Correspondence to Zvi Selinger: selinger{at}vms.huji.ac.il
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Abbreviations used in this paper: ERG, electroretinogram; GDP, guanosine diphosphate.
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Introduction |
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Membrane attachment of heterotrimeric G proteins has been extensively investigated, and the effect of lipid modification on membrane localization has been addressed by several studies (Wedegaertner, 1998; Resh, 1999; Chen and Manning, 2001; Kosloff et al., 2002, 2003; Smotrys and Linder, 2004). All G protein subunits (with the exception of transducin) are palmitoylated, and some are additionally modified by myristoylation. The
subunits of Gq/G11, including the Drosophila melanogaster eyespecific Gq
, as well as Gs
, G12
, and G13
are modified only by palmitoylation. The corresponding ß
subunits undergo isoprenylation of a cysteine residue at the so-called CAAX box of the
subunit (for review see Wedegaertner, 1998; Resh, 1999; Chen and Manning, 2001). Plasma membrane attachment of the
subunits Gs
and Gq
is dependent on coexpression with the ß
subunits (Evanko et al., 2000, 2001). Furthermore, the ß
subunits, having only one membrane attachment signal on the
subunit, are poorly targeted to the plasma membrane and require coexpression of the
subunit for efficient plasma membrane attachment (Evanko et al., 2001; Michaelson et al., 2002; Takida and Wedegaertner, 2003). Altogether, these studies led to a model of two membrane attachment signals that are needed for plasma membrane attachments and localization of heterotrimeric G protein subunits (Wedegaertner, 1998; Resh, 1999). It should be noted, however, that most of these studies have been performed by using various culture cells that were transfected with vectors yielding overexpressed proteins (usually the
and ß
subunits of the heterotrimeric G protein). This procedure is bound to cause distortion of the original stoichiometry of
and ß
subunits, which is difficult to control under these conditions. The extensively studied Drosophila visual system combined with the large repertoire of Drosophila visual mutants offer a unique opportunity to study in vivo the various roles of the ß
dimer, its cellular localization, and the functional consequences of altering
/ß
stoichiometry.
The Drosophila visual system is a specialized system that is composed of highly polarized and compartmentalized cells that sequester the phototransduction machinery in a specific signaling compartment called the rhabdomere (Minke and Hardie, 2000; Hardie and Raghu, 2001). This signaling compartment is functionally equivalent to the vertebrate rod photoreceptor outer segment, which also sequesters the phototransduction machinery in a specific cell compartment. Phototransduction in Drosophila is initiated upon the activation of rhodopsin by light and proceeds through a photoreceptor-specific Gq protein (Gqe; Scott et al., 1995), which, in turn, activates the phospholipase C enzyme effector (Devary et al., 1987). Upon activation, the eye-specific Gq subunit (Gq
e) dissociates from the eye-specific ß
dimer (Gß
e) and translocates, at least in part, from the membrane to the cytosol (Kosloff et al., 2003; Cronin et al., 2004).
In this study, we show (by using a series of eye-specific Gße hypomorph mutants) that the ß dimer has a crucial role in both membrane attachment and rhabdomeral targeting of the
subunit that can account for the decreased light sensitivity previously observed in these mutants (Dolph et al., 1994). On the other hand, by using the almost null mutant for the eye-specific Gq
subunit G
q1, we found that the ß
dimer is dependent on the
subunit for membrane attachment but not for targeting to the rhabdomere, suggesting a role for the ß
dimer in targeting the heterotrimer to the photoreceptor signaling compartment (the rhabdomere). An analysis of the protein levels of Gq
e and Gße subunits revealed a surprising twofold excess of the Gße subunit over the Gq
e subunit. Mutants that eliminated this excess showed a dramatic increase in spontaneous activity of the phototransduction cascade. Conversely, double mutations that also reduced the level of Gq
e and, thereby, restored the excess of Gße over Gq
e completely reversed this phenotype. Together, these results provide a significant insight into the strategy used by the photoreceptor cell in vivo to avoid spontaneous activity at the G protein level.
