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First published online 30 May 2006
doi: 10.1242/jcs.02986

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Subcellular translocation of the eGFP-tagged TRPL channel in Drosophila photoreceptors requires activation of the phototransduction cascade

¹ Department of Biosensorics, Institute of Physiology, University of Hohenheim, 70599 Stuttgart, Germany
² Department of Physiology and The Kühne Minerva Center for Studies of Visual Transduction, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel

View larger version (42K): [in a new window]   Fig. 1. Expression of TRPL-eGFP in photoreceptors of transgenic Drosophila. (A) Western blot analysis of protein extracts obtained from heads of wild-type flies (wt), TRPL-null mutant (yw; trpl302), and TRPL-eGFP-expressing flies in TRPL-null (yw; trpl302; trpl-eGFP) or wild-type background (yw; trpl-eGFP). The same blot was probed with antibodies directed against TRPL, TRP and GFP as indicated. Proteins obtained from three Drosophila heads were loaded per lane. (B) The green fluorescing deep pseudopupil of TRPL-eGFP-expressing flies (trpl-eGFP) raised in the dark (left panel). Fluorescence of the pseudopupil was not observed when the flies were raised in the light (middle panel), nor was it observed in wild-type eyes (right panel). (C) Subcellular localization of TRPL-eGFP in dark- and light-raised transgenic flies (yw; trpl-eGFP). eGFP-fluorescence was detected in intact eyes after optical neutralization of the cornea by water immersion. Flies were kept in the dark (left panel) or under orange light (right panel) for 16 hours. The insets show the central area of the eye at higher magnification. Scale bar, 15 µm.

View larger version (13K): [in a new window]   Fig. 2. Time course of TRPL-eGFP translocation. At the indicated time points fluorescence images of intact eyes of flies expressing TRPL-eGFP (yw; trpl-eGFP) were obtained using the water immersion technique. From these images the percentage of TRPL-eGFP present in the rhabdomeres at different times after switching the flies from darkness to light (A) or vice versa (B) was calculated (see Materials and Methods). Each data point represents the mean value ± s.d. of at least five independent measurements. (C) Dark-(dark l/d) or light-adapted flies (light d/l) expressing TRPL-eGFP (yw; trpl-eGFP) were subjected to alternating light and dark intervals of 30 minutes each for 16 hours. The percentage of TRPL-eGFP present in the rhabdomeres was determined. The values of dark- and light-adapted flies are shown additionally. Mean values ± s.d. of at least five experiments are shown.

View larger version (23K): [in a new window]   Fig. 3. Fusion of the TRPL channel to eGFP did not affect the physiological properties of the channel in vivo and in situ. (A, upper) ERG recordings from wild-type (WT) flies in response to a pair of orange light stimuli (Schott OG 590 edge filter) with maximal intensity attenuated by 1.0 log unit. (A, middle) ERG recordings from transgenic Drosophila expressing TRPL-eGFP fusion protein on null trpl; trp background (yw trpl-eGFP; trpl302; trpP343) in response to the same pair of orange light stimuli used for trace A (upper). There is a transient receptor potential, which declines to baseline within 5 seconds. This light response is typical for trp mutants and it is expected from light activation of TRPL channels without the presence of TRP channels. An unusual large response to the second light stimulus after a 10-second dark interval can be observed. (A, bottom) ERG recordings from the null trp mutant (yw;; trpP343) in a paradigm identical to that of the traces in A (upper and middle). (B) Intensity-response relationship (V-log I curve) measured from WT and the two mutants of Fig. 2A. The peak ERG amplitude was measured as a function of the orange light intensities. The error bars indicate ± s.d. (n=9). (C) The peak amplitude of the response to the second stimulus (see traces A middle and bottom) was divided by the peak amplitude of the response to the first stimulus and the averaged ratio calculated from six flies per mutant is presented for the trpl-eGFP and trpP343 mutants. One minute of dark adaptation was used between the pairs of stimuli. (D) The light-induced currents at different membrane potentials are similar in both the TRPL-eGFP-expressing fly and the null trp mutant. Whole-cell patch-clamp recordings from isolated ommatidium of yw trpl-eGFP; trpl302; trpP343 fly. Voltage clamp responses to identical orange light pulse of 100 milliseconds duration (Schott OG 590 edge filter with maximal intensity attenuated by 1.0 log unit) delivered at the time indicated by the arrow. The photoreceptor was voltage clamped at membrane potentials between -120 mV and +80 mV in steps of 20 mV. A reversal potential of -1 mV was determined by interpolation after plotting the peak amplitude of the light-induced current as a function of the membrane potential. Very similar reversal potential and similar strongly outward rectifying current voltage relationship was reported for the trp mutant (Hardie and Minke, 1992).

