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First published online 31 August 2004
doi: 10.1242/jcs.01371

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Light-dependent subcellular translocation of G_q in Drosophila photoreceptors is facilitated by the photoreceptor-specific myosin III NINAC

Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA

View larger version (78K): [in a new window]   Fig. 1. Time-course of Gq translocation from the rhabdomere to the cell body. (A) 1-µm-thick retinal cross-sections of wild-type flies that were dark-raised (0 minutes) or light exposed for 5 minutes or 60 minutes. Sections were stained with anti-Gq antibodies. In dark-raised flies, Gq displays complete rhabdomeric localization. Dark-raised flies exhibited Gq immunoreactivity that was non-uniform across any one rhabdomere, displaying areas of higher concentration that appear to be randomly localized. By contrast, after 5 minutes of illumination, Gq was progressively concentrated towards the base of the rhabdomere (arrowheads). After 60 minutes, we often observed localization of Gq at the plasma membrane of the cell body. (B) A 1-µm-thick retinal cross-section of a single wild-type Drosophila ommatidium stained with phalloidin conjugated to rhodamine. The rhabdomere and cell body of a single photoreceptor cell are indicated by the letters R and C, respectively. For further clarification, the cell body is outlined. (C) Quantitation of Gq in the rhabdomeres from cross-sections of dark-raised wild-type flies, exposed to light for 5 minutes, 10 minutes, 20 minutes or 60 minutes. Images used were taken at the same exposure time as dark-raised samples. Values are expressed as a percentage of Gq signal measured in the rhabdomeres of dark-raised flies, in which Gq protein is exclusively localized to the rhabdomeres. After 5 minutes of illumination at 50.7x103 lumens meter-2, 54.4±0.63% of Gq translocates out of the rhabdomere; no additional Gq appears to be translocated out of the rhabdomere with longer light exposures. SEMs are indicated.

View larger version (76K): [in a new window]   Fig. 2. Time course of Gq translocation from the cell body to the rhabdomere. (A) Retinal cross-sections of dark-raised wild-type flies that were light exposed for 2.5 hours with a light intensity of 50.7x103 lumens meter-2 and then incubated in the dark for 20 minutes or 150 minutes. Tissue sections were immunostained with an antibody against Gq. (B) Quantitation of Gq in the rhabdomeres from cross-sections of wild-type flies that were light-exposed for 2.5 hours and then incubated in the dark for 20 minutes, 60 minutes, 150 minutes or 240 minutes. Values are expressed as a percentage of Gq signal in the rhabdomeres of the corresponding dark-raised flies. In the wild type, 89.3±1.76% of Gq is localized to the rhabdomeres within 60 minutes of dark incubation. SEMs are indicated.

View larger version (61K): [in a new window]   Fig. 3. (A) The quantity of Gq translocated from the rhabdomere to the cell body is dependent on the intensity of illumination. Cross-sections of dark-raised wild-type flies were illuminated under the indicated light intensities for 1 hour. Sections were stained with anti-Gq antibody and quantified. Percentages of Gq remaining in the rhabdomeres after exposure to the indicated light intensities (I) were calculated, where initial light intensity (I0)=70.0x103 lumens meter-2. Exposure to light intensities with log(I/I0) of -5 to 0 resulted in translocation of 20-75% Gq out of the rhabdomeres. SEMs are indicated. (B) Photoisomerization of metarhodopsin to rhodopsin is required for Gq transport from the cell body to the rhabdomere. Shown are retinal cross-sections immunostained for Gq. Dark-raised wild-type flies were exposed to: (1) blue (bandpass 470 nm > > 490 nm) light for 2 hours; (2) blue light for 2 hours followed by 2 hours of orange (long-pass > 580 nm) light; or (3) blue light for 2 hours followed by 2.5 hours of dark incubation. Dark-raised flies showed normal, rhabdomeric localization of Gq whereas flies illuminated with blue light displayed translocation of Gq into the cell body. Flies exposed to blue light followed by orange light displayed almost full recovery of Gq to the rhabdomeres. Flies exposed to blue light followed by dark incubation showed no recovery of Gq to the rhabdomeres. These results indicate that photoisomerization of metarhodopsin to rhodopsin is absolutely required for normal translocation of Gq from the cell body to the rhabdomeres.

