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. 2016 Oct 27;11(10):e0165518.
doi: 10.1371/journal.pone.0165518. eCollection 2016.

Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans

Tomoyo Ujisawa  1   2 , Akane Ohta  1   3   2 , Misato Uda-Yagi  2 , Atsushi Kuhara  1   3   2
Affiliations

Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans

Tomoyo Ujisawa et al. PLoS One. .

Abstract

Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Genetic redundancy of Gα protein in cold tolerance.
(A) Molecular model for the G-protein-mediated pathway of light sensation by the ASJ sensory neuron [19]. Gα, a trimeric G-protein α subunit (goa-1 or gpa-3), GC, guanylyl cyclase (daf-11 or odr-1), cGMP, cyclic guanosine monophosphate, PDE, phosphodiesterase (pde-1, pde-2 or pde-5). (B) Schema indicating cold tolerance of C. elegans. Wild-type animals died after a cold stimulus of 2°C for 24 hours, when they were cultivated at 20°C. In contrast, wild-type animals cultivated at 15°C survived. (C) Cold-tolerance survival phenotypes of Gα mutants. Full allele names of the double and triple mutants are the same as those of the single mutants. For each assay, n ≥ 12. Analysis of variance followed by Dunnett's post-hoc test was used for multiple comparisons. *P < 0.05; **P < 0.01.
Fig 2
Fig 2. Functional redundancy of Gα at ASJ ciliated endings.
(A) Intracellular localization of each Gα protein in the ASJ sensory neuron. The wild type with ASJ-specific expression of dsRedm and each Gα (GPA-1, GPA-3 or GOA-1,)::Venus were analyzed by confocal microscopy. The top panel is a schematic diagram of the ASJ sensory neuron. The left-hand images indicate localization of Gα::Venus in ASJ. The center images indicate dsRedm localization in ASJ. The right-hand panels are merged images of the left and center images and the bright-field image. The arrows in the panels indicate co-localization sites of Gα::Venus and dsRedm in ASJ. Scale bar, 10 μm. (B) Specific expression of Gα genes in the ASJ sensory neuron partially rescues the abnormal cold tolerance of the Gα triple mutant. 20°C-cultivated Gα triple mutants showed abnormal enhancement of cold tolerance, which was partially rescued by expressing individual Gα genes in ASJ. In this figure, we used goa-1p for goa-1’s own promoter, gpa-1p for gpa-1’s own promoter, gpa-3p for gpa-3’s own promoter, gcy-5p as a promoter for expressing genes in the ASER gustatory neuron, ceh-36p as a promoter for expressing genes in the AWC sensory neuron of the thermotaxis neural circuit, and trx-1p as a promoter for specifically expressing genes in the ASJ thermosensing neuron. For each assay, n ≥ 10. Error bars indicate standard errors of the means. Analysis of variance followed by Dunnett's post-hoc test was used for multiple comparisons. **Significantly different (P < 0.01).
Fig 3
Fig 3. Calcium concentration changes in ASJ of Gα mutants with warming stimuli.
(A) Wild type with ASJ specific expression of YC3.60 [trx-1p::yc3.60] used for calcium imaging. The schematic diagram indicates an ASJ sensory neuron in the head. The lower pair of images shows the response to a warming stimulus of the ASJ cell body in the 20°C-cultivated wild type. Arrows indicate ASJ cell body. (B) In vivo calcium imaging of ASJ from the wild type cultivated at 20°C. Relative calcium concentrations were measured as the yellow/cyan fluorescence ratio of YC3.60. The calcium concentration in ASJ changed upon the warming stimulus (n = 18). (C–G) Calcium imaging of ASJs in Gα mutants cultivated at 20°C. The transgene of trx-1p::yc3.60 was introduced into each Gα mutant and we measured relative calcium concentrations under warming stimuli, as in the wild-type experiment (panel B, shown as pale blue lines in panels C–G; these experiments were performed simultaneously; n = 18–20). The temperature change during this test is indicated in the bottom chart. (H) The bar chart shows the average ratio changes in the period 5 s before the maximum point to 5 s after the maximum point in the experiments shown in graphs B–G. (I) The bar chart shows the average ratio changes around the minimum point during 10 s from 280 s to 290 s in the experiments shown in graphs B–G. (J) The bar chart shows the differences between the average maximum and minimum values shown in graphs H and I. Error bars indicate SEM (B–J). Analysis of variance followed by Dunnett's post-hoc test was used for multiple comparisons (H–J, compared with the wild type). *P < 0.05; **P < 0.01. Colors used in the bar graphs in H–J, are the same as those used for the corresponding response curves in B–G.