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Results |
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The hypomorph Gße mutants Gße1, Gße2, and the heterozygote of the most severe mutant, Gße1/+, express the Gße subunit protein at levels of 4, 13, and 50% of wild-type flies, respectively (Fig. 1, A and B). Despite the progressive decrease in the Gße subunit level in these mutants, the level of the subunit was undiminished and is the same level as in wild-type flies (Fig. 1 A). Similarly, in the G
q1 mutant, which expresses negligible levels of the
subunit, the level of the Gße subunit was the same as in wild-type flies (Fig. 1 C). Although the levels of the Gße subunit that we found in the Gße1 and Gße2 mutants were higher than those previously reported (Dolph et al., 1994), the progressive decrease of the Gße subunit protein among these mutants was similar (Fig. 1 B). The eye-specific G
e subunit, which forms an extremely tight complex with the Gße subunit, completely disappeared in the severe Gße1 mutant but, like Gße, was undiminished in the G
q1 mutant (Fig. 1 D). Therefore, we can conclude that the Gße mutants are, in fact, ß
mutants and that the effects observed in Gße mutants can be ascribed to a decrease in the level of the ß
subunit dimer without effecting the level of the
subunit.
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The reduced light sensitivity of Gße mutants is caused by the mislocalization of Gqe
One of the major advantages of Drosophila for the study of phototransduction in vivo is the ability to examine the electrophysiological response in detail and characterize the phenotype that results from a decrease in a specific phototransduction component, which is caused by mutation. Two physiological phenotypes were observed for Gße mutants (Dolph et al., 1994). The first phenotype was a dramatic loss of light sensitivity (reaching a decrease by two orders of magnitude in the Gße1 mutant), and the second phenotype was a slow termination of the light response. To address the possibility that the reduced sensitivity to light in Gße mutants arises from a reduction in membrane-bound Gqe, we reexamined the sensitivity to light in four Drosophila mutants with reduced levels of Gße. Figs. 2 and 3 show a correlation between the level of membrane-bound Gq
e (Fig. 2) and the sensitivity of the response to light (Fig. 3) in which low levels of membrane-bound Gq
e correspond to low light sensitivity. The latter was accompanied by a modified waveform of the light-induced current (Fig. 3, inset). The fact that heterozygous Gße1/+ showed only a minor reduction in the sensitivity to light is consistent with previous results showing that 50% of Gq
e is sufficient to maintain normal sensitivity to light (Scott et al., 1995). Together, these results indicate that the loss of light sensitivity is caused by the effect of Gße mutants on membrane attachment and targeting of the Gq
e subunit to the signaling compartment (the rhabdomere). Clearly, when rhodopsin and Gq
e are present in different cellular compartments, the Gq
e subunit cannot transfer signals from rhodopsin to the phospholipase C enzyme.
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The Gße subunit is present in excess over the Gqe subunit
The presence of 80% of Gqe in a membrane-bound form in wild-type dark-adapted flies (Fig. 2 B, left), whereas only 50% of Gße is membrane bound (Fig. 4 B, left), raised the question of the stoichiometry of these two components. To determine the levels of the subunits in vivo, we performed immunoblot analysis with a mixture of Gq
e- and Gße-specific antibodies at a concentration five times that required for their saturation. Furthermore, two different anti-Gße antibodies that were raised against two different sequences of the Gße protein gave similar results (see SDS-PAGE and immunoblotting). In wild-type flies, the amount of Gße was
2.5 times higher than the amount of Gq
e (Fig. 5, A and B). To verify the excess of Gße over Gq
e subunits in wild-type flies, which was determined by Western blotting, we calibrated the immunoblot with the use of purified recombinant Drosophila Gq
e and Gße proteins. We determined the concentrations of the recombinant proteins spectrophotometrically by using calculated extinction coefficients of Gq
e = 42,350 cm1 M1 and Gße = 60,000 cm1 M1 at 280 nm. This quantitative analysis of two samples of wild-type fly head homogenate again revealed an excess of Gße over Gq
e of
2.5 times (Fig. 5 C).
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The unexpected excess of Gße over Gqe was almost completely abolished in the Gße1 heterozygous mutant (Gße1/+). The ratio between Gße and Gq
e in this mutant was
1:1. The decrease in Gße levels of this mutant did not change the ratio between membrane-bound Gq
e and Gße, which remained
1:1. In the soluble fraction, however, we found a large decrease of excess Gße. Although the ratio between soluble Gße and Gq
e in wild-type flies was
7:1, the ratio in the Gße1/+ mutant was reduced to
2.5:1 (Fig. 5, A and B).
