Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA
* Author for correspondence (e-mail: tsunoda{at}bu.edu)
Accepted 21 June 2004
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Summary |
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Key words: Light-dependent, Translocation, Drosophila, G-protein, NINAC, Myosin
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Introduction |
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In the rhabdomeres, light stimulation of rhodopsin leads to the activation of a Gq protein, which activates a phospholipase Cß (PLC), eventually leading to the opening of the light-activated ion channels, transient receptor potential (TRP) and TRP-like (TRPL). Multiple components mediate deactivation of the light response, including the eye-specific protein kinase C (eye-PKC), calmodulin, Arrestin-2 and calmodulin-dependent protein kinase II (Byk et al., 1993
; Hardie and Minke, 1993
; Kahn and Matsumoto, 1997
; Ranganathan et al., 1991
; Scott et al., 1997
; Smith et al., 1991
).
Recent reports have suggested that translocation of TRPL channels in Drosophila photoreceptors and the G-protein transducin in vertebrate photoreceptors contributes to long-term light-adaptation (Bahner et al., 2002; Sokolov et al., 2002
). In the dark, TRPL and transducin are localized to the rhabdomeres of photoreceptors and the outer segment of rods, respectively. In the light, both display massive translocation to the cell body and inner segment (Bahner et al., 2002
; Brann and Cohen, 1987
; Broekhuyse et al., 1987
; Broekhuyse et al., 1985
; Mangini and Pepperberg, 1988
; McGinnis et al., 1992
; Mendez et al., 2003
; Philp et al., 1987
; Sokolov et al., 2002
; Whelan and McGinnis, 1988
).
While our study was in progress, a report by Kosloff et al. (Kosloff et al., 2003) showed that Drosophila Gq
also translocates between the rhabdomere and cell body in a light-dependent manner. Here, we provide further characterization of Gq
translocation and provide the first evidence implicating the photoreceptor-specific class-III myosin NINAC in Gq
transport. We show that as much as 50% of Gq
translocates from the rhabdomere to the cell body within 5 minutes of illumination. When light intensity is increased over five orders of magnitude, translocation of Gq
out of the rhabdomere increases from 20% to 75%, consistent with a role in light adaptation. We then examine a range of genetic mutants to gain insight into the signaling pathway leading to Gq
translocation. We show that the translocation of Gq
requires the light activation of rhodopsin but does not require any of the known signaling components downstream of the G-protein. Finally, we show that, although ninaC mutants display normal translocation of Gq
from the rhabdomere to the cell body, they exhibit a significantly reduced rate of Gq
transport from the cell body to the rhabdomere. We suggest that NINAC might serve as a motor for the light-dependent redistribution of Gq
from the cell body to the rhabdomere.
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Materials and Methods |
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Immunolocalization studies
Flies less than 5 days old were placed 9.3 cm from a white light source (Lambda LS 175W xenon arc lamp with 400-700 nm bandpass filter; Sutter Instruments, Novato, CA, or equivalent) for the indicated time. Intensities are indicated, as measured by an Extech 403125 digital light meter. All experiments were conducted at 24°C. After illumination, fly heads were immediately removed, placed on minutien pins (Fine Scientific Tools, Foster City, CA), and fixed in 3% paraformaldehyde in PBS for 1 hour on ice. Heads were then washed with PBS and incubated in 2.3 M sucrose overnight at 4°C. For dark-raised samples, identical fixing procedures were performed under dim red-light conditions. Heads were halved, oriented with the eyes facing upwards on an ultramicrotomy pin (Ted Pella, Redding, CA), and frozen in liquid nitrogen. 1-µm-thick sections were then cut using a Leica Ultracut UCT attached to an EM FCS cryo unit (Leica Microscopy and Scientific Instruments Group, Heerbrugg, Switzerland) at -81°C. Sections were incubated in blocking solution (1% bovine serum albumin and 0.1% saponin in PBS) for 30 minutes at room temperature and incubated with an antibody against Gq (1:400) (Scott et al., 1995
), TRPL (1:50) (Niemeyer et al., 1996
), Arr2 (1:100) (Dolph et al., 1993
) or rhodamine-conjugated phalloidin (1:500) (Molecular Probes, Eugene, OR) overnight at 4°C. Antibodies were received as a gift from C. S. Zuker (University of California, San Diego, CA). Rhodamine-conjugated goat-anti-rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used at 1:200 for 1 hour at room temperature in the dark. Slides were mounted with 90% glycerol and p-phenylenediamine (Sigma Aldrich, St Louis, MO). Light-exposed images in the figures were taken at the same exposure as dark-raised samples and brightened for illustration purposes to indicate Gq
localization in the cell body.