View larger version (78K): [in a new window]   Fig. 4. Gq translocation requires the light-activation of rhodopsin, but does not require the activation of the phototransduction components downstream of Gq. (A) Cross-sections of the wild type (WT) and null mutants in Rh1 (ninaEI17), PLC (norpAP41), TRP (trp343) and PKC (inaC109), all stained with anti-Gq antibody. All flies were either dark raised or light exposed for 2 hours (3000 lumens meter-2). In ninaEI17, Gq is rhabdomeric in both dark and light conditions. A low level of Gq signal was seen in cell bodies, consistent with previous reports showing that ninaE mutants display subrhabdomeric invaginations into the cell bodies (Leonard et al., 1992; O'Tousa et al., 1989). The remaining null mutants displayed rhabdomeric localization of Gq in the dark and translocation of Gq upon illumination. This suggests that rhodopsin is necessary for translocation of Gq, whereas PLC, TRP and PKC are not. (B) Gq undergoes a light-dependent shift to membranes in trp343 null mutants similar to WT. Dark-adapted WT and trp343 flies were exposed to white light (50.7x103 lumens meter-2) for 2.5 hours. Membrane and corresponding cytosolic fractions were isolated for immunoblot analysis. The bar graph shows the proportions of total Gq present in the membrane and cytosolic fractions for dark-adapted and light-exposed flies. DC, dark-adapted cytosolic fraction; DM, dark-adapted membrane fraction; LC, light-exposed cytosolic fraction; LM, light-exposed membrane fraction. The trp343 null mutants displayed a light-dependent shift of Gq from the membrane-associated fraction to the cytosolic fraction that was not significantly different from the wild type. WT: DM, 81.5±6.0%; LM, 13.2±9.6%; trp: DM=81.5±10.5%, LM=4.2±1/3%. A representative immunoblot is shown above the graph. SEMs are indicated

View larger version (70K): [in a new window]   Fig. 5. (A) The translocation of Gq is independent of TRPL and Arrestin-2. Cross-sections from trpl302 and arr25 null mutants were stained with an antibody for Gq. All flies were either dark-raised or light exposed for 2 hours. Gq displayed normal, light-dependent translocation in both the trpl and the arr2 mutants, indicating their non-involvement in Gq's translocation. (B) The translocation of TRPL and Arr2 is independent of Gq. Cross-sections from dark-raised and light-exposed dgq1 mutants were immunostained for TRPL and Arr2. The dgq1 mutant is a severe hypomorph that produces 1% of the wild-type levels of Gq (Scott et al., 1995). TRPL displays normal translocation, moving from the rhabdomere to the cell body upon illumination. Likewise, Arr2 displays normal light-dependent translocation from the cell body to the rhabdomere. These results indicate that the light-regulated translocation of both Arrestin2 and TRPL do not require Gq.

View larger version (75K): [in a new window]   Fig. 6. The mechanism of Gq translocation does not involve Shibere-mediated endocytosis. Dark-raised shiberets1 flies were first incubated at 30°C for 1-2 minutes in the dark to block endocytosis, as indicated by paralysis. Paralyzed shiberets1 flies continued to be incubated at 30°C but were either kept in the dark or transferred to the light for 1 hour. This incubation did not harm the flies because paralysis was reversible. Cross-sections were stained for Gq. When endocytosis is disrupted, Gq still displays light-regulated translocation from the rhabdomere to the cell body.

View larger version (34K): [in a new window]   Fig. 7. The mechanism of Gq translocation from the cell body to the rhabdomere involves the photoreceptor-specific myosin NINAC. (A) Cross-sections from wild-type (WT) and the null mutant ninaC5 were immunostained for Gq. Flies were light-exposed for 2.5 hours (Lex) and then dark-incubated (LD) for 0 minutes, 60 minutes or 240 minutes. Light-exposed WT and ninaC5 flies displayed normal translocation of Gq to the cell bodies. After 60 minutes of dark incubation, near-complete recovery of Gq to the rhabdomeres was observed in the WT, whereas ninaC5 mutants required up to 240 minutes of dark incubation for complete recovery of Gq to the rhabdomeres. Total Gq protein remained constant in light-exposed and dark-incubated samples as tested by immunoblot analysis (data not shown). (B) Quantitation of Gq in the rhabdomeres from cross-sections of dark-raised wild-type (filled circles) and ninaC5 (open squares) flies, exposed to light for 5 minutes or 60 minutes. Images used for quantitation were taken at the same exposure time as dark-raised samples. Values are expressed as a percentage of Gq signal measured in the rhabdomeres of dark-raised flies. In both dark-raised WT and ninaC5 flies, Gq protein is exclusively localized to the rhabdomeres, whereas, after 5 minutes of illumination at 50.7x103 lumens meter-2, the maximum amount (50%) of Gq translocates out of the rhabdomere. (C) Quantitation of Gq in the rhabdomeres from cross-sections of WT and ninaC5 flies that were light exposed for 2.5 hours and then incubated in the dark for 60 minutes, 150 minutes or 240 minutes. Values are expressed as a percentage of Gq signal in the rhabdomeres of the corresponding dark-raised flies. The recovery of Gq to the rhabdomeres was significantly slower in ninaC5 mutants. In the WT flies, 90% of Gq is localized in the rhabdomeres within 60 minutes, whereas, in ninaC5 mutants, only 50% is localized in the rhabdomeres. After 60 minutes and 150 minutes of dark incubation, differences between WT and ninaC5 were statistically significant (Student's t-test, P<0.05). Full recovery of Gq to the rhabdomeres in ninaC5 mutants was complete by 240 minutes.

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