C. elegans phototransduction requires a G protein-dependent cGMP pathway and a taste receptor homolog

Abstract

The eyeless animal C. elegans is able to sense light and engages in phototaxis behavior that is mediated by photoreceptor cells. However, the molecular and cellular mechanisms underlying phototransduction in C. elegans remain largely unclear. By recording the photoreceptor neuron ASJ in wild-type and various mutant worms, we found that phototransduction in ASJ is a G protein-mediated process and requires membrane-associated guanylate cyclases, but not typical phosphodiesterases. In addition, we found that C. elegans phototransduction requires LITE-1, a candidate photoreceptor protein known to be a member of the invertebrate taste receptor family. Our genetic, pharmacological and electrophysiological data suggest a model in which LITE-1 transduces light signals in ASJ via G protein signaling, which leads to upregulation of the second messenger cGMP, followed by opening of cGMP-sensitive CNG channels and stimulation of photoreceptor cells. Our results identify a phototransduction cascade in C. elegans and implicate the function of a 'taste receptor' in phototransduction.

Figure 1. Phototransduction in ASJ is a G-protein-mediated process

(a) Light-induced conductance in ASJ. Clamping voltage: −70 mV. Light stimulus: 350 ± 25 nm, 5 s, −1.75 log I/I_o. Worm photoreceptor cells are most sensitive to UV-A light. The downward “spikes” in this trace and many others in the paper are typical for many worm neurons that are very small (~1 pF; ~2 µm in diameter) and exhibit high input resistance. (b) Blocking G-protein signaling blocked phototransduction. mSIRK (50 µM) is membrane-permeable. (c–d) cGMP-evoked currents were not affected by mSIRK. cGMP: 1 mM. (e) Bar graph summarizing the data in (a–d). n ≥ 6 (photocurrents); n ≥ 4 (cGMP-induced currents). Error bars: SEM. **P < 0.002 (t test). (f) Activation of G-proteins opened CNG channels in the dark. GTPγS: 100 µM. Alleles: p671 for tax-2; p678 for tax-4. (g) Activation of Gi/o opened CNG channels in the dark. Mastoparon: 5 µM. (h) Bar graph summarizing the data in (f–g). n ≥ 6. **P < 0.0003 (ANOVA with Dunnett test). (i) Blocking Gi/o blocked phototransduction. PTX was expressed as a transgene in ASJ. (j) PTX blocked GTPγS- (top trace) but not cGMP-induced current (bottom trace). (k) The goa-1(n1134);gpa-3(pk35) double mutant lacked photocurrents. See supplementary figure 1 for single mutant data. (l) Mutations in goa-1 and gpa-3 blocked GTPγS- (top trace) but not cGMP-induced current (bottom trace). (m) Histogram summarizing the data in (i–l). n ≥ 5. Error bars: SEM. **P < 0.005 (ANOVA with Dunnett test).

Figure 2. Phototransduction in ASJ requires membrane-associated GCs

(a) Dendrogram of C. elegans and human PDEs (hPDEs). (b) The light-induced current was greatly potentiated in the pde-1,2,5 triple mutant pde-1(nj57)pde-5(nj49);pde-2(nj58). (c) The light-induced current was greatly potentiated in the pde-1,2,4,5 quadruple mutant pde-1(nj57)pde-5(nj49);pde-3(nj59);pde-2(nj58). A similar result (51.7 ± 3.28 pA pF⁻¹; n = 5) was also obtained with another quadruple mutant strain: pde-1(nj57)pde-5(nj49);pde-3(nj59);pde-2(tm3098). (d) The light-induced current was normal in the pde-4(nj60);pde-6(ok3410) double mutant. (e) Bar graphs summarizing the data in (b–d). n ≥ 7. Error bars: SEM. **P < 0.0001 (ANOVA with Dunnett test; compared to WT). (f) No light-induced current was detected in the GC mutants daf-11(ks67) and daf-11(m47). (g) The light-induced current in the GC mutant odr-1(n1936) was greatly reduced. (h) Bar graph summarizing the data in (f–g). daf-11(ks67) is temperature-sensitive, and all recordings involving this allele were done at 25°C in this study. All other recordings were performed at 20°C. The photocurrent density in wild-type recorded at 25°C was similar to that at 20°C (data not shown). n ≥ 7. Error bars: SEM. **P < 0.0005 (ANOVA with Dunnett test; compared to WT).

