Fig 1.
Mutations in inx-19 and inx-18 result in hypersensitivity to quinine.
A,B) Diagram of inx-19 and inx-18 alleles used. Innexin genes code for proteins that consist of 4 transmembrane helices with intracellular N and C tails. Inx-19(ky634) is a SNP resulting in an E>K substitution within the first extracellular loop, while inx-19(tm1896) is an in-frame deletion of 546bp that removes most of the intracellular loop and a portion of the third transmembrane domain. Inx-18(ok2454) is a ~1800bp deletion that removes the second-fourth transmembrane domains and a portion of the C-terminus. C) Quinine Drop Test with 1 mM quinine. Inx-19(ky634), inx-19(tm1896), and inx-18(ok2454) mutant animals are hypersensitive to 1 mM quinine, responding a greater percentage of the time. N2 (wild-type) = 18%, n = 510; inx-19(ky634) = 65%, n = 120, p<0.0001; inx-19(tm1896) = 44%, n = 390, p<0.0001; inx-18(ok2454) = 44%, n = 350, p<0.0001.
Fig 2.
Expression of inx-19 and inx-18 in ASK and ASH restores wild-type quinine sensitivity.
A) Expression of inx-19 isoform A cDNA under the native promoter in inx-19(tm1896) animals rescued quinine sensitivity to N2 (wild-type) levels. Expression in ASK (Psra-9, which expresses solely in ASK [46]) or ASH (Posm-10, which also expresses in the tail neurons PHA and PHB as well as weakly in ASI [47, 48]) alone did not significantly rescue the behavior, while simultaneous expression did. N2 = 15%, n = 220; inx-19(tm1896) = 46%, n = 210; inx-19;Pinx-19::inx-19cDNA = 18%, n = 100, p = 0.62 vs N2, p<0.0001 vs inx-19; inx-19;Psra-9::inx-19cDNA = 32%, n = 100, p = 0.0009 vs N2, p = 0.02 vs inx-19; inx-19;Posm-10::inx-19cDNA = 37%, n = 110, p<0.0001 vs N2, p = 0.13 vs inx-19; inx-19;Psra-9::inx-19cDNA; Posm-10::inx-19cDNA = 22%, n = 110, p = 0.16 vs N2, p<0.0001 vs inx-19. B) Expression of inx-18 gDNA in inx-18(ok2454) animals rescued the quinine hypersensitivity phenotype, as did expression of inx-18 cDNA in ASK (Psra-9). N2 = 13%, n = 120; inx-18(ok2454) = 48%, n = 120; inx-18;inx-18gDNA = 12%, n = 100, p = 0.84 vs N2, p<0.0001 vs inx-18; inx-18;Psra-9::inx-18cDNA = 14%, n = 120, p>0.99 vs N2, p<0.0001 vs inx-18.
Fig 3.
INX-19 and INX-18 colocalize in the nerve ring when expressed in ASK and ASH.
A) Diagram of the C. elegans head in a dorsal view. Dashed box indicates the location of imaging of ASK and ASH axons in the nerve ring. B-D) INX-19 expressed in both ASK (where it is tagged with GFP) (B) and ASH (where it is tagged with mCherry) (C) forms multiple puncta that colocalize along the ASK-ASH axons in wild-type animals. Points of colocalization are indicated with white arrowheads. ASK and ASH are additionally expressing cytosolic mTagBFP2, seen in the axons that traverse the image, highlighted in D. E-G) INX-19 tagged with mCherry expressed in ASH (E) colocalizes in the nerve ring with GFP-tagged INX-18 expressed in ASK in wild-type animals (F). A white arrowhead indicates a point of colocalization. Cytosolic BFP fills the ASK-ASH axons, highlighted in G. H) Quantification of colocalization. In worms expressing INX-19 in ASK and ASH, 67% of nerve ring puncta colocalize (n = 144 puncta in 14 animals). In worms expressing INX-18 in ASK and INX-19 in ASH, ~4% of nerve ring puncta colocalize (n = 81 puncta in 10 animals). Each dot represents an individual worm, and error bars are ±SEM.
Fig 4.
inx-18 and inx-19 play distinct roles in ASK-ASH electrical synapse localization and function.
A) inx-19 cDNA was expressed using Psra-9 and fluorescent puncta in the nerve ring were counted in N2 (wild-type), inx-18(ok2454) and inx-19(tm1896) backgrounds. Each dot represents an individual worm and error bars are ±SEM. Ordinary one-way ANOVA between three groups showed significant differences (F[2,12] = 5.763, p = 0.02, α = 0.05). Dunnett’s multiple comparison test showed that INX-19 ASK puncta were decreased in inx-18(ok2454) (n = 5, p = 0.01) and in inx-19(tm1896) (n = 5, p = 0.05) in comparison to N2 (n = 5). B) inx-19 cDNA was expressed using Psrd-10 and puncta in the nerve ring were counted in N2, inx-18(ok2454) and inx-19(tm1896) backgrounds. Each dot represents an individual worm and error bars are ±SEM. Ordinary one-way ANOVA between three groups showed no significant differences (F[2,14] = 0.814, p = 0.46, α = 0.05). C) inx-18 cDNA was expressed using Psra-9 and puncta in the nerve ring were counted in N2, inx-18(ok2454) and inx-19(tm1896) backgrounds. Each dot represents an individual worm and error bars are ±SEM. Ordinary one-way ANOVA between three groups showed no significant differences (F[2,13] = 1.637, p = 0.23, α = 0.05). D) Inx-18(ok2454);inx-19(tm1896) double mutant animals were assayed for sensitivity to 1 mM quinine using the quinine drop test. Double mutants responded at higher rates than either inx-18 or inx-19 single mutants. N2 = 18%, n = 510; inx-19(tm1896) = 44%, n = 390; inx-18(ok2454) = 44%, n = 350; inx-19;inx-18 = 53%, n = 180, p = 0.05 vs inx-19, p = 0.05 vs inx-18.