Spontaneous activity of Gße mutants
A new and striking phenotype of Gße mutants was revealed in this study. Whole cell patch-clamp recording of dark-adapted mutant photoreceptor cells showed spontaneous, unitary, inward currents that were similar in shape to the single photon responses known as quantum bumps (Fig. 6; Henderson et al., 2000). The frequency of these spontaneous responses was different for the various Gße mutants. For the Gße1 mutant, only a low frequency of spontaneous bumps was observed, which was not much different from the frequency of spontaneous bumps observed in wild-type flies. A higher frequency of spontaneous bumps was clearly noted for the Gße2 mutant, whereas the most dramatic increase in the frequency of spontaneous bumps was observed for the Gße1 heterozygous mutant (Gße1/+). The high frequency of spontaneous bumps in the heterozygous Gße1 mutant is surprising because this mutant has normal sensitivity to light in contrast to the Gße1 homozygote, which is the most severe mutant but has an almost normal frequency of spontaneous bumps (Figs. 3 and 6). This complex behavior can be explained by the decreased levels of Gqe observed in the signaling compartment of these mutants (Fig. 2). Indeed, when the bump frequency was normalized to the number of rhabdomeral Gq
e, a similar bump frequency per rhabdomeral Gq
e was observed for all of the Gße mutants, whereas the wild-type bump frequency remained much lower (Fig. 6 C).
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Discussion |
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We also examined how a decrease in the subunit of the G
q1 mutant influences membrane attachment and targeting of the ß
subunits to the rhabdomere. In this case, the ß
dimer is soluble and not membrane attached but is still targeted to the rhabdomere. The presence of ß
in the rhabdomeral cytosol may be physiologically important for preventing spontaneous activity because the ß
subunits are in close proximity to the membrane-bound signaling molecules.
We have previously shown that the eye-specific Gqe subunit translocates from rhabdomeral membranes to the cytosol in response to illumination (Kosloff et al., 2003). Gq
e behaves like many other G
subunits, which demonstrate activity-dependent translocation from the membrane to the cytosol (for review see Resh, 1999; Chen and Manning, 2001; Smotrys and Linder, 2004). The Drosophila eyespecific ß
dimer behaves differently from the Gq
e subunit, as it does not show any significant change in its distribution even after prolonged illumination (Fig. 4). A possible reason for this result might be an interaction between the
subunit of the ß
dimer and the photoactivated rhodopsin. Such an interaction has been reported for the transducin
subunit and the active form of vertebrate rhodopsin (Kisselev and Downs, 2003). Both vertebrate and invertebrate photoreceptor cells contain high concentrations of rhodopsin, and even a weak interaction could be significant as a result of mass action. It should be noted, however, that studies in rat retina detected light-dependent movement of both the
and ß
subunits from the rod outer to inner segment, although the ß
subunits moved more slowly than the
subunit, suggesting that it might be caused by an interaction of the ß
complex with phosducin (Sokolov et al., 2002, 2004). The different behavior of Gß
subunits in vertebrate and Drosophila might be caused by the difference in stability of the active rhodopsin in these two systems. Whereas vertebrate rhodopsin undergoes bleaching and inactivation, the activated rhodopsin of Drosophila is stable for hours (Minke and Selinger, 1996).
Spontaneous, dark photoreceptor activity in Gße mutants
A functional hallmark of visual photoreceptors is utmost sensitivity of the capacity for single photon detection. This sensitivity is achieved by very high concentrations of the photoreceptor rhodopsin and its target G protein as well as by the large amplification that is generated during the phototransduction process. High sensitivity also depends on an exceedingly low spontaneous activity (low, dark noise) that sets the limit on the absolute sensitivity of this signaling system. Rhodopsin is the only G proteincoupled receptor that has covalently linked 11 cis-retinal that behaves like a "quasi" antagonist in the dark, preventing spontaneous activity. The visual G protein, however, needs special mechanisms to prevent spontaneous activation, but these mechanisms remain unknown.