Figure 3. Guanylate cyclases act downstream of G-proteins and upstream of CNG channels to mediate phototransduction

(a–b) GC/DAF-11 acted downstream of G-proteins. Mutation in daf-11 blocked the ability of GTPγS in stimulating ASJ. Shown in (a) is a sample trace. n ≥ 5. Error bars represent SEM. **P < 0.00001 (t test). (c–d) GC/DAF-11 acted upstream of CNG channels. cGMP can efficiently open CNG channels in ASJ of daf-11 mutant worms. Shown in (c) is a sample trace. n ≥ 5. Error bars represent SEM.

Figure 4. Light, GTPγS and cGMP activate the same type of CNG channels in photoreceptor cells

(a) Light evoked an inward current in the pde-1,2,5 triple mutant under the classic whole-cell mode. (b) GDPβS blocked phototransduction. GDPβS (100 µM) was dialyzed into ASJ through the recording pipette. (c) Light and GTPγS acted on the same type of CNG channels. In the pde triple mutant, once CNG channels were activated by GTPγS, light cannot further induce an inward current. (d) Light and cGMP activated the same type of CNG channels. In the pde triple mutant, once CNG channels were activated by cGMP, light cannot further induce an inward current. (e) Bar graph summarizing the data in (a–d). n ≥ 6. Error bars: SEM. **P < 0.0001 (ANOVA with Dunnett test).

Figure 5. LITE-1 is required for phototransduction in photoreceptor cells

(a) Three mutants showed a strong defect in phototaxis behavior. Head avoidance response to UV-A light (2 s, −1.43 log I/I_o) was scored as previously described, . The response rate in xu7 and xu10 was similar to that of no-light control and likely resulted from spontaneous reversals. n ≥ 10. Error bars: SEM. **P < 0.00001 (ANOVA with Dunnett test; compared to wild-type). (b) lite-1 genomic structure and mutations identified in lite-1. We have identified two lite-1 isoforms. An SL1 sequence was found before the ATG in the second exon, indicating that it represents a short form of lite-1. This isoform was used in the current study. (c–e) LITE-1 was required for phototransduction in ASJ. Shown are sample traces of ASJ in wild-type (c), lite-1(xu7) (d), and lite-1(xu7) expressing a wild-type lite-1 transgene specifically in ASJ under the trx-1 promoter (e). See supplementary figure 5 for ASK traces. (f) Bar graph summarizing the data in (c–e). Error bars: SEM. n ≥ 7. **P < 0.00002 (ANOVA with Dunnett test; compared to WT). (g) Expression of a wild-type lite-1 transgene specifically in ASJ or ASK showed a rescuing effect on phototaxis behavioral defect in lite-1(xu7) mutant worms. The trx-1 and srg-8 promoter was used to drive expression of the transgene in ASJ and ASK, repectively, . Error bars: SEM. n ≥ 10. *P < 0.05 (ANOVA with Bonferroni test; compared to lite-1).

Figure 6. LITE-1 functions upstream of G-proteins

(a–b) LITE-1 acted upstream of G-proteins. GTPγS (a) and cGMP (b) can induce an inward current in ASJ of lite-1(xu7) mutant worms. (c) Bar graph summarizing the data in (a–b). The density of GTPγS and cGMP induced currents in ASJ of lite-1(xu7) mutant worms are similar to those in wild-type. n ≥ 6. Error bar: SEM. (d) LITE-1 acted upstream of GCs and CNG channels. Wild-type lite-1 was expressed as a transgene under the tax-2Δ promoter in the photoreceptor cells ASJ, ASK and AWB. This transgene can rescue the phototaxis defect in lite-1(xu7) mutant worms. This rescuing effect requires the GC DAF-11 and CNG channel TAX-2 and TAX-4. **P < 0.001 (ANOVA with Dunnett test; compared to the rescue). n ≥ 10. Error bars: SEM.

Figure 7. Transgenic expression of LITE-1 can confer photo-sensitivity to the photo-insensitive neuron ASI

(a) The ASI neuron was photo-insensitive. No photocurrent could be detected in ASI. (b) Expression of LITE-1 in ASI turned it into photo-sensitive. LITE-1 was expressed as a transgene in ASI under the sra-6 promoter that labels both ASI and ASH in the head . ASI recordings performed in ASH-ablated worms and non-ablated worms yielded similar results (9.1 ± 1.3 pA pF⁻¹ vs. 9.4 ± 1.8 pA pF⁻¹; n = 5). (c–e) The function of LITE-1 in ASI also required daf-11, tax-2 and tax-4, as mutations in these genes blocked LITE-1-dependent photocurrents in ASI. (f) Bar graph summarizing the data in (a–e). n ≥ 5. Error bars: SEM. **P < 0.00001 (ANOVA with Dunnett test; all compared to WT without transgene).