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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 (ninaE^I17), PLC (norpA^P41), TRP (trp^P343) and eye-PKC (inaC^P109) 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).

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