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First published online 20 June 2006
doi: 10.1242/jcs.03049
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Research Article |
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA
Author for correspondence (e-mail: tsunoda{at}bu.edu)
Accepted 16 May 2006
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: TRP Channels, TRPL Channels, Arrestin, Translocation, Drosophila, Phototransduction
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Introduction |
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60,000 tightly packed microvilli and houses most of the signaling components. Here, the G-protein-coupled receptor (GPCR) rhodopsin is photo-converted to the activated meta-rhodopsin state. Meta-rhodopsin activates the Gq
-protein, which in turn activates phospholipase-Cß (PLC), leading to the opening of two light-activated ion channels, the TRP and TRPL channels (Hardie and Raghu, 2001
; Ranganathan et al., 1995; Tsunoda and Zuker, 1999). Visual arrestin-2 (Arr-2) and an eye-specific protein kinase-C (eye-PKC) have been shown to be required for normal deactivation of the light-response (Dolph et al., 1993; Hardie et al., 1993; Ranganathan et al., 1991; Smith et al., 1991).
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subunit (Cronin et al., 2004; Kosloff et al., 2003), TRPL channels (Bahner et al., 2002) and visual Arr-2 (Kiselev et al., 2000; Lee et al., 2003; Orem and Dolph, 2002), have been reported to display lightdependent translocation between the rhabdomere and cell body. Vertebrate and crayfish photoreceptors have also been reported to display a similar light-dependent translocation of the G-protein, transducin and visual Arr-2 (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; Terakita et al., 1998; Whelan and McGinnis, 1988). Dynamic localization of components in and out of the signaling compartment is probably a mechanism for regulating the quantity of components available for signaling, thereby contributing to long-term light adaptation (Bahner et al., 2002; Sokolov et al., 2002). Here, we show that TRPL channels translocate out of the rhabdomere in two distinct stages. In the first stage, lightinduced translocation occurs within 5 minutes, whereas the second stage requires over 6 hours. Each stage exhibits a distinct apical versus basolateral localization, implicating different transport mechanisms. Genetic analyses suggest that activation of the other light-activated channel TRP and an eyespecific PKC are both required for the second stage of TRPL translocation, whereas only PLC is required for the first stage. Finally, we show that Arr-2 is required for the localization and stability of TRPL channels.
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). The underlying mechanism of this translocation, however, remains unknown. Since TRPL channels are integral membrane proteins, we predict that TRPL channels must mobilize in a membrane-restricted manner. Fig. 1B shows that TRPL channels are indeed associated with membrane fractions in the dark and after light-exposure. One possibility is that TRPL channels are incorporated into vesicles at the base of the rhabdomere, similar to rhodopsin-Arr-2 complexes that accumulate in norpA and rdgC mutants (Alloway et al., 2000; Kiselev et al., 2000; Orem and Dolph, 2002). Alternatively, TRPL channels might diffuse laterally through the membrane, out of the rhabdomere and into the supporting stalk membrane. To gain insight into how TRPL channels are transported out of the rhabdomere, we set out to examine the light-dependent progression of TRPL translocation in more detail. We performed immunolocalization studies for TRPL channels in retinal cross-sections from dark-adapted and light-exposed flies. In the dark, TRPL channels consistently localized to the rhabdomeres of photoreceptors, with a higher concentration found at the base of the rhabdomeres (Fig. 1). For all genotypes, flies raised in the dark were indistinguishable from dark-adapted (>10 hours) flies. With only 5 minutes of light-exposure, TRPL channels translocated out of the rhabdomere and into the adjoining stalk membrane (Fig. 1C). TRPL protein was present both at the base of the rhabdomere and in the stalk membrane, giving an apical, ringlike localization pattern for each ommatidium (Fig. 1). The speed of the light-induced redistribution of TRPL protein suggests that TRPL channels do indeed undergo light-induced translocation, rather than light-induced synthesis and insertion in the stalk membrane. Interestingly, translocated TRPL channels appear to be restricted to the apical membrane since no TRPL immunostaining was present in the basolateral membrane of the photoreceptor cell at this time. We increased the duration of light-exposure and found that at more than 4 hours of light-exposure, translocated TRPL channels were only localized to the stalk membrane, displaying the same apical localization pattern seen after 5 minutes of light-exposure (Fig. 1C). We refer to this localization pattern as the first stage of TRPL-channel translocation (Fig. 1A).
