Research ArticleGENETICS

Cold acclimation via the KQT-2 potassium channel is modulated by oxygen in Caenorhabditis elegans

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Science Advances  06 Feb 2019:
Vol. 5, no. 2, eaav3631
DOI: 10.1126/sciadv.aav3631
  • Fig. 1 KQT-2 in sensory neurons regulates cold acclimation.

    (A) Cold acclimation assay. Wild-type animals cultivated at 25°C fail to survive at 2°C, whereas animals cultivated at 15°C survive at 2°C. When wild-type animals cultivated at 25°C are transferred to and conditioned at 15°C for 5 hours, they exhibit increased survival at 2°C. (B to D) Cold acclimation of kqt-2(ok732) mutants assayed by the 25°C→15°C→2°C protocol. kqt-2(ok732) exhibited supranormal cold acclimation. Number of assays ≥ 12. (E to G) Cold acclimation of kqt-2(ok732) mutants assayed by the 15°C→25°C→2°C protocol. Number of assays ≥ 10. (B to G) Error bar indicates SEM. Comparisons were performed using the unpaired t test (Welch). *P < 0.05, **P < 0.01. (H) Two kqt-2 loss-of-function mutants exhibited supranormal cold acclimation. Number of assays ≥ 11. Error bar indicates SEM. Comparisons were performed using Dunnett’s test. *P < 0.05, **P < 0.01. (I) Transgenic rescue of kqt-2(ok732) with a genomic fragment encompassing the wild-type kqt-2(+) gene and native promoter sequence. Number of assays ≥ 8. (J) Rescue of kqt-2 mutants by tissue-specific expression of kqt-2 cDNA. Number of assays ≥ 9. (I and J) Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01. (K to N) kqt-2::GFP driven by the kqt-2 promoter is expressed in ADL and ASK head sensory neurons. GFP fluorescence of ADL and ASK (yellow) neurons are colabeled with DiI, which labels only six pairs of amphid sensory neurons in the head with red fluorescence (magenta). (K) Lateral view. A, anterior; P, posterior; D, dorsal; V, ventral. (L) Ventral view. L, left; R, right. (M) GFP driven by the kqt-2 promoter is expressed in fan and ray sensory neurons in the tail of males. (N) Wild-type animal expressing kqt-2cDNA::gfp (8 ng/μl) using a 9.0-kb kqt-2 promoter. KQT-2::GFP is observed in whole sensory neurons and is especially localized to sensory endings and cell bodies of head sensory neurons.

  • Fig. 2 KQT-2 in ADL neurons is necessary for cold acclimation and a Ca2+ response in response to temperature stimuli.