The Gß subunits are known to bind to G
-GDP switch regions, thereby stabilizing the binding of GDP and suppressing spontaneous receptor-independent activation (Itoh and Gilman, 1991; Lambright et al., 1996; Sondek et al., 1996; Preininger and Hamm, 2004). To find out whether this interaction is relevant to the Drosophila eyespecific Gq heterotrimer, whose three-dimensional structure has not been determined, we have constructed a homology model of the DGqe heterotrimer (
ß
) based on the crystal structure of transducin (unpublished data). It appears from the model that the Gß
e subunit complex directly contacts the switch I and switch II space regions of Gq
e as was previously reported for other G proteins. Therefore, it is conceivable that the Drosophila Gß
e complex prevents the spontaneous activation of Gq
e by binding to Gq
e switch regions. It should be pointed out, however, that the physiological consequences of this effect in vivo have not been described previously. Furthermore, the mechanism that suppresses the spontaneous activity of G proteins under physiological conditions is unknown. In this study, we report (Fig. 6) the observation of a high frequency of spontaneous activity in the heterozygous Gße1/+ mutant in which the level of the ß subunit was decreased to 50% of its level in wild-type flies. Surprisingly, a further decrease in the level of Gße in the Gße2 and Gße1 mutants did not increase the frequency of spontaneous activity but rather decreased the frequency. This is easily seen in the most severe mutant (Gße1), which has only 4% of Gße, as the spontaneous activity is not much different from the low frequency in wild-type flies. This is probably the reason why the increase in spontaneous activity was not detected in the initial characterization of Gße mutants (Dolph et al., 1994). These results indicate that the relationship between the level of Gße and the spontaneous activity is not straightforward. We suggest that the observed spontaneous activity of ß mutant photoreceptor cells is regulated by two opposite effects of the ß
dimer. On the one hand, the decrease in ß
levels leaves some G
-GDP unassociated with ß
, and this free G
-GDP undergoes spontaneous exchange of the bound GDP for free GTP, leading to spontaneous activity. On the other hand, the decreased level of ß
leads to a proportional decrease of G
in the signaling compartment, resulting in a diminished ability to activate the phototransduction process. To test this notion, we normalized the observed rate of spontaneous activity to the number of Gq
e subunits in the rhabdomeres of Gße mutants that lack excess Gße over Gq
e. We found (Fig. 6 C) similar frequencies of normalized spontaneous activity for all of the Gße mutants, which is consistent with a role of excess ß
over Gq
e in suppressing spontaneous activity.
The presence of excess Gße over Gqe in the Drosophila photoreceptor cell
One of the unexpected and novel findings of this study is the presence of the Drosophila eyespecific Gße subunit in 2.5-fold excess over the Gq
e subunit. Because the levels of
and ß subunit proteins are maintained independently of one another, unequal levels of these subunits are mechanistically possible. Our calibration curves using purified recombinant Gße and Gq
e proteins (Fig. 5 C) verified the excess of Gße over Gq
e subunits, which was determined by immunoblot analysis with a mixture of Gq
e and Gße antibodies (Fig. 5 A). Furthermore, we have shown that as long as the two antibodies are maintained at saturating concentrations and determinations are performed in the same gel, levels of the
and ß subunits are obtained that nicely fit the expected results from gene dosage effects (Fig. 5 A). Furthermore, according to the "two-signal model" for membrane attachment of peripheral membrane proteins, one expects to find equal amounts of membrane-bound Gq
e and Gße subunits. In accord with this notion, although we found about twofold excess of total Gße over Gq
e, an analysis of these subunits in the membrane-bound fraction gave a ratio of 1:1.
In the heterozygous Gße1/+ mutant, in which there is a reduction of 50% in the level of the ß subunit, yielding a ß/ ratio of
1, we found a dramatic increase in the spontaneous activity of photoreceptor cells (Figs. 5 and 6). The critical role of the excess of Gße over Gq
e was revealed in the G
q1/+;Gße1/+ double heterozygous mutant, in which the rate of spontaneous activity was dramatically reduced by restoring the excess of Gße over Gq
e. This indicates that the excess of Gße, rather than its absolute amount, is important to maintain a low frequency of spontaneous activity. Furthermore, this mutant rules out the possibility that the spontaneous activity we observed was caused by side effects of the Gße mutation. Altogether, this is the first demonstration of the strategy of excess ß
over the
subunit in vivo for the suppression of spontaneous activity at the G protein level.