Quantitative analyses
Tissue sections were visualized using an Olympus BX51 upright fluorescent microscope, and 10-bit images were acquired using an Olympus MagnaFire 2.0 digital camera S99806 for quantitation. Images were analysed using the `Manual Tag' option provided by ImagePro Express (Media Cybergenetics, Silver Springs, MD). A fixed circular area that encompassed approximately 90-100% of the area of an individual rhabdomere was used to measure fluorescent signal intensity within the rhabdomeres and also to measure background intensity levels. Circles used to measure background intensity were placed just off the section to detect any autofluorescence coming from the mounting medium or slide. Because ommatidia are so densely packed in the retina and photoreceptor cell bodies occupy most of the section (Fig. 1), we were unable to measure background intensity levels from the tissue section itself. For quantification in the rhabdomeres, the circle was placed with its bottom edge at the base of the rhabdomere. Identical images acquired in phase-contrast mode were used as an aid to positioning the circle on fluorescent images. For each time point, 240-480 rhabdomeres were measured from eight to twelve sections, from three to eight flies. The R7 photoreceptor cell was not included in any of these analyses. After subtracting average background levels of signal, an average signal intensity and standard errors were calculated for each time point using Microsoft Excel (Microsoft) and Origin 6.0 (Microcal, Northampton, MA).
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Isolation of retinal membranes
For each light condition, 30 fly heads were collected on minutien pins, placed in Eppendorf tubes, and stored at -80°C. To prepare retinal extracts, fly heads were homogenized in buffer (20 mM Hepes, 30 mM NaCl, 5 mM EDTA, protease inhibitors, pH 7.5) and centrifuged (5000 rpm, 4°C, 1-2 minutes) to remove chiton (this process was repeated three times). Membranes were separated from cytosol by ultracentrifugation (55,000 rpm, 4°C, 30 minutes). Cytosolic fractions were collected and concentrated using Centricon-10 columns (Millipore, Bedford, MA). Membrane fractions were resuspended in the above buffer solution, without EDTA. To quantify Gq in fractions, 8-bit images were generated of immunoblots comparing membrane and cytosolic fractions. ImageQuant (Molecular Dynamics, Sunnyvale, CA) was used to quantify signal in protein bands. Background intensities were taken from the same blots, just near the band of interest.
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Results |
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Translocation of Gq between the rhabdomere and cell body is reversible, because light-exposed flies returned to the dark displayed rhabdomeric localization (Fig. 2). To examine the time-course of translocation of Gq
from the cell body to the rhabdomere, we placed light-exposed flies in the dark for increasing time periods and quantified rhabdomeric Gq
immunostaining in tissue sections (Fig. 2). Movement of Gq
from the cell body to the rhabdomere was slower than translocation in the opposite direction.
90% of Gq
returned to the rhabdomeres within 1 hour and full recovery required
2.5 hours of dark incubation (Fig. 2).
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If Gq translocation is a mechanism for light adaptation then we would expect that higher background light intensities would lead to an increase in the quantity of Gq
transported out of the rhabdomeres and a decrease in the Gq
available for signaling. To test this, we examined Gq
translocation after 1 hour of exposure to different light intensities. Indeed, we found that the proportion of Gq
remaining in the rhabdomeres decreases with increasing intensities of light (Fig. 3A). 20-75% of Gq
translocated when exposed to light intensities increased over five orders of magnitude. These results demonstrate that light intensity regulates the quantity of Gq
protein available for signaling. Because a reduction in the amount of Gq
has been shown to decrease amplification (Hardie et al., 2002
), Gq
translocation might be a mechanism underlying light adaptation.
|
Rhodopsin is required for translocation of Gq but PLC, TRP, TRPL and eye-PKC are not
The signaling pathway triggering translocation of Gq is likely to include at least part of the phototransduction cascade. Kosloff et al. (Kosloff et al., 2003
) showed that a constitutively activated form of the major rhodopsin Rh1 results in the persistence of non-membrane-bound Gq
. In order to confirm that Rh1 is required for the subcellular translocation of Gq
, we tested for Gq
translocation in a null mutant of Rh1, ninaEI17. We performed immunostaining using an antibody against Gq
on tissue sections from the eyes of dark-raised and light-exposed mutant flies. Because ninaEI17 mutants display retinal degeneration with age (Leonard et al., 1992
; O'Tousa et al., 1989
), we used young (<24-hour-old) flies whose photoreceptor structure was closer to normal. We found that Gq
is localized to the rhabdomeres of photoreceptors in both dark-raised and light-exposed ninaEI17 flies (Fig. 4A). These results demonstrate that Rh1 is required for Gq
's subcellular translocation.