    (A) Abnormal kqt-2(ok732) cold acclimation was rescued by expressing kqt-2cDNA in ADL, not ASK neurons. Number of assays ≥ 18. Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01. (B) Avoidance behavior against 1-octanol received by ADL, AWB, and ASH sensory neurons. Number of assays ≥ 9. Error bar indicates SEM. Comparisons were performed using Dunnett’s test. *P < 0.05, **P < 0.01. (C to L) Ca2+ imaging of ADL neurons in animals cultivated at 15° or 25°C in response to temperature stimuli from 17° to 23°C. The graphs indicate the change in yellow fluorescent protein (YFP)/cyan fluorescent protein (CFP) ratio in response to temperature stimuli. The bar graphs indicate the average change in ratio from 160 to 170 s (D and H) or from 170 to 180 s (F, J, and L). (C and D) In vivo calcium imaging of ADL neurons in the wild type cultivated at 25° or 15°C. n ≥ 16. (E and F) The responsiveness of ADL neurons to temperature change is abrogated in the kqt-2 mutant and restored by ADL-specific expression of kqt-2(+). n ≥ 18. (G and H) In vivo calcium imaging of ADL neurons in the wild type and kqt-2 mutant cultivated at 15°C. n ≥ 16. (I and J) Overexpression of kqt-2(+) in wild-type ADL neurons increases Ca2+ response to temperature stimuli. n ≥ 25. (K and L) Increased Ca2+ response in ADL neurons in kqt-3(aw1) and kqt-3(aw1);kqt-2(ok732) mutants. n ≥ 16. (D, H, and J) Error bar indicates SEM. Comparisons were performed using the unpaired t test (Welch). *P < 0.05, **P < 0.01. (F and L) Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01 NS, not significant. Analysis by two-way analysis of variance (ANOVA) confirmed that there was a significant effect of the kqt-2 genotype (P = 0.042) and also a significant effect of the kqt-3 genotype (P = 2.20 × 10−8), but there was no significant interaction between kqt-2 and kqt-3 genotypes (P = 0.133; see Supplementary raw data file). Data of wild-type animals cultivated at 25°C, shown in (C), are shared with (E), (I), and (K), Figs. 3 (D, F, and H) and 4D, and fig. S6A, given that the experiments were conducted simultaneously (C, E, I, and K). Data of wild-type animals cultivated at 15°C, shown in (C), are shared with (G), given that the experiments were conducted simultaneously (C and G). Data of kqt-2(ok723) cultivated at 25°C, shown in (E), were shared with (K) and Figs. 3H and 4D, given that the experiments were conducted simultaneously (E and K). (M) Epistasis analyses of cold acclimation in kqt-2 and kqt-3 mutants. All mutant strains exhibit supranormal cold acclimation, but the kqt-3(aw1) cold acclimation phenotype appears epistatic to kqt-2(ok732). Number of assays ≥ 12. Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01.

  • Fig. 3 OCR-1 is a negative regulator of the thermal response in ADL sensory neurons.

    (A and B) ocr-1(ok132) and ocr-2(ak47) mutants did not show abnormal cold acclimation. Number of assays ≥ 13. Error bar indicates SEM. Comparisons were performed using Dunnett’s test. (C) Supranormal cold acclimation of kqt-2(ok732) was suppressed by ocr-1(ok132). Number of assays ≥ 8. Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01. (D to I) Ca2+ imaging of ADL neurons in animals cultivated at 25°C in response to a defined temperature ramp. The graph indicates YFP/CFP ratio change in response to temperature stimuli. The bar graph indicates the average ratio change from 140 to 150 s (E, G, and I). (D and E) ocr-1(ok132) increases the Ca2+ response to temperature stimuli. n ≥ 17. (F and G) Increased Ca2+ thermal response of ADL neurons in ocr-1(ok132) is suppressed by ocr-2(ak47) or osm-9(ky10) mutations. n ≥ 18. The Ca2+ thermal responses in ocr-2(ak47) and osm-9(ky10) single mutants are described in fig. S6. (H and I) Abrogation of thermal response by kqt-2(ok732) loss of function is suppressed in ocr-1(ok132);kqt-2(ok732) double mutants. n ≥ 18. (E) Error bar indicates SEM. Comparisons were performed using Dunnett’s test. *P < 0.05, **P < 0.01. (G and I) Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01. Wild-type data in (D) are shared with (F) and (H), Figs. 2 (C, E, I, and K) and 4D, and fig. S6A, given that the experiments were conducted simultaneously (D, F, and H). kqt-2(ok723) data in (H) are shared with Figs. 2 (E and K) and 4D, given that the experiments were conducted simultaneously. ocr-1 data in (D) are shared with (F) and (H), given that the experiments were conducted simultaneously (D, F, and H).

  • Fig. 4 Oxygen signaling modulates ADL-mediated cold acclimation and thermal responsiveness.