Fig 4
Fig 4. Calcium concentration changes in ASJ of Gα mutants with cooling stimuli.
ASJ specifically expressing YC3.60 in wild-type worms and Gα mutants. (A) The schematic diagram indicates an ASJ sensory neuron in the head. The bottom panel shows the response to cooling stimuli of the ASJ cell body in 20°C-cultivated wild type, with a scale showing pseudo-color images depicting the fluorescence ratio of cameleon before and during temperature change. Arrows indicate ASJ cell body. (B) In vivo calcium imaging of ASJ from wild-type samples cultivated at 20°C. Relative calcium concentrations were measured as the yellow/cyan fluorescence ratio of YC3.60. The calcium concentration in ASJ changed upon temperature warming stimuli. n = 14. (C–G) relative calcium concentrations under cooling stimuli in Gα mutants, as in the wild-type experiment (panel B; shown as pale blue lines in C–G; these experiments were performed simultaneously; n = 18–20). Time and temperature in the experimental field during this test are indicated in the bottom chart. (H) The bar chart shows the average ratio during 5 s before the maximum point to 5 s after the maximum point in graphs B–G. (I) The bar chart shows the average ratio change around the minimum point during 10 s from 280 to 290 s in graphs B–G. (J) The bar chart shows the average ratio change of the difference value between the maximum and minimum points in graphs B–G. Error bars indicate SEM (B–J). Analysis of variance followed by Dunnett’s post-hoc test was used for multiple comparisons (H–J, compared with the wild type). *P < 0.05; **P < 0.01. Colors used in the bar graphs in H–J, are the same as those used for the corresponding response curves in B–G.
Fig 5
Fig 5. Calcium concentration changes in ASJ of PDE mutants with warming stimuli.
(A–F) Calcium imaging of ASJ from PDE mutants cultivated at 20°C. The transgene of trx-1p::yc3.60 was introduced into each PDE, mutant and the relative calcium concentration changes under warming stimuli were measured as in the wild-type experiment shown in Fig 3B (pale blue lines in panels A–F.; the experiments were performed simultaneously; n = 15–19). The temperature change during the experiment is indicated in the bottom chart. (G) The bar chart shows the average ratio changes from 5 s before the maximum point to 5 s after the maximum point in Figs 3B and 5A–F. (H) The bar chart shows the average ratio changes from around minimum point during 10 s from 280 to 290 s of the experiments shown in Figs 3B and 5A–F. (I) The bar chart shows the average ratio changes of the difference values between maximum and minimum points of the experiments shown in Figs 3B and 5A–F. Error bars indicate standard error of mean (A–I). Analysis of variance followed by Dunnett’s post-hoc test was used for multiple comparisons (G–I, compared with the wild type). *P < 0.05; **P < 0.01. Colors used in the bar graphs in G–I, are the same as those used for the corresponding response curves in A–F.
Fig 6
Fig 6. Calcium concentration changes in ASJ of PDE mutants with cooling stimuli.
ASJ specifically expressing YC3.60 in wild-type worms and PDE mutants. (A–F) we measured relative calcium concentrations under cooling stimuli as in the wild-type experiment shown in Fig 4B (pale blue lines; these experiments were performed simultaneously; n = 13–21). Temperature changes during the experiment are indicated in the bottom chart. (G) The bar chart shows the average ratio changes from 5 s before the maximum point to 5 s after the maximum point in Figs 4B and 6A–F. (H) The bar chart shows the average ratio changes from around minimum point during 10 s from 280 to 290 s of the experiment in Figs 4B and 6A–F. (I) The bar chart shows the average ratio changes of the difference values between maximum and minimum points of the experiment in Figs 4B and 6A–F. Error bars indicate SEM (A–I). Analysis of variance followed by Dunnett’s post-hoc test was used for multiple comparisons (G–I, compared with the wild type). *P<0.05; **P < 0.01. Colors used in the bar graphs in G–I, are the same as those used for the corresponding response curves in A–F.