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Activation of rhodopsin to meta-rhodopsin is required for Gq translocation
Unlike vertebrate rhodopsin, the major Drosophila rhodopsin Rh1 is maximally photoisomerized to the active metarhodopsin state with a wavelength (max) of 480 nm (blue) light, whereas meta-rhodopsin is photoconverted back to rhodopsin with a
max of 580 nm (orange) light (Minke, 1986
). Because rhodopsin is required for Gq
translocation, it is possible that light activation either of rhodopsin to metarhodopsin or of metarhodopsin to rhodopsin triggers Gq
translocation. Because the white-light spectrum contains both blue and orange wavelengths, we used blue light alone (470 nm <
< 490 nm) to test whether conversion of rhodopsin to meta-rhodopsin was sufficient to trigger translocation of Gq
. In agreement with previous reports (Kosloff et al., 2003
; Sokolov et al., 2002
), there was no noticeable difference between Gq
translocation triggered by 480 nm light and that triggered by white light (Fig. 3B). These results show that photoisomerization of rhodopsin to meta-rhodopsin leads to Gq
translocation from the rhabdomere to the cell body.
The white light used in Fig. 2 also transmits orange light, so it was not clear whether dark incubation alone or photoisomerization of meta-rhodopsin to rhodopsin is required for Gq translocation back into the rhabdomeres. To test this, we exposed wild-type flies to blue light (470 nm <
< 490 nm) followed by orange light (
> 570 nm) (Fig. 3B). Our results show that most, but not all, of Gq
was translocated back into the rhabdomere after 2 hours of orange-light exposure (Fig. 3B). Full recovery is not reached, probably because consistent activation of a small quantity of rhodopsin to meta-rhodopsin at 580 nm (Kirschfeld and Franceschini, 1977
; Ostroy et al., 1974
; Salcedo et al., 1999
) prolongs the overall return of Gq
to the rhabdomere. Flies that were exposed to blue light followed by 2.5 hours of dark incubation displayed no translocation of Gq
back to the rhabdomere, suggesting that photoisomerization of meta-rhodopsin to rhodopsin is required for Gq
translocation from the cell body to the rhabdomere.
PLC, TRP, TRPL and eye-PKC are not required for Gq translocation
To determine which, if any, of the components downstream of Gq in the phototransduction cascade are required for Gq
translocation to the cell body, we used null mutants for different components of the phototransduction cascade. Kosloff et al. (Kosloff et al., 2003
) showed that, in PLC mutant (norpA) photoreceptors, Gq
loses its membrane association with illumination similar to the wild type, suggesting that PLC and activation of all downstream signaling components are not required for Gq
translocation. Surprisingly, however, Gq
translocation was blocked in a null mutant for the downstream TRP channel (Kosloff et al., 2003
). Only Gq
's membrane association was tested in norpA mutants previously, and the requirement for TRP but not PLC is unclear because TRP acts downstream of PLC. To resolve these conflicting findings, we examined the subcellular localization of Gq
in retinal tissue sections from null mutants of: the effector PLC (norpAP41), the two light-activated ion channels TRP (trp343) and TRPL (trpl302), and the eye-specific PKC required for deactivation of the cascade (inaC209). We found that Gq
translocated from the rhabdomere to the cell body upon light exposure in all of these mutants (Fig. 4A, Fig. 5A). Unlike TRPL translocation, which requires the presence of the TRP protein but not its function (Bahner et al., 2002
), we find that translocation of Gq
does not require TRP in either capacity.
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Our results for trp mutants contrast with those reported by Kosloff et al. (Kosloff et al., 2003), so we also compared the Gq
's light-dependent shift from membrane-association to cytosol in the wild type and in trp mutants. In wild-type flies, 82% of Gq
is present in dark-adapted membrane fractions, with only 13% present in light-exposed membrane fractions (Fig. 4B). In trp mutants, the shift of Gq
from the membrane to the cytosolic fraction was not significantly different from wild type (Fig. 4B; Student's t-test P>0.05), supporting our immunocytochemical data. Altogether, our results show that the signaling pathway leading to Gq
translocation begins with the light activation of rhodopsin to meta-rhodopsin but does not require the activation of phototransduction components downstream of Gq
, including PLC, TRP, TRPL and eye-PKC.