Fig 5.
ASK and ASH architecture is unaltered in inx-18 and inx-19 mutant animals.
A) Diagram of neural architecture of ASK, ASH, and ASI in the C. elegans head. The dendrites reach out to the nose while the axons extend from the cell body into the nerve ring around the isthmus of the pharynx. B-D) Representative confocal images of the worm head with Psra-9::mCherry (ASK) and Posm-10::bfp (ASH and weakly in ASI) show cell bodies, dendrites extending to the nose, and axons projecting into the nerve ring. Images on the left include maximum intensity projections of the mCherry and BFP images superimposed upon a brightfield image to show location of cells; images on the right are maximum intensity projections of the mCherry and BFP channels without the brightfield image to show details of the cell architecture. Comparison between wild-type, inx-19(tm1896), and inx-18(ok2454) (15–20 animals per genotype were imaged) show no major differences in cell architecture.
Fig 6.
ASK Ca2+ responses to quinine presentation are unaltered in inx-18 and inx-19 mutant animals while ASH Ca2+ responses are heightened in both.
A) GCaMP6s fluorescence intensity in ASH in response to 10 mM quinine. Cells were imaged for 30s with presentation of quinine at 10s. The lite-1(ce314) mutation was included to eliminate blue-light induced calcium responses in ASK and ASH. All genotypes showed an increase in ASH GCaMP6s fluorescence in response to quinine presentation, though for lite-1;inx-19(tm1896) and lite-1;inx-18(ok2454) animals the response is larger and faster than that of lite-1(ce314). Averaged GCaMP6s traces are shown and error bars are ±SEM. n = 48 animals for all genotypes tested. B) GCaMP6s fluorescence intensity in ASK in response to 10 mM quinine. ASK showed small increases of GCaMP6s signals and there were no significant differences between genotypes. Averaged GCaMP traces are shown and error bars are ±SEM. n = 24, n = 21 and n = 22 animals imaged for lite-1(ce314), lite-1;inx-19 and lite-1;inx-18, respectively. C, D) Heatmaps showing individual traces from all worms analyzed. Data points in the heatmaps represent GCaMP6s signals normalized to the averaged fluorescence intensity of the first 3 seconds of imaging. E) Quantification of ASH fluorescence change at four seconds after quinine stimulation. One-way ANOVA between three groups showed significant differences (F[2,141] = 3.89, p = 0.02, α = 0.05), and Dunnett’s multiple comparison test showed that mean ASH GCaMP6s fluorescence change in lite-1(ce314) animals (n = 48) differed from both lite-1;inx-19 (n = 48, p = 0.02) and lite-1;inx-18 (n = 48, p = 0.05) animals. F) Quantification of ASK fluorescence change four seconds after quinine stimulation. One-way ANOVA between three groups showed no significant differences in ASK GCaMP6s fluorescence (F[2,64] = 0.202, p = 0.817, α = 0.05) between lite-1(ce314) (n = 24), lite-1;inx-19 (n = 21) and lite-1;inx-18 animals (n = 22).
Fig 7.
Mutations in inx-18 and inx-19 disrupt endogenous cGMP levels in ASK and ASH.
A) Diagram of FlincG3. The cGMP binding domains of PKG 1α (blue) are followed by circularly permuted EGFP (green) and a short PKG 1α tail (blue). WingG2 increases in brightness in response to cGMP. B) Example of FlincG3 and mScarlet expression within ASH. Ellipses were drawn around the cell body to measure fluorescence intensity. C) cGMP levels within the ASH cell body. The ratio between mean fluorescence intensity of FlincG3 and mScarlet signals was determined for each genotype. Decreases in ASH FlincG3 fluorescence were found in inx-18(ok2454) and inx-19(tm1896) mutant animals when compared to wild-type worms. Each data point was obtained from a single cell; error bars are ±SEM. One-way ANOVA between three groups showed significant differences (F[2,68] = 3.643, p = 0.03, α = 0.05), and Dunnett’s multiple comparison test showed that mean fluorescence intensity in lite-1(ce314) (n = 24) cells differed from both lite-1;inx-18 cells (n = 24, p = 0.05) and lite-1;inx-19 cells (n = 23, p = 0.04). D) cGMP levels within the ASK cell body. ASK FlincG3 fluorescence was not altered in inx-18(ok2454) mutant animals, and increased in inx-19(tm1896) mutant animals when compared to wild-type animals. Each data point was obtained from a single cell; error bars are ±SEM. One-way ANOVA between three groups showed significant differences (F[2,72] = 8.115, p = 0.0007, α = 0.05), and Dunnett’s multiple comparison test showed that mean fluorescence intensity in lite-1(ce314) cells (n = 26) did not differ from lite-1;inx-18 cells (n = 25, p = 0.87) but was increased in lite-1;inx-19 cells (n = 24, p = 0.0008).
Fig 8.
Model of ASK-ASH electrical synapse facilitation of ASH modulation.
Table 1.
DNA constructs.