu 839/2-5).

membrane and to the apical stalk membrane. EPs containing TRPL and rhodopsindosome. From the endosome, rhodopsin is targeted to lysosomal degradation anddomere during dark-adaptation. AJ, adherence junction; EP, endocytic particle; SC,

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ejcb.2011.02.003.

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