Gq translocates independently of Arrestin and TRPL
Another component that acts upstream of Gq and that might play a role in signaling Gq
translocation is Arrestin-2. In Drosophila, there are two eye-specific arrestin proteins, Arrestin-1 (Arr1) and Arrestin-2 (Arr2) (Hyde et al., 1990
; LeVine et al., 1990
; Smith et al., 1990
; Yamada et al., 1990
). Arr2 is five to seven times more abundant than Arr1 and arr2 mutants display defects in deactivation (Dolph et al., 1993
), consistent with its role of binding and deactivating metarhodopsin. The role of Arr1 is unclear, because arr1 mutants do not exhibit any significant defects in their light responses except as an arr1 arr2 double mutant (Dolph et al., 1993
). Vertebrate visual arrestin has been reported to undergo light-dependent movement between the outer and inner segments of rods (Mendez et al., 2003
), whereas Arr2 in Drosophila translocates between the rhabdomere and cell body of photoreceptors (Kiselev et al., 2000
; Lee et al., 2003
). Similar light-dependent translocation was also reported in crayfish (Terakita et al., 1998
).
Because Arr2 acts upstream of the G-protein, it might also play a role in signaling the translocation of Gq. ß-Arrestin has been reported to have multiple functions, including serving as a scaffold for two different signaling pathways (Luttrell et al., 1999
; Zuker and Ranganathan, 1999
). Because Arr2 and TRPL also undergo light-dependent translocation between the rhabdomere and cell body, albeit in different directions, (Bahner et al., 2002
; Kiselev et al., 2000
; Kosloff et al., 2003
; Lee et al., 2003
), we tested the possibility that Gq
's translocation might be dependent on Arr2 or TRPL. We found that Gq
can translocate independently of TRPL and Arr2 (Fig. 5A), and that TRPL and Arr2 can translocate independently of Gq
(Fig. 5B). This is similar to visual arrestin and transducin in vertebrate rods (Mendez et al., 2003
; Zhang et al., 2003
). Although transport mechanisms for Gq
, Arr2 and TRPL might or might not be shared, activation of components downstream of Gq
is not required for signaling translocation of Gq
, Arr2 or TRPL.
Translocation of Gq is not regulated by shibere-mediated endocytosis
What is the molecular mechanism underlying Gq translocation? Because vertebrate transducin displays light-dependent translocation to detergent-resistant lipid rafts (Nair et al., 2002
), it is possible that Gq
translocation involves a membrane-associated type of transport, such as endocytosis. Previous studies have suggested that endocytosis mediates the movement of rhodopsin-arrestin complexes from the rhabdomeres to the cell body in the retinal degeneration mutant rdgC (Kiselev et al., 2000
) and in norpA mutants (Alloway et al., 2000
; Orem and Dolph, 2002
). To test whether translocation of Gq
occurs through endocytosis, we used the temperature-sensitive shibere mutant shits1. The shibere gene product is a homolog of the dynamin GTPase required in the `pinching off' of vesicles during endocytosis (van der Bliek and Meyerowitz, 1991
). At 25°C, shits1 mutants are indistinguishable from wild type, whereas, at 29-30°C, shits1 mutants display rapid paralysis as a result of disrupted endocytosis (Grigliatti et al., 1973
; van der Bliek and Meyerowitz, 1991
). To determine whether Gq
translocates from the rhabdomere to the cell body in photoreceptors via endocytosis, we examined the immunolocalization of Gq
in dark-raised and light-exposed shits1 mutants incubated at the restrictive temperature. We found that Gq
was localized to the rhabdomeres in dark-raised shits1 flies, whereas light exposure resulted in translocation of Gq
to the cell body similar to wild-type flies (Fig. 6). These results suggest that the molecular mechanism underlying Gq
translocation does not involve shibere-mediated endocytosis.