    (A) Cold acclimation of animals cultivated on 3.5- and 6.0-cm-diameter plates. Number of assays ≥ 8. Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01. (B) Oxygen concentration at the surface of 3.5- and 6.0-cm-diameter agar plates with bacterial lawns and worms cultured from 24 to 64 hours after egg laying. Larger 6.0-cm-diameter plates exhibit higher sustained O2 concentrations. For each point, n = 10. Error bar indicates SEM. Comparisons were performed using the unpaired t test (Welch). *P < 0.05, **P < 0.01. (C) gcy-35 mutant, lacking the intracellular O2 sensor, suppresses the supranormal cold acclimation of kqt-2(ok732). Number of assays ≥ 11. Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01. (D and E) Abrogation of Ca2+ thermal response in ADL neurons of kqt-2(ok732) is suppressed in kqt-2(ok732);gcy-35(ok769). n ≥ 18. Error bar indicates SEM. Comparisons were performed using the Tukey-Kramer method. *P < 0.05, **P < 0.01. The bar graph indicates the average ratio change from 160 to 170 s (E). Wild-type data in (D) are shared with Figs. 2 (C, E, I, and K) and 3 (D, F, and H) and fig. S6A, given that the experiments were conducted simultaneously (D). kqt-2(ok723) data in (D) are shared with Figs. 2 (E and K) and 3H, given that the experiments were conducted simultaneously (D). (F) Enhanced cold acclimation of kqt-2 was recovered to a normal level after cultivation under 5% O2. Number of assays ≥ 9. Error bar indicates means SEM. Comparisons were performed using Dunnett’s test. *P < 0.05, **P < 0.01. (G) Model of molecular mechanisms regulating activity in ADL neurons. OCR-1 acts as a negative regulator of OCR-2/OSM-9 thermally responsive TRP channels in ADL neurons. KQT-2 acts as a negative regulator of KQT-3 potassium channels. (H) Model of neuronal circuitry integrating ADL temperature sensing with oxygen signaling by URX visceral oxygen sensory neurons via RMG hub interneurons. In wild-type worms, URX neurons may indirectly modulate temperature signaling of ADL neurons in cold acclimation. Left: Sensing of high O2 levels by URX neurons, which is dependent on GCY-35, is signaled through RMG to ADL neurons via chemical and electrical synapses to promote cold acclimation, presumably by inhibiting ADL excitability. Middle: Loss of KQT-2 in ADL neurons results in decreased neuronal activity in response to temperature change and increased cold acclimation. Loss of GCY-35 blocks active O2 signaling from URX neurons, mimicking a low O2 environment. This increases ADL excitability sufficiently to suppress the inhibitory effect of KQT-2 loss of function. Right: Supranormal cold acclimation observed in kqt-2 mutants is thus suppressed by gcy-35 mutations or low O2 concentration.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/2/eaav3631/DC1

    Supplementary Results and Discussion

    Supplementary Methods

    Fig. S1. Cold acclimation assays of selected mutant animals defective in genes identified by previous DNA microarray analysis.

    Fig. S2. Localization analysis of KQT-2.

    Fig. S3. Lipid composition of wild-type and kqt-2(ok732).

    Fig. S4. Behavioral assays of kqt-2(ok732).

    Fig. S5. Cold acclimation assays of kqt-1(ok413).

    Fig. S6. Ca2+ imaging of ADL neurons in mutants defective in TRP channels expressed in ADL neurons.

    Fig. S7. Cold acclimation of animals cultivated with O2 concentrations of 20, 10, and 5%.

    Fig. S8. Cold acclimation assays with mutants defective in GSP-4, a sperm-specific protein phosphatase (PP1).

    Supplementary raw data file

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Results and Discussion
    • Supplementary Methods
    • Fig. S1. Cold acclimation assays of selected mutant animals defective in genes identified by previous DNA microarray analysis.
    • Fig. S2. Localization analysis of KQT-2.
    • Fig. S3. Lipid composition of wild-type and kqt-2(ok732).
    • Fig. S4. Behavioral assays of kqt-2(ok732).
    • Fig. S5. Cold acclimation assays of kqt-1(ok413).
    • Fig. S6. Ca2+ imaging of ADL neurons in mutants defective in TRP channels expressed in ADL neurons.
    • Fig. S7. Cold acclimation of animals cultivated with O2 concentrations of 20, 10, and 5%.
    • Fig. S8. Cold acclimation assays with mutants defective in GSP-4, a sperm-specific protein phosphatase (PP1).

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