QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 20 June 2006
doi: 10.1242/jcs.03049

This Article Summary Full Text Full Text (PDF) A correction has been published Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cronin, M. A. Articles by Tsunoda, S. Search for Related Content PubMed PubMed Citation Articles by Cronin, M. A. Articles by Tsunoda, S.

Two stages of light-dependent TRPL-channel translocation in Drosophila photoreceptors

Michelle A. Cronin^*, Minh-Ha Lieu^* and Susan Tsunoda

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

View larger version (64K): [in a new window]   Fig. 1. TRPL channels display two stages of light-induced translocation. (A) Diagram of a single ommatidium and cross-sections of single wild-type ommatidia. (Left panel) Single ommatidium illustrating apical and basolateral membranes of the photoreceptor cell. Black features, apical membrane, including the rhabdomere (R) and the supporting stalk membrane (arrowheads); gray, basolateral membrane; , 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 244 lux. Representative ommatidia are shown here and in Figs 3, 5 and 7 from multiple wild-type retinal sections taken from 37 eyes of 35 flies (Dark), 45 eyes of 37 flies (Light, 2hrs), 28 eyes of 26 flies (Light, 12hrs). (B) Representative immunoblot analysis of retinal membrane (M) and cytosolic (C) fractions isolated from wild-type flies that were dark-adapted (DR) or light-exposed for 2 or 12 hours (Lex 2hrs or Lex 12hrs, respectively). In contrast to Gq, which displays a light-dependent shift from membrane to cytosol as previously reported (Cronin et al., 2004), TRPL is found in the membrane fraction during each light condition examined. Rhodopsin (Rh1) was used as a loading control for the membrane fractions. An immunoblot representative for seven different experiments is shown. (C) Time course of the two stages of TRPL-channel translocation from the rhabdomere to the basolateral membrane. Shown are wild-type retinal cross-sections immunostained for TRPL after light-exposures of increasing duration. Dark-raised wild-type flies show rhabdomeric TRPL localization. Within 5 minutes of light-exposure, TRPL rapidly translocates to the stalk membrane, producing the same apical ring-like pattern as seen after 2 and 4 hours of light-exposure; we designate this translocation to the stalk membrane as stage 1. After 6 hours of light-exposure, TRPL is first found localized to the basolateral membrane in some, but not all photoreceptor cells. With 10 hours of light exposure, TRPL is consistently found localized to the basolateral membrane of all photoreceptors. TRPL localization to the basolateral membrane is designated as stage 2 of TRPL translocation. Shown are representative ommatidia from multiple retinal sections, taken from five eyes of five flies (Dark), six eyes of five flies (Light, 5min), seven eyes of six flies (Light, 2hrs), eight eyes of five flies (Light, 4hrs), six eyes of four flies (Light, 6hrs), five eyes of four flies (Light, 10hrs).

View larger version (116K): [in a new window]   Fig. 2. (A,B) Time course of TRPL channel relocalization to the rhabdomere after (A) stage 1 and (B) stage 2 of light-induced translocation. (A) Wild-type flies were light-exposed for 2 hours to induce translocation to the stalk membrane, and then dark-incubated for increasing durations. Full recovery of TRPL localization to the rhabdomere required 6 hours of dark-incubation. Shown are representative ommatidia from multiple retinal sections taken from ten eyes of eight flies (Light, 2hrs), ten eyes of eight flies (Dark, 4hrs), ten eyes of seven flies (Dark, 6hrs). (B) Wild-type flies were light-exposed for 10 hours to induce translocation to the basolateral membrane, and then dark-incubated for increasing durations. After 6 hours of dark-incubation, TRPL gradually appeared in the stalk membrane in some photoreceptor cells. Full recovery of TRPL localization to the rhabdomeres was seen after 10 hours of darkincubation. For all of the above, wild-type retinal cross-sections were immunostained for TRPL. Shown are representative ommatidia from multiple retinal sections taken from five eyes of three flies (Light, 10hrs), five eyes of four flies (Dark, 6hrs), six eyes of six flies (Dark, 10hrs).