Fig 7
Fig 7. Genetic epistasis between PDE and Gα mutations on temperature signaling.
ASJ specifically expressing YC3.60 in wild-type worms and Gα or/and PDE mutants. (A) we measured relative calcium concentrations under warming stimuli. The blue, green, and magenta lines indicate calcium concentration changes in wild-type animals, gpa-1 and pde-5 mutants, respectively. The yellow line indicates calcium concentration in the pde-5; gpa-1 mutants (n = 9–14). Temperature changes during the experiment are indicated in the lower chart. (B) The bar chart shows the average ratio changes from 5 s before the maximum point to 5 s after the maximum point of the experiment shown in panel A. (C) The bar chart shows the average ratio changes from around minimum point during 10 s from 280 to 290 s of the experiment shown in panel A. (D) The bar chart shows the average ratio changes of the difference values between maximum and minimum points of the experiment shown in panel A. Colors used in graphs B–D are the same as those used for the corresponding response curves in A. (E) We measured relative calcium concentrations under cooling stimuli. The blue, green, and magenta lines indicate calcium concentration changes in wild-type animals, gpa-1 and pde-3 mutants, respectively. The yellow line indicates calcium concentration in the pde-3; gpa-1 mutants. n = 11–13. Temperature changes during the experiment are indicated in the bottom chart. (F) The bar chart shows the average ratio changes from 5 s before the maximum point to 5 s after the maximum point in Fig 7E. (G) The bar chart shows the average ratio change from around minimum point during 10 s from 280 to 290 s of the experiment in Fig 7E. (H) The bar chart shows the average ratio change of the difference value between maximum and minimum points of the experiment in Fig 7E. Colors used in graphs F-H are the same as those used for the corresponding response curves in E. Error bars indicate SEM (A–H). Analysis of variance followed by Dunnett’s post-hoc test was used for multiple comparisons. *P < 0.05; **P < 0.01. NS, not significant (P > 0.05).
Fig 8
Fig 8. A molecular model for light and temperature signaling in ASJ sensory neuron, which controls cold tolerance.
Some molecules are thought to be common, and some specific, to the temperature- and light-signaling pathways of ASJ. Gene name shown in bold indicate molecules specific to temperature signaling. Because the temperature response was not completely extinguished in the mutants in this study, unidentified signaling molecules such as Gα, GC and PDE may also be required for temperature signaling. The temperature receptor has not been identified.
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Grants and funding

AK was supported by the Naito Foundation, the Uehara Memorial Foundation, the Cosmetology Research Foundation, the Hirao Taro Foundation of KONAN GAKUEN for Academic Research, a JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (15H04404) and Grant-in-Aid for Challenging Exploratory Research (26640018), and a Grant-in-Aid for Scientific Research on Innovative Areas, Thermal Biology (15H05928), from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. AO was supported by the Kato Memorial Bioscience Foundation, the Shiseido Female Researcher Science Grant, the Japan Science Society, JSPS KAKENHI, a Grant-in-Aid for Young Scientists (B) (15K18579), and a Grant-in-Aid for JSPS Fellows PD (16J00123), Japan. TU was supported by a Grant-in-Aid for JSPS Fellows DC (15J04977), Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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