Two possible mechanisms can explain how the excess of Gße over Gqe prevents spontaneous activity. One mechanism could be through participation of the soluble pool of rhabdomeral Gße in accelerating the hydrolysis of Gq
e-GTP if spontaneous exchange occurs. This mechanism is currently under investigation. The second mechanism could be through the stabilization of Gq
e-GDP, thus preventing the exchange of bound GDP for free GTP. In an insightful, theoretical paper dealing with the spontaneous activity of G proteins by using thermodynamic model simulations, it was found that the concentration of ß equal to that of
is barely sufficient to suppress spontaneous activity, whereas a twofold excess of ß
over the
subunit produces a large decrease in spontaneous activity (Onaran et al., 1993). Altogether, our in vivo studies point to the importance of ß
subunits as principle modulators of spontaneous activity and to the relevance of this strategy in vivo.
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Materials and methods |
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Assay of light-dependent Gße localization
Assay for the light-dependent localization of Gße was performed as described previously (Kosloff et al., 2003). In short, dark-adapted flies were subjected to illumination with activating blue light (18-W white light lamp with a 1-mmthick wide band filter [Schott BG 28; Bes Optics] 12 cm away from the flies) for various durations at 22°C. Termination was performed by moving the flies to 4°C in the dark and promptly separating the fly heads. 10 flies were used for each time point.
Preparation of Drosophila head homogenate and fractionation
Heads were separated from 10 flies that were dark adapted overnight (except in Fig. 4) and homogenized in 1 ml isotonic homogenization buffer (20 mM Tris, pH 7.5, 120 mM KCl, 0.1 mM MgCl2, 0.1 mM PMSF, and 5 mM ß-mercaptoethanol). Homogenate was either directly precipitated with 5% TCA or subjected to fractionation. Membranes and cytosol fractions were separated by centrifugation (15,800 g for 15 min at 4°C). The pellet was washed and centrifuged again, and the supernatants were combined. Ultracentrifugation at 150,000 g for 30 min did not change the distribution of and ß subunits between the fractions. The proteins were precipitated by 5% TCA, ran on SDS-PAGE, and subjected to quantification as described in SDS-PAGE and immunoblotting.
Preparation of recombinant Drosophila Gqe and Gße proteins
cDNA clones of Gqe and Gße genes were obtained from the Medical Research Council UK gene service. Gq
e cDNA was amplified and cloned into pQE-80 vector (QIAGEN) that contained an NH2-terminal 6x His tag and was expressed in Rosetta bacterial cells (Novagen). The recombinant (His)6-Gq
e protein was then purified on a Ni-Sepharose column (GE Healthcare) and eluted with a 20250-nm imidazole gradient using fast protein liquid chromatography Akta explorer (GE Healthcare).
Gße cDNA was amplified and cloned into pHis-parallel 1 (pET22) vector (obtained from P. Sheffield, University of Virginia, Charlottesville, VA) that contained an NH2-terminal 6x His tag and was expressed in HMS174 bacterial cells (Novagen). Purified recombinant (His)6-Gße was extracted from inclusion bodies by applying 6 M guanidine HCl on a bacterial membrane extract that had been washed three times with 1% Triton X-100. Both proteins were 95% pure as determined by SDS-PAGE and Coomassie blue staining. Recombinant protein concentrations were determined spectrophotometrically by using a calculated molar extinction coefficient of 42,350 for Gq
e and 60,000 for Gße at 280 nm.
SDS-PAGE and immunoblotting
Equal protein amounts that were determined by Bradford assay were loaded on the specified gel. For detection of the or ß subunits of DGqe, a 10% SDS-PAGE was used. To detect both subunits (
and ß) on the same gel, proteins were separated on a gradient 7.515% SDS-PAGE. For the detection of G
e, a 20% SDS-PAGE with 4 M urea was used. The urea was needed for separation of the
subunit from the ß subunit. Subsequent to SDS-PAGE separation, proteins were subjected to Western blot analysis using the specified antibodies.