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Photoreceptor-specific myosin NINAC is involved in the transport of Gq from the cell body to the rhabdomere
Another possible mechanism of Gq translocation is via a motor protein. In vertebrate photoreceptors, kinesin II and myosin VIIa have been suggested to function in the transport of opsin and arrestin from the inner segment to the outer segment (Marszalek et al., 2000
; Williams, 2002
). Because the rhabdomere is composed of microvilli, the major cytoskeletal component is actin. Therefore, a myosin is likely to be the molecular motor for Gq
translocation. NINAC is the only photoreceptor-specific myosin that has been identified thus far in Drosophila (Montell and Rubin, 1988
). NINAC is a class-III myosin that is made up of an N-terminal protein-kinase domain, followed by a myosin head domain and two calmodulin-binding sites (Montell and Rubin, 1988
; Porter et al., 1993
). NINAC has been suggested to play a role in phototransduction (Porter et al., 1992
; Wes et al., 1999a
) as well as in linking the axial cytoskeleton to the microvillar membrane (Hicks et al., 1996
; Hicks and Williams, 1992
). Although mechanoenzymatic activity has not yet been demonstrated for Drosophila NINAC, a recent report by Komaba et al. (Komaba et al., 2003
) showed that human myosin III functions as an actin-based motor protein with a translocating activity of 0.11 µm second-1, as determined in an in vitro actin gliding assay. Because human myosin III acts as a plus-end motor (Komaba et al., 2003
) and microvilli consist of bundled actin filaments polarized with their plus ends oriented away from the cell body (Arikawa et al., 1990
; Mooseker et al., 1982
), we expected that NINAC might transport Gq
from the cell body to the rhabdomere.
To test whether either of the NINAC isoforms (Montell and Rubin, 1988; Porter et al., 1992
) is involved in the light-dependent translocation of Gq
, we examined the time course of Gq
translocation in the ninaC5 (also called ninaCP235) null mutant. Because ninaC mutants have been shown to display light-dependent and age-dependent retinal degeneration, all experiments were performed using dark-raised, newly eclosed (<8 hours old) flies that display the minimum amount of degeneration (Porter et al., 1992
). Our results show that the time course of Gq
transport from the rhabdomere to the cell body was similar to the wild type (Fig. 7): maximal translocation was complete within 5 minutes. The rate of Gq
transport from the cell body to the rhabdomere, however, was significantly reduced (Fig. 7). The proportion of Gq
in the rhabdomeres after 2.5 hours of light exposure was similar between wild-type and ninaC5 mutants: 51.1±0.9% and 46.5±0.5%, respectively. After 1 hour of dark incubation, we found that, in wild-type flies, 89.3±1.8% of Gq
is localized to the rhabdomeres, whereas only 50.3±0.6% of Gq
is localized to the rhabdomeres in ninaC5 mutants. Almost full recovery of Gq
to the rhabdomeres was reached within 1 hour for the wild type, whereas full recovery of Gq
to the rhabdomeres of ninaC5 mutants was observed after 4 hours of dark incubation. Our findings are consistent with a model in which NINAC functions as a plus-end-directed motor protein facilitating Gq
transport.
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Discussion |
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Signaling pathway triggering translocation of Gq
Our genetic analyses show that Gq translocation from the rhabdomere to the cell body requires the activation of rhodopsin to meta-rhodopsin, but not any of the known signaling components downstream of the G-protein, including PLC, TRP, TRPL, eye-PKC and Arr2. In agreement with our studies, Kosloff et al. (Kosloff et al., 2003
) showed that a constitutively activated rhodopsin leads to the persistence of non-membrane-bound Gq
and that norpA mutants display a light-dependent shift of Gq
from the membrane-associated fraction to the soluble fraction of head homogenates. In contrast to our data, however, Kosloff et al. (Kosloff et al., 2003
) report a requirement for the TRP channel in Gq
translocation. The reasons for these different results are unclear. Different experimental conditions, including light intensity, illumination time or fixation procedures following illumination, might have contributed to these conflicting results. We further demonstrate that the translocation of Gq
from the cell body to the rhabdomere requires the photoconversion of meta-rhodopsin to rhodopsin.
The signaling pathways following the light activation of rhodopsin and leading to Gq translocation between the rhabdomere and cell body remain to be determined. Activation of Gq
might or might not be necessary for translocation. Pharmacological agents that block Gq
activation might be useful in determining whether activation of Gq
is required for translocation. One possibility is that, even without activating Gq
, meta-rhodopsin might signal some other unidentified component leading to the mobilization of Gq
. Another possibility is that activation of Gq
by rhodopsin is the trigger for translocation itself. Activated Gq
remains in the rhabdomeres to activate PLC and might then become a target for transport. Alternatively, activated Gq
might signal the translocation of inactive G-protein heterotrimers. In vertebrates and crayfish, Gß
has been reported to translocate out of the outer segment and rhabdomeres, respectively, with illumination (McGinnis et al., 2002
; Sokolov et al., 2002
; Terakita et al., 1998
; Zhang et al., 2003
). The half-time to complete translocation of the transducin-
subunit from the outer to the inner segment was reported to be three times faster than that for the transducin-ß subunit (Sokolov et al., 2002
), suggesting that the
and ß
subunits translocate separately. In knockout mice lacking the transducin-
subunit, however, transducin-ß
was unable to redistribute to the inner segment with light, suggesting that transducin translocates as a heterotrimer (Zhang et al., 2003
). It has not yet been demonstrated whether the Gß
subunit in Drosophila photoreceptors also moves out of the rhabdomere with illumination. If so, association with Gq
before, during and after transport will need to be assayed.