View larger version (47K): [in a new window]   Fig. 4. Functional rhodopsin is required for the light-dependent translocation of TRPL-eGFP. Wild-type (A,B), yw; ninaE17; trpl-eGFP (C,D), yw/w; ninaE17/P[Rh1+3]; trpl-eGFP (E,F) and w; trpl-eGFP; ninaBP315 (H-J) flies expressing TRPL-eGFP were raised in the dark or in orange light for 16 hours. ninaE17 + Rh3 was also illuminated with UV for 16 hours (G). Representative images of the eGFP fluorescence in intact eyes obtained by the water immersion technique are shown. For light-raised ninaBP315 two individuals are shown that revealed different levels of TRPL-eGFP internalization (I,J). Bar, 15 µm. In K, dark-raised flies were transferred to white, blue, green or orange light for 16 hours. Then the percentage of TRPL-eGFP present in the rhabdomeres was determined. Mean values ± s.d. of at least five experiments are shown.

View larger version (43K): [in a new window]   Fig. 5. Role of arrestins in the translocation of TRPL-eGFP. Water immersion images of eGFP-fluorescence in the eyes of the following arrestin mutants raised for 16 hours in the dark or in orange light are shown: (A,B) arr11 cn bw; trpl-eGFP, (C,D) w; trpl-eGFP; arr23 st, (E,F) yw trpl-eGFP; arr11 cn bw; arr23 (arr11 arr23 double mutant). Bar, 15 µm. (G) The rhabdomeral amount of TRPL-eGFP in the dark-raised (black bars) and orange light-exposed (white bars) arrestin mutants was quantified as in Figs 4, 5. For comparison, the relative amount of TRPL-eGFP in the rhabdomeres of dark-raised and orange light-exposed wild-type flies (WT), as determined in Fig. 5, is shown. Values are expressed as a percentage of the R values of dark-raised wild-type flies.

View larger version (111K): [in a new window]   Fig. 6. Components of the phototransduction cascade are required for TRPL-eGFP translocation. Water immersion images of eGFP fluorescence in the eyes of the following mutants raised for 16 hours in the dark or in orange light are shown: (A,B) w; Gq1; trpl-eGFP, (C,D) w; Gq1/Df(2R)vg135; trpl-eGFP/+, (E,F) w norpAP24; trpl-eGFP/+, (G,H) norpAP57; bw/trpl-eGFP; st/+ (note: this mutant has red eyes, hence eGFP-fluorescence in the cell body was shielded by screening pigments), (I,J) yw; trpl-eGFP; trpP343, (K,L) yw; inaD1 cn bw; trpl-eGFP, (M,N) w inaFP106x; trpl-eGFP/+, (O,P) w; trpl-eGFP/+; trpP365/+. Bar, 15 µm.

View larger version (113K): [in a new window]   Fig. 7. Removal of extracellular Ca2+ inhibited light-induced translocation of TRPL-eGFP. Optical sections of live isolated retinas of flies expressing TRPL-eGFP on a null trpl background (yw; trpl-eGFP trpl302) are shown. The images were obtained by confocal microscopy using a water immersion objective (Olympus 60x/0.9 w LUMPLan F1). (A,D) Images obtained from sliced heads illuminated for 4 hours (Schott OG 590 unattenuated orange light) and incubated in oxygenated extracellular solution with 1 mM EGTA supplemented with 1% FBS and 5 mM sucrose. (B,E) Images obtained from illuminated sliced heads incubated in oxygenated extracellular solution with 1 mM Ca2+ supplemented with 1% FBS and 5 mM sucrose. (C) Image obtained from isolated retina of dark-raised flies. Bars, 10 µm (A,B) and 2 µm (C-E).

View larger version (121K): [in a new window]   Fig. 8. TRPL-eGFP translocation is independent of dynamin. (A,B) In the temperature sensitive mutant w shi ts1; trpl-eGFP/+ kept in the dark or in orange light, at a temperature of 29°C localization of TRPL-eGFP resembled that in the wild type. (C,D) In the myosin III mutant w; ninaC5; trpl-eGFP endocytosis of TRPL-eGFP but not its translocation from the cell body to the rhabdomere, was partially inhibited. Bar, 15 µm.

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