Two different anti-Gße polyclonal antibodies were made in rabbit as described previously (Palczewski et al., 1993). One antibody was made against a peptide from the COOH terminus of the protein (residues 333346), and the other was made against a peptide from the NH2 terminus (residues 313).
For Gqe detection, we used anti-Gq
e polyclonal antibodies that were previously made by us (Kosloff et al., 2003), and for G
e detection, rabbit polyclonal antibodies that were directed against the Calliphora G
e protein were used (obtained from A. Huber, University of Karlsruhe, Karlsruhe, Germany; Schulz et al., 1999).
To determine the ratio between Gqe and Gße subunits, we performed Western blot analysis using a mixture of anti-Gq
e and anti-Gße each at a 1:1,000 dilution, which is five times higher than their saturating concentration. To rule out the possibility that the Gße excess we observed is caused by the antibodies, we repeated these experiments with the two different Gße antibodies and obtained the same results. To further ensure that the Gße excess over Gq
e was not a result of the antibody concentrations, we repeated this procedure with a higher concentration of anti-Gq
e or with a twofold dilution (1:2,000) of anti-Gße. In all of these cases, an excess of Gße over Gq
e was observed.
Relative protein amounts on the same gel were determined by quantification of the ECL signal by using the Plus Gel system (LAS-1000; Fuji).
Immunogold EM
Immunogold EM was performed as described previously (Kosloff et al., 2003). All sections were made from flies that were dark adapted overnight. Sections were incubated with either Gqe antibodies (dilution of 1:80) or Gße affinity-purified antibodies (dilution of 1:20). Gße antibodies were affinity purified by using Affi-Gel 10 gel (Bio-Rad Laboratories) according to the manufacturer's instructions for anhydrous coupling followed by elution with glycine-HCl, pH 2.5. The secondary antibody used was goat antirabbit conjugated to 18 nm of gold particles.
Sections were observed and photographed with a transmission electron microscope (Technai-12; Philips) equipped with a CCD camera (MegaView II; Soft Imaging System) and were visualized with analySIS 3.0 image processing software (Soft Imaging System).
Electroretinogram (ERG) and M potential
ERG recordings were performed on intact flies as described previously (Peretz et al., 1994). Orange light (OG-590 Schott edge filter; Bes Optics) from a Xenon high pressure lamp (operating at 50 W; model LPS 220; Photon Technology International) was delivered to the compound eye by an optic fiber and was attenuated by natural density filters. The maximal luminous intensity at the eye surface was 12.5 mW/cm2. M potential recordings were performed as described previously (Minke and Kirschfeld, 1980). In brief, an adapting light of maximal intensity 20-s blue light (Schott BG-28) from the Xenon high pressure lamp was delivered 1 min before each white test stimulus (70 jouls of photographic flash light).
Whole cell recording
Dissociated ommatidia were prepared from newly eclosed white-eyed adult flies (<1 h after eclosion; Hardie, 1991) that were maintained in a 12-h dark/12-h light cycle and kept in the dark 24 h before the experiment. Whole cell patch-clamp recordings were performed as previously described (Hardie and Minke, 1992). Signals were amplified with a patch-clamp amplifier (Axopatch 200B; Axon Instruments, Inc.), sampled at 2,000 Hz, and filtered below 1,000 Hz. The bath solution contained 120 mM NaCl, 5 mM KCl, 10 mM N-Tris buffer, pH 7.15, 4 mM MgSO4, and 1.5 mM CaCl2. The pipette solution contained 120 mM K gluconate, 2 mM MgSO4, 10 mM N-Tris buffer, pH 7.15, 4 mM MgATP, 0.4 mM Na2GTP, and 1 mM NAD+.
Transillumination of the halogen light source (100 W) was used as previously described (Peretz et al., 1994). The orange stimulating light (Schott OG-590) was applied via a condenser lens (Carl Zeiss MicroImaging, Inc.) and was attenuated by neutral density filters.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (EY-03529 to B. Minke and Z. Selinger), the Israel Science Foundation (to Z. Selinger and B. Minke), the German-Israel Foundation (to B. Minke), and the Minerva Foundation (to B. Minke and Z. Selinger).
Submitted: 14 June 2005
Accepted: 4 October 2005
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References |
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