14 research outputs found
C. elegansの低温耐性における温度情報伝達の分子制御機構
温度は生物の生存にとって欠かせない環境情報である。生物は、めまぐるしく変化する環境温度下において生存するために、複雑な体内メカニズムを巧みに働かせている。しかし、この分子制御メカニズムには未知の点が残されている。本研究では、シンプルなモデル生物である線虫Caenorhabditis elegansを用いて動物の温度応答メカニズムの解明を目指し解析を行った。動物の低温に対する応答メカニズムを解析するための実験系として、C. elegansの低温耐性現象を見つけた。低温耐性とは、15℃で飼育された個体は2℃の低温刺激後も生存できるが、20℃や25℃で飼育された個体は2℃の低温刺激後に死滅する現象である。また、25℃飼育個体は、わずか3時間15℃に置くことで2℃の低温刺激後も生存できるようになる。本研究では、この現象を指標にして低温耐性を制御する分子生理機構と組織ネットワークの解析をおこなった。低温耐性を制御する細胞を同定するために、神経系や腸などの組織に異常をもつ変異体の低温耐性を解析したところ、感覚ニューロンで機能するcGMP依存性チャネルTAX-4の変異体において低温耐性の異常が観察された。さらに、tax-4変異体の頭部のASJ感覚ニューロンでtax-4遺伝子を発現させたところ、低温耐性の異常が回復した。これまでに、ASJ感覚ニューロンは光を感じる感覚ニューロンとして知られていた。そこで、ASJ感覚ニューロンが光だけでなく温度をも感知しているのかを、カルシウムイメージング解析を用いて調べたところ、ASJの温度に対する応答性が確認された。ASJ感覚ニューロンの光情報伝達に関わる3量体Gタンパク質経路の変異体を調べたところ、低温耐性の異常がみられた。つまり、光と温度は同じ細胞において共通の分子経路を介して情報を下流へ伝達することが示唆された。低温耐性の表現型とカルシウムイメージングによる解析から、ASJ感覚ニューロンにおいて、複数のGタンパク、グアニル酸シクラーゼ、ホスホジエステラーゼが各々共同的に機能していることが示唆された。ASJはシナプス部位からインスリンを分泌し、腸や神経系がインスリンを受容することが示唆された、さらに、インスリン情報伝達経路の下流で機能するFOXO型転写因子DAF-16による遺伝子の発現制御などを受けて全身の低温耐性が制御されることが見つかった。甲南大学平成28年(2016年度
Regulation of temperature habituation through neuron and intestine in nematode <i>Caenorhabditis elegans</i>
Sperm Affects Head Sensory Neuron in Temperature Tolerance of Caenorhabditis elegans
Tolerance to environmental temperature change is essential for the survival and proliferation of animals. The process is controlled by various body tissues, but the orchestration of activity within the tissue network has not been elucidated in detail. Here, we show that sperm affects the activity of temperature-sensing neurons (ASJ) that control cold tolerance in Caenorhabditis elegans. Genetic impairment of sperm caused abnormal cold tolerance, which was unexpectedly restored by impairment of temperature signaling in ASJ neurons. Calcium imaging revealed that ASJ neuronal activity in response to temperature was decreased in sperm mutant gsp-4 with impaired protein phosphatase 1 and rescued by expressing gsp-4 in sperm. Genetic analysis revealed a feedback network in which ASJ neuronal activity regulates the intestine through insulin and a steroid hormone, which then affects sperm and, in turn, controls ASJ neuronal activity. Thus, we propose that feedback between sperm and a sensory neuron mediating temperature tolerance
Calcium concentration changes in ASJ of PDE mutants with warming stimuli.
<p>(A–F) Calcium imaging of ASJ from PDE mutants cultivated at 20°C. The transgene of <i>trx-1p</i>::<i>yc3</i>.<i>60</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.g003" target="_blank">Fig 3B</a> (pale blue lines in panels A–F.; the experiments were performed simultaneously; <i>n</i> = 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.g003" target="_blank">3B</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.g003" target="_blank">3B</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.g003" target="_blank">3B</a> and 5A–F. Error bars indicate standard error of mean (A–I). Analysis of variance followed by Dunnett’s <i>post-hoc</i> test was used for multiple comparisons (G–I, compared with the wild type). *<i>P</i> < 0.05; **<i>P</i> < 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.</p
Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in <i>Caenorhabditis elegans</i>
<div><p>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 <i>Caenorhabditis elegans</i>. 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.</p></div
Genetic redundancy of Gα protein in cold tolerance.
<p>(A) Molecular model for the G-protein-mediated pathway of light sensation by the ASJ sensory neuron [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.ref019" target="_blank">19</a>]. Gα, a trimeric G-protein α subunit (<i>goa-1</i> or <i>gpa-3</i>), GC, guanylyl cyclase (<i>daf-11</i> or <i>odr-1</i>), cGMP, cyclic guanosine monophosphate, PDE, phosphodiesterase (<i>pde-1</i>, <i>pde-2</i> or <i>pde-5</i>). (B) Schema indicating cold tolerance of <i>C</i>. <i>elegans</i>. 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 <i>post-hoc</i> test was used for multiple comparisons. *<i>P</i> < 0.05; **<i>P</i> < 0.01.</p
A molecular model for light and temperature signaling in ASJ sensory neuron, which controls cold tolerance.
<p>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.</p
Genetic epistasis between PDE and Gα mutations on temperature signaling.
<p>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, <i>gpa-1</i> and <i>pde-5</i> mutants, respectively. The yellow line indicates calcium concentration in the <i>pde-5; gpa-1</i> mutants (<i>n</i> = 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, <i>gpa-1</i> and <i>pde-3</i> mutants, respectively. The yellow line indicates calcium concentration in the <i>pde-3; gpa-1</i> mutants. <i>n</i> = 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 <i>post-hoc</i> test was used for multiple comparisons. *<i>P</i> < 0.05; **<i>P</i> < 0.01. NS, not significant (<i>P</i> > 0.05).</p
Calcium concentration changes in ASJ of PDE mutants with cooling stimuli.
<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.g004" target="_blank">Fig 4B</a> (pale blue lines; these experiments were performed simultaneously; <i>n</i> = 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.g004" target="_blank">4B</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.g004" target="_blank">4B</a> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165518#pone.0165518.g004" target="_blank">4B</a> and 6A–F. Error bars indicate SEM (A–I). Analysis of variance followed by Dunnett’s <i>post-hoc</i> test was used for multiple comparisons (G–I, compared with the wild type). *<i>P</i><0.05; **<i>P</i> < 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.</p
Functional redundancy of Gα at ASJ ciliated endings.
<p>(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 <i>goa-1p</i> for <i>goa-1</i>’s own promoter, <i>gpa-1p for gpa-1</i>’s own promoter, <i>gpa-3p for gpa-3</i>’s own promoter, <i>gcy-5p</i> as a promoter for expressing genes in the ASER gustatory neuron, <i>ceh-36p</i> as a promoter for expressing genes in the AWC sensory neuron of the thermotaxis neural circuit, and <i>trx-1p</i> 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 <i>post-hoc</i> test was used for multiple comparisons. **Significantly different (<i>P</i> < 0.01).</p