We were, however, puzzled as to why we did not see TRPL translocation to the plasma membrane of the entire cell body – including the basolateral membrane – as previously reported (Bahner et al., 2002). Since this earlier study used light conditions of lower intensity and longer duration (12 hours), we examined whether similar light conditions would induce the translocation of TRPL channels to the basolateral membrane. We found that for light intensities spanning more than three orders of magnitude (from 31 lux to 57x103 lux), TRPL channels still translocated into a similar apical ring-like pattern after 5 minutes of light-exposure (data not shown), suggesting that light intensity was not responsible for the difference in TRPL localization. We found, however, that – for both highand low-light intensities – TRPL channels localized to the entire plasma membrane, including the basolateral membrane, with longer durations of light-exposure. Although there was some variability from fly to fly, the first appearance of TRPL immunostaining in the basolateral membrane of photoreceptors was observed after 6 hours of light-exposure (Fig. 1C). TRPL displayed consistent localization to the basolateral membrane after 10 hours of light-exposure (Fig. 1C). Thus, longer lightexposure induces TRPL channels to somehow bypass the AJs and localize in the basolateral membrane; we now refer to this as the second stage of TRPL-channel translocation (Fig. 1A). Together, our results show two temporally separable stages of light-induced TRPL translocation with distinct subcellular localization patterns.
We also examined the time-course of TRPL channel relocalization to the rhabdomere with dark-incubation following each stage of light-induced translocation. Wild-type flies were first exposed to light for 2 or 10 hours, inducing translocation to the first or second stage, respectively, and then dark-incubated for increasing times before retinas were sectioned and immunostained for TRPL. Surprisingly, the restoration of TRPL channels to the rhabdomere after the first stage of translocation occurred much more slowly than expected: even after 4 hours of dark incubation, some TRPL staining was still evident in the stalk membrane, and not until 6 hours of dark incubation was TRPL observed exclusively in the rhabdomeres (Fig. 2A). The relocalization of TRPL channels to the apical membrane after the second stage of translocation was first seen after 6 hours of dark incubation, and full recovery of TRPL to the rhabdomeres required 10 hours of dark incubation (Fig. 2B). Since recovery of TRPL channels to the rhabdomere is quite lengthy from either stage of TRPL localization, it is unclear – based on the reported time of TRPL synthesis (Bahner et al., 2002) – whether TRPL channels do indeed undergo translocation back to the rhabdomere or whether newly synthesized TRPL channels are targeted to the rhabdomeres.
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Signaling the first stage of TRPL translocation is independent of TRP-channel activation
The finding that light-induced translocation of TRPL channels occurs in two distinct stages creates a new framework in which to examine TRPL translocation. We set out to determine which components and events of the phototransduction cascade are required for triggering each stage of translocation. To do this, we examined the localization of TRPL channels in retinal tissue sections from null-mutants of the major rhodopsin (Rh1), the effector PLC and the other light-activated channel TRP (ninaEI17, norpAP41 and trpP343, respectively). All flies were either dark-raised or light-exposed for 2 or 12 hours. ninaEI17 mutants display retinal degeneration (Leonard et al., 1992; O'Tousa et al., 1989) and, although we used young (<24hour-old) flies to minimize the effects of degeneration, retinal sections still showed significant degeneration, resulting in a higher level of background TRPL immunostaining (see Fig. 3). TRPL immunostaining, however, appeared to be localized primarily in the rhabdomeres of both dark-raised and lightexposed ninaEI17 flies (Fig. 3), suggesting that the light receptor Rh1 is required in signaling both stages of TRPL translocation (Fig. 3).
We next examined the requirement for PLC and TRP channels in the first stage of TRPL-channel translocation. As expected, dark-raised norpAP41 and trpP343 mutants displayed normal rhabdomeric localization of TRPL. After 2 hours of light-exposure, we found that TRPL channels remained entirely rhabdomeric in norpAP41 mutants (Fig. 3), suggesting that activation of the effector PLC is required for the first stage of TRPL translocation. By contrast, we found that TRPL channels translocated normally to the stalk membrane in trpP343 mutants light-exposed for 2 hours, demonstrating that the first stage of TRPL-channel translocation is independent of TRP channels (Fig. 3).