Molecular mechanism underlying Gq translocation
How is Gq transported between the rhabdomere and cell body? We envisioned three potential mechanisms: (1) endocytosis of membrane-associated Gq
; (2) transport by a myosin; and/or (3) diffusion of free Gq
from one compartment to the other. Because Gq
returns to the rhabdomeres significantly more slowly than it leaves, translocation in each direction might involve different components and/or mechanisms.
Our results show that translocation of Gq is unaltered in the shits1 mutant at the restrictive temperature, suggesting that shibere-mediated endocytosis is an unlikely mode of translocation. Endocytosis might instead eliminate unwanted components from the rhabdomere, because accumulated rhodopsin-arrestin complexes in norpA and rdgC mutants are removed by endocytosis (Alloway et al., 2000
; Kiselev et al., 2000
; Orem and Dolph, 2002
).
Another possibility for transport of Gq is a mechanism involving myosin(s). In this study, we show that the photoreceptor-specific myosin NINAC is required for a normal rate of Gq
transport from the cell body to the rhabdomere. We cannot, however, rule out the possibility that NINAC, which contains a protein-kinase domain and has been implicated as a signaling protein in phototransduction (Hofstee et al., 1996
; Porter et al., 1995
; Porter and Montell, 1993
; Wes et al., 1999b
), is involved in signaling Gq
translocation. ninaC null mutants have also been shown to exhibit a loss of the axial cytoskeleton from rhabdomeres and undergo retinal degeneration (Hicks and Williams, 1992
; Matsumoto et al., 1987
), making it possible that slowed Gq
transport is due in part to rhabdomeric cytoskeletal degeneration. However, if slowed Gq
transport is indeed a secondary effect of retinal degeneration, we would expect the effect to be rather non-specific. Because our results show that only plus-end-directed translocation is affected in ninaC mutants, whereas minus-end-directed translocation is unaltered, we suggest that the slowed rate of Gq
transport is a direct effect of the loss of NINAC protein. Future analyses of additional ninaC mutant alleles will determine whether NINAC does indeed function as a motor protein in Gq
transport.
Interestingly, ninaC mutants do not display a complete blockage of Gq translocation from the cell body to the rhabdomere. One possibility is that multiple myosins contribute to the transport of Gq
. There are 12 other myosins in Drosophila (Tzolovsky et al., 2002
). It will be important to determine whether any of these myosins are expressed in photoreceptors. Alternatively, in the absence of NINAC, Gq
might ultimately be translocated to the rhabdomeres by another molecular mechanism, such as diffusion. In wild type, NINAC might function in concert with diffusion to increase the rate of Gq
transport. Diffusion might contribute to translocation of Gq
in either direction. For example, when meta-rhodopsin activates the Gq protein in phototransduction, the concentration of free Gq
is suddenly increased in the rhabdomeres, perhaps driving Gq
down its concentration gradient into the cell body. If diffusion drives Gq
back into the rhabdomeres with dark incubation then we expect that proteins binding to Gq
might play a role in regulating the concentration of free Gq
. It will be important to determine whether, in Drosophila photoreceptors, Gq
ß
translocates as an inactive heterotrimer, Gq
and Gß
translocate independently or Gß
does not translocate. Other binding proteins of Gq
and Gß
might also play essential roles in regulating the concentration of free G-protein subunits and their direction of translocation.
Subcellular localization of transduction proteins has proved to be crucial for signaling because mislocalization of components often results in the severe impairment of function. Dynamic regulation of protein localization might be an important strategy for controlling the quantity of transduction components available for signaling. In this way, cells can adjust their sensitivity and prevent overstimulation. The subcellular translocation of Gq in Drosophila photoreceptors provides an attractive model for further investigation into the signaling pathway leading to translocation and the molecular mechanisms of transport.
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Acknowledgments |
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