View larger version (87K): [in a new window]   Fig. 3. Distinct signaling pathways trigger each stage of TRPL translocation. Shown are representative cross-sections of single wildtype ommatidia and null-mutants of Rh1 (ninaEI17), PLC (norpAP41), TRP (trpP343) and eye-PKC (inaCP109) immunostained for TRPL. R1 - R7/R8 photoreceptor cells are indicated as1-7/8 in the dark-raised wild-type (WT) ommatidia. (Dark) For all dark-adapted mutants, TRPL channels localized to the rhabdomeres, similar to wild type. Note that, despite using newly eclosed flies, ninaE mutants display light-independent retinal degeneration. Because of this degeneration, TRPL immunostaining is not as clear in these mutants as in the others; localization of TRPL in light-exposed flies, however, is no different than in dark-raised flies, suggesting that translocation is blocked in ninaE mutants as expected. (Stage 1) After a 2-hour lightexposure, TRPL channels in trp and inaC mutants translocated to the stalk membrane, similar to wild type, whereas TRPL channels in ninaE and norpA mutants remained localized in the rhabdomeres. These results signify a requirement for Rh1 and PLC but not TRP or eye-PKC in the first stage of TRPL translocation. (Stage 2) In contrast to wild-type flies, TRPL channels did not translocate to the basolateral membrane in the R1-R6 photoreceptor cells of any of the mutants after a 12-hour light-exposure (see labeling in the WT Stage 2 panel for reference). In ninaE and norpA mutants, TRPL remained rhabdomeric, whereas in trp and inaC mutants, TRPL remained restricted to the apical membrane including the stalk membrane. These results show that Rh1, PLC, TRP and eye-PKC are all required for signaling the second stage of TRPL-channel translocation to the basolateral membrane. Shown for each genotype and light condition are representative ommatidia from multiple retinal tissue sections; wild type: see Fig. 1; ninaE: 12 eyes of ten flies (Dark), 12 eyes of seven flies (Stage 1), 13 eyes of eight flies (Stage 2); norpA: 17 eyes of 14 flies (Dark), 28 eyes of 21 flies (Stage 1), 28 eyes of 21 flies (Stage 2); trp: 17 eyes of 13 flies (Dark), six eyes of five flies (Stage 1), 13 eyes of nine flies (Stage 2); inaC: seven eyes of four flies (Dark), 14 eyes of nine flies (Stage 1), 12 eyes of seven flies (Stage 2).

View larger version (94K): [in a new window]   Fig. 4. The second stage of TRPL translocation is induced in darkraised TrpP365/+ mutants. Shown are representative cross-sections of single ommatidia from dark-raised wild-type (WT) and TrpP365/+ newly eclosed adult flies (<4 hours old), 18-22 hours after eclosion (AE), and 40-44 hours AE, immunostained for TRPL. Wild-type photoreceptors exhibited rhabdomeric localization of TRPL channels in the dark, regardless of age. TrpP365/+ flies less than 4 hours old also displayed rhabdomeric TRPL localization, indicating that TRPL channels are initially trafficked to the rhabdomeres similar to wild type. By 18-22 hours AE, TRPL channels in TrpP365/+ mutants had translocated to the basolateral membrane of photoreceptors. Shown for each genotype are representative ommatidia from multiple retinal tissue sections; wild-type: two eyes of two flies (<4hrs AE), two eyes of two flies (18-22hrs AE), three eyes of two flies (40-44hrs AE); TrpP365/+: five eyes of five flies (<4hrs AE), five eyes of five flies (18-22hrs AE), six eyes of five flies (40-44hrs AE).