Activation of TRP channels is essential for the second stage of TRPL-channel translocation
We next investigated whether the second stage of TRPLchannel translocation could be induced in norpAP41 and trpP343 mutants. We found that TRPL channels did not translocate to the basolateral membrane of R1-R6 photoreceptor cells in either mutant after a 12-hour light-exposure. TRPL channels remained exclusively rhabdomeric in norpAP41 mutants, wheras TRPL channels remained restricted in the stalk membrane in trpP343 mutants, similar to TRPL localization after a 2-hour light-exposure (Fig. 3). Clear basolateral staining of TRPL was not observed in any R1-R6 photoreceptor cells from trpP343 mutants after 12 hours of light-exposure. Altogether, in contrast to the first stage of TRPL translocation, these findings suggest that the activation of TRP channels is required for signaling apical to basolateral translocation of TRPL channels.
Conversely, Bahner et al. (Bahner et al., 2002) found that TRPL translocation after a 12-hour light-exposure was independent of TRP-channel activation, based on TRPL immunostaining of norpA mutants. These findings, however, were contradicted by their own experiments with a TRPL-eGFP transgenic line (`note added in proof') (Bahner et al., 2002), also indicated by Frechter and Minke (Frechter and Minke, 2006), and Minke and Parnas (Minke and Parnas, 2006). Given this discrepancy, we set out to determine whether activation of TRP channels alone is sufficient to trigger translocation of TRPL channels to the basolateral membrane. To do this, we used the TrpP365 mutant, which contains a gainof-function mutation in the trp gene that results in constitutive activation of TRP channels and subsequent Ca2+ influx into the photoreceptor cells (Hong et al., 2002; Yoon et al., 2000). Since TrpP365 mutants display massive photoreceptor degeneration, we used young adult [<44-hours after eclosion (AE)] TrpP365/+ heterozygous flies, which display an almost normal photoreceptor structure (Hong et al., 2002). We examined whether the one copy of TrpP365 in TrpP365/+ heterozygotes was sufficient to induce TRPL translocation to the basolateral membrane. We first examined young TrpP365/+ mutant flies (18-22 hours AE) that had been raised in complete darkness. We found that TRPL channels were indeed localized to the basolateral membrane (Fig. 4). To determine whether TRPL channels were initially trafficked and localized to the rhabdomeres of TrpP365/+ mutants, we examined dark-raised, newly eclosed (<4 hours AE) TrpP365/+ mutants. We found that, indeed, TRPL channels displayed rhabdomeric localization (Fig. 4), demonstrating that TRPL channels in TrpP365/+ mutants do not exhibit any defect in trafficking TRPL channels to the rhabdomeres. Taken altogether, we propose that TRP channel activation is both essential and sufficient for signaling apical to basolateral mobilization of TRPL channels.
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Eye-PKC is also required for signaling leading to the second stage of TRPL-channel translocation
To further investigate the signaling pathway that triggers the second stage of TRPL-channel translocation, we sought to identify the signaling events downstream of TRP channel activation. Since activation of TRP channels leads to an influx in Ca2+ ions, we tested whether eye-PKC, which is activated by Ca2+ and diacyl glycerol (DAG), is also required for signaling the translocation of TRPL channels to the basolateral membrane. Although eye-PKC has been shown to function in deactivation of the light-response and light-adaptation (Hardie et al., 1993; Smith et al., 1991), the exact mechanisms of action as well as additional roles of eye-PKC are unknown. We examined eye-PKC null-mutants (inaCP209) that were darkraised, or light-exposed for 2 or 12 hours. Similar to trpP343 mutants, TRPL channels in R1-R6 photoreceptors cells of inaCP209 mutants were localized to the rhabdomeres in the dark and translocated to the stalk membrane with 2 hours of light, but were unable to translocate to the basolateral membrane after 12 hours of light exposure (Fig. 3). These results indicate that eye-PKC is required for the second stage of TRPL-channel translocation in R1-R6 photoreceptor cells, specifying perhaps a signaling pathway for basolateral localization that is distinct from the first stage of TRPL translocation.