View larger version (66K): [in a new window]   Fig. 5. Arr-2 is required for rhabdomeric localization of TRPL channels. (A) Shown are representative cross-sections of single ommatidia from wild-type, arr11, arr25, and arr21 mutants that were either dark-adapted (Dark), or light-exposed for 2 hours (Stage 1) or 12 hours (Stage 2), and then immunostained for TRPL. Whereas wild-type flies, arr11 and arr21 mutants displayed rhabdomeric localization of TRPL in the dark, arr25 mutants exhibited a mislocalization of TRPL channels in a pattern similar to TRPL channels in wild-type photoreceptors after a 2-hour light-exposure. All arrestin mutants displayed TRPL staining in the stalk membrane after a 2-hour light-exposure and TRPL staining in the basolateral membrane after a 12-hour light-exposure. For stage 1, arr25 mutants were light-exposed using a 50.7x103 lux white-light source. Shown for each genotype and light condition are representative ommatidia from multiple retinal tissue sections; wild-type: see Fig. 1; arr11: five eyes of four flies (Dark), six eyes of five flies (Stage 1), four eyes of three flies (Stage 2); arr25: 19 eyes of 13 flies (Dark), five eyes of five flies (Stage 1), eight eyes of seven flies (Stage 2); arr21: 11 eyes of ten flies (Dark), eight eyes of six flies (Stage 1), eight eyes of six flies (Stage 2). (B) Representative immunoblot showing the presence of wild-type Arr-2 protein and the C-terminal truncated Arr-2 (39 kD), expressed in wild type and arr21 mutants, respectively. The immunoblot was probed with a polyclonal antibody against the Nterminal sequence of Arr-2 (see Materials and Methods). Anti-INAD was used as a loading control.

View larger version (63K): [in a new window]   Fig. 6. (A) Arr-2 is required for the stability of TRPL channels. Representative immunoblot of TRPL, eye-PKC (PKC), rhodopsin (Rh1), and inositol polyphosphate 1-phosphatase (IPP) in newly eclosed (0d) and 10 day old (10d) dark-raised wild type (WT) and arr25 mutant (arr25). In arr25 mutants is an age-dependent decline in the steady-state level of TRPL protein. By contrast, levels of eye-PKC and rhodopsin remain constant in wild type and arr25 mutant. Anti-IPP was used on all blots as a loading control. Representative immunoblot from a total of 20 different experiments.

View larger version (150K): [in a new window]   Fig. 7. The localization of TRPL channels in arr25 pupae. Representative cross-sections of single ommatidia from dark-adapted wild type and arr25 null-mutants at 72 hours and 90 hours APF, and post-eclosion, immunostained for TRPL. At 72 hours APF, the rhabdomeres of photoreceptors were visible (phase) with low levels of TRPL staining present in the cell body of photoreceptors. At 90 hours APF, TRPL was localized at the base of the rhabdomeres of wild-type photoreceptors, similar to the adult. At 90 hours APF, TRPL staining in the arr25 pupae appeared in both the rhabdomeres and dispersed in the cell body. This is in contrast to the adult arr25 null-mutant, which displayed TRPL staining in the stalk membranes and at the base of the rhabdomeres. Interestingly, puncta of TRPL staining were present in arr25 pupae 90 hours APF that were not seen in wild type. The corresponding phase-contrast images for the arr25 mutants are shown below (phase). Shown for each genotype and light condition are representative ommatidia from multiple retinal tissue sections; wild-type: five eyes of four flies (72hrs APF), six eyes of five flies (90hrs APF); arr25: four eyes of three flies (72hrs APF), seven eyes of six flies (90hrs APF). For number of eyes and flies used for adult ommatidia, see Figs 1 and 5.

View larger version (18K): [in a new window]   Fig. 8. Proposed model for the two stages of light-induced TRPLchannel translocation. A diagram of a single photoreceptor cell with rhabdomere (R) and cell body (C) is shown. The apical (gray) and basolateral (orange) membranes are also indicated. Arr-2 is required for maintaining TRPL channels (green) in the rhabdomeres. During Stage 1, TRPL channels are released from the rhabdomere and translocate by lateral diffusion into the stalk membrane, where the channels are restricted to the apical membrane by the AJs (open circles). Stage 1 requires Rh1 and the effector PLC. In Stage 2, TRPL channels bypass the AJs and translocate to the basolateral membrane. Stage 2 requires Rh1, PLC, TRP and eye-PKC.