Arr-2 is required for the rhabdomeric localization of TRPL channels
In the dark, TRPL channels are anchored exclusively in the rhabdomere by a yet unknown mechanism. One possibility is that light-exposure triggers the disruption or release of this anchor, thus allowing TRPL channels to diffuse out of the rhabdomere and into the stalk membrane during the first stage of TRPL translocation. We hypothesize that there is a lightdependent scaffold protein for TRPL channels in the rhabdomere. Although the scaffolding protein INAD (inactivation-no-afterpotential-D) is required for the rhabdomeric localization of some phototransduction components (reviewed in Hardie and Raghu, 2001; Tsunoda and Zuker, 1999), TRPL channel localization is not affected by the loss of INAD (Tsunoda et al., 1997). Since arrestin proteins in other systems have recently been shown to function as molecular scaffolds (Miller and Lefkowitz, 2001; Perry and Lefkowitz, 2002), we tested whether either of the two photoreceptor-specific arrestin proteins Arr-1 and Arr-2 (Hyde et al., 1990; LeVine et al., 1990; Smith et al., 1990; Yamada et al., 1990), are involved in the localization of TRPL channels in the rhabdomere. We performed immunolocalization studies for TRPL channels in retinal tissue sections from dark-adapted arr1 and arr2 mutants. We first examined the arr11 mutant allele, which expresses 10% of wild-type levels of Arr-1 protein (Dolph et al., 1993). We found that TRPL channels displayed clear rhabdomeric localization (Fig. 5A), suggesting that Arr-1 is not required for the rhabdomeric localization of TRPL channels. We next examined a null mutant for Arr-2, arr25. In contrast to arr11 mutants, TRPL channels in darkraised arr25 null-mutants were severely mislocalized (Fig. 5A). These results show that Arr-2 is required for the rhabdomeric localization of TRPL channels in the dark. Interestingly, the subcellular localization of TRPL channels in dark-raised arr25 mutants was strikingly similar to the localization pattern of TRPL channels after the first stage of light-induced translocation in wild-type photoreceptors (Fig. 1, Fig. 5A).
Since Arr-2 contains an extended C-terminal tail that is absent from Arr-1 (LeVine et al., 1990), we tested whether the C-terminal tail of Arr-2 confers the rhabdomeric localization of TRPL channels. To do this, we examined the arr21 mutant allele, which encodes a truncated Arr-2 protein with the last 46 amino acids of its C-terminus deleted (Dolph et al., 1993). We found, however, that TRPL channels were localized in the rhabdomeres of dark-adapted arr21 mutants similar to wildtype (Fig. 5), indicating that the C-terminal tail of Arr-2 is not required for the rhabdomeric localization of TRPL channels. Future structure-function studies should reveal how Arr-2 functions in the rhabdomeric localization of TRPL channels.
To examine whether Arr-1 or Arr-2 play a role in the translocation of TRPL channels, we exposed arr11, arr25 and arr21 mutants to 2 and 12 hours of light. With 2 hours of light-exposure, all of the arrestin mutants displayed TRPL localization similar to that observed in dark-adapted arr25 mutants. After 12 hours of light-exposure, we found that TRPL channels were able to translocate to the basolateral membrane in arr11, arr25 and arr21 mutants (Fig. 5). These results demonstrate that Arr-2 functions in the maintenance of TRPL channels in the rhabdomeres in the dark, but is not required for their translocation to the basolateral membrane.
Arr-2 is required for the stability of TRPL channels
The localization of TRPL channels in arr25 mutants is not only similar to the localization of TRPL channels in the first stage of translocation in wild-type photoreceptors, but it is also reminiscent of the mislocalization of TRP channels in inaD null-mutants. In these mutants, TRP channels are primarily located in the stalk membrane flanking each rhabdomere (Tsunoda et al., 1997). These similar localization patterns suggest that the relationship between Arr-2 and TRPL channels is analogous to the relationship between INAD and TRP channels. Since TRP protein levels decay with age in inaD mutants (Tsunoda et al., 1997), we examined whether the same is true for TRPL protein levels in arr25 mutants. Because arr25 mutants display light-dependent degeneration (Dolph et al., 1993), flies were raised in the dark to prevent degeneration. Indeed, we found that levels of TRPL protein in arr25 mutants declined from newly eclosed flies to 10-day-old flies, whereas TRPL levels remained unchanged in wild-type flies (Fig. 6). By contrast, levels of other transduction proteins, including rhodopsin and eye-PKC, remain stable in wild-type flies and arr25 mutants (Fig. 6). Thus, Arr-2 is required for both the rhabdomeric localization and stability of TRPL channels.
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Discussion |
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For the second stage of light-induced TRPL translocation, we propose that activation of TRP channels is both essential and sufficient for signaling TRPL channel mobilization from apical to basolateral membrane. In addition, we show that eye-PKC is required for this mobilization. Our results specify a light-induced signaling pathway for the second stage of TRPLchannel translocation that is distinct from that of the first stage. One possibility is that influx of Ca2+ through the more Ca2+ selective TRP channels activates eye-PKC, whose targets are likely to play key roles in the mobilization of TRPL channels to the basolateral membrane. Since the major role of eye-PKC has been thought to be in the deactivation of the phototransduction cascade, studies have investigated the phosphorylation of known phototransduction components by eye-PKC, identifying TRP and INAD as targets (Huber et al., 1996; Liu et al., 2000). Future studies, however, searching for new targets of eye-PKC, might reveal important players and mechanisms involved in the transport of TRPL channels to the basolateral membrane. Fig. 8 illustrates a model of the two stages of light-induced TRPL translocation and the signaling proteins required for each stage.
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The two stages of light-induced TRPL-channel translocation may also serve different physiological functions. For example, TRPL channels in the stalk membrane may undergo bidirectional transport as a mechanism for regulating the number of channels available for signaling, whereas TRPL channels in the basolateral membrane may be targeted for degradation. The extended duration of dark-incubation required for full recovery of TRPL channels to the rhabdomeres from either stage was unexpected and thus allows for the possibility that TRPL channels do not undergo translocation back to the rhabdomeres. These results also suggest that TRPL translocation plays physiological roles other than lighta-daptation.
In this report, we also demonstrate that TRPL channels are mislocalized and unstable in arr2 null-mutants. How does Arr2 function in the localization of TRPL channels? Interestingly, the localization of TRPL channels in arr2 mutants is similar to the localization of TRPL channels after the first stage of light-induced translocation in wild-type photoreceptors. This observation suggests that the requirement for Arr-2 in the rhabdomeric localization of TRPL channels is mechanistically tied to the first stage of light-induced translocation. That is, when Arr-2 is not present to maintain TRPL channels in the rhabdomere, TRPL channels appear to undergo the same migration as in the first stage of light-dependent translocation. One possibility is that Arr-2 functions as part of a lightdependent anchoring complex for TRPL channels in the rhabdomere. The dissolution of this anchor may then allow free diffusion of TRPL channels in the apical membrane, leading to the apical localization pattern seen after the first stage of TRPL translocation in wild-type flies and dark-incubated arr2 null-mutants.
The classical function of arrestin proteins is to deactivate GPCRs: visual arrestins deactivate meta-rhodopsin, whereas ßarrestins deactivate ß-adrenergic receptors (Arshavsky, 2002; Claing et al., 2002; Dolph, 2002; Pierce and Lefkowitz, 2001). Recent studies have shown that arrestin proteins can also function as molecular scaffolds (Miller and Lefkowitz, 2001; Perry and Lefkowitz, 2002). The relationship between Arr-2 and TRPL is complicated, however, by the fact that Arr-2 and TRPL undergo light-dependent translocation between the rhabdomere and cell body in opposite directions (Kiselev et al., 2000; Lee et al., 2003). Although a previous study has found that 35% of Arr-2 is present in the rhabdomeres of darkadapted photoreceptors (Lee et al., 2003) that could, in theory, act as a scaffold for TRPL channels, no such binding between Arr-2 and TRPL has yet been detected. Another possibility is that Arr-2 functions indirectly in the rhabdomeric localization of TRPL channels. ß-arrestin, which binds and deactivates the ß2 adrenergic GPCRs, has been shown to recruit the receptor tyrosine kinase Src, thereby establishing a link with MAP kinase signaling pathways (Luttrell et al., 1999; Luttrell and Lefkowitz, 2002; Miller and Lefkowitz, 2001; Pierce and Lefkowitz, 2001; Zuker and Ranganathan, 1999). Thus, another possibility is that the loss of Arr-2 affects other signaling proteins in photoreceptors that might interact with Arr-2 and play a role in the localization of TRPL channels.
The major phenotype of arr2 mutants is impaired deactivation of active metarhodopsin (Dolph et al., 1993). Taking the results of this study, we now know that arr2 mutants also exhibit a loss of TRPL channels from the rhabdomeres of photoreceptors, which may have additional, more subtle, effects on the arr2 phenotype. Since trpl mutants display defects in light-adaptation (Leung et al., 2000) and the lightdependent translocation of TRPL channels has been shown to correlate with long-term light-adaptation (Bahner et al., 2002), defects in adaptation displayed by arr2 mutants (Lee et al., 2003) might be partially due to the loss of TRPL channels from the rhabdomeres.
Recent findings in vertebrates and invertebrates have shown that the subcellular localization of some phototransduction components is in fact dynamic. These studies have shed new light on how signaling is regulated by the subcellular translocation of transduction components. The identification of two distinct stages of TRPL translocation has given us a framework in which to examine the signaling pathways triggering each stage of TRPL translocation, and the transport mechanisms involved. Future studies are likely to identify additional signaling and structural proteins involved in these processes, as well as the different physiological roles played by each stage of translocation.
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Materials and Methods |
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), norpAP41 (Bloomquist et al., 1988; Lindsley and Zimm, 1992), trpP343 (Scott et al., 1997), inaCP209 (Smith et al., 1991), arr25 (Alloway and Dolph, 1999), arr21 (Dolph et al., 1993), arr11 (Dolph et al., 1993), TrpP365 (Yoon et al., 2000). All flies were raised at 25°C by standard techniques. Mutant alleles were crossed into a w- or cn bw background to eliminate retinal pigments that autofluoresce.
Antibodies
To generate an N-terminal Arr-2 and TRPL polyclonal antibody, we used peptide sequences corresponding to Arr-2 residues 9-25 (KKATPNGKVTFYLGRRD) and TRPL residues 1083-1097 (DNSNFDIHVVDLDEK); an additional N-terminal cysteine was added to each peptide for conjugation to the KLH carrier protein. Immunization of rabbits was performed by ProSci, Inc. (Poway, CA). Sera were ammonium sulfate precipitated and affinity-purified. Specificity was tested using wild-type and null mutant controls for both tissue sections and immunoblots. The Arr-2 C-terminal antibody (Dolph et al., 1993), and antibodies against eye-PKC, rhodopsin, INAD and IPP were used as previously described (Tsunoda et al., 1997).
Immunolocalization studies
Flies were placed under a white-light source (Lambda LS 175W Xenon-arc lamp with 400-700 nm bandpass filter, Sutter Instruments, Novato, CA, or equivalent), at 57x103 lux (unless otherwise indicated), for given times. Light intensity was measured by an EXTECH 403125 digital light-meter. All experiments were conducted at 24°C. After illumination, fly heads were fixed in 3% paraformaldehyde in PBS, washed four times with PBS, infiltrated with 2.3 M sucrose overnight at 4°C, and frozen on stubs in liquid nitrogen as previously described (Cronin et al., 2004). 1-1.5 µm thick sections were cut from retinas using a Leica Ultracut UCT with EM FCS cryo unit at –82°C (Leica Microscopy and Scientific Instruments Group, Heerbrugg, Switzerland). Dark-adapted flies and pupae were fixed under a dim red light before sectioning. Sections were blocked in 1% BSA, 0.1% sapponin in PBS for 30 minutes and immunostained as previously described (Cronin et al., 2004). Anti-TRPL antibody (1:500) was used overnight at 4°C, followed by Rhodamine-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA) (1:200, 1 hour, room temperature). Slides were mounted with 90% glycerol and p-phenylenediamine (Sigma Aldrich, St Louis, MO).
Membrane and cytosol isolation
For each light condition, 30 fly heads were collected on minutien pins, placed in Eppendorf tubes, and stored at –80°C. Membranes were separated from cytosol as previously described (Cronin et al., 2004).
SDS-PAGE and immunoblot analysis
10% polyacrylamide gels were used for all immunoblot analyses. Samples contained three fly heads sonicated in 20 µl SDS loading buffer (Figs 5, 6) or membrane and/or cytosol isolated from the equivalent of 3.75 fly heads (Fig. 1) as described above. Protein was separated by standard SDS-PAGE, transferred to nitrocellulose membranes that were subsequently blocked in 5% dried milk in PBS and probed with the indicated antibodies.
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Acknowledgments |
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Footnotes |
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References |
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, adherens junctions separating apical and basolateral membranes. (Right panels) Cross-sections of single wild-type ommatidia, immunostained for TRPL, shown to illustrate typical localization of TRPL channels in the dark, after the first or the second stage of light-induced TRPL translocation. In each ommatidium, seven photoreceptor cells are visible. The rhabdomere (R) and cell body (C) of a single photoreceptor cell are indicated in one ommatidium (Dark); the approximate boundary of the cell body is outlined. TRPL channels are exclusively localized to the rhabdomeres of dark-adapted photoreceptors; arrowheads indicate the concentration of channels at the base of rhabdomeres. After 2 hours of light-exposure (stage 1), TRPL moves into the supporting stalk membrane adjacent to each rhabdomere (arrowheads), forming an apical ring-like localization pattern for each ommatidium. With 12 hours of light-exposure (stage 2), TRPL translocates to the basolateral membrane (arrowheads); light